Ethnopharmacological Approaches to Wound Repair · 2019. 8. 7. · Ethnopharmacological Approaches to Wound Repair Guest Editors: Esra Küpeli Akkol, Fatma U. Aﬁﬁ, Saringat Hj

Ethnopharmacological Approaches to Wound RepairGuest Editors: Esra Küpeli Akkol, Fatma U. Afifi, Saringat Hj. Baie, Ashok D. Taranalli, and Shrikant Anant

Evidence-Based Complementary and Alternative Medicine

Ethnopharmacological Approaches toWound Repair

Evidence-Based Complementary and Alternative Medicine

Ethnopharmacological Approaches toWound Repair

Guest Editors: Esra Kupeli Akkol, Fatma U. Afifi,Saringat Hj. Baie, Ashok D. Taranalli, and Shrikant Anant

Copyright © 2013 Hindawi Publishing Corporation. All rights reserved.

This is a special issue published in “Evidence-Based Complementary and Alternative Medicine.” All articles are open access articlesdistributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in anymedium, provided the original work is properly cited.

Editorial Board

Terje Alraek, NorwayShrikant Anant, USASedigheh Asgary, IranHyunsu Bae, Republic of KoreaLijun Bai, ChinaSarang Bani, IndiaVassya Bankova, BulgariaWinfried Banzer, GermanyVernon A. Barnes, USADebra L. Barton, USAJairo Kenupp Bastos, BrazilSujit Basu, USADavid Baxter, New ZealandAndre-Michael Beer, GermanyAlvin J. Beitz, USAPaolo Bellavite, ItalyYong Chool Boo, Republic of KoreaFrancesca Borrelli, ItalyGloria Brusotti, ItalyArndt Bussing, GermanyWilliam C. S. Cho, Hong KongLeigh F. Callahan, USARaffaele Capasso, ItalyOpher Caspi, IsraelShun-Wan Chan, Hong KongIl-Moo Chang, Republic of KoreaChun-Tao Che, USAYunfei Chen, ChinaTzeng-Ji Chen, TaiwanKevin W. Chen, USAJuei-Tang Cheng, TaiwanEvan Paul Cherniack, USAJen-Hwey Chiu, TaiwanJae Youl Cho, KoreaShuang-En Chuang, TaiwanEdwin L. Cooper, USAVincenzo De Feo, ItalyRocio De la Puerta, SpainAlexandra Deters, GermanyDrissa Diallo, NorwayMohamed Eddouks, MoroccoAmr E. Edris, EgyptTobias Esch, GermanyYibin Feng, Hong KongJosue Fernandez-Carnero, Spain

Juliano Ferreira, BrazilPeter Fisher, UKRomain Forestier, FranceJoel J. Gagnier, CanadaM. N. Ghayur, PakistanAnwarul Hassan Gilani, PakistanMichael Goldstein, USASvein Haavik, NorwaySeung-Heon Hong, KoreaMarkus Horneber, GermanyChing Liang Hsieh, TaiwanBenny Tan Kwong Huat, SingaporeRoman Huber, GermanyAngelo Antonio Izzo, ItalyKanokwan Jarukamjorn, ThailandStefanie Joos, GermanyZ. Kain, USAOsamu Kanauchi, JapanKenji Kawakita, JapanJongYeol Kim, KoreaCheorl-Ho Kim, Republic of KoreaYoun Chul Kim, Republic of KoreaYoshiyuki Kimura, JapanToshiaki Kogure, JapanChing Lan, TaiwanAlfred L?ngler, GermanyLixing Lao, USACharlotte Leboeuf-Yde, DenmarkTat leang Lee, SingaporeJang-Hern Lee, Republic of KoreaMyeong Soo Lee, Republic of KoreaChristian Lehmann, CanadaMarco Leonti, ItalyPing-Chung Leung, Hong KongShao Li, China Xiu-Min Li, USAChun Guang Li, AustraliaSabina Lim, KoreaWen Chuan Lin, ChinaChristopher G. Lis, USAGerhard Litscher, AustriaI-Min Liu, Taiwan Ke Liu, ChinaYijun Liu, USAGaofeng Liu, ChinaCynthia R. Long, USAIrne Lund, Sweden

Gail B. Mahady, USASubhash C. Mandal, IndiaJeanine L. Marnewick, South AfricaFrancesco Marotta, ItalyVirginia S. Martino, ArgentinaJames H. McAuley, AustraliaAndreas Michalsen, GermanyDavid Mischoulon, USAHyung-In Moon, Republic of KoreaAlbert Moraska, USAMark Moss, UKMinKyun Na, Republic of KoreaRichard L. Nahin, USAVitaly Napadow, USAF. R. F. Nascimento, BrazilIsabella Neri, ItalyTelesphore Benoıt Nguelefack, CameroonMartin Offenbacher, GermanyKi-Wan Oh, Republic of KoreaY. Ohta, JapanOlumayokun A. Olajide, UKThomas Ostermann, GermanyStacey A. Page, CanadaTai-Long Pan, TaiwanBhushan Patwardhan, IndiaBerit Smestad Paulsen, NorwayAndrea Pieroni, ItalyRichard Pietras, USAXianqin Qu, AustraliaCassandra L. Quave, USARoja Rahimi, IranKhalid Rahman, UKCheppail Ramachandran, USAKe Ren, USAMee-Ra Rhyu, Republic of KoreaJose Luis Rıos, SpainPaolo Roberti di Sarsina, ItalyBashar Saad, Palestinian AuthorityAndreas Sandner-Kiesling, AustriaAdair Roberto Soares Santos, BrazilGuillermo Schmeda-Hirschmann, ChileRosa Schnyer, USAAndrew Scholey, AustraliaVeronique Seidel, UKDana Seidlova-Wuttke, Germany

Senthamil R. Selvan, USATuhinadri Sen, IndiaRonald Sherman, USAKaren J. Sherman, USAKan Shimpo, JapanByung-Cheul Shin, KoreaJian-nan Song, ChinaRachid Soulimani, FranceElisabet Stener-Victorin, SwedenMohd Roslan Sulaiman, MalaysiaVenil N. Sumantran, IndiaToku Takahashi, USATakashi Takahashi, JapanRabih Talhouk, LebanonJoanna Thompson-Coon, UKMei Tian, ChinaYao Tong, Hong Kong

K. V. Trinh, CanadaVolkan Tugcu, TurkeyYew-Min Tzeng, TaiwanCatherine Ulbricht, USADawn M. Upchurch, USAAlfredo Vannacci, ItalyMani Vasudevan, MalaysiaJoseph R. Vedasiromoni, IndiaCarlo Ventura, ItalyWagner Vilegas, BrazilPradeep Visen, CanadaAristo Vojdani, USADietlind Wahner-Roedler, USAChenchen Wang, USAChong-Zhi Wang, USAShu-Ming Wang, USAY. Wang, USA

Kenji Watanabe, JapanWolfgang Weidenhammer, GermanyJenny M. Wilkinson, AustraliaV. C N Wong, Hong KongHaruki Yamada, JapanNobuo Yamaguchi, JapanHitoshi Yamashita, JapanYong Qing Yang, ChinaKen Yasukawa, JapanE. Yesilada, TurkeyM. Yoon, Republic of KoreaBoli Zhang, ChinaHong Q. Zhang, Hong KongHong Zhang, SwedenRuixin Zhang, USAHaibo Zhu, China

Contents

Ethnopharmacological Approaches to Wound Repair, Editors: Esra Kupeli Akkol, Fatma U. Afifi,Saringat Hj. Baie, Ashok D. Taranalli, and Shrikant AnantVolume 2013, Article ID 804039, 2 pages

Evaluation of Topical Tocopherol Cream on Cutaneous Wound Healing in Streptozotocin-InducedDiabetic Rats, Teoh Seong Lin, Azian Abd Latiff, Noor Aini Abd Hamid, Wan Zurinah bt Wan Ngah,and Musalmah MazlanVolume 2012, Article ID 491027, 6 pages

Pilot Study with regard to the Wound Healing Activity of Protein from Calotropis procera (Ait.) R. Br.,Ramar Perumal Samy and Vincent T. K. ChowVolume 2012, Article ID 294528, 11 pages

Plectranthus amboinicus and Centella asiatica Cream for the Treatment of Diabetic Foot Ulcers,Yuan-Sung Kuo, Hsiung-Fei Chien, and William LuVolume 2012, Article ID 418679, 9 pages

Astragaloside IV Downregulates β-Catenin in Rat Keratinocytes to Counter LiCl-Induced Inhibition ofProliferation and Migration, Fu-Lun Li, Xin Li, Yi-Fei Wang, Xiu-Li Xiao, Rong Xu, Jie Chen, Bin Fan,Wen-bin Xu, Lin Geng, and Bin LiVolume 2012, Article ID 956107, 12 pages

Characteristics and Clinical Managements of Chronic Skin Ulcers Based on Traditional ChineseMedicine, Fu-Lun Li, Yi-Fei Wang, Xin Li, Feng Li, Rong Xu, Jie Chen, Lin Geng, and Bin LiVolume 2012, Article ID 930192, 6 pages

Dexamethasone Resisted Podocyte Injury via Stabilizing TRPC6 Expression and Distribution,Shengyou Yu and L. YuVolume 2012, Article ID 652059, 7 pages

A Study of the Wound Healing Mechanism of a Traditional Chinese Medicine, Angelica sinensis, Using aProteomic Approach, Chia-Yen Hsiao, Ching-Yi Hung, Tung-Hu Tsai,and Kin-Fu ChakVolume 2012, Article ID 467531, 14 pages

Fractionation of an Extract of Pluchea odorata Separates a Property Indicative for the Induction of CellPlasticity from One That Inhibits a Neoplastic Phenotype, Mareike Seelinger, Ruxandra Popescu,Prapairat Seephonkai, Judith Singhuber, Benedikt Giessrigl, Christine Unger, Sabine Bauer,Karl-Heinz Wagner, Monika Fritzer-Szekeres, Thomas Szekeres, Rene Diaz, Foster M. Tut,Richard Frisch, Bjorn Feistel, Brigitte Kopp, and Georg KrupitzaVolume 2012, Article ID 701927, 11 pages

Inhibitory Effect of Nelumbo nucifera (Gaertn.) on the Development of Atopic Dermatitis-Like SkinLesions in NC/Nga Mice, Rajendra Karki, Myung-A Jung, Keuk-Jun Kim,and Dong-Wook KimVolume 2012, Article ID 153568, 7 pages

Topical Application of Chrysanthemum indicum L. Attenuates the Development of AtopicDermatitis-Like Skin Lesions by Suppressing Serum IgE Levels, IFN-γ, and IL-4 in Nc/Nga Mice,Sunmin Park, Jung Bok Lee, and Suna KangVolume 2012, Article ID 821967, 8 pages

Wound Healing and Anti-Inflammatory Effect in Animal Models of Calendula officinalis L. Growing inBrazil, Leila Maria Leal Parente, Ruy de Souza Lino Junior, Leonice Manrique Faustino Tresvenzol,Marina Clare Vinaud, Jose Realino de Paula, and Neusa Margarida PauloVolume 2012, Article ID 375671, 7 pages

A Therapeutic Approach for Wound Healing by Using Essential Oils of Cupressus and Juniperus SpeciesGrowing in Turkey, Ibrahim Tumen, Ipek Suntar, Hikmet Keles, and Esra Kupeli AkkolVolume 2012, Article ID 728281, 7 pages

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013, Article ID 804039, 2 pageshttp://dx.doi.org/10.1155/2013/804039

Editorial

Ethnopharmacological Approaches to Wound Repair

Esra Küpeli Akkol,1 Fatma U. Afifi,2 Saringat Hj. Baie,3

Ashok D. Taranalli,4 and Shrikant Anant5

1 Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey2 Faculty of Pharmacy, University of Jordan, Amman 11942, Jordan3Department of Pharmaceutical Technology, School of Pharmacy, Universiti Sains Malaysia, 11800 Minden, Malaysia4Department of Pharmacology, Faculty of Pharmacy, KLE University, Belgaum, Karnataka 590010, India5 Department of Molecular and Integrative Physiology, and Medicine, University of Kansas School of Medicine,3901 Rainbow Boulevard, MS 3040, Kansas City, KS 66160, USA

Correspondence should be addressed to Esra Kupeli Akkol; [email protected]

Received 2 December 2012; Accepted 2 December 2012

Copyright © 2013 Esra Kupeli Akkol et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Wound is breaking of the skin by a physical injury. Woundhealing is a connective tissue response along with the repairprocess which immediately comes after the injury. It occursas a sequence of phases such as haemostasis, inflammation,proliferation, and remodelling and causes series of interac-tions between the extracellular matrix, cytokine mediators,and different cell types. For rapid healing several medicinalplants were reported in ethnobotanical studies. Traditionalremedies which claimed to have wound healing potential arewidely used in developing countries due to their accessibilityand low cost. However, these remedies should be evaluatedfor their efficacy and safety before their utilization. In thiscontext, the papers selected for this special issue includescientifically evaluated information and lead to developmentof novel drugs for rapid healing of wounds. We would like tothank the authors for their contributions for this special issue.

This special issue contains twelve papers. T. Lin et al.investigated the wound healing effect of tocopherol in dia-betic rats. This study has proven the wound healing potentialof tocopherol cream by increasing the rate of wound closureand total protein content significantly in diabetic condition.

R. Samy and V. Chow provide a scientific basis forthe use of Calotropis procera for treating skin and woundinfections in traditional medicine. The aqueous extract ofstem bark of C. procera exhibited more pronounced potentantimicrobial activity. Calo protein isolated from the aqueous

extract of C. procera showed broad-spectrumactivity as wellas significant wound healing activity.

In the paper entitled “Plectranthus amboinicus and Cen-tella asiatica cream for the treatment of diabetic foot ulcers,” Y.Kuo et al. investigated the effects of a topical cream containingP. amboinicus (Lour.) Spreng. (Lamiaceae) andC. asiatica (L.)Urban for diabetic foot ulcers. P. amboinicus and C. asiaticacream was found to be a safe alternative to hydrocolloid fiberdressing without significant difference in effectiveness.

F. Li et al. used an in vitro model of ulcer-like woundprocesses, lithium-chloride-(LiCl-) induced cultured mousekeratinocytes, to investigate the effects of astragaloside IVtreatment, and they concluded that astragaloside IV canpromote ulcerated wound healing by downregulating 𝛽-catenin to increase keratinocyte migration and proliferation.

F. Li et al. discuss the classification andpathogenic processof chronic skin ulcers and strategies of traditional Chinesemedicine. This study has shown a good approach to woundmanagement bymeans of the strategies of traditional Chinesemedicine for different wound types.

The results of the paper by S. Yu and L. Yu entitled “Dex-amethasone resisted podocyte injury via stabilizing TRPC6expression and distribution” revealed that dexamethasonemay maintain the structure and function integrity of slitdiaphragm by blocking TRPC6 signal pathway and played animportant role in mechanisms of antiproteinuria.

2 Evidence-Based Complementary and Alternative Medicine

C. Y. Hisao et al. investigated the wound healing effect ofAngelica sinensis in the paper entitled “A study of the woundhealing mechanism of a traditional Chinese medicine, Angelicasinensis, using a proteomic approach.” The wound healingpotential ofAngelica sinensiswas confirmedby proteomic andbiochemical analysis in scientific platform.

M. Seelinger et al. showed the antineoplastic and woundhealing potential of Pluchea odorata according to thebioactivity-guided fractionation assay.

The other paper was on the inhibitory activity ofNelumbonucifera (Gaertn.) on the development of atopic dermatitis byKarki et al. The results of the study suggested that Nelumbonucifera (Gaertn.) leaf may be a useful natural resource forthe management of atopic dermatitis, which is a chronicinflammatory skin disease.

S. Park et al. evaluated the healing effect of Chrysanthe-mum indicum L. on skin lesions. This study revealed thatChrysanthemum indicum reduced interleukin- (IL-) 4 and IL-13 in 2,4-dinitrochlorobenzene-treated HaCaT cells and maybe an effective alternative substance for the management ofthe atopic dermatitis.

The paper, by L. Parente et al., evaluated the woundhealing and anti-inflammatory activity of Calendula offici-nalis in animal models.This experimental study revealed thatC. officinalis possesses anti-inflammatory and antibacterialactivities as well as angiogenic and fibroblastic propertiesacting in a positive way on the inflammatory and proliferativephases of the healing process.

And the paper by I. Tumen et al. evaluated the woundhealing and anti-inflammatory activities of the essential oilsobtained from some Juniperus species, growing in Turkey,by using linear incision and circular excision experimentalwound models, hydroxyproline estimation, and acetic-acid-induced capillary permeability tests. The results showedthat J. oxycedrus subsp. oxycedrus and J. phoenicea displayremarkable wound healing and anti-inflammatory effectswhich support the folkloric use of the plants.

Esra Kupeli AkkolFatma U. Afifi

Saringat Hj. BaieAshok D. Taranalli

Shrikant Anant

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2012, Article ID 491027, 6 pagesdoi:10.1155/2012/491027

Research Article

Evaluation of Topical Tocopherol Cream on Cutaneous WoundHealing in Streptozotocin-Induced Diabetic Rats

Teoh Seong Lin,1 Azian Abd Latiff,1 Noor Aini Abd Hamid,2

Wan Zurinah bt Wan Ngah,3 and Musalmah Mazlan3

1 Department of Anatomy, Faculty of Medicine, Universiti Kebangsaan Malaysia,Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia

2 Faculty of Medicine, Cyberjaya University College of Medical Sciences, 63000 Cyberjaya, Malaysia3 Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz,50300 Kuala Lumpur, Malaysia

Correspondence should be addressed to Musalmah Mazlan, [email protected]

Received 28 March 2012; Revised 13 September 2012; Accepted 13 September 2012

Academic Editor: Shrikant Anant

Copyright © 2012 Teoh Seong Lin et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Diabetes is a common cause of delayed wound healing. The aim of the study was to determine the effect of topical administration oftocopherol cream on the wound healing process in diabetic rats. The study was conducted using 18 male Sprague Dawley rats whichwere divided into three groups: (I) diabetic rats receiving control cream (n = 6), (II) diabetic rats receiving 0.06% tocopherol cream(n = 6), and (III) diabetic rats receiving 0.29% tocopherol cream (n = 6). Four cutaneous wounds were created at the dorsal regionof the rats. Wound healing was assessed by total protein content, rate of wound closure estimation, and histological studies onthe tenth day after wounding. Tocopherol treatment enhanced the wound healing process by increasing rate of wound closure andtotal protein content significantly (P < 0.05) compared to the control group. Histological observation also showed better organizedepithelium and more collagen fibers in the tocopherol treated groups. Application of tocopherol cream enhances wound healingprocess in diabetic condition which is known to cause delay in wound healing.

1. Introduction

Wound can be defined as a disruption of the normal cellular,anatomical, and functional continuity of a structure. Thus,wound healing is a complex process which aims to restorethe structural and functional integrity of the wounded tissue[1]. Wound healing can be divided into 3 stages, inflam-mation, proliferation, remodeling and maturation phaseswhich involved the interaction of various cells, cytokines,and growth factors [2]. In some pathological disorders likediabetes mellitus, renal failure, malnutrition, wound healingis greatly impaired [3]. In diabetic patients, the prevalenceof diabetic foot ulcers are 4–10%, and the treatment of footulcers are expensive and extensive [4]. Previous researchstudy has shown that free radical inhibits the wound healingprocess [5]. Thus, the wound healing process can be acceler-ated by using antioxidants.

Recently, research has focused on the use of naturalantioxidants like herbal extracts and vitamins on woundhealing. The beneficial effects of vitamins on wound healinghave mainly been studied using animal models. Only vitaminC has been shown to accelerate healing in human subjects[6]. Oral and topical application of vitamin A has beenshown to enhance healing in diabetic, immunocompro-mised, and malignant tumor animal models [7–10]. Thepositive effect of vitamin E oral administration on woundhealing has also been well documented. Previously, we hadreported benefit of oral administration of palm-vitamin Eon wound healing in aging and diabetic rat models [11, 12].Raxofelast, a hydrophilic vitamin-E-like compound injectedintraperitoneally has shown its promising wound healingproperties in an incisive wound model of diabetic rats [13].However, considering the poor effusion and microcircula-tion insufficiency in diabetic patients, topical application of


vitamin E might be more effective in accelerating woundhealing compared to oral administration. Hence, in thisstudy, we aimed to evaluate the effect of tocopherol topicalapplication in the form of cream on wound healing ofstreptozotocin-induced diabetic rats.

2. Methods

2.1. Animals. Healthy male Sprague-Dawley rats (weighing250–300 g) bred in Laboratory Animal Resource Unit, Uni-versiti Kebangsaan Malaysia were used throughout the exper-imental period. They were housed under controlled environ-mental conditions with free access to rat pellets and cleanwater, caged individually. Prior ethical approval was obtainedfrom the Universiti Kebangsaan Malaysia Animal EthicsCommittees (UKMAEC).

2.2. Diabetes Induction. Streptozotocin (STZ, Sigma, Ger-many) was dissolved in normal saline. Following this, 45 mg/kg dose of STZ was injected to the overnight fasted rats vialateral tail vein under mild diethyl ether anesthesia [2]. Threedays later, blood samples were drawn from the tail of theserats to determine fasting blood glucose level using glu-cometer (Advantage, Germany). The rats with fasting bloodglucose levels more than 8 mmol/L were labeled diabetic andwere included for the experiment.

2.3. Experimental Groups. The diabetic rats were randomlydivided into 3 groups with 6 rats in each group. Group Iconsidered as diabetic control group receiving vehicle cream.Whereas group II and III served as diabetic treatment group,treated with 0.06% and 0.29% tocopherol cream (GoldenHope Bioorganics, Malaysia), respectively.

2.4. Rat Excision Wound Model and Treatment. The fur onthe back of the anesthetized rats was shaved with electricalshaver and cleaned with alcohol swab. Four full thicknessexcision wounds were made on the dorsum of each rat withsterile 6 mm punch biopsy needles (Stiefel, Ireland) [12].The excision included the epidermis, dermis, and panniculuscarnosus. The wounds were left undressed and were treatedwith 0.1 g cream according to their respective groups topi-cally once daily for a period of 10 days.

2.5. Rate of Wound Closure Determination. The wound witha centimeter scale was photographed after wounding day 0,day 6, and day 10. The photographs were used for the meas-urement of the wound areas using image analyzer software[2]. The rate of wound closure, that is, percentage of woundreduction from the original wound was calculated using thefollowing formula [14]:

Rate of wound closure

= wound area day 0−wound area day (n=1, 6, 10)×100%wound area day 0

.

(1)

0

5

10

15

20

25

30

Day 3

Fast

ing

bloo

d gl

uco

se (

mm

ol/L

)

∗

∗∗ ∗

∗∗

Day 1(prior to injection)

Day 10(prior to sacrifice)

Group 1Group 2Group 3

Figure 1: Fasting blood glucose level of the experimental rats. Therats experience elevated blood glucose level three days after theinjection of STZ, and remain high throughout the experimentalperiod (∗P < 0.01).

2.6. Termination of Experiment. After 10 days of daily treat-ment of topical cream (20 days after STZ), the rats wereeuthanized by cervical dislocation under anesthesia. A pairof wound was excised using the same-sized punch biopsyneedle and stored at −70◦C for Bradford assay. The otherpair was excised, along with a 5 mm margin of the surround-ing unwounded skin preserved in 10% formalin for lightmicroscopy.

2.7. Bradford Assay. Bradford method was used for quantita-tion of total protein content in the wound [15]. The woundtissue was homogenized in 1.15% potassium chloride at aratio of 1 : 5 (wt/vol). Five mL of protein reagent (0.01%Coomassie Brilliant Blue G250, 4.7% ethanol, 8.5% phos-phoric acid) was added and mixed into 0.1 mL of homo-genate. Absorbance was measured at 595 nm against a blankprotein reagent prepared by mixing 0.1 mL normal saline and5 mL protein reagent. A standard curve was prepared using 0,20, 40, 60, 80, and 100 μg/mL of BSA in normal saline treatedsimilarly.

2.8. Histological Observation. The formalin-fixed tissueswere routinely processed by standard procedures and serialsections of 5 μm were cut and stained with hematoxylin andeosin (H&E) and Verhoeff ’s van Gieson (VVG). The slideswere examined and photomicrographs were obtained usingdigital camera (Pixelink, Canada) attached to a light micro-scope (Leica, Germany).

2.9. Statistical Analysis. All results were expressed as mean ±SEM. The means of the group was compared using analysisof variance (ANOVA) followed by Scheffe’s test. Data analysiswas performed using statistical package programme SPSS11.5. A P value <0.05 was considered as statistically signifi-cant.

Evidence-Based Complementary and Alternative Medicine 3

Group II

Group III

Group I

Day 0 Day 6 Day 10

Figure 2: Photograph taken on the excision wounds made on the experimental rats. All groups showed complete wound closure on day 10.

0102030405060708090

100

Day 6 Day 10

Rat

e of

wou

nd

clos

ure

(%

) ∗∗

∗

Group 1Group 2Group 3

Figure 3: Effect of tocopherol on rate of wound closure. Rate ofwound closure was measured as percentage of reduction of originalwound size. Topical tocopherol treatment given to the diabetic ratsincreased the rate of wound closure (∗P < 0.05). Treatmentwith 0.29% tocopherol cream showed higher rate of wound closurecompared to the 0.06% tocopherol cream.

3. Results

3.1. Diabetic Induction. Following injection of STZ intra-venously, the rats showed 3-4-fold increase of fasting bloodglucose level compared to their normal level before injec-tion (Figure 1, P < 0.01). The fasting blood glucose levelremained elevated throughout the experimental period. Top-ical treatment given to the rats wound did not exhibit anyeffect on the fasting blood glucose level (P > 0.05).

3.2. Rate of Wound Closure. Figure 2 showed the contractionrate of the wounds on different days of the experimental

0

5

10

15

20

25

30

35

40

Group 1 Group 2 Group 3

Tota

l pro

tein

con

ten

t(m

g pr

otei

n/g

wet

wei

ght)

∗∗

Figure 4: Effect of tocopherol on total protein content. Total pro-tein content was measured according to Bradford’s assay. There wassignificant increase in the total protein content in the wound tissuesharvested when given treatment with tocopherol cream (∗P < 0.05).Treatment with 0.29% tocopherol cream showed the highest totalprotein content amongst other groups.

rats. Rate of wound closure was calculated as the percentageof wound reduction from the original wound (Figure 3).The rate of wound closure was significantly increased inboth the diabetic rat groups receiving treatment (P < 0.05).Diabetic rats receiving treatment of 0.29% tocopherol creamhave a higher rate of wound closure compared to the 0.06%tocopherol cream.

3.3. Bradford’s Assay. Total protein content of the wounds(Figure 4) harvested from experimental rats on day 10 wasestimated using Bradford’s assay. Treatment with tocopherolcream increased the total protein content of the woundsignificantly (P < 0.05).


I

S

E

(a)

II

S

E

(b)

IIIE

S

(c)

Figure 5: H&E stained section on the 10th day wound (10X). Complete epithelialization of the wound area was observed in all groups. Newlyformed epithelium in group I appeared thin. There were interdigitation between epithelium and dermis of the skin in group III suggestingstronger integrity of the skin. E: epidermis and S: scar/granulation tissue.

I

E

(a)

II

S

GrC

G

(b)

III

S

GrC

G

(c)

Figure 6: H&E stained section on the 10th day wound (20X). Evaluation on the newly formed epithelium revealed that group I does notcontain all the strata of an epithelium. Whereas group II and III showed the presence of strata germinativum (G), spinosum (S), granulosum(Gr), and corneum (C). E: epidermis.

I

(a)

II

(b)

III

(c)

Figure 7: VVG stained section on the 10th day wound (40X). The collagen fibres (arrows) found in the scar tissue deep to the epithelium ingroup I appeared thin and scanty. Treatment with tocopherol cream increased the abundance of collagen fibres found in the scar tissue.

3.4. Histological Findings. H&E staining was performed onthe wounds harvested on day 10 (Figures 5 and 6). All groupsof experimental rats showed complete epithelialization ofthe wound area. The new epithelial layer formed in group Iappeared thin and did not show all four strata structure.Tocopherol cream treated group showed a well-formed epi-thelium. Treatment with 0.29% tocopherol cream increasedthe interdigitation between the epithelium and dermis layers.VVG staining (Figure 7) showed the abundant collagen fibers

formed in both the treated group compared to the scantydeposition of fibres in the control group.

4. Discussion

Tocopherol and tocotrienol which are structurally similar aresubfamily of vitamin E [12]. α-Tocopherol is known as themost abundant and active form of vitamin E in humans [16,17]. Previous studies have demonstrated the beneficial effects


of vitamin E in wound healing process when given orally[11, 12]. The present study shows that topical application oftocopherol cream also enhances wound healing in diabeticrats.

In this present study, STZ was used to induce diabetes inexperimental rats intravenously. Three days after the injec-tion of STZ, all the rats showed increase of fasting bloodglucose level, reduced body weight, polydipsia, and polyuria.The fasting blood glucose remained constantly elevatedduring the entire experimental period. These sign and symp-toms were shown in a previous study as well [18]. The topicalapplication of tocopherol cream in group II and III exhibitedno effect on the fasting blood glucose.

Rate of wound closure is a useful measurement to assessthe progress of wound healing. It had been shown that thereis a decrease in rate of wound contraction in diabetic ratscompared to the normal rats [2]. In the present study, appli-cation of both 0.06% and 0.29% tocopherol cream was ableto increase the rate of wound contraction. This is in accor-dance to a previous study which showed increased rate ofwound contraction following oral supplementation of toco-pherol [12].

The total protein content is an indicator for the proteinlevel and cellular proliferation of the wound tissue [2]. In thisstudy, tocopherol cream was able to increase the total proteincontent in the diabetic rats, which is in accordance to a previ-ous study [12]. This could indicate that tocopherol enhancesprotein synthesis, cellular proliferation, and migration in thewound tissue. Interestingly, a previous study showed thatphysiologically relevant concentrations of α-tocopherol inhi-bits cell proliferation in vascular smooth muscle cells whileproliferation of fibroblast was not inhibited [19].

Histological observation revealed complete epithelializa-tion of all diabetic rats, with or without tocopherol creamtreatment after 10 days of wounding. However, untreateddiabetic rats showed immature and thin epithelial layer.Tocopherol treated group showed that the newly formedepithelium contains the strata germinativum, spinosum,granulosum, and corneum. In group III, there was presenceof interdigitation between the epithelium and dermis. Theseinterdigitations were important as it offered resistance toseparation of the epidermal layer due to shreding. It was alsoshown that with tocopherol treatment, collagen fibers weremore numerous in the scar tissue which was also reflected inthe total protein content.

Diabetes mellitus, a common endocrine disease, is oneof the leading causes of impaired healing. Delayed woundhealing in diabetes is multifactorial which includes hypergly-caemia, infections, suppressed immunity, local ischemia, andoxidative stress [20]. In diabetic condition, hyperglycaemiadecreases cell proliferation and collagen deposition. Thechronic inflammation which occurs at the wound site causeslocal ischemia, generation of reactive oxygen species (ROS),vascular stasis in microcirculation, decreased chemotaxis andphagocytosis, reduced level of growth factors, inhibition offibroblast proliferation, and decreased deposition of extra-cellular matrix molecules which then leads to delay of woundhealing in diabetic patients [20, 21]. Previous study hasshown that tocopherol stimulates the production of cyclic

adenosine monophosphate (cAMP) which often associatedwith immunomodulatory effects by modulating the inflam-matory responses of a variety of immune cell types includingmacrophages [22]. Tocopherol also has been shown to pos-sess anti-inflammatory effect by attenuation of pro-inflam-matory cytokine and chemokine production [22]. Thus,application of tocopherol is beneficial to wound healing indiabetic rats which might be due to its antioxidant and anti-inflammatory property.

In conclusion, the administration of topical tocopherolcream has positive effect on wound healing process in dia-betic animal model. In the present study, the higher dose of0.29% tocopherol cream showed better wound healing com-pared to the 0.06% tocopherol cream.

Funding

This study was funded under the Sime Darby Research Grantno. JJ-001-2008.

Conflict of Interests

The authors declare that they have no conflict of interests.

References

[1] R. Thakur, N. Jain, R. Pathak, and S. S. Sandhu, “Practices inwound healing studies of plants,” Evidence Based Complemen-tary and Alternative Medicine, vol. 2011, Article ID 438056, 17pages, 2011.

[2] S. L. Teoh, A. A. Latiff, and S. Das, “The effect of topical extractof Momordica charantia (bitter gourd) on wound healing innondiabetic rats and in rats with diabetes induced by strepto-zotocin,” Clinical and Experimental Dermatology, vol. 34, no.7, pp. 815–822, 2009.

[3] A. K. Deodhar and R. E. Rana, “Surgical physiology of woundhealing : a review,” Journal of Postgraduate Medicine, vol. 43,no. 2, pp. 52–56, 1997.

[4] J. Majtan, “Methylglyoxal—a potential risk factor of manukahoney in healing of diabetic ulcers,” Evidence-based Com-plementary and Alternative Medicine, vol. 2011, Article ID295494, 5 pages, 2011.

[5] D. Foschi, E. Trabucchi, M. Musazzi et al., “The effects ofoxygen free radicals on wound healing,” International Journalof Tissue Reactions, vol. 10, no. 6, pp. 373–379, 1988.

[6] W. M. Ringsdorf and E. Cheraskin, “Vitamin C and humanwound healing,” Oral Surgery Oral Medicine and Oral Pathol-ogy, vol. 53, no. 3, pp. 231–236, 1982.

[7] E. Seifter, G. Rettura, J. Padawer, F. Stratford, D. Kambosos,and S. M. Levenson, “Impaired wound healing in streptozo-tocin diabetes. Prevention by supplemental vitamin A,” Annalsof Surgery, vol. 194, no. 1, pp. 42–50, 1981.

[8] M. F. Trevisani, M. A. Ricci, J. T. Tolland, and W. C. Beck,“Effect of vitamin A and zinc on wound healing in steroid-treated mice,” Current Surgery, vol. 44, no. 5, pp. 390–393,1987.

[9] M. Haws, R. E. Brown, H. Suchy, and A. Roth, “Vitamin A-soaked gelfoam sponges and wound healing in steroid-treatedanimals,” Annals of Plastic Surgery, vol. 32, no. 4, pp. 418–422,1994.


[10] J. Weinzweig, S. M. Levenson, G. Rettura et al., “Supplementalvitamin A prevents the tumor-induced defect in woundhealing,” Annals of Surgery, vol. 211, no. 3, pp. 269–276, 1990.

[11] A. H. Noor Aini, I. Illyana, W. N. Wan Zurinah, M. T.Gapor, and M. Musalmah, “Relationship between antioxidantenzymes activity with wound closure and effects of palmvitamin E supplementation during aging,” Malaysian Journalof Biochemistry and Molecular Biology, vol. 8, pp. 59–62, 2003.

[12] M. Musalmah, M. Y. Nizrana, A. H. Fairuz et al., “Comparativeeffects of palm vitamin E and α-tocopherol on healing andwound tissue antioxidant enzyme levels in diabetic rats,”Lipids, vol. 40, no. 6, pp. 575–580, 2005.

[13] M. Galeano, V. Torre, B. Deodato et al., “Raxofelast, a hydro-philic vitamin E-like antioxidant, stimulates wound healing ingenetically diabetic mice,” Surgery, vol. 129, no. 4, pp. 467–477, 2001.

[14] D. Gopinath, M. R. Ahmed, K. Gomathi, K. Chitra, P. K.Sehgal, and R. Jayakumar, “Dermal wound healing processeswith curcumin incorporated collagen films,” Biomaterials, vol.25, no. 10, pp. 1911–1917, 2004.

[15] M. M. Bradford, “A rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the princi-ple of protein dye binding,” Analytical Biochemistry, vol. 72,no. 1-2, pp. 248–254, 1976.

[16] J. J. Thiele, S. N. Hsieh, and S. Ekanayake-Mudiyanselage,“Vitamin E: critical review of its current use in cosmetic andclinical dermatology,” Dermatologic Surgery, vol. 31, no. 7, part2, pp. 805–813, 2005.

[17] Y. Yoshida, E. Niki, and N. Noguchi, “Comparative studyon the action of tocopherols and tocotrienols as antioxidant:chemical and physical effects,” Chemistry and Physics of Lipids,vol. 123, no. 1, pp. 63–75, 2003.

[18] S. L. Teoh, A. A. Latiff, and S. Das, “A histological study ofthe structural changes in the liver of streptozotocin-induceddiabetic rats treated with or without Momordica charantia(bitter gourd),” Clinica Terapeutica, vol. 160, no. 4, pp. 283–286, 2009.

[19] D. Boscoboinik, A. Szewczyk, C. Hensey, and A. Azzi, “Inhibi-tion of cell proliferation by α-tocopherol: role of protein kinaseC,” Journal of Biological Chemistry, vol. 266, no. 10, pp. 6188–6194, 1991.

[20] A. A. Latiff, S. L. Teoh, and S. Das, “Wound healing indiabetes mellitus: traditional treatment modalities,” ClinicaTerapeutica, vol. 161, no. 4, pp. 359–364, 2010.

[21] W. K. Stadelmann, A. G. Digenis, and G. R. Tobin, “Imped-iments to wound healing,” American Journal of Surgery, vol.176, no. 2, pp. 39S–47S, 1998.

[22] S. Salinthone, A. R. Kerns, V. Tsang, and D. W. Carr, “α-Tocopherol (vitamin E) stimulates cyclic AMP productionin human peripheral mononuclear cells and alters immunefunction,” Molecular Immunology, vol. 53, no. 3, pp. 173–178,2012.


Research Article

Pilot Study with regard to the Wound Healing Activity of Proteinfrom Calotropis procera (Ait.) R. Br.

Ramar Perumal Samy and Vincent T. K. Chow

Infectious Diseases Programme, Department of Microbiology, Yong Loo Lin School of Medicine,National University of Singapore, Singapore 117597

Correspondence should be addressed to Ramar Perumal Samy, [email protected]

Received 5 March 2012; Revised 16 May 2012; Accepted 28 May 2012

Academic Editor: Fatma U. Afifi

Copyright © 2012 R. Perumal Samy and V. T. K. Chow. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

We provide the scientific basis for the use of Calotropis procera for treating skin and wound infections in traditional medicine. Theaqueous extract of stem-bark of C. procera exhibited more pronounced potent antimicrobial activity. Calo-protein was purifiedand identified from the most-active aqueous extracts of C. procera and showed broad-spectrum activity. Calo-protein inhibitedthe growth of S. aureus and E. aerogenes effectively at 25 μg/ml concentration. Mice topically treated with Calo-protein revealedsignificant wound healing after 14 days comparable to fusidic acid (FA) as positive control. This protein was devoid of cytolyticeffect even at higher concentrations on skin cells after 24 h. Further investigation of this Calo-protein of C. procera on bacterialinhibition may provide a better understanding of the scientific basis and justification for its use in traditional medicine.

1. Introduction

Staphylococcus aureus is a leading cause of skin and soft-tissueinfections worldwide [1]. S. aureus infections are increasinglycaused by methicillin-resistant S. aureus (MRSA) that hasdeveloped resistance to β-lactam antibiotics. MRSA is amajor problem in healthcare settings [2], with reportedincidence rate of invasive MRSA infections of 31.3 per100,000 individuals, and 20% of these infections resultingin death [3]. World-wide, the rate of methicillin resistance35.9% is high for surgical [4], chronic (7.4 million) [5], andtraumatic wounds (1.5 million). Wound healing is a complexprocess that involves various inflammatory, proliferative,and remodeling phases [6]. In particular, chronic woundsare major concerns for patient. Since they affect a largenumber of patients and may reduce their life span [7].Chronic wounds such as leg ulcers, diabetic foot ulcers,and sores are common in both acute and communityhealthcare settings [8]. Chronic wounds may be infected bybacteria such as Streptococcus pyogenes, Enterococcus faecalis,Staphylococcus aureus/MRSA [9], Pseudomonas aeruginosa,

Enterobacter aerogenes, and Escherichia coli, while fungi andviruses may also cause skin and wound infections [10].

Many bacteria have developed resistance due to the abuseuse of antibiotics, while the existing drugs have also causedserious adverse effects in humans [11]. Various preventiveand treatment options of wounds are available [8]. However,drugs capable of promoting the wound repair process arestill limited. Other considerations include the higher costfor producing synthetic drugs and the various side effectsassociated with their use [12]. To overcome these issues, thesearch for alternative agents from plants used in traditionalmedicine is justified. World-wide, research is conducted toidentify new potent, nontoxic wound-healing agents frommedicinal plants. Plants are important potential sourcesof drugs for the biomedical or pharmaceutical industry.Countries such as India and China have rich resources ofvaluable medicinal plants for the treatment of wound andburns [13]. Approximately 80% of the world population stillrelies on traditional medicine for the treatment of commondiseases [14, 15]. Medicinal plants offer significant potentialfor the development of novel antibacterial therapies and


adjunct treatments [16]. Plant-derived drugs serve as pro-totypes to develop more effective and less toxic medicines.In previous studies, few attempts were made to confirmthe antimicrobial activity of indigenous medicinal plants[17, 18]. Various extracts of medicinal plants were shown topossess antimicrobial activity against Staphylococcus aureus[18].

Calotropis procera (Ait.) R. Br. is well known for its toxicas well as medicinal properties. The milk weed has beenfound to be effective in the treatment of leprosy, fever, men-orrhagia, malaria, and snake bites. Previous investigationstudy demonstrated various biological activities of C. procerasuch as anti-inflammatory potential in rats [19, 20], and itsosmotin proteins exert antifungal activity [21]. C. proceralatex administered to rats revealed toxic, wound healing, andpain-killing effects [22]. Chemical compounds in the latexare calotropagenin glycosides/derivatives [23], cardenolides,flavonoids, and saponins [24]. However, the C. procera stembark has hitherto not been well studied completely. Hence,in the present study C. procera was evaluated and furthercharacterized for antimicrobial activity and wound-healingpotential in mouse model.

2. Materials and Methods

2.1. Ethnomedicinal Survey. The stem bark of Calotropis pro-cera (Ait.) R. Br. (family Asclepiadaceae) plant was collectedin May 2004, from the Thiruthani, Tiruvallur district, (nearChennai), Tamil Nadu, India. Plant was identified by ataxonomist with help of Matthew [25]. Voucher specimenwas prepared and stored at Entomology Research Institute,India.

2.2. Preparation of Extracts. The stem bark was collected inthe field and cleaned by a sterile muslin cloth, cut into smallpieces by sterile razor blade, and stored in a sterile polythenebags. All the parts were dried under shade at ambienttemperature (31◦C) and ground to small powdery granulesby electric blender (Preethi, Chennai, India). Using a soxhletapparatus, the shade-dried and powdered plant material(200 g of each) was extracted with 1000 mL of hexanefor 10 h, and successive extracts were done using differentorganic solvents such as ethyl acetate, dichloromethane, andmethanol individually and then finally extracted twice with600 mL of sterile distilled water (H2O) using a shaking waterbath at 65◦C for 3 h. All the collected extracts were filteredusing Whatman number 1 filter paper and evaporated witha rotary evaporator (Buchi, Labortechnik AG, Switzerland)and freeze dryer (lyophilized) to obtain the crude extracts.The dried crude extracts were stored at 4◦C for antimicrobialassays [18].

2.3. In Vitro Antimicrobial Activity. The standard bacterialcultures of Gram-negative (Escherichia coli, Enterobacteraerogenes, Proteus vulgaris, and Proteus mirabilis) and Gram-positive (Pseudomonas aeruginosa and Staphylococcus aureus)bacterial strains were obtained from the Department ofMicrobiology, National University of Singapore, Singapore.

The strains were stored at −70◦C, subcultured on 20 mLMueller Hinton (MH) agar plates (pH 7.4), and incubatedovernight at 37◦C prior to use. The antimicrobial propertywas tested using the disc-diffusion method [26]. Five youngcolonies of each strain of bacteria taken from their respectivecultures grown overnight on MH agar plates (Oxoids, UK)were suspended in 5 mL of sterile saline (0.9%), and thedensity of the suspension adjusted to approximately 3 ×108 colony forming unit (CFU). The swab was used toinoculate the dried surface of MH agar plate by streakingfour times over the surface of the agar and rotating the plateapproximately 90◦C to ensure an even distribution of theinoculums. The medium was allowed to dry for about 3 minbefore adding a 6 mm diameter sterile paper disc (BecktonDickson, USA) on the surface. Each disc was tapped gentlydown onto the agar to provide a uniform contact. 100 μg/mLof each plant extract (lyophilized aqueous residues) wasweighed and dissolved in 1 mL of water, and 20 μL of theextracts (containing 100 μg of residues) were applied oneach disc (3 replicates). The sterile blank disc served as anormal control. The antimicrobial activity of the extracts onthe clinical isolates was determined in comparison with thereference antibiotic (chloramphenicol 30 μg/disc), which wasused as a positive control. The plates were incubated at 37◦Cfor 24 h, and the inhibition zones measured and calculated.

2.4. Phytochemical Screening of Extracts. The phytochemicalscreening was done on the extracts using the chemicalmethod previously reported for the detection of secondarymetabolites [27].

2.5. Extraction and Isolation. The most active aqueousextract of stem bark of C. procera was used for thepurification of antimicrobial agents [27]. The residue 0.5 gmwas dissolved completely in 5 mL of 50 mM Tris-HCl (pH7.4), after centrifugation at 12,000 rpm for 15 min, and clearsolution was filtered by a membrane filter (0.22 μm). Sample1 mL diluted (1 : 5 ratio) in 4 mL of Tris-HCl and injectedinto a Superdex G-75 column linked to high-performanceliquid chromatography (HPLC AKTA, Denmark) resolvedfive fractions (P1–P5). Fraction (1 mL) was monitored at280 nm and collected. All the fractions were screened againstbacteria, of which fraction (P2) exerted higher antimicrobialactivity. The most active fraction (P2) was further separatedby C18 reverse-phase (RP)-HPLC column, which gavefour fractions (CF-1–CF-4). Resultant separation of activefraction (CF-1) displayed maximum inhibitory effect againstbacteria that was eluted by C8 RP-HPLC column, final yieldsof the fraction purity were determined by matrix-assistedlaser desorption ionization-time of flight/mass (MALDI)-TIF/MS and designated as “Calo-protein.” The concentratedprotein was then tested (100 μg/mL) against bacteria in vitro.

2.6. Animals. Eight-week-old male Swiss Albino mice, bodyweight ranging from 25–30 gm, were obtained from the NUSanimal breeding center Sembawang, Singapore. All animalswere kept in individual cages (Darzon, Laboratory, USA)with standard laboratory conditions with dark/light cycle


(24 ± 2.0), food pellet and water ad libitum used in woundhealing model of experiments.

2.6.1. Mice Model of Wound. Mice were divided into threegroups, each group consists of three mice each (n = 3),respectively, (IACUC Protocol number 691/04). The dorsalskin of the mice was marked by permanent marker andthen cleaned with 70% ethyl alcohol (Merck, Germany)for surface sterilization before shaved by sterile surgicalblade [28]. The animals were anesthetized (75 mg/kg ofketamine + 0.1 mg/kg of medetomidine) and full thicknessof 16 mm (8 × 8 mm) for excision wound created and 50 μLof bacteria (1 × 105 colony forming units per mL). Group Iinfected mice wound received only saline served as a control,Group II infected mice wound treated with 5 mg/kg, bodyweight of Calo-protein mixed with aqueous cream (ICMPharma Pte Ltd., Kallang Place, Singapore) formulationswere topically applied on the dorsal side of the mice, GroupIII infected mice applied 5 mg/kg, b.w of fusidic acid (FA)sodium salts (Sigma Co., St. Louis, USA) served as anantibiotic control. The wounds were left open without anydress and kept individually in separate cages. The wound areamonitored and measured every day up to 14 days (2-weektime).

2.6.2. Measurement of Wound Area. Treatment with Calo-protein and standard drug were performed by topicalapplication on the wound surface up to 4 days. The woundareas were traced on millimeter in diameter (mm2) by tracerpaper (Merck Co., Germany) immediately after the woundcreated and every day until the healing was completed.The percentage of wound reduction in the wound size wascalculated according to the following formula:

Wound contraction (%) = Wc −Wt

Wc× 100, (1)

where Wc is the wound area immediately after woundcreation as a control (c), Wt is the wound area on day oftreatment (t).

2.6.3. Histological Evaluation. The experiment was termi-nated after 14 days and the wound removed by surgicallyfor histopathological examination. The tissues were cut andtrimmed down and processed in dehydration in alcoholseries 50–90% for 30 min each, 100% ethyl alcohol (I/II), andhistoclear 100% (I) for 60 min each, and 100% histoclear (II)for overnight. The decalcification was done with molted waxat 55◦C for 1 h in each jar. The blocks were prepared by usingwax, 5 μm thick sections were stained with haematoxylinand eosin (H&E) and imaged by Olympus Light microscope(Olympus America Inc., PA, USA).

2.6.4. Measurement of Collagen. Healed tissues of thewounds were cut, weighed (100 mg), and homogenized with1 mL of T-PER buffer (Thermo Scientific, IL, USA). Thehomogenates were centrifuged at 10,000 rpm for 10 minat 4◦C. The supernatant collected and collagen contentin the Calo-protein, FA-treated, and wound control mice

were determined by ELISA using mouse antitype I collagenassay kit (Chondrex, Inc., Redmond, WA, USA) as per themanufactures instructions.

2.7. Cytotoxicity Assay. The cytotoxic potential of differentcrude extracts of C. procera was evaluated against humanmacrophage (U-937) cell lines by XTT assay at 2000,1000, 500, 250, 100, 50, 25, and 12.5 μg/mL [29]. The cellproliferation assayed in 96-well microtitre plates, confluentcells (5 × 106 cells per well) were incubated with diverseextracts for 24 h and the inhibitory concentrations recorded.Further, cytotoxic and cytolytic effects of purified proteintested on human skin fibroblast cells (HEPK) were evaluatedby measuring the release of lactate dehydrogenase (LDH)enzyme using a cytotoxicity detection kit (Roche Mannheim,Germany) at various concentrations (1000–0.001 μg/mL).After 24 h, 200 μL of aliquot of the cell supernatant obtainedfrom each 96-well plate was used for the quantification ofcell death and cell lysis, based on the measurement (450 nm).LDH activity was released from the cytosol of damaged cellsinto the supernatant. The assay was performed in triplicate(n = 3), percentage cell proliferation and cytolytic effectswere calculated.

2.8. Statistical Analysis. The data presented as mean ±standard deviation (SD), each concentration tested threereplicates (n = 3), inhibitory concentrations millimeter(mm) in diameter. The percentage reduction of wound datawas compared using one-way analysis of variance (ANOVA)followed by Dunnett’s tests. Values with P less than ∗P >0.01, and ∗∗P > 0.05 compared with the control group wereconsidered as statistical significance.

3. Results

3.1. Ethnobotanical Survey and Application in Healthcare.This study attempts to provide the scientific basis for thetraditional beliefs of this herbal remedy. We surveyed thetraditional medical knowledge on C. procera from the localpractitioners for preparing home remedies for their routinehealthcare (Figures 1(a), 1(b), 1(c), and 1(d)). In TamilNadu, decoction of the stem bark is traditionally used forthe treatment of skin diseases, while the aerial parts of C.procera are used for treating fever, pain, muscular spasm, andso forth. The milky latex wet with a clean cloth was appliedmainly on affected areas of cut wounds, thorn injuries,and inflamed swellings. The botanical/family name, yield ofextracts, and their activities are provided.

3.2. In Vitro Antimicrobial Activity. Table 1 shows the invitro antimicrobial, cytotoxic, and phytochemical proper-ties of organic and aqueous extracts of stem bark of C.procera as a traditional medicinal plant. Among the testedextracts, ethyl acetate, methanol, and aqueous extractsdisplayed high antimicrobial activity against the testedbacteria. The methanol and aqueous extracts revealed rela-tively broad-spectrum activity against bacteria at 100 μg/mLconcentration. Aqueous extracts exhibited more pronounced


Habitat

st

(TT

: TN

, In

dia)

(a)

wl

Leaf

C. p

roce

ra(A

it.)

R.B

r.

(b)

f

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(TT

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, In

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Fruit

fp

wlC

. pro

cera

(Ait

.) R

.Br.

(d)

Figure 1: The giant milkweed Calotropis procera growing as a spreading shrub or small tree has a simple stem with only few branches. (a,b) Large, dark-green leaves in opposite pairs along smooth stem. (c) Beautiful waxy white flowers have deep purple spots or blotches at thebase of each five petals. (d) It produces a fleshy fruit with inflated pod containing several brown seeds with white long silky hair. It exudes amilky white latex when cut or broken. St: stem, f: flower, fp: fruit pod, and wl: waxy leaf surface.

antimicrobial activity with the largest inhibitory zones of28 mm diameter compared to the standard chloramphenicolantibiotic. Overall, methanol and aqueous extracts of C.procera exerted a broad activity against both Gram-negativeand Gram-positive bacteria. However, the hexane anddichloromethane extracts exhibited weaker activity againstthe tested bacteria, while the hexane, ethyl acetate, anddichloromethane extracts did not exhibit any effect againstE. coli and P. aeruginosa.

The most active aqueous crude extract of C. procerawas purified by superdex G-75 (Figure 2(a)), reverse-phase HPLC columns C18 (Figure 2(b)). The final frac-tion was designated as Calo-protein (Figure 2(c)), thepurity of its molecular weight of the isolated Calo-protein determined by MALDI-TOF/MS (Figure 2(d)).The Calo-protein was tested against bacteria at a widerange of concentrations (100–6.25 μg/mL). The purifiedCalo-protein showed broad-spectrum activity against testedbacteria (Figure 2(c)). However, the Calo-protein inhibited

the growth of S. aureus and E. aerogenes effectively at25 μg/mL concentrations. It showed largest inhibitory zones(30 mm) equal to chloramphenicol (31 mm). Even at thelowest concentration of Calo-protein, the growth of S.aureus, E. aerogenes, P. vulgaris, P. mirabilis, and E. coli waseffectively inhibited. P. aeruginosa was weakly inhibited byCalo-protein at all the tested concentrations. Finally, thepurified protein from the C. procera stem bark exerted thestrongest antimicrobial activity against S. aureus than thecrude extracts (Figure 3).

3.3. Phytochemical Screening. Phytochemical screening ofthe stem bark of C. procera indicated the presence of varioussecondary metabolites such as alkaloids, flavonoids, tannins,coumarins, anthraquinones, saponins, cardiac glycosides,sterols, and teriterpenes. The crude extracts of this plant arerelatively rich in glycosides, alkaloids, tannins, sterols, andterpenes which may inhibit the growth of organisms.


Table 1: Antimicrobial activity of different extracts of stem-bark of C. procera tested on Gram-positive and Gram-negative bacteria at100 μg/mL concentration and their cytotoxic potential was evaluated against human macrophage (U-937) cell lines by XTT assay at 2000,1000, 500, 250, 100, 50, 25, and 12.5 μg/mL doses.

Crude extracts Phytochemicalscreening

Yield E. coli E. aerogenes P. vulgaris P. mirabilis P. aeruginosa S. aureus Cytotoxicity(μg/mL)

(1) Hexane Flovanoids 5.36 gm — 11± 0.94 7± 0.1 9± 0.06 — 8± 0.12 <250

(2) Ethyl acetate Steroids 3.42 gm — 18± 0.06 17± 0.27 15± 0.2 — 25± 0.06 <500

(3) Dichloromethane Polyphenols 4.78 gm — 12± 0.4 8± 0.23 11± 0.48 — 13± 0.21 <500

(4) Methanol Terpenoids 5.98 gm 14± 0.31 19± 0.2 23± 0.4 20± 0.6 8± 0.12 27± 0.06 >2000

(5) AqueousGlycosides;

Tannins;saponins

7.15 gm 11± 0.14 22± 0.7 25± 1.3 20± 0.12 9± 0.23 28± 0.15 >2000

(6) Chloramphenicol Standardantibiotic

30 μg 21± 0.34 24± 0.3 23± 0.12 26± 0.23 10± 0.35 29± 0.12 —

Clinical isolates of Gram-positive and Gram-negative bacteria were used. Values are mean ± SD of three replicates (n = 3), inhibition zones in diameters(mm) produced by the extract around the discs. Size of disc was 6 mm. Absence of bacterial inhibition denoted by (—), that is, no inhibition zone around thedisc by crude extract of plants.

500

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%)

0 6825.2 12651.4

12539.4688

18477.6 24303.8

25047.1563

104.1

Mass (m/z)

30130

(d)

Figure 2: (a) The most active stem bark extract was separated by Superdex G-75 column and resolved into five fractions (P1–P5). (b) Theactive fraction (P2) was further eluted and gave fractions CF-1–CF-F4. (c) The final pure fraction was separated by reverse-phase HPLCcolumn (C8) using CF-1 fraction and designated as Calo-protein (Clo-p) of C. procera. Calo-p was tested for in vitro antimicrobial efficacyat various dose and in wound healing studies in mouse model. (d) The molecular mass of the Calo-protein was determined by MALDI-TOF/MS.


0

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(f)

Figure 3: Antimicrobial properties of purified protein from the most active C. procera extract tested against bacteria that cause skininfections. (a–f) Results are expressed as the mean ± SD (n = 3) of the inhibition zones (diameter around the discs). The original sizeof disc was 6 millimeter in diameter. The bacterial growth inhibitory efficacy was compared with chloramphenicol used as a positive control.


3.4. Cytotoxicity of Crude Extracts. The hexane, ethyl acetate,and dichloromethane crude extracts of C. procera showedtoxicity to human macrophages (U-937) to 250 and500 μg/mL, However, methanol and aqueous extracts wereless toxic up to >2000 μg/mL, whereas the lower concen-trations (100–12.5 μg/mL) were devoid of toxic effects andmorphological changes of cells (Table 1). However, variedtoxic effects were observed in the C. procera crude extractsin a dose-dependent manner compared to control cells.

3.5. Cytotoxic Effect of Protein. Normal human skin fibroblast(HEPK) cells were exposed to Calo-protein to assay forcytotoxicity varying concentrations. However, this proteindid not exert any toxic effect on skin cells even at higherconcentrations (1000 μg/mL) (Figure 4(a)). Furthermore,the Calo-protein did not display any cytolytic effects at all thetested concentrations after 24 h compared with control cells(Figure 4(b)). The purified bioactive protein of C. procera didnot show toxic effects on skin cells up to 1000 μg/mL. Lightmicroscopic images of human skin fibroblasts were exposedto the Calo-protein at varying doses (1000–0.001 μg/mL).There was no alteration in the control cells (Figure 4(c)).Whereas the lower dose (100 μg/mL) of protein did not affectthe cell morphology (Figure 4(d)), but the higher dose ofprotein (1000 μg/mL) showed some changes on HEPK cellsafter 24 h (Figures 4(e) and 4(f)).

3.6. Wound Healing Effect of Calo-Protein. Significantwound healing activity was recorded in the mice treatedwith Calo-protein after 14 days compared to control(Figures 5(a) and 5(b)). There was a considerable reductionin wound area from day 4 onwards in treated mice comparedto mice receiving fusidic acid. The treatment with Calo-protein accelerated the rate of wound closure or repairmuch faster than the control mice. In the histopathologicalexamination, there was notably less noticeable infiltration ofinflammatory cells, greatly increased blood vessel formation,and proliferation of cells by the Calo-protein. Interest-ingly, there was full thickness of reepithelialization in theepidermis, well-organized granular layer, compared to thecontrol. The wounds of animal treated with Calo-proteinshowed full-thickness of epidermal regeneration that coveredcompletely the wounded area. The epidermis was thick anddisorganized especially compared with the adjacent normalskin and complete epithelialization, vascularization, and hairfollicles formation were observed in treated mice (Figures5(c), 5(d), 5(e), and 5(f)). This protein exerted a positiveimpact on wound healing by enhanced cellular proliferation,granular tissue formation and epithelialization, and earlydermal and epidermal regeneration. In addition, topicalapplication of the Calo-protein of mice pronounced in morecollagen content than the FA-treated mice versus controlmice (Figures 5(g), 5(h), and 5(i)).

4. Discussion

Our findings confirmed the antimicrobial property of C.procera used in traditionally medicine for the treatment of

skin and wound-related disorders. We previously reportedthe antimicrobial activity [30] of certain traditional medic-inal plants used in Tamil Nadu, India. Of these, the aqueousextracts of C. procera, exhibited showed the most significantantimicrobial effect against S. aureus and E. aerogenes.Interestingly, the growth of several wound causing bacterialwas controlled by organic and aqueous extracts. Out of5 different extracts that were examined, 3 extracts werefailed to show any antimicrobial effect against E. coli andP. aeruginosa at the tested concentrations. This negativeeffect may be attributed to the different climatic and edaphicfactors influencing the secondary metabolites. Another studyreported the lack of antimicrobial compounds inadequateconcentrations in the extracts [31].

Both the organic and aqueous extracts showed antimi-crobial activity against wound-causing bacteria. Notably, theaqueous extract of C. procera exerted comparatively strongereffect against S. aureus, E. aerogenes, and P. mirabilis. Inaddition, the inhibitory activities of C. procera extracts werecomparable to that of the reference antibiotic. Previously,it was reported that the apical twigs and latex of C.procera produced greatest inhibition zones against S. aureus[32]. Furthermore, we demonstrated the high antimicrobialpotency of the C. procera extract against all the testedbacteria may be due to the high content of glycosides andvarious proteins present in the aqueous extracts [23, 24].The differences in the antimicrobial activity of variousextracts may be directly related to the diversity of compoundsaccumulated in C. procera: proteins [21], calotropageninglycosides [23], cardenolides, flavonoids, and saponins [24]thus corroborating our results. Such compounds can bindthe Gram-negative bacteria to form a heavy soluble complexon the cell surface that subsequently disturbs the interactionbetween bacteria and cell receptors.

Previously, Freitas et al. [33] studied the enzymaticactivities and protein profile of latex of the C. procera.The C. procera protein showed broad-spectrum activityagainst tested bacteria at 100–6.25 μg/mL concentrations.We showed that the Calo-protein inhibited the growthof S. aureus and E. aerogenes, and it was equivalentto chloramphenicol. Even at the lowest dose of protein,the growth of S. aureus, E. aerogenes, P. vulgaris, P.mirabilis, and E. coli was inhibited effectively. The ethylacetate and dichloromethane crude extracts of C. procerashowed toxicity to human macrophage (U-937) cells at250 and 500 μg/mL doses than the methanol and aqueousextracts. However, the pure protein failed to show toxiceffects on human skin (HEPK) cells at doses of up to1000 μg/mL. Magalhaes et al. [34] studied that the variousorganic extracts of the stem of C. procera and foundthat ethyl acetate and acetone extracts displayed highercytotoxic potential against tumor cells (IC 0.8–4.4 mL),but the methanolic extract was weakly cytotoxic. Similarly,laticifer proteins (LPs) obtained from the latex of C.procera displayed considerable cytotoxic effects (IC 0.42–1.36 μg/mL) on SF295 and MDA-MB435 cells. In addition,LP was shown to inhibit DNA synthesis and to targetDNA topoisomerase I triggering apoptosis in cancer cells[35].


Cell cytotoxicity on human skin fibroblasts (HEPK)

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Figure 4: Cytotoxic effect of Calo-protein from the stem bark of C. procera assayed on human skin cells and their morphological changesobserved for 24 h. (a) Calo-protein was tested against skin cell up to 1000 μg/mL concentrations for 24 h. There were no toxic effectsrecorded at high concentrations. (b) The protein did not produce severe cytolytic effects after 24 h exposure of the plant protein in vitro.Light microscopic images showing the morphological changes of human skin fibroblast cells were exposed to the Calo-protein obtained fromthe stem bark of C. procera tested at varying doses (1000–0.001 μg/mL). (c) There was no alteration in the control cells, (d) while lower dose(100 μg/mL) of protein did not affect the cell morphology (e, f), the higher dose of protein (1000 μg/mL) showed some changes on HEPKcells after 24 h. Untreated cells served as a control (ctrl).


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Figure 5: Wound-healing activity by C. procera protein was determined in a mouse model. (a) Reduction of wound area was compared withantibiotic (FA) up to 14 days. (b) Percentage of wound healing effect of Calo-protein was compared with the control group. (c, d) The proteintreatment showed full thickness of reepithelialization in the epidermis, well-organized granular layer, compared to the wound control. (e, f)Masson’s Trichrome (MT) staining showed less deposition of collagen in the wound control mice after 14 days. (g–i) Biochemical analysisshowed that protein treated enhanced more collagen synthesis than the FA and WCtrl after 14 days. ep: epidermis, de: dermis, ct: cortex, ne:neutrophils, mf: muscle fiber, and co: collagen.


Wound Healing Effect of Calo-Protein. The latex of C. procerahas been used in traditional medicine to treat differentinflammatory diseases. The anti-inflammatory activity oflatex proteins has been well documented using differentinflammatory models [36]. Wound healing events involvedifferent phases such as preventing blood loss, inflammation,epithelial repair such as proliferation [7], mobilization,migration, differentiation, tissue remodeling, and collagendeposition. The acute inflammatory phase involved inwound healing is accompanied by the synthesis of extra-cellular matrix that is remodeled to the formation of scartissue, connective tissue, collagenization, and acquisition ofwound strength [37]. Our mouse model revealed enhancedrate of wound contraction than the control. The micetreated with 5 mg/kg Calo-protein showed favorable resultscompared with other groups. The treated wound exhibitedmarked dryness of wound margins with tissue regenerationat 14 days. The healing potential of C. procera on dermalwounds in guinea pigs has been documented [37]. In thisstudy, the topical application of Calo-protein constituentaccelerated wound repair, with full-thickness coverage of thewound area by organized epidermis. The wound healingpotential of Calo-protein is corroborated by documentedanti-inflammatory effect of the C. procera plant in rats[19, 20]. The Calo-protein led to potent wound contractioncomparable with FA treatment. In addition, it had favorableeffects on inflammation and wound healing.

Histological examination showed that increased cellularinfiltration in treated cases may be due to the chemotacticeffect enhanced by the extract that attracts inflammatory cellstowards the wound site. Augmented cellular proliferationmay be due to the mitogenic activity of the Calo-proteinthat may have contributed significantly to the repair process.Dermal regeneration in treated mice also implied that theprotein exerted a positive impact on cellular proliferation,granular tissue formation and epithelization. The finalstage of the healing process involves collagen depositionand remodeling within the dermis [38]. Thus, the MTstaining clearly supported the wound repair process inCalo-protein-treated mice that showed more pronouncedcollagen accumulation than the control. Several types ofsecondary metabolites and active compounds isolated fromplants have been documented in animal models as the activeprinciples for influencing wound healing. The latex of C.gigantean was previously shown to have wound healingeffects in rats comparable to those of nitrofurazone, andthe extract contains high content of glycosides, flavonoids,phenolic, and triterpenoid compounds with antimicrobialand antioxidant properties [28]. In our study, Calo-proteinisolated from the aqueous extract of C. procera possesseswound-healing property against bacterial infections. It waspreviously reported that calactin, mudarin, and calotropainare active constituents of C. procera latex [39], withantimicrobial and antioxidant properties.

5. Conclusions

The therapeutic potency of C. procera demonstrated thatthe wound-healing activity of this protein was effective for

inhibition of bacterial pathogens. We conclude that the Calo-protein from the C. procera stem bark has potential to bedeveloped into new therapeutic agent(s) for wound healingagainst bacterial infections.

Abbreviations

Calo-p: Calotropis procera proteinXTT: (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-

tetrazolium-5-carboxanilide)FA: Fusidic acid (sodium salts)MRSA: Methicillin resistant Staphylococcus aureusMH: Mueller Hinton agarCFU: Colony forming unitCHL: ChloramphenicolHPLC: High-performance liquid chromatographyCF: Calotropis procera fractionWc: Wound controlWt: Wound treatmentT-PER: Tissue protein extraction reagentELISA: Enzyme-linked immunoabsorbance assayU-937: Human macrophageHEPK: Human skin fibroblastLDH: Lactate dehydrogenase enzymeSBE: Stem-bark extractLP: Latex proteins.

Acknowledgments

The authors are grateful to the Academic Research Fund(ARF), National University of Singapore for the financialsupport (Grant no. R-181 000 078 122). They also thankthe Department of Microbiology, NUS for providing thebacterial cultures used in this study.

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Research Article

Plectranthus amboinicus and Centella asiatica Cream forthe Treatment of Diabetic Foot Ulcers

Yuan-Sung Kuo,1 Hsiung-Fei Chien,1, 2 and William Lu3

1 Department of Surgery, National Taiwan University Hospital, Taipei 100, Taiwan2 Department of Surgery, National Taiwan University College of Medicine, No. 1, Section 1, Jen-Ai Road, Taipei 10051, Taiwan3 Oneness Biotech Co., Ltd., 7F-1, No. 3-1, Yuan Qu Street, Nangang District, Taipei 115, Taiwan

Correspondence should be addressed to Hsiung-Fei Chien, [email protected]

Received 17 February 2012; Accepted 5 April 2012

Academic Editor: Esra Kupeli Akkol

Copyright © 2012 Yuan-Sung Kuo et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Effects of a topical cream containing P. amboinicus (Lour.) Spreng. (Lamiaceae) and C. asiatica (L.) Urban (Umbelliferae) wereevaluated and compared to effects of hydrocolloid fiber wound dressing for diabetic foot ulcers. A single-center, randomized,controlled, open-label study was conducted. Twenty-four type 1 or type 2 diabetes patients aged 20 years or older with Wagnergrade 3 foot ulcers postsurgical debridement were enrolled between October 2008 and December 2009. Twelve randomly assignedpatients were treated with WH-1 cream containing P. amboinicus and C. asiatica twice daily for two weeks. Another 12 patientswere treated with hydrocolloid fiber dressings changed at 7 days or when clinically indicated. Wound condition and safety wereassessed at days 7 and 14 and results were compared between groups. No statistically significant differences were seen in percentchanges in wound size at 7- and 14-day assessments of WH-1 cream and hydrocolloid dressing groups. A slightly higher proportionof patients in the WH-1 cream group (10 of 12; 90.9%) showed Wagner grade improvement compared to the hydrocolloid fiberdressing group but without statistical significance. For treating diabetic foot ulcers, P. amboinicus and C. asiatica cream is a safealternative to hydrocolloid fiber dressing without significant difference in effectiveness.

1. Introduction

Development of foot ulcers is a common complication ofdiabetes. Approximately 15% to 20% of 16 million peoplewith diabetes in the United States are hospitalized forfoot ulceration and subsequent infection during the diseasecourse [1]. Foot ulcers are primarily caused by peripheralneuropathy that reduces protective sensations for pain andtemperature associated with trauma, and also by motorneuropathies that result in foot deformity, local pressure,and eventual ulceration [2]. A multicenter study identifiedperipheral sensory neuropathy, trauma and deformity ascausative factors in 63% of diabetes patients; ischemia, callusformation, and edema were other prominent factors in ulceretiology [3]. Infection often follows diabetic foot ulcerationdue to vascular insufficiency and reduced leukocyte function.Diabetic foot infections range from simple cellulitis tomore complicated chronic osteomyelitis and are associated

with increased frequency of infection, high morbidity withincreased hospitalization, and lower extremity amputation[1, 4]. In diabetes patients, mild, moderate, and severe footinfections are all considered serious because the combinationof an open wound and reduced immune defenses mayquickly spread infection into the subcutaneous tissue anddeeper structures [5]. Impaired microvascular circulationcharacteristic of diabetes limits transport of phagocytic cellsand antibiotics to the affected area, predisposing diabetespatients to more frequent and potentially more severe footlesions that are more difficult to treat. Treatment of footulcers and resultant infection may involve aggressive surgicaldebridement [4], oral or intravenous antibiotics [6, 7], andwound management with topical applications [8]. Metabolicabnormalities also require correction if foot ulcer treatmentcan be expected to be effective [2].

Prompt, effective treatment of foot ulcers is essentialto reduce risk of exacerbation and amputation. Wound


closure is the main treatment goal and the type of treatmentemployed depends on wound severity, vascularity, andwhether infection is present. Treatment may include footelevation, pressure relief, debridement of necrotic tissue,and application of various topical treatments, including wet-to-dry dressings, topical antiseptics, semipermeable films,foams, hydrocolloids, and calcium alginate swabs, amongothers [1]. Botanical and herbal treatments have been usedfor centuries in traditional Chinese medicine (TCM) asevidenced by recent reports of TCM drug use patternsin a general hospital in Taiwan [9]. We formulated aninvestigational botanical product designated as WH-1 creamfor the treatment of diabetic foot ulcers. WH-1 cream con-tains extracts from two botanical raw materials, Plectranthusamboinicus (Lour) Spreng (Lamiaceae) and Centella asiatica(Linn) Urban (Umbelliferae). P. amboinicus and C. asiaticahave exhibited anti-inflammatory and healing propertiesapplicable to wound treatment. P. amboinicus is one ofnearly 300 botanical species in the Plectranthus genus of theLamiaceae family, which is noted for its diversity of use, par-ticularly as medicines for skin, infective, digestive, and res-piratory problems [10]. In its native environment in Kenya,Africa, it is the most frequent Plectranthus species applied totreat burns, wounds, sores, insect bites, and skin allergies.Evaluation of the in vivo (rat model) anti-inflammatoryand antitumor activities of an extract from the leaves of P.amboinicus results showed significant reduction of edemaand confirmed the anti-inflammatory properties at specificdosage levels [11]. In Taiwan and other East and SouthAsian countries, where Plectranthus species have been usedfor treating cough, fever, sore throat, mumps, and mosquitobites, Chiu et al. [12] investigated the anti-inflammatory andanalgesic properties of P. amboinicus extract in vivo and invitro. The extract inhibited pain induced in mice by aceticacid and formalin injection and inflammation induced bycarrageenan. The anti-inflammatory effect was attributedto modulating antioxidant enzyme activity in the liver andproduction of tumor necrosis factor alpha (TNF-α). Shuklaet al. [13] isolated asiaticoside from C. asiatica and found thathydroxyproline increased 56%, tensile strength increased57% and both collagen and epithelialization increased aftertopical application of 0.2% asiaticoside solution to punchwounds in guinea pigs. The active constituents of C.asiatica include pentacyclic triterpene derivatives shown tobe effective in treating venous insufficiency and striae gravi-darum (pregnancy-related stretch marks) [14]. Its sedative,analgesic, antimicrobial, antiviral, and immunomodulatoryeffects have also been studied but without confirmation ofspecific effects.

To investigate the effectiveness of WH-1, we comparedits effects to a relatively new hydrocolloid fiber dressing withdemonstrated effectiveness (Aquacel Hydrofiber Dressing,ConvaTec, Valencia, CA, USA). This occlusive, adhesivedressing combines absorbent carboxy-methyl-cellulose non-woven fiber to manage partial- and full-thickness wounds.It is reported to absorb up to three times its weight inexudate while providing an optimum healing environmentand minimal bacterial cross-contamination [15]. Although it

is one of the standard treatments applied by plastic surgeons,healing time results have varied [15–17].

While botanical and herbal therapies are usually appliedsafely and effectively, severe adverse effects of these agentshave been reported when used either directly or combinedwith standard Western medical approaches [18, 19]. There-fore, such treatments must be subjected to scientific studyto support their continued use by demonstrated safety andefficacy. This study aimed to evaluate the effects of a topicalcream containing P. amboinicus and C. asiatica comparedto the effects of hydrocolloid fiber wound dressing whenapplied topically to diabetic foot ulcers in patients withdiabetes type 1 and type 2.


A single-center, randomized, controlled, open-label studywas conducted between October 2008 and December 2009.

2.1. Subjects. Twenty-four type 1 or type 2 diabetes patientsaged 20 years and older with Wagner grade 3 foot ulcerspostsurgical debridement were enrolled. Wagner grade 3was defined as “deep ulcer involving osteitis, abscess, orosteomyelitis” according to the Wagner classification system[20].

Inclusion criteria were patients with type 1 or type2 diabetes, aged 20 years or older, and having Wagnergrade 3 foot ulcers postsurgical debridement. Patients withpoor nutritional status (albumin <3 g/dL), poor diabeticcontrol (HbA1c >10%), anemia (hemoglobin <10 g/dL),and leukocyte counts <1,000/cu mm were excluded. Otherexclusion criteria were presence of connective tissue diseaseor known or suspected malignancy local to the study ulcer;renal failure insufficiency (serum creatinine >1.5 mg/dL) orabnormal liver function (AST, ALT >2.5 × upper limit ofnormal range); requiring treatment with immunosuppres-sive agents, corticosteroids, chemotherapy or radiotherapy;female patients with positive pregnancy test or breastfeedingor unwilling to use appropriate contraceptive methodsduring study; patients with known sensitivity to essential oilsor lanolin cream. Withdrawal criteria included withdrawalof consent, lost to follow up, treatment failure (defined asworsening of Wagner grade or serious infection in ulcersite), and requiring the use of a prohibited medication.Withdrawal was also a consideration if any other medicalcondition was present that, in the opinion of the principalinvestigator, indicated that continued treatment with studytherapy and participation in this trial was not in the bestinterest of the patient. The 24 patients were randomlyassigned to two equal groups comprising 12 patients in aWH-1 cream group treated with WH-1 cream containingP. amboinicus and C. asiatica twice daily for two weeks andanother 12 patients in a hydrocolloid group treated withhydrocolloid fiber dressings during the same time period.

2.2. Ethical Considerations. All enrolled patients were ad-vised about their participation in the study and all providedsigned informed consent. The study protocol was reviewed


and approved by the Institutional Review Board (IRB) ofNational Taiwan University Hospital (IRB no. 200801067 M).

2.3. Formulation of Investigational Product: WH-1 Cream.WH-1 cream contained extracts from two botanical rawmaterials, P. amboinicus and C. asiatica. The plants ofP. amboinicus were collected on 2007 according to goodagricultural and good collection practices. C. asiatica. extractwas sourced commercially with certificate of analysis of theextract and herbal material. Guided by previous pharma-cological wound healing studies [11–14], the most activefractions, PA-F4 from P. amboinicus and S1 from C. asiatica,were combined in a 1 : 4 ratio to form the drug substanceWH1-DS. The final drug product, WH-1 cream, contained1.25% of drug substance in a cream base, 15 g per tube.The cream base contained cetostearyl alcohol, ireine, liquidpetrolatum, methyl paraben propyl paraben, Span 60, Tween60, white petrolatum, water, and pigments.

2.4. Treatment Administration. During the two-week studyperiod, WH-1 cream was applied topically twice daily to 12WH-1 group patients in an amount to fully cover the ulcerarea in a thin and even layer. The maximum amount mustnot exceed 2 millimeters in thickness. After each application,the comparative product, hydrocolloid fiber wound dressing(AQUACEL Hydrofiber Dressing) was applied to 12 patientsin the hydrocolloid fiber dressing group and left in place forup to 7 days or changed earlier as clinically indicated. Afterapplying WH-1 cream or hydrocolloid fiber dressing, thewound was covered with a transparent, adhesive, waterproofdressing (Opsite, Smith & Nephew, Taipei, Taiwan). Afterwound dressing for two weeks, the wounds in both groupswere all reconstructed by split-thickness skin graft or primaryclosure.

Treatment allocation was performed before site initia-tion. Permuted-block treatment allocation was used to assignparticipants to each group. A list of sequential numbers wasgenerated using a permuted-block randomization procedurewith a block size of 4 in SAS 9.1, with each number randomlyassigned to one group. Patients meeting the inclusion andexclusion criteria were randomly assigned in a 1 : 1 ratio tothe WH-1 cream group or the hydrocolloid fiber dressinggroup according to a predefined randomization schedule.After random assignment, subjects received the study med-ication at the baseline visit and were assessed for woundcondition and safety during the 2-week treatment period(follow-up visits at the 7th and 14th day). Hydrocolloidfiber dressing group patients had the dressing applied atbaseline and changed at the 7th day or earlier if clinicallyindicated.

2.5. Wound Assessment. Prior to beginning the study, allpatients in both treatment groups had received surgicaldebridement with complete reduction of necrotic tissueand debris to visualization of bleeding healthy tissue. Afterthe study treatment period, the primary endpoint was thepercentage change in wound size defined as wound size atendpoint (days 7 and 14) compared to wound size at baseline

(postsurgical debridement). The secondary endpoint was theimprovement in Wagner Grade from baseline (all patientswere Wagner grade 3 at baseline postdebridement) to finalvisit at day 14. Safety assessment involved the observation ofpresence or absence of adverse events for the study period.

Laboratory parameters measured included: hemoglobin,HbA1c, WBC with differential counts, AST, ALT, albumin,creatinine, fasting plasma glucose, BUN, and urine protein,which were tested at screening/baseline and at the end ofstudy (day 14). A pregnancy test was indicated for femalesof child-bearing potential before the study began.

Physical examination and vital signs were done atbaseline and at days 7 and 14. Concomitant treatments werepermitted as follows: sharp surgical debridement (includingresection of necrotic soft tissue and bone, sinus tracts,fistulae, undermined borders, callus) to form viable woundmargins was performed before randomization and repeatedas needed during the dosing period. Systemic antimicrobialagents were allowed for treatment of infections. Non-weight bearing or offloading was required for all subjects.Prohibited treatments during the study period includedimmunosuppressive agents, corticosteroids, chemotherapyand radiotherapy.

2.6. Statistical Analysis. Demographic parameters and otherbaseline characteristics were analyzed by treatment groupfor the randomized population. The intent-to-treat (ITT)population contained all randomized patients who hadreceived at least one dose of study medication and had at leastone follow-up efficacy endpoint evaluation. Additionally,only patients with a Wagner grade 3 foot ulcer were includedin the ITT population. Analysis of primary and secondaryendpoints was based on the ITT population. The safetypopulation was defined as all randomized patients who hadtaken any dose of study medications and was the primarypopulation for analyzing the safety data.

Comparability among the two treatment groups wasevaluated using Mann-Whitney U test for continuous vari-ables and Fisher’s exact test for categorical variables. Data arepresented as a median (interquartile range) for continuousdata and numbers (percentages) for categorical data. TheWilcoxon signed ranks test was used to compare differencesbefore and after treatment in each group. All statisticalassessments were two-sided and evaluated at the 0.05 levelof significant difference. Statistical analyses were performedusing SPSS 15.0 statistics software (SPSS Inc., Chicago, IL,USA) and SAS 9.1 (SAS Institute Inc., Cary, NC, USA).

3. Results

A total of 24 patients were screened and enrolled intothe study between October 29, 2008 and December 14,2010. Of these, 12 patients were randomly assigned to theWH-1 cream group and 12 patients to the hydrocolloidfiber dressing group. No participants were excluded duringscreening. Figure 1 shows a flow chart of the trial thatpresents the reasons for early termination and protocolviolations in detail. Only one patient in the hydrocolloid


Patients screened(n = 24)

Randomized(n = 24)

WH-1 cream group

(n = 12)

Completed study(n = 12) (n = 0)

Withdrawn Completed study

(n = 11) (n = 1)Withdrawn

Analyzed(n = 11)

Analyzed(n = 10)

Protocol violation(n = 1)

Protocol violation(n = 1)

Aquacel Hydrofiber dressing group

(n = 12)

Figure 1: Patients’ flow chart.

Table 1: Demographics and baseline characteristic of patients with Wagner grade 3 foot ulcers (n = 24).

WH-1 cream(n = 12)

AQUACELHydrofiber dressing (n = 12)

P value

Age (years)1 75.5 (50.0, 82.0) 69.5 (59.0, 79.5) 0.862

Gender, n (%)2 1.000

Male 4 (33.3) 5 (41.7)

Female 8 (66.7) 7 (58.3)

Target side, n (%)2

Right 5 (41.7) 6

Left 7 (58.3) 6

Height (cm)1 160 (144, 166) 160 (151, 168) 0.758

Weight (kg)1 64 (50, 101) 67 (63, 77) 0.758

BMI (kg/m2)1 30.82 (24.01, 36.68) 25.04 (22.97, 30.68) 0.356

Wound size (cm2)1 3.92 (1.18, 10.64) 4.47 (2.57, 14.57) 0.386

P values are from 1Mann-Whitney U test and 2Fisher’s exact test.Values are 1median (interquartile) and 2number (percentage).

fiber dressing group did not complete the study becausethe patient withdrew consent and decided to receive full-thickness skin grafts. Two other patients (1 in WH-1 groupand 1 in hydrocolloid fiber dressing group) were protocolviolating because their ulcers were not Wagner grade 3 atenrollment.

WH-1 cream group patients included 4 men and 8women with a median age of 75.5 years (range, 36–87 years)and the hydrocolloid fiber dressing group included 5 menand 7 women with a median age of 69.5 years (range,44–89 years). No statistically significant differences werefound between the two groups in patients’ demographics andbaseline characteristics (P > 0.05) (Table 1).

The ITT population comprised 21 patients; 11 patientstreated with WH-1 and 10 patients treated with hydrocolloidfiber dressing. The primary endpoint, percent changes inwound size from baseline at the final visit (day 14, end week2) in the ITT population are shown in Table 2. No statisticallysignificant differences were found in the percent changesin wound size between the WH-1 cream and hydrocolloidfiber dressing groups (P = 0.673). Analysis of results withintreatment groups showed that only the hydrocolloid fiberdressing group had significant decreases in wound sizes frombaseline to the final visit (P = 0.037, Figure 2).

After 2 weeks of treatment, 4 (36.4%) patients hadWagner grades 2 and 6 (54.6%) patients had Wagner grade


Table 2: Efficacy issue-primary and secondary endpoints in intent-to-treat population (n = 21).

WH-1 cream(n = 11)

AQUACELHydrofiber dressing

(n = 10)P value

Primary endpoint

Percent changes in wound size (%)1 −27.18 (−38.86, 36.10) −22.64 (−36.90, −3.20) 0.673

Secondary endpoint

Improvement on Wagner Grade, n (%)2 10 (90.9) 7 (70.0) 0.311

P values are from 1Mann-Whitney U test and 2Fisher’s exact test.Values are 1median (interquartile) and 2number (percentage).

Figure 2: Changes of wound size.

1 in the WH-1 cream group; 4 (40.0%) patients had Wagnergrade 2 and 3 (30.0%) patients had Wagner grade 1 in thehydrocolloid fiber dressing group. The secondary endpointwas the proportion of patients who had improvements inWagner grade, and improvement in the WH-1 group (10of 11 patients, 90.9%) was slightly higher than that inthe hydrocolloid fiber dressing group (7 of 10 patients,70.0%), but the difference was not statistically significant(P = 0.311). Figure 3 shows photographs demonstratingimprovements in wound size and Wagner grades of the WH-1 cream group.

All adverse events (AEs) were collected by patients ormonitored by the investigator throughout the study andwere coded using the 13th quarter Medical Dictionary forRegulatory Activities (MedDRA version 13.1). The numbers(%) of patients with at least one adverse event of maximumseverity during the treatment period are summarized inTable 3. During the treatment period, 5 of 12 patients inthe WH-1 cream group (41.7%) and 5 of 12 patients in thehydrocolloid fiber dressing group (41.7%) reported at least

one adverse event. According to the investigator’s judgment,none of these events was study treatment related. Nosignificant difference in adverse events was found betweenthe WH-1 cream and hydrocolloid fiber dressing groups(P > 0.05). In addition, there was no death or serious adverseevents in any patient during this clinical study.

4. Discussion

In this study, we compared the effects of a botanicalcream containing P. amboinicus and C. asiatica with theeffects of hydrocolloid fiber wound dressing when appliedtopically to diabetic foot ulcers. No statistically significantdifferences were found in the percent changes in wound sizeand improvements in Wagner grade between two groupsof diabetes patients who were treated with either WH-1 cream or hydrocolloid fiber dressings for the two-weekstudy period. Although the proportion of patients whoshowed improvement in Wagner grade was slightly higher inpatients treated with WH-1 cream than in those treated with


Wound size: 4.58 cm2, Wagner grade 3



(a)




(b)

Figure 3: The improvement in wound size and Wagner grades of three patients in the WH-1 cream group. (a) Before WH-1 cream treatment,(b) the 14th day after treatment.

hydrocolloid fiber dressing (90.9% versus 70%, resp.), thedifference was not significant. However, within-group anal-yses showed that only the hydrocolloid fiber dressing grouphad significant decreases in wound sizes from baseline to thefinal visit. Safety assessment was similar in both groups, withonly minimal adverse events and no serious adverse eventsreported or observed during the study period. Also, no pro-nounced changes were seen in mean SBP, DBP, pulse rate andbody temperature in any patient. Few changes were noted inclinical laboratory data (i.e., hematology, serum chemistries,and urinalysis) and none were clinically significant.

Taking Chinese herbs is a common self-treatment behav-ior in Asian society as well as a frequently prescribed

treatment, especially for patients with major diseases [9]. Itis not unusual that we apply a botanical medicine to thetreatment of diabetic foot ulcers and achieve good resultswithout adverse events. However, reports of severe adverseeffects induced by botanical or herbal agents directly or bycombined use with standard Western medicine are found inthe literature [18, 19]. Systemic absorption of those agentsremains problematic. In this study, results showed that thesystemic absorption of WH-1 cream was minimal and itappeared to be safe and well tolerated when administered topatients with diabetic foot ulcers. Our results provide helpfulevidence and insight into the use of extracts from Chineseherbs for modern treatment applications.


Table 3: Summary of adverse events (n = 24).

WH-1 cream(n = 12)

AQUACELHydrofiber dressing

(n = 12)P value

Any adverse event(s) 5 (41.7%) 5 (41.7%) 1.000

Constipation 2 (16.7%) 3 (25.0%) 1.000

Tinea pedis 22 (16.7%) 0 (0.0%) 0.478

Hypoglycemia 1 (8.3%) 0 (0.0%) 1.000

Dizziness 1 (8.3%) 0 (0.0%) 1.000

Visual field defect 1 (8.3%) 0 (0.0%) 1.000

Conjunctivitis 0 (0.0%) 1 (8.3%) 1.000

Diarrhoea 0 (0.0%) 1 (8.3%) 1.000

Gastritis 0 (0.0%) 1 (8.3%) 1.000

Vomiting 0 (0.0%) 1 (8.3%) 1.000

Oedema 0 (0.0%) 1 (8.3%) 1.000

P values are from Fisher’s exact test.

The insignificant differences between WH-1 and hydro-colloid fiber dressing can be explained in part becauseof the relatively short observation period in this prelim-inary study. Most short-term studies showed preliminary“healing” signals such as reepithelization, which can onlybe confirmed through advanced confirmatory research. Forexample, “Regranex” (becaplermin, the only FDA-approvedprescription platelet-derived growth factor gel for deepneuropathic diabetic foot ulcers) demonstrated only a slightimprovement in ulcer volume (P = 0.056) when used in41 patients in a 28-day dose-ranging phase II study [21].However, Regranex was reported to have a 50% incidenceof complete wound closure in the pivotal 20-week phaseIII study [22]. Other studies of topical applications for footulcers have been longer but have not necessarily yieldedbetter results. For example, in a comparative study betweenhoney and povidone iodine applied as dressing solutions forWagner grade 2 diabetic foot ulcers, patients were followedfor 6 weeks and a mean healing time of 14.4 days (range7 to 26 days) was achieved for patients receiving honeytreatment compared to 15.4 days (range 9–36 days) forpovidone iodine patients; even without significant differencein healing time, it could still be concluded that honey wasa safe alternative dressing for Wagner grade 2 foot ulcers[23]. Another multicenter trial investigating three dressingsfor managing diabetic foot ulcers had a duration of 12 weeksand significant differences were noted in results between thethree treatment groups but none directly related to meanhealing time [15]. Nevertheless, in future research expandingour present preliminary study results, we will apply longercontinuous treatment and observation of diabetic footulcers.

Another possible explanation for lack of significantdifferences between WH-1 cream and a topical cream witha hydrocolloid preparation could be the comparison of“apples and oranges.” However, the hydrocolloid wounddressing used in our study (Aquacel Hydrofiber Dressing)was one of three dressings investigated in a large, multicentertrial to determine whether modern dressings were more

clinically effective than traditional dressings in the treatmentof diabetes-related foot ulcers [15]. In that “apples to apples”comparison, no differences in effectiveness were found inthe three dressings in terms of percentage healed at 24weeks or in the mean time to healing or quality of healing(i.e., incidence of recurrence). While that study comparedparallel treatments over a longer period of time, the onlysignificance was that the hydrocolloid fiber dressing provedto be significantly more costly per treatment even thoughfewer changes of dressing were required to achieve thesame results. This suggests that more information about theeffectiveness of WH-1 could be gained through comparisonswith a wider range of topical medicines as well as newerproducts in widespread use.

Further investigation of P. amboinicus and C. asiat-ica cream must include other clinical parameters suchas pulsation, Doppler ultrasound, and patients’ subjectivesymptoms. Also, in the present study, only wound sizesand improvements in Wagner grades were compared andother signs and symptoms associated with diabetic footulcers, including pain, numbness, pallor, and pulses, werenot compared. We also did not focus on infection of footulcers since our investigational product was not intended asan antimicrobial measure; however, infection was noted andrecorded in some patients and systemic antimicrobial agentswere allowed for treatment of infections. When diabetic footulcers of different Wagner grades and infection by differentorganisms were investigated by culture and sensitivity andsusceptibility to citric acid as the sole antimicrobial agent,citric acid gel was 94% effective in controlling foot infectionsfor Wagner grades 1 and 2 and also in Wagner grade 3without deep osteomyelitis [8]. One drawback of the Wagnerclassification system is that it does not factor in the presenceof infection [4]. Since infection may be another factorin the insignificant results between the investigated WH-1cream versus a highly absorbent hydrocolloid fiber dressing,future studies may expand our approach to determinepossible relationships between infection, wound sizes, andimprovements in Wagner grade.


5. Limitations

This study has certain limitations, including that it isa single-site study with a small sample and a shortobservation period for wound reconstruction. It isessentially a pilot study conducted to observe safetyand effectiveness of the botanical cream designated as WH-1in treating Wagner grade 3 foot ulcers since the investigatedproduct is currently undergoing a phase II clinical trialfor treatment of Wagner grade 1 diabetes foot ulcers[http://clinicaltrials.gov/ct2/show/NCT00709514?term=wh1&rank=1]. Future study will include comparisons with awider range of topical treatments for diabetic foot ulcers andan expanded sample and longer study period.

6. Conclusion

For treating diabetic foot ulcers, P. amboinicus and C. asiaticacream is a safe alternative to hydrocolloid fiber wounddressing without significant differences in effectiveness. P.amboinicus and C. asiatica cream is a safe and effectivealternative for use in patients for whom hydrocolloid fiberwound dressing may be contraindicated.

Abbreviations

AE: Adverse eventsIRB: Institutional Review BoardITT: Intent to treatTNF-α: Tumor necrosis factor alphaTCM: Traditional Chinese medicine.


The authors declare that they have no conflict of interests.

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Research Article

Astragaloside IV Downregulates β-Catenin inRat Keratinocytes to Counter LiCl-Induced Inhibition ofProliferation and Migration

Fu-Lun Li,1 Xin Li,1 Yi-Fei Wang,1 Xiu-Li Xiao,2 Rong Xu,1 Jie Chen,1 Bin Fan,1

Wen-bin Xu,1 Lin Geng,1 and Bin Li1

1 Department of Dermatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine,Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China

2 Baoshan Branch, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine and Pharmacy, Shanghai 201900, China

Correspondence should be addressed to Bin Li, [email protected]

Received 2 October 2011; Accepted 5 February 2012


Copyright © 2012 Fu-Lun Li et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Re-epithelialization is a crucial step towards wound healing. The traditional Chinese medicine, Astragalus membranaceus (Fisch)Bge, has been used for hundreds of years for many kinds of ulcerated wounds. Recent research has identified the active compoundin this drug as astragaloside IV (AS-IV), but the underlying molecular mechanisms of its therapeutic action on keratinocytesremain poorly understood. In this study, we used an in vitro model of ulcer-like wound processes, lithium chloride (LiCl)-inducedcultured mouse keratinocytes, to investigate the effects of AS-IV treatment. The effects on cell proliferation were evaluated by theMTS/PMS colorimetric assay, effects on cell migration were determined by a wound-healing scratch experiment, effects on thecell cycle were analyzed by flow cytometry, and effects on protein expression were analyzed by immunoblotting and immuno-fluorescence. LiCl strongly inhibited cell proliferation and migration, up-regulated β-catenin expression, and down-regulatedproliferating cell nuclear antigen (PCNA) expression. AS-IV treatment attenuat the inhibition of proliferation and migration,significantly reducing the enhanced β-catenin expression, and recovering PCNA and β-tubulin expression. Thus, AS-IV mediatesmouse keratinocyte proliferation and migration via regulation of the Wnt signaling pathway. Down-regulating β-catenin toincrease keratinocyte migration and proliferation is one mechanism by which AS-IV can promote ulcerated wound healing.

1. Introduction

The skin of all animals provides a protective barrier againstentry by pathogens and environmental toxins. Skin cellsalso encompass a rich molecular infrastructure that involvesimmune system processes, which further defend againstdisease or injury to the underlying organs. Thus, cutaneouswounds not only affect skin homeostasis, but also represent asystemic threat; thus, the wound healing process is criticalfor general health and well-being [1]. The wound healingprocess is intricate and not yet fully understood; however, itis known that many underlying conditions, such as diabetes,cancer, and vascular disease, can prolong or block theprocess. In those cases, normal trauma caused by a simpleaccident or fall can become an ulcerated chronic wound,

significantly impacting one’s quality of life and increasing therisk of death.

For many centuries, the world’s physicians have soughtout methods and therapeutic agents that will promote andaccelerate wound healing [2]. Generally, the cutaneouswound healing process can be divided into three overlapping,but distinct, phases: the inflammatory phase, the prolifera-tive phase (neoangiogenesis, tissue formation, reepithelial-ization) and the tissue remodeling phase [3–5]. Reepithelial-ization is considered a crucial step towards wound healing,and impairment of this process has been demonstrated toresult in the development of chronic wounds [6].

The traditional Chinese medicine, Astragalus mem-branaceus (Fisch) Bge, has long been used to treat ulceratedwounds. Recently, the main active compound from this drug,


O

O

O

O

O

OH

OHOH

OH

OH

OH

OH

OH

H3C

H3C

HOH2CCH3

CH3

CH3

CH3

CH3

CH3

Figure 1: Chemical structure of astragaloside IV.

astragaloside IV (AS-IV), a 3-O-β-D-xylopyranosyl-6-O-β-D-glucopyranosylcycloastragenol (chemical structure shownin Figure 1), was purified. Research studies into the biologicalproperties of AS-IV have revealed strong anti-inflammatoryactivity, which involves inhibition of NF-κB activationand downregulation of adhesion molecule expression [7].In addition, many other pharmacological activities havebeen demonstrated, including antidiabetic and antioxidativeactivities [8]. However, the precise mechanisms by whichAS-IV promotes wound healing remain to be elucidated. Inorder to develop this Chinese herb as a bona fide therapeuticagent to treat aberrant wound healing, it is necessary togain a detailed understanding of the involved cellular andmolecular mechanisms.

It is known that activation of β-catenin/c-myc pathwayscan inhibit keratinocyte migration and differentiation,thereby impairing wound healing [9]. Prior studies usingembryological models indicated that administration oflithium chloride (LiCl) can induce signaling pathways thatmimic Wnt/β-catenin activation. Specifically, LiCl treatmentinhibits GSK-3β activity by inducing phosphorylation atSer-9 [10], which in turn, stabilizes free cytosolic β-catenin.Thus, in vitro treatment of cultured keratinocytes with LiClallows for the controlled experimental analysis of the molec-ular processes underlying impeded wound healing.

Activated β-catenin and c-myc have been clinicallydetected in the epidermis of chronic wounds. As such, it hasbeen proposed that these molecules may serve as markersof impaired healing and may represent useful targets oftherapeutic intervention [9]. In a previous study, we demon-strated that diabetic skin ulcers can be efficiently managedby treatment with a Chinese medicine cocktail that includesAstragalus membranaceus (Fisch) Bge. We hypothesized thatthe mechanism of action might involve inhibition of theWnt signaling pathway [11]. The study presented hereinwas designed to investigate the potential effects of AS-IV

on Wnt/β-catenin signaling in primary keratinocytes underLiCl-induced conditions. These findings reveal a positive rolefor AS-IV in cutaneous wound healing via downregulation ofβ-catenin expression.


2.1. Chemicals and Reagents. AS-IV (99.2% purity) was pur-chased from the National Institute for the Control of Phar-maceutical and Biological Products (Shanghai, People’sRepublic of China) and completely dissolved in room tem-perature DMSO at a stock concentration of 20 mg/mL. DAPIwas purchased from Roche Diagnostic Corp. (Indianapolis,IN, USA). Bromodeoxyuridine (BrdU) detection kit waspurchased from BD Biosciences (San Diego, CA, USA).LiC1 (pro analysis grade) was from Sigma-Aldrich (St.Louis, MO, USA). Antibodies against β-catenin and GAPDHwere purchased from Cell Signaling Technology (Beverly,MA, USA). Guinea pig anti-K14 was purchased from RDI-Fitzgerald (Oaklyn, NJ, USA). Antibody against β-tubulinwas purchased from Sigma-Aldrich. Immunofluorescencesecondary antibodies, Alexa Fluor 488-conjugated (green)and Alexa Fluor 594-conjugated (red), were from Invitrogen(Carlsbad, CA, USA). Fetal calf serum (FCS) and other cellculture reagents, unless otherwise indicated, were fromGibco (Invitrogen).

2.2. Cell Culture. Primary mouse keratinocytes were pre-pared from newborn mice, as previously described [12],and cultured at 37◦C in an atmosphere of 5% CO2 in Cnt-07 medium with appropriate supplements (Cellntec, Berne,Switzerland).

2.3. Cell Proliferation (MTS/PMS) and BrdU IncorporationAssays. Cell proliferation was assayed using the MTS/PMS


method. Briefly, cells (1 × 103/well) were plated in 96-wellculture plates (Nunclon; Nunc, Roskide, Denmark). Afterovernight incubation, cells were treated in the absence orpresence of LiCl and AS-IV. Each concentration was regardedas a single treatment group, while the control group onlycontained DMSO. Each group contained six replicate wells.After culture plates were incubated for 0, 24, 48, or 72 h,100 μL of MTS/PMS working solution was added to eachwell, and the keratinocytes were then incubated continuouslyfor another 4 h. The absorbance (A value) of each wellwas measured using a spectrophotometer (Beckman Coulter;Miami, FL, USA) at 490 nm. The effect of AS-IV on thecell growth inhibitory rate in keratinocytes was calculatedaccording to the following formula: % inhibitory rate = [A490

value of treated group]/[( A490 value of control group) ×(100%)]. The 50% inhibitory concentration (IC50) wasdetermined from dose-response data from at least threeindependent experiments. Cell number and cell viabilitywere determined daily using a Coulter Counter (BeckmanCoulter-Z2) and the trypan blue dye exclusion test. For the5-bromo-2′-deoxyuridine (BrdU) incorporation assay, cellswere seeded and serum starved as above. After treatmentwith PBS or LiCl at different concentrations for 12 h in 10%FBS-containing media, cells were incubated with BrdU foran additional 2 h. BrdU-labeled cells were detected using anenzyme-linked immunosorbent assay- (ELISA-) based col-ormeric kit (BD Biosciences; San Diego, CA, USA) accordingto the protocol provided by the manufacturer.

2.4. Western Blot Analysis. Lysates from cultured cells wereprepared as previously described [13]. Whole cell proteinextracts were resolved by SDS-PAGE and electrotransferredto membranes for specific antibody detection as previouslydescribed [14].

2.5. In Vitro Scratch Assay. When mouse keratinocytesreached 100% confluency, cells were transferred to basalKBM medium (Life Technologies, Inc., Grand Island, NY,USA) and incubated for 24 h. Cells were then treated with8 μg/mL mitomycin (ICN, Irvine, CA, USA) for 1 h and thenwashed with basal media. LiCl, an inhibitor of glycogensynthase kinase 3 (GSK3), was applied to activate Wnt sig-naling. Since LiCl can also efficiently block EGF-stimulatedmigration, EGF-induced cell migration was used as a positivecontrol. Scratches were performed in treated cell cultures aspreviously described, and cultures were photographed. Then,mixtures of 20 μmol/L LiCl with or without 25 ng/mL EGFand 80 μg/mL AS-IV were added to the cultures and incu-bated for 24 h, 48 h, or 72 h. The cells were rephotographed,and comparisons to the pre-treatment images were usedto quantitatively determine the extent of cell migration aspreviously described. Briefly, the distance covered by a cellmoving into the scratch wound area was measured. Thirtymeasurements were taken for each experimental condition.Three images were analyzed per condition and per timepoint.

2.6. Flow Cytometry for Cell Cycle Analysis. Cells were platedat a density of 1.5 × 106 cells per 6 cm diameter dish and

allowed to grow for 24 h, after which the medium waschanged to serum-free medium. After 16–18 h of starvation,cells are synchronized in the G0 phase of the cell cycle. Dif-ferent experimental reagents were then added to the serum-free medium and incubated for the indicated times. Cells(1 × 106) were harvested by trypsinization, washed twice incold PBS, and fixed in 70% alcohol for 30 min on ice. Afterwashing in cold PBS three times, cells were resuspended in1 mL Krishan staining solution [15] and left overnight at 4◦C.The next day, cells were filtered through a 96-micron poresize nylon mesh, and a total of 10000 stained nuclei wereanalyzed with a flow cytometer (BD Biosciences). DNAhistograms were prepared using the accompanying ModFitanalysis program (BD Biosciences), which plots and statis-tically fits the fractions of cells in the G0-G1, S, and G2-Mphases. Each condition was repeated in triplicate.

2.7. Immunofluorescence Staining. Cells were cultured on8-well chamber slides (Thermo Fisher Scientific, Waltham,MA, USA) and treated with different concentrations of AS-IV for 24 h. Cells were then fixed with 4% paraformaldehyde,permeabilized with 0.3% Triton X-100, and digested inpepsin solution (Lab Vision, Thermo Fisher Scientific). Non-specific binding in crude protein extracts was blocked byincubating cells in 10% normal goat serum plus 0.1% NP-40 for 1 h. Detection of specific proteins was carried out byincubating with primary antibodies overnight. Alexa Fluor488- or 594-conjugated secondary antibodies were used forimmunofluorescence staining, and images were obtainedwith a Leica SP2 confocal microscope (Deerfield, IL, USA).

2.8. Statistical Analysis. Data are presented as the mean± SDof at least three independent experiments, unless designatedotherwise. Statistical analysis was carried out using Student’st-Test, and a value of P < 0.05 was considered to be signifi-cant.

3. Results

3.1. AS-IV Attenuates LiCl-Induced Inhibition of KeratinoctyeGrowth. To investigate the normal function of the Wntsignaling pathway in keratinocytes, we used LiCl to activatethe Wnt/β-catenin signaling pathway. The effect of LiCl onkeratinocyte proliferation was examined by the MTS/ PMSassay and measurement of BrdU incorporation into DNAduring DNA synthesis. As shown in Figure 2(a), kerat-inocytes were treated with different concentrations (0∼40.0 mM/L) of LiCl for 24, 48, and 72 h. Controls were sub-confluent and synchronous keratinocytes were treated withDMSO solvent alone for the indicated times. Compared tothe control group, LiCl-treated keratinocytes exhibited con-sistent growth retardation. The half maximal (50%) inhi-bitory concentration (IC50) values (Figure 2(a), dotted line)of growth achieved after LiCl-treatment for 24, 48, and 72 hwere 25.85 ± 5.03, 16.16 ± 0.95, and 12.35 ± 1.38 mM,respectively. Lower concentrations of LiCl (<5 mM) did notinhibit keratinocyte growth. Higher concentrations of LiCl


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Figure 2: Cell proliferation of keratinocytes in response to LiCl and AS-IV treatment. (a) LiCl inhibits proliferation of mouse keratinocytes,as evidenced by the MTS/PMS assay. Data are expressed as a percentage of the control (100%) and represent the mean ± SD of threeexperiments. ∗P < 0.05, ∗∗P < 0.01. (b) LiCl induced a senescent-like morphology in live keratinocytes, as evidenced by phase-contrastmicroscopy. (c) Growth curve of keratinocytes in response to LiCl and AS-IV treatments. (d) OD at 490 nm of keratinocytes treated with orwithout LiCl and AS-IV for 72 h. Scale bar = 100 μm.


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Figure 3: BrdU staining of cells treated with LiCl in the absence of presence of AS-IV. (a) LiCl treatment produced a concentration-dependent decrease in BrdU-positive cells compared to control cells. (b) Histograms represent the mean± SD of the percent of BrdU-positivecells treated with different concentrations of LiCl. (c) Changes in β-catenin protein expression in response to LiCl. (d, e, f) Comparison ofthe effects of LiCl ± AS-IV on cell proliferation, by detection of BrdU incorporation and β-catenin protein expression. AS-IV resolved LiCl-induced inhibition of cell proliferation and downregulation of β-catenin. ∗P < 0.05. Expression of K14 and GAPDH were detected as proteinloading controls Scale bar = 100 μm.

(>5 mM) produced significant and dose-dependent inhibi-tion of keratinocyte proliferation (Figure 2(a), circle). TheBrdU incorporation assay also revealed a concentration-dependent decrease in BrdU-positive cells in response to dif-ferent dosages of LiCl. As shown in Figure 3(a), the amountof BrdU-positive cells following LiCl-treatment of 10 mM

(57.91 ± 6.57%), 20 mM (23.48 ± 3.19%), and 40 mM(9.67 ± 2.45%) was significantly less than those in theDMSO-treatment control group (71.81 ± 6.79%; all P <0.01). Cell viability was unaffected by LiCl-treatment at alldoses (Figure 4(c)). These data unambiguously confirm thatLiCl is able to exert potent growth inhibitory effects on


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Range: 5 microns–50 micronsNumber 2172Number percent 100%Mean 14.57 micronsStandard deviation 3.31 micronsMode 16.41 microns

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Subrange statisticsRange: 5 microns–50 micronsNumber 2534Number percent 100%Mean 16.49 micronsStandard deviation 3.78 micronsMode 17.05 microns

Subrange statisticsRange: 5 microns–50 micronsNumber 2046Number percent 100%Mean 18.89 micronsStandard deviation 4.92 micronsMode 19.62 microns

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Figure 4: LiCl affects keratinocyte morphology but not cell viability. Treatment with 20 mM and 40 mM LiCl caused cell size to enlarge (a,red arrow). (b) Average cell size from three experiments. (c) LiCl treatment had no effect on keratinocyte viability, as evidenced by the cellviability assay.

keratinocytes in a concentration- and time-dependent man-ner. These results also indicate that LiCl-treatment inhibitskeratinocyte proliferation without affecting their viability.

To investigate the function of AS-IV in keratinocytesoverexpressing β-catenin, we incubated the cells with AS-IV and LiCl for 72 h. As shown in Figures 2(c) and 2(d),AS-IV treatment attenuated LiCl-induced inhibition of cellproliferation, as evidenced by the fact that optical density(OD) at 490 nm was significantly higher in the LiCl groupthan in the DMSO control group (P < 0.05). Similarly, theBrdU assay showed that AS-IV treatment was able to enhancethe percentage of positive cells (from 23.48% to 31.05%;Figure 3(e)), suggesting that AS-IV was able to promoteDNA synthesis.

3.2. LiCl Induces Senescence-Like Changes in Keratinocytes.LiCl treatment also led to obvious morphological changesin keratinocytes, including increased size, spreading, andflattened appearance. There were no rounded cells, char-acteristic of apoptosis, observed in LiCl-treated cultures(Figure 2(b)). Using the cell viability assay, we measured the

change in diameter of keratinocytes in response to exposureto different doses of LiCl (from 10 mM to 40 mM) for 48 h.As shown in Figure 4, LiCl treatment at doses of 20 mMand 40 mM resulted in an enlarged cell size (Figure 4(a),red arrow), which indicated that high concentrations of LiClmay change keratinocyte morphology. The average diametersof LiCl-treated keratinocytes were greater (10 mM, 16.45 ±1.36 μm; 20 mM, 16.59 ± 0.36 μm; 40 mM, 17.72 ± 1.01 μm)than that in the control group (15.23 ± 0.97 μm).

3.3. AS-IV Attenuates LiCl-Induced S Phase Cell Cycle Arrest inKeratinocytes. LiCl-induced inhibition of growth may resultfrom cell cycle effects. Thus, the cell cycle distribution ofLiCl-treated and control cells was determined by measuringthe DNA content of Krishan-stained cells using fluorescence-activated cell sorting. As shown in Figure 5, LiCl-treatedcells exhibited a higher proportion of cells in the S phasethan control cells. Histograms were generated to visualize thepercent of cell cycle distributions produced by different LiClconcentrations (Figure 5(b)) and different exposure times(Figure 5(c)). Interestingly, when LiCl-treated cells were


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Figure 5: LiCl induces S phase arrest in keratinocytes. (a) Keratinocytes were exposed to various concentrations of LiCl for 24 h. (b)Keratinocytes were exposed to 20 mM LiCl for 24 h, 48 h, and 72 h. (c) Histogram showing the average amounts of cells in various cell cyclestages from three experiments following a 24 h exposure to different LiCl concentrations. (d) Histogram showing the average distribution ofcells in various cell cycle stages from three experiments with LiCl ± AS-IV, and LiCl + EGF treatments.

exposed to AS-IV, there was remarkable attenuation ofinduced cell cycle arrest in the S phase. Specifically, the per-centage of S phase cells in LiCl-treated keratinocytes cultureswent from 48.81± 7.43 to 35.28± 2.14 upon AS-IV exposure(P < 0.05).

3.4. AS-IV Attenuates LiCl-Induced Overexpression of β-Cat-enin in Keratinocytes. We investigated the effect of AS-IV onβ-Catenin expression in primary keratinocytes using twostandard assays, namely immunofluorescence staining andWestern blot analysis. As expected, keratinocytes incubated


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Figure 6: β-catenin is differentially regulated by LiCl and AS-IV. Stabilization of β-catenin in keratinocytes was followed by immuno-fluorescence staining with a β-catenin-specific antibody (a) and Western blot analysis (b). (a) DMSO (negative control) and EGF (positivecontrol) treated keratinocytes exhibited normal β-catenin membrane expression. The LiCl-treated group showed stabilized β-catenin, whichwas downregulated following AS-IV treatment. (b) GAPDH protein expression was used as the protein loading control. (c) Western blotanalysis revealed that PCNA expression, which is related to cell proliferation, was downregulated 50% by LiCl compared to the controlcells, and upregulated to 80% by AS-IV. GAPDH expression was used as a protein loading control. Asterisks indicate statistically significantdifferences between control and LiCl-treated cells. The scale bar in the first panel of (a) represents 100 μm, and is applicable to both sections.

with EGF showed normal β-catenin membrane expression,but those treated with LiCl exhibited prominent nuclearβ-catenin staining. LiCl-induced nuclear β-catenin was at-tenuated by AS-IV exposure, as evidenced by remarkablyless β-catenin nuclear staining (Figure 6(a)). Similarly, West-ern blot analysis indicated that LiCl-treated keratinocytesexpressed over 5-fold more β-catenin than the control cells.In addition, AS-IV exposure of LiCl-treated cells led tonearly 3-fold less β-catenin that that in the control group(Figure 6(b)) and additionally, PCNA, which indicating thecell proliferation, was downregulated 50% by LiCl comparedwith DMSO control, and upregulated to 80% by AS-IV(Figure 6(c)).

3.5. AS-IV Attenuates LiCl-Induced Migration Inhibition ofKeratinocytes. To test if LiCl-induced overexpression of β-catenin can affect keratinocyte migration during wound

healing, we used the in vitro wound scratch assay. Ker-atinocytes were incubated with LiCl, LiCl and EGF (positivecontrol), and/or AS-IV for 72 h. Keratinocyte migration intothe scratch area was observed and the distance was quan-tified. As shown in Figure 7, LiCl treatment inhibited ker-atinocyte migration by almost 80%, whereas LiCl and AS-IVpromoted migration by 60%, presumably via downregula-tion of activated β-catenin.

3.6. AS-IV Attenuates LiCl-Induced Downregulation of β-Tubulin. Tubulin protein is a key mediator of cell migration[16]. To determine whether LiCl treatment affected β-tubulin expression, we incubated primary keratinocytes withLiCl alone or with AS-IV. Changes in β-tubulin expressionlevels were evaluated by immunofluoresence following stain-ing of treated cells with a β-tubulin-specific antibody (Figure8(a)), and by immunoblotting (Figure 8(c)). β-tubulin was


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Figure 7: AS-IV attenuates LiCl-induced inhibition of keratinocyte migration. (a) Mouse primary keratinocytes with LiCl-inducedactivation of β-catenin were used in the scratch assay. LiCl inhibited keratinocyte migration compared to untreated cells. Inhibition wasprominent at 48 h and was sustained through 72 h (∗∗P < 0.01). EGF (positive control) stimulated migration was significant after 72 h,as evidenced by the nearly closed wound. AS-IV attenuated the LiCl-induced keratinocyte migration inhibition effect, as evidenced by thedifference in wound closure after 72 h (∗P < 0.05, compared with LiCl-treated group). Straight lines demarcate the initial wound area, anddotted lines indicate the migrating front of cells. (b) β-catenin was upregulated at the scratch wound edge. Insets show enlarged images ofthe dotted rectangle. The white circle indicates the keratinocytes starting to migrate from the nonnuclear β-catenin-positive cell. Scale bar =100 μm. (c) Histograms showing the average coverage of scratch wounds widths as percentages of the baseline wound width at time of scratch(0) and 24 h, 48 h, and 72 h after LiCl, LiCl + AS-IV, and LiCl + EGF treatments. ∗P < 0.05, ∗∗P < 0.01. Scale bar = 100 μm.

expressed in most of the untreated primary keratinocytes,but LiCl-treatment reduced β-tubulin expression. As shownin Figure 8(b), β-tubulin was expressed in only 15% of LiCl-treated cells but was expressed in 27% of cells treated withLiCl and AS-IV. In the DMSO-treated control cultures, 80%of cells expressed β-tubulin. Together, our findings indicatethat LiCl is able to cause downregulation of β-tubulin, whichwas resolved, at least in part, by treatment with AS-IV. Thus,overexpression of β-catenin during the wound healing pro-cess may impair healing by reducing β-tubulin expression,

and subsequently inhibiting keratinocyte migration. SinceAS-IV can attenuate LiCl-induced downregulation of β-tubulin, it may represent a promising therapeutic agent forimproving migration and promoting wound healing.

4. Discussion

In developed countries, 0.2 to 1% of the population is af-fected by chronic venous ulcers [17]. In China, it hasbeen reported that approximately 1.5%–3.0% of surgical


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Figure 8: AS-IV attenuates LiCl-induced β-tubulin downregulation. Keratinocytes were treated with LiCl (20 mM) and LiCl (20 mM) +AS-IV (80 μg/mL) for 24 h, and β-tubulin was determined by immunofluorescence (a, merged images of K14/β-tubulin/DAPI were shown)and immunoblotting ((c), GAPDH protein expression was used as the protein loading control). (b) Cell numbers of β-tubulin-positivekeratinocytes per 100 total cells are presented. AS-IV significantly attenuated LiCl-induced β-tubulin downregulation. ∗P < 0.05, ∗∗P < 0.01.Scale bar = 100 μm.

inpatients are hospitalized for chronic refractory wounds.Nonhealing chronic wounds not only decrease the quality oflife of the sufferer and increase risk of further infection anddeath, but also represent a significant economic burden onthe healthcare system. The experiments described in thepresent study show that AS-IV is able to repair chronic cut-anous wounds through regulation of cell migration and cellproliferation.

Among the many biological molecules known to influ-ence wound healing, Wnt signaling is believed to be crucial,as it mediates skin development throughout life and canexert both positive and negative effects on the wound healingprocess. LiCl is a well-characterized inhibitor of GSK3, andas such can effectively activate Wnt signaling in vitro [10].Numerous studies have shown that LiCl can elicit remarkablyvarious effects on different cell types. For instance, LiCl wasshown to stimulate proliferation of cultured erythrocytesvia activation of the Wnt/β-catenin signaling pathway [18].In addition, LiCl-stimulated proliferation of MCF-7 humanbreast cancer cells and caused a remarkable change in theexpression of phosphoinositide metabolites [19]. In con-trast, administration of LiCl suppressed prostate cancer cellproliferation by disrupting the E2F-DNA interaction andsubsequent E2F-mediated gene expression [20]. LiCl alsoinhibited proliferation of the human esophageal cancer cell

line, Eca-109, by inducing G2/M cell cycle arrest [21]. Maoet al. reported that LiCl treatment induced cell cycle arrestin G2/M in primary bovine aortic endothelial cells withoutaffecting cell viability [22]. These studies demonstrate thatLiCl can mediate a wide variety of cellular functions depend-ing on the context. Here, we demonstrate that LiCl caninduce cell proliferation and migration inhibition and causeS phase arrest in mouse primary keratinocytes (Figure 5).

The important role of Wnt signaling pathway activationin wound healing is well recognized [23, 24]. The Wntsignaling pathway is known to regulate a variety of cell func-tions, including cell fate, polarity, and differentiation, via thecanonical or β-catenin stabilization pathway and/or the pla-nar cell polarity or noncanonical pathway [25]. In addition,Wnt signaling was previously shown to mediate proliferationand migration of fibroblasts and keratinocytes [26]. Ker-atinocytes, the major cellular component of the epidermis,have several critical roles in the wound healing process. Thekeratinocytes that appear at the nonhealing edges of chronicwounds have been shown to overexpress β-catenin proteinand are believed to differ from normal, healthy keratinocytes.

During the normal wound healing process, the re-epithelialization stage involves covering the wound surfacewith a layer of epithelium, which involves differentiation,proliferation, and migration of epidermal keratinocytes.


Research has shown that overexpression of β-catenin andc-myc contributes to impaired wound healing by inhibitingkeratinocyte migration and altering their differentiation.

A previous study from our laboratory also indicated thata Chinese formula that included Radix astragali ingredientsmight be effective in regulating the Wnt signaling pathway[11]. However, the underlying mechanisms have remainedambiguous. AS-IV is the major phytochemical compoundof the well-known Chinese herbal medicine Huangqi, whichis extracted from the dried root of Radix astragali, and isa widely used herbal remedy in traditional Chinese medi-cine for the treatment of diabetes and inflammation [7].Therefore, the goal of the present study was to determine theeffect of AS-IV on keratinocyte proliferation and migrationin wound healing.

In this report, we show that treatment of primary ker-atinocytes with LiCl, an inhibitor of GSK-3β and an acti-vator of the Wnt signaling pathway, induced stabilizationand nuclear translocation of β-catenin associated with cellproliferation and migration inhibition. Moreover, LiCl treat-ment significantly induced cell cycle arrest in the S phase.MTS/PMS and cell viability assays showed that LiCl inducedcell growth retardation without affecting cell viability; itappeared that this effect was a result of induction of asenescent-like phenotype. AS-IV treatment of LiCl-inducedkeratinocytes resulted in attenuated growth retardation andreduced amounts of cells in the S phase. These new findingsled us to hypothesize that β-catenin overexpression duringimpaired wound healing may account for the dysfunctionalkeratinocyte proliferation and migration.

In our study, the BrdU incorporation assay was usedto determine DNA synthesis activity as an indirect methodof evaluating cell proliferation. BrdU analysis showed thatAS-IV treatment was able to increase the percentage ofBrdU-positive cells (Figure 3(e)), indicating that AS-IV caneffectively promote DNA synthesis activity in keratinocytes.Keratinocyte migration is essential for skin wound healing.Treatment with LiCl alone inhibited keratinocyte migrationabilities in a wound scratch assay. Interestingly, β-cateninwas upregulated at the scratch wound edge in LiCl-inducedkeratinocytes cultures. AS-IV effectively attenuated this LiCl-induced migration inhibition effect (Figure 7). Moreover, wefound that keratinocytes started to migrate from the non-nuclear β-catenin positive cell, indicating that the migrationof β-catenin-positive cells is blocked and AS-IV treatmentimproves cell migration associated with β-catenin expres-sion.

In summary, using LiCl as a tool to activate Wnt/β-catenin signaling, we have shown the functional relevanceof Wnt/β-catenin signaling in keratinocyte proliferation andmigration. Importantly, we have also provided evidence forthe application of AS-IV in the treatment of wound healingand revealed its role in the wound healing reepithelializationstage. These AS-IV related discoveries provide novel insightsinto the molecular mechanisms underlying the significantregulation of the proliferative effects of AS-IV in keratino-cytes, which can enhance epithelialization and healing ofchronic wounds [27]. Future studies are needed to furtherelucidate the effect of AS-IV on other molecules and cells

involved in wound healing, as this will help to advance thedevelopment of this drug for clinical application.

Abbreviations

AS-IV: Astragaloside IVLiCl: Lithium chlorideDMSO: Dimethyl sulfoxideGSK-3: Glycogen synthase kinase-3MTS/PMS: 3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazineethosulfate

PI: Propidium iodideBrDU: 5-bromo-2′-deoxyuridinePBS: Phosphate buffered salineIC50: Half maximal (50%) inhibitory concentrationPCNA: Proliferating cell nuclear antigen.

Acknowledgments

This study was supported by grants (nos. 81102596 and30973751) from the National Science Foundation (NSFC) ofChina. It was also supported by grants (no. 11XD1404700)from Shanghai Science and Technology Committee, Innova-tion Program of Shanghai Municipal Education Commission(no. 12YZ067) and Innovative Research Team at the Univer-sity of Shanghai Municipal Education Commission (phaseII).

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Review Article

Characteristics and Clinical Managements ofChronic Skin Ulcers Based on Traditional Chinese Medicine

Fu-Lun Li, Yi-Fei Wang, Xin Li, Feng Li, Rong Xu, Jie Chen, Lin Geng, and Bin Li

Department of Dermatology, Yueyang Hospital of Integrated Traditional Chinese and Western Medicine,Shanghai University of Traditional Chinese Medicine, Shanghai 200437, China

Correspondence should be addressed to Bin Li, [email protected]

Received 19 February 2012; Revised 9 March 2012; Accepted 10 March 2012


Copyright © 2012 Fu-Lun Li et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chronic skin ulcer (CSU), including diabetic ulcers, venous ulcers, radiation ulcers, and pressure ulcers, remains a great challengein the clinic. CSU seriously affects the quality of life of patients and requires long-term dedicated care, causing immensesocioeconomic costs. CSU can cause the loss of the integrity of large portions of the skin, even leading to morbidity and mortality.Chinese doctors have used traditional Chinese medicine (TCM) for the treatment of CSU for many years and have accumulatedmuch experience in clinical practice by combining systemic regulation and tropical treatment of CSU. Here, we discuss theclassification and pathogenic process of CSU and strategies of TCM for the intervention of CSU, according to the theories of TCM.Particularly, we describe the potential intervenient strategies of the “qing-hua-bu” protocol with dynamic and combinational TCMtherapies for different syndromes of CSU.

1. Introduction

A chronic skin ulcer (CSU) is defined as a wound lesionthat lasts more than four weeks without remarkable healingtendency or as a frequently recurrent wound [1]. TraditionalChinese medicine (TCM) considers that CSU belongs to the“ulcer” branch of the Ulcer and Sore diseases. There are morethan 8 million patients who have been diagnosed with CSUeach year in the United States [2], which costs more than10 billion dollars to treat this serious disease each year [3].In China, patients with CSU account for 1.5%–3% of thetotal hospitalized patients in the surgical departments [4].Therefore, the development of therapeutic strategies for theintervention of CSU patients is of great significance.

TCM has been used for the prevention and treatmentof CSU for many years. Historically, there are several TCMtheories for the intervention of CSU and they include the“wei-nong-zhang-rou (keeping the right amount of puson the surface of the ulcer to stimulate the growth ofgranulation),” “qu-fu-sheng-xin (removing necrotic tissuesto stimulate the growth of new skin),” and “ji-ping-pi-zhang (inhibition of inflammation to promote skin wound

recovery).” These TCM theories have been used as theguidelines for the intervention of CSU. The principles ofTCM intervention for CSU mainly focus on (1) systemicconsideration, (2) treatment based on syndrome differenti-ation, (3) differentiation of diseases and determination ofthe disease stage, (4) combination of systemic with topicaltreatments, (5) interior and exterior treatments together, and(6) treatment of symptoms as well as the causes. Accordingly,a therapeutic method should not only facilitate the woundhealing but also effectively decrease or relieve the scaring.Indeed, TCM has been used for the successful treatmentof many cases with CSU. Here, we discuss the currentapproaches on TCM treatment of CSU, particularly bycentering on the interventional strategies of “qing-hua-bu,”a dynamic and combinational therapy of TCM for differenttypes of CSU.

2. Theoretical Understanding of CSUDevelopment

In TCM, the pathogenesis of CSU is theoretically causedby “Re (heat) evil.” The pathogenic process of CSU was


described first in “Lingshu: yongju” as follows: “cold evilaccumulates in the meridian and results in a stiffness inblood flow and body jam, which inhibits the circulationof defensive energy, leading to inflammation. Subsequently,cold evil changes into heat evil, which causes tissue damagesand then pus formation.” Accordingly, the damaged tissuesin ulcers are the main factor contributing to the pathogenicprogression of CSU. Conceivably, “removing necrotic tissuesto stimulate the growth of new skin” has been used as a goldstandard for the intervention of CSU in TCM [4]. This isconsistent with the traditional view that “the fresh skin willnot grow until the removal of the necrotic tissues.” Actually,“removing necrotic tissues to stimulate the growth of newskin” has been demonstrated to be successful in the treatmentof CSU for many years in the clinic [5].

The “Fu (rotten)” tissues in the “Qu-Fu-Sheng-Ji” (re-moving necrotic tissues to stimulate the growth of newskin) protocol are comprised of infected necrotic tissues,overhyperplasia of pathological granulation and pathogens,which effectively obstruct the repair of an ulcer [5]. However,during the clinical practice, we found that treatment withTCM, according to the principle of “removing necrotictissues to stimulate the growth of new skin,” had variableoutcomes in patients with CSU, although they had ulcerswith similar shape, color, secreted fluid, and location. Similartreatment resulted in the rapid recovery of ulcers in somepatients, but did not in other patients. Although we alwaysremoved the damaged and necrotic tissues as much aspossible, we did not observe the regeneration of fresh tissues,even with the recurrence of tissue damage in some patients.In the TCM history, there are many TCM doctors, who haveinvestigated this phenomenon and described the potentialreasons. In the Song dynasty, Ziming Chen described inhis famous book of “wai-ke-jing-yao (essence of diagnosisand treatment of external diseases)” that “ulcer that hasbeen treated for a long time without remarkable recoveringdisplays the traits of white meat with little pus in thewound, which indicates a body deficiency in both Qi (vitalenergy) and Blood, a cold wound without sufficient qi andblood circulation.” Similarly, Dongxuan Buddhist in theSong dynasty wrote in the “wei-ji-bao-shu” (the guidelinesof human health) that “if the carbuncle has progressed intoulceration, there are always “Yu” (obstructed blood supplyand necrotic tissues) around the wound, which can invadeinto deep tissues to destroy muscles and bones. Hence,clearance of “Yu” should be of the primary strategy for thetreatment of the disease.” Professor Hanjun Tang, a well-known Chinese doctor at the Department of Surgery ofLonghua Hospital at Shanghai University of TCM, considersthat the skin ulcer is a “Re (heat)” syndrome because theskin ulcers usually begin with “redness and swelling aroundthe wound, which progressively forms pus.” In contrast,CSU is always at the middle or later stage of the diseaseprocess with the characteristics of “(1) the dim dark skinaround the wound, (2) subsiden wound, (3) a little pusand secretion, and (4) gray and pale granulation.” Thesecharacteristics belong to the syndromes of “Xu (deficiency)”

and “Yu (stasis).” The pathologic mechanisms underlying therefractory skin ulcers are that “long term disease results ina deficiency and stasis in both qi and blood, leading to adisorder of ying (nutrition) and wei (immunity) and skindystrophy.” In addition, it is well-known that “long termillness contributes to the development of Yu (stasis) and Xu(deficiency) syndromes.” Indeed, the “Yu” syndrome in anulcer is an external manifestation of the deficiency in the five“zang” organs and the stasis of qi and blood [6]. Hence, threepathologic factors of the “Re (heat),” “Xu (deficiency),” and“Yu (stasis)” sequentially or simultaneously contribute to thedevelopment and progression of CSU. The “Re (heat)” is thesymptom of an ulcer, while the “Xu (deficiency)” and “Yu(stasis)” are the causative factors of CSU. Sometimes, theyare reciprocal causation [7] because “Yu causes Xu and viceversa.” Apparently, the “Xu” and “Yu” are two key pathologicfactors of the development of CSU. Therefore, clearance of“Fu” (removal of necrotic tissues) is just one way to retardthe disease. In contrast, combination of supplement vacuityof “Xu (qi blood and ying yang)” with the removal of “Yu” isthe fundamental basis for the treatment of CSU. From longterm clinical practice and scientific researches, we believe thatthe “qu-yu (removal of stasis)” is crucial for the treatment ofCSU, according to the therapeutic principle of “bu-xu-qu-yu(tonification of weakness and removal of stasis),” because theremoval of the local stasis can eliminate the source of rottentissue formation, promote the growth of new tissues, andlimit the formation of scars [8]. Hence, we further emphasizethe view that “removal of stasis is beneficial for the growth ofnew tissues with little scar” [9].

3. The Characteristics of DifferentSyndromes of Ulcers

In TCM, a syndrome is a complex disharmony pattern ofsigns and symptoms at a given stage of the developmentand progression of a disease, and it reflects the internal andexternal conditions of individuals as well as the pathogenesis,location, instincts, healthy energy-evil struggle, and degree ofthe disease. The syndromes of CSU are the comprehensivemanifestation of systemic and local pathogenesis. Wangand Que [10] reported 338 patients with CSU at thelower limbs and divided the clinical syndromes into 3types of qi and blood deficiencies, spleen deficiency withdampness encumbrance, and qi deficiency with blood stasis.Patients with qi and blood deficiencies are characterizedby subsidence wound with pale or dim purple granulation,stiff dull skin around the ulcer, and fatigue to talk. Patientswith spleen deficiency and dampness encumbrance usuallyhave a swelling granulation on the wet wound, light yellowand thin pus, sallow complexion, and little defecation, whilepatients with qi deficiency and blood stasis commonly havepus immersion or pus covering tongue fur, lots of pus orrotten tissues with odor smell, heat pain or itch pain in centerplace, warm or a little hot skin, and dull-red skin aroundthe wound. Our clinical researches have classified these ulcerpatients into qi and blood deficiencies, spleen deficiency withdampness encumbrance, and qi deficiency with blood stasis.


4. Strategies for Systemic Treatment of Patientswith CSU by the “Qing-Hua-Bu” Protocol

Modern medical science has divided the healing processof ulcers into the three phases of inflammation, tissueformation, and tissue remolding. During the healing process,there are two main aspects of the growth of new granulationin the wound and the migration of the epithelial tissueto cover the wound. In the history of TCM, there aredifferent strategies for the treatment of different stages ofCSU. Treatment of a CSU with TCM still follow the principleof “syndrome differentiation and treatment,” combiningsystemic regulation with local treatment and consideringsimultaneous interior and exterior treatments and thesymptoms and causes together, according to the disease andits progressive stage. Methodologically, we should considerthree major pathologic factors of “Re,” “Yu,” and “Xu” inthe three stages of the pathogenic process of a CSU. On thebasis of the common principle of “qu-fu-sheng-ji (removalof necrotic tissues to stimulate the growth of fresh skin)”,we should pay special attention to the supplement of “Xu”and the removal of “Yu” using the “qing-re (clearing heat),hua-yu (resolve stasis), and bu-xu (tonification)” methodsto treat the syndromes at earlier, middle, and later stages ofthe disease, respectively. These three strategies can be appliedindependently, sequentially, or simultaneously.

4.1. Clearance of Heat and Promotion of Diuresis to TreatDampness Invasion at the Early Stage of CSU. At the earlystage of CSU, foreign particles and bacteria in the woundedarea will be cleared by the activated infiltrating neutrophilsand be phagocytosed by macrophages. Patients with CSUat this stage usually show skin inflammation with localskin itching and pain, red swelling and diffusion, andprogressively form an ulcer following the broken lesionand fluid secretion (Figure 1(a)). Inappropriate treatmentmay deteriorate the ulcer with fresh red meat accompaniedby odorous pus, thick or thin ulcer edge, red or purpleskin around the ulcer as well as heat burning and pain.Sometimes, patients with ulcers may have a red tongue witha thin or thick yellow coating and a slippery and string pulse.These symptoms result mainly from long-term standing orheavy loading that damage qi and blood circulation, leadingto poor nutrition of the skin. This, together with the woundand the invasion of dampness heat and pathologic evil, willeventually cause the symptoms of ulcers. Hence, treatmentof an ulcer at this stage should follow the principle of“clearing away heat and removing dampness, harmonizingying for detumescence” using the modified Simiao Bolusplus Biye Shenshi decoction for drying dampness. We canuse Jin Yin Hua (Lonicera japonica Thunb), Zi Hua DiDing (Viola chinensis) for clearing away heat and detoxify;Cang Chu (Atractylodes lancea), Fu Ling (Poria cocos), YiYi Ren (Semen coicis), Han Fang Ji (Stephania tetrandra S.Moore) as the subordinate medicines to clear away dampnessand detumescent, as well as to reduce the side effects ofthe principal medicines, including bitter cold and stomach-hurting. Dang Gui (Angelica sinensis), Chi Shao (Platycodi

(a)

(b)

(c)

Figure 1: Typical cases with CSU. (a) A case with dampness-heat diffusing downward syndrome of CSU manifested with redgranulation and yellow pus. (b) A case with qi-stagnancy andblood-stasis syndrome of CSU manifested with dark redness ordim purple skin around granular tissues. (c) A case with vital-Qideficiency and blood stasis syndrome of CSU manifested with gray-white and swelling granulation.

Radix), and Dan Pi (Moutan Cortex) are used as the assistantmedicines to cool blood and resolve stagnation. Chuan NiuXi (Cyathula officinalis), Huang Bo (Phellodendri ChinensisCortex), and Sheng Gan Cao (Glycyrrhizae Radix) are usedfor leading heat downward flow and harmonizing all themedicines.

Recommendations for Topical Treatments of an Ulcer. If thewound is covered by pus or rotten tissues, we can spreadJiuyi, Ba’er, Qisan, or even Wuwu powder on the surface,


depending on the thickness and tightness of the pus androtten tissues. Subsequently, we cover the wound with a thinlayer of Hongyou ointment (red greasy ointment) and fixthe wound daily until the pus and decayed tissues in theulcer disappear and the color of the granulation changes intored. If an ulcer is surrounded by red swelling and heat pain,we may use Ruyi golden ointment to compress around theskin, but not directly on the wound. Once the wound is redswelling with itching and produces a lot of fluid, we will usethe Qing dai ointment. If an ulcer produces a lot of fluid, wewill use the diluted skin health washing liquid. If there is asmall wound with empty vesicle under the wound and a lotof fluid, we will use a drug thread to drain out the liquid.

4.2. Promoting Blood Circulation to Dissipate Blood Stasis forthe Treatment of Patients with a CSU and a Qi-Stagnancyand Blood-Stasis Syndrome at the Middle Stage of the Wound-Healing Process. At the middle stage of the wound-healingprocess, inflammation reactivity in an ulcer is reducedand new stroma, which is often called granulation tissue,begins to grow until the wound space is filled. There isno decayed tissue and pus on the ulcer, but may havea crusting scar on the surface of the ulcer. The woundusually displays dark redness or dim purple around thegranular granulation tissues (Figure 1(b)) with a hard base,but the wound does not minimize within days. The woundis surrounded by dim purple or dark gray skin with a dulland stiff feeling. The edge of the wound uplifts with a shapeof the “mouth of water tank in China,” Meanwhile, thegranulation tissues are usually hyperplastic, accompaniedby numb and dull pain. Patients usually have stasis spotsand ecchymosis on their dark red tongue with thick-whitegrease or yellow grease, and their pulse becomes stringyand slow or thin and obscure. Patients with a long-lastingulcer at this stage usually have the evil qi in their bodiesfor a while, which stagnates the meridian and causes wounddystrophy. These alterations will result in growth stagnation,which in turn leads to the migration of the wound anda vicious circle of the healing process. Treatment shouldprimarily consider to promote qi and blood circulation andto clear the stagnation by using the modified Taohong SiwuDecoction. In this decoction, Tao Ren (Peach seed), HongHua (Carthamus tinctorius), and Dang Gui (Angelica sinensis)are used as the monarch drugs to resolve stasis and circulatethe blood. Chuan Xiang (Aucklandiae Radix), Ru Xiang(Olibanum), Mo Yao (Comiphora Myrrha), Chuang Xiong(Rhizoma Chuanxiong), and Di Long (Pheretima) are usedas the ministerial drugs to circulate the meridian. Chi Shao(Paeoniae Radix), Dan Shen (Salviae miltiorrhizae Radix),and Sheng Di (Rehmanniae Radix) are used as the adjuvantdrugs to cool the blood and for detoxifying. Gan Cao(Glycyrrhizae Radix) is used to harmonize all the medicinesinvolved.

Recommendations for Topical Treatments of an Ulcer at ThisStage. If we observe a wound covered with the crust or drynecrotic tissues, we would initially treat the wound withVaseline greasy ointment to soften the crust within days,

and then employ a minimal debridement method, just likea silkworm eating food to clear the crust. If we see an ulcerwithout pus and crust, we will spread Shengji Powder orBabao pill on the wound, followed by covering the woundwith a gauze with Shengji Yuhong ointment, Shengji Bayuointment, or red greasy ointment. If we observe a woundsurrounded by the “mouth of the water tank in China” witha dim dark and stiff skin, we usually use a percussopunctatorto stick the edge and the skin around the ulcer moderatelyand then use the strategies described above. If we detecthyperplastic granulation that is higher than the edge of thewound, which may block the epithelial growth, we would siftPingnu pill or Wumei powder to the wound, use 10% NaClhydropathic gauzes to compress the ulcer for several days, ordirectly use debridement method.

4.3. Supplements for Deficiency to Promote Granulation forthe Treatment of Patients with a CSU and Vital-Qi Deficiencyand Blood Stasis Syndrome at Later Stage of the Wound-Healing Process. At later stage of the wound-healing process,the wound will contract and be subjected to extracellular-matrix reorganization. Patients with a vital-qi deficiency andblood stasis syndrome usually display a subsided wound withpale or swelling granulation, but without obvious necrotictissues. Their wounds can be surrounded by the “mouthof water tank in China” and purple, hard, and stiff skin,particularly in the afternoon. Their tongues are pale andfatty with teeth marks around the brim and ecchymosis aswell as thin and white grease, and their pulses are string fineor heavy fine. These pathogenic characteristics usually comefrom long-term diseases. It is wellknown that “the long illnesscauses Xu, and the long illness causes Yu.” Actually, “Yu” canlead to “Xu” and “Xu” can enhance “Yu.” The qi deficiencyinhibits the growth of fresh tissues and is associated withwound subsidence and gray-white and swelling granulation(Figure 1(c)). The qi deficiency also disturbs normal cir-culation and leads to wound swelling, particularly in theafternoon. Similarly, the heave “Yu” promotes the formationof the “mouth of water tank in China” around the woundwith purple, hard, and stiff skin. The principle strategies forthe treatment of CSU at this stage are to use medicines forthe Yi-qi-huo-xue (supplementing qi and activating blood)and bu-xu-sheng-ji (tonification deficiency to promote tis-sue regeneration). A modified Buyang Huanwu Decoctionshould be applied for patients with a CSU at this stage. Inthis decoction, Huang Qi (Astragalis Raw Radix) and Tai ZiShen (Pseudostellariae Radix) are used as the monarch drugsto strengthen healthy energy. Dang Gui (Angelicae sinensisRadix), Dan Shen (Salviae miltiorrhizae Radix), Tao Ren(Semen Persicae), Hong Hua (Carthami Flos), and ChuangXiong (Rhizoma Chuanxiong) are used as the ministerialdrugs to reduce stasis and to circulate the blood. Bai Zhu(Rhizoma Atractylodis Macrocephalae) and Fu Ling (PoriaCocos) are used as the adjuvant drugs to induce diuresisthrough strengthening the spleen. A leech is used to circulatethe meridian. Chuan Niu Xi (Cyathulae Radix) is used to leadthe downward drug flow. Gan Cao (Glycyrrhizae Radix) willharmonize all the medicines involved.


Recommendations for Topical Treatments of an Ulcer at ThisStage. Due to the lack of necrotic tissues and pus on thewound at this stage, we can spread Sheng Ji powder andBabao pill on the wound and cover with a gauze withHongyou ointment or Shengji Yuhong ointment. If thereis a vesicle under the wound, we can compress the woundby a bound tied therapy. If the ulcer subsides, we can addcotton to ensure that the wound be exposed to drugs. Ifthe wound displays dim-dry, hard, and stiff granulation,we can use ointments, such as Hongyou ointment, ShengjiYuhong ointment, and Shengji Baiyu ointment, as well asTangchuang oil or Shirun Shao shang oil. If there are a lot offluids on the ulcer, we can cover the wound with a gauze withKangfuxin liquid or recombinant bovine fibroblast growthfactor. In parallel with these treatments, physical treatments,such as microwave, far infrared energy, UV, and laser can beused as the assistant methods.

5. Treatment of the CSU-Related Primary andAssociated Diseases

The refraction of CSU is mainly related to its primary diseaseand its related comorbid factors. Actually, CSU is not anindependent disease, rather a syndrome of other diseasesat a certain stage. Hence, inquiring a detailed history ofthe disease, careful physical examination, and completingauxiliary examinations, including etiologic examination andpathologic biopsy, to confirm the diagnosis and to detect therefractory factors prior to treatment will be helpful. Thesepreliminary works will play critical roles in the treatmentof patients with a CSU and preventing the formation of ahyperplasic scar. In addition, these procedures will help inthe diagnosis of malignant diseases or specific infection andin making appropriate decisions for the treatment of a CSU.

It is important to change position of patients with apressure ulcer at least every 1-2 hours to relieve the pressureusing pillows, sheepskin, foam padding, and others, becausethe long-term pressure against the skin reduces blood supplyto the area, accompanied by active treatment of patientswho have malnutrition, imbalance of electrolytes, and otherbasic diseases simultaneously. For a venous ulcer, we usuallysuggest that the patients reduce the standing time and avoidwalking for a long time. Actually, simple elevation of theirlegs above heart level for 30 minutes three to four timesper day can also reduce edema and improve blood flow inthe veins, which can accelerate the healing process. We alsosuggest that the patients wear elastic socks and bandagesin combination with some drugs that improve venousblood circulation, such as Aescuven forte, melilotus extracttablet, and Diosmin. Moreover, we pay special attention totreatment of deep vein thrombosis and lower limb embolism.

If patients have arterial ulcers, we first differentiate ulcersthat stem from diabetic angiopathies and thromboangiitisobliterans from arteriosclerosis obliterans. Subsequently,we treat the patients with a vasodilator and plateletaggregation inhibitors such as Alprostadil, cilostazol, orlow molecular dextran as well as TCM with a functionof promoting blood circulation. If necessary, we will

provide an operation and interventional therapy for thosepatients. If patients have an ulcer secondary to allergiccutaneous vasculitis or pyoderma gangrenosum, we willtreat the primary disease actively. If bacterial test is positivefor a specimen from the ulcer, we will distinguish thecolonization of a bacterium from infective bacteria in thewound, because the presence of bacteria in a wound canbe a sample contamination, bacterial colonization, criticalcolonization, or infection. Actually, all chronic woundsare contaminated, and they contain at least nonreplicatingmicroorganisms within or on the surface of the woundbed. Microorganisms live in a wound, such as bacterialcolonization, and can be harmless because host defensescan clear them out. Colonization is defined as replicatingmicroorganisms that adhere to the wound surface, but donot cause damage to the host (Wound and Skin Care Center;http://www.vnaa.org/vnaa/g/?h=html/wound center July).Hence, it is not necessary to provide special treatmentfor them. Infection characterized by the presence ofreplicating microorganisms within a wound will result ina subsequent host response. If bacterial infection causeshigh fever and accounts of white blood cells as well aserythema, warmth, swelling, pain, odor, and purulentdrainage around the wound, these suggest that the infectedbacteria may have entered into the body, leading to asystemic infection. Accordingly, we usually treat thesepatients with antibiotics or other medicines, according tothe antibiotic sensitivity of the microorganism, particularlyfor those with fungus, Mycobacterium tuberculosis, oratypical Mycobacterium infection. Patients with diabetesfor a long period usually develop vascular complicationsand neuropathy so that their ulcers are easily secondary tobacterial infection. Therefore, therapeutic strategies shouldinclude hyperglycemic correction, peripheral vascularvasodilation, neuron nutrition, and controlling infection. Inaddition, we should recognize that long-term unsealed skinulcers may undergo malignant transformation. Similarly,an ulcer can be a symptom of some skin malignant tumors.Hence, it is critical to recognize the possibility of malignanttumor with marginal swelling, cauliflower shape, andhardness around the ulcer tissue, particularly for a long-termunsealed ulcer. We recommend multipoint and multitimepathological biopsies for a highly suspicious ulcer to affirmthe diagnosis, and then perform an operation or other radioand chemotherapies for those patients.

Numerous data from many animal and clinical studiesimplicate the efficacy of TCM in the treatment of a CSU [11–16]. However, there is a little evidence to demonstrate theefficacy of TCM in the treatment of a diabetes-related CSU inthe clinic due to the lack of high-quality clinical researches.Therefore, further high-quality clinical investigations arewarranted to verify the beneficial activity of TCM in thetreatment of a CSU [17].

Abbreviations

TCM: Traditional Chinese medicineCSU: Chronic skin ulcers.


Acknowledgments

This study was supported by grants from the NationalScience Foundation of China (NSFC) (81102596 toFulun Li, 30973751 to Bin Li), Innovation Program ofShanghai Municipal Education Commission (12YZ067 toFulun Li), Shanghai Science and Technology Committee(11XD1404700 to Bin Li), and Shanghai Municipal HealthBureau (ZYSNXD-CC-ZDYJ018 to Bin Li) as well as thegrant from Innovative Research Team in University ofShanghai Municipal Education Commission (phase II).

References

[1] M. A. Fonder, G. S. Lazarus, D. A. Cowan, B. Aronson-Cook,A. R. Kohli, and A. J. Mamelak, “Treating the chronic wound:a practical approach to the care of nonhealing wounds andwound care dressings,” Journal of the American Academy ofDermatology, vol. 58, no. 2, pp. 185–206, 2008.

[2] A. Harsha, O. Stojadinovic, H. Brem et al., “ADAM12: apotential target for the treatment of chronic wounds,” Journalof Molecular Medicine, vol. 86, no. 8, pp. 961–969, 2008.

[3] D. J. Margolis, W. Bilker, J. Santanna, and M. Baumgarten,“Venous leg ulcer: incidence and prevalence in the elderly,”Journal of the American Academy of Dermatology, vol. 46, no.3, pp. 381–386, 2002.

[4] X. B. Fu, “The further emphasis on the development andtreatment of chronic refractory skin ulcers,” Chinese Journalof Traumatology, vol. 20, no. 8, pp. 449–451, 2004.

[5] M. Zhang and Y. Wang, “The discussion of using Qu Fu ShengJi theory to treat chronic refractory skin ulcers,” GuanmingJournal of Traditional Medicine, vol. 24, no. 5, pp. 794–796,2009.

[6] Z. Y. Wang and Y. Zhang, “The experience of Qu Yu Sheng Jimethod in treating of chronic ulcers used by professor YunjunTang,” New Chinese Traditional Medicine, no. 6, pp. 13–14,2002.

[7] B. Li, F.-L. Li, and K.-Q. Zhao, “Investigation of connectivemathematics model on chronic cutaneous ulcer in traditionalChinese medicine syndrome differentiation,” Chinese Journalof Dermatovenerology of Integrated Traditional and WesternMedicine, vol. 9, no. 1, pp. 4–7, 2010.

[8] B. Li, “The observation of the effects of Shengji Huayu recipein the treatment of limber chronic ulcer,” Liaoning Journal ofTraditional Chinese Medicine, no. 8, pp. 359–360, 2000.

[9] B. Li, Z. Y. Wang, X. L. Xiao, F. L. Li, and B. Fan, “Effectsof Shengji Huayu recipe and its decomposed formulas onsynthesis of collagen types I and III in granulation tissue ofrats in early wound healing,” Journal of Chinese IntegrativeMedicine, vol. 3, no. 3, pp. 216–219, 2005.

[10] Y. J. Wang and H. F. Que, “Clinical study of syndromedifferentiation standards for chronic skin ulcer of lowerlimbs,” Journal of Chinese Integrative Medicine, vol. 7, no. 12,pp. 1139–1144, 2009.

[11] S. Schreml, R. M. Szeimies, L. Prantl, S. Karrer, M. Landthaler,and P. Babilas, “Oxygen in acute and chronic wound healing,”British Journal of Dermatology, vol. 163, no. 2, pp. 257–268,2010.

[12] F. L. Li, H. Deng, H. W. Wang et al., “Effects of externalapplication of chinese medicine on diabetic ulcers and theexpressions of β-catenin, c-myc and K6,” Chinese Journal ofIntegrative Medicine, vol. 17, no. 4, pp. 261–266, 2011.

[13] J. N. Xu, H. F. Que, and H. J. Tang, “Effects and actionmechanisms of Buyang Huanwu decoction in wound healingof chronic skin ulcers of rats,” Journal of Chinese IntegrativeMedicine, vol. 7, no. 12, pp. 1145–1149, 2009.

[14] X. L. Xiao, Z. Y. Wang, H. J. Tang, and X. D. Liu, “Effects ofFuhuang Shengji Yuchuang ointment on expressions of typesI and III collagens in granulation tissue of wound in rats withdiabetes,” Zhong Xi Yi Jie He Xue Bao, vol. 7, no. 4, pp. 366–371, 2009.

[15] F. L. Li, B. Li, Z. Y. Wang, B. Fan, W. B. Xu, and R. Xu, “Effectof resolving stagnation and promoting granulation therapy onexpressions of bax and Bcl-2 in granulation tissue of diabeticrats during wound healing,” Zhong Xi Yi Jie He Xue Bao, vol. 5,no. 6, pp. 661–664, 2007.

[16] Z. Y. Wang, X. L. Luo, and C. P. He, “Management of burnwounds with Hippophae rhamnoides oil,” Nan Fang Yi Ke DaXue Xue Bao, vol. 26, no. 1, pp. 124–125, 2006.

[17] M. Chen, H. Zheng, L. P. Yin, and C. G. Xie, “Is oraladministration of Chinese herbal medicine effective and safeas an adjunctive therapy for managing diabetic foot ulcers?A systematic review and meta-analysis,” Journal of Alternativeand Complementary Medicine, vol. 16, no. 8, pp. 889–898,2010.


Research Article

Dexamethasone Resisted Podocyte Injury via Stabilizing TRPC6Expression and Distribution

Shengyou Yu and L. Yu

Guangzhou Medical College, Guangzhou First Municipal People’s Hospital, Guangdong Province, Guangzhou 510180, China

Correspondence should be addressed to L. Yu, [email protected]

Received 23 July 2011; Accepted 19 January 2012


Copyright © 2012 S. Yu and L. Yu. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

TRPC6, a member of the canonical transient receptor potential channel (TRPC) subfamily, is an important cation selective ionchannel on podocytes. Podocytes are highly differentiated cells located on the visceral face of glomerular basement membraneand featured by numerous foot processes, on which nephrin, podocin, and TRPC6 locate. Podocytes and the slit diaphragm (SD)between adjacent foot processes form a selective filtration barrier impermeable to proteins. TRPC6 is very critical for normalpodocyte function. To investigate the function of TRPC6 in podocytes and its relation to proteinuria in kidney diseases, we over-expressed TRPC6 in podocytes by puromycin aminonucleoside (PAN) and observed the changes of foot processes, TRPC6 proteindistribution, and mRNA expression. Accordingly, in this study, we further investigated the role of specific signaling mechanismsunderlying the prosurvival effects of dexamethasone (DEX) on podocyte repair. Our results showed that podocytes processesof overexpressing TRPC6 were reduced remarkably. These changes could be rescued by DEX via blocking TRPC6 channel.Additionally, our results also showed an improvement in TRPC6 arrangement in the cells and decrease of mRNA expressionand protein distribution. From these results, we therefore proposed that overexpression of TRPC6 in podocytes may be one of thefundamental changes relating to the dysfunction of the SD and proteinuria. DEX may be maintained the structure and functionintegrity of SD by blocking TRPC6 signal pathway and played an important role in mechanisms of anti-proteinuria.

1. Introduction

TRPC6 is determined in recent years which is closely relatedwith proteinuria and positioned in the structure proteinmolecules of SD. Proteinuria is a common feature of kidneydysfunction of glomerular origin and is itself a risk factor forrenal disease [1]. There is a growing body of experimentaland clinical literature showing that podocyte number isa critical determinant for the development of proteinuriaand glomerulosclerosis [2]. PAN is used to induce a well-established cell model for podocytes injury. DEX is widelyused for the treatment of a variety of glomerular diseasescharacterized by podocyte injury and proteinuria, includingmembranous nephropathy, minimal change disease, FSGS,and lupus nephritis. However, the signaling mechanismsunderlying the antiproteinuria effects of DEX have not beenwell defined. In this study, we further investigated the roleof specific signaling mechanisms underlying the protectioneffects of DEX on podocyte injury. Our results showed

that, in podocytes overexpressing TRPC6, cell processes werereduced remarkably. These changes could be rescued by thetreatment of the cells with DEX to block TRPC6 channel.Additionally, the podocytes overexpressing TRPC6 treatedwith DEX showed an improvement in TRPC6 arrangementin the cells and decrease of mRNA expression and proteindistribution.


2.1. Cells in Culture. Conditionally immortalized mousepodocyte clone (a kindly gift from Professor Peter Mundel,USA, and Professor Jie DING, Peking University FirstHospital) was cultured at 33◦C in RPMI-1640 containing10% fetal bovine serum (Gibco, Gaithersburg, MD, USA),100 U/mL penicillin/streptomycin, and 10 U/mL of mouserecombinant r-interferon (PEPRO Tech, London, UK) andthen shifted to 37◦C for differentiation by removal of r-interferon [3]. had typical character of mature podocyte after


two weeks. In the studies described below, all experimentswere performed in growth-restricted podocytes.

2.2. Experimental Design. In order to examine the effect ofDEX on PAN-induced podocyte injury, podocytes grownunder growth restrictive conditions for 12 days were incu-bated with media containing 10% FBS in the presence of1 μmol/L DEX (Sigma Chemical Co). DEX was not removeduntil the end of each experiment. To determine if DEXreduced a range of injuries, the following experiments wereundertaken after 1 h of DEX incubation: PAN. To determinethe mechanisms of podocyte injury induced by PAN, weexposed podocytes to PAN (Sigma Chemical Co) at aconcentration of 50 μg/mL. Following a 48 h incubation withPAN in the presence or absence of DEX, then observed andharvested at 8 h, 24 h, and 48 h, respectively. The experimentswere all repeated three times.

2.3. RT-PCR Analysis. Total RNA was extracted frompodocyte using Trizol reagent according to the man-ufacturer’s instruction, and the RNA concentration wasdetermined after the sample was dissolved in diethyl-pyrocarbonate-treated water. Isolated RNA (1 μg) of eachsample was subjected to reverse transcription by usingRever Tra Ace (TOYOBO Co., Japan) according to themanufacture’s protocol. The resulting cDNA (3 μL) wasused for PCR amplification. The sequence-specific primerswere designed and synthesized by Shanghai InvitrogenBiotechnology Co, Ltd. Primers used were as follows. TRPC6upstream and downstream primers, respectively, were asfollows. Forward: 5 GTTAATTGCGATGATCAATAGTT-3.Reverse: 5-GACTTGGTACAAGATTGAAGG-3. Probe: 5-FAM-CCAGGAAATTGAGGATGATGCGGACGTG-BHQ1-3, product length being 143 bp. GAPDH upstream anddownstream primers were as follows. Forward: 5-GGTGAAGGTCGGTGTGAACGGAT-3. Reverse: 5-CCACTTTGCCACTGCAAATGGCAG-3. Probe: FAM-CTGGTGACCAGGCGCCCAATACGGCC-BHQ1, product length being118 bp. The PCR amplification was started with 2 min of de-naturation at 94◦C, which was followed by 34 cycles (forGAPDH, 30 cycles) of denaturation at 94◦C for 30 s,annealing at 55◦C for 30 s (GAPDH, 55◦C for 30 s), andpolymerization at 72◦C for 75 s (GAPDH, 30 s). The finalextension lasted 7 min at 72◦C and then ended at 4◦C. PCRproducts (5 μL) were separated on 1% ethidium bromide-stained agarose gels and later scanned with gel imagingsystem (Bio Rad Company A). Independent experiment wasrepeated 3 times.

2.4. Western Blot Analysis. Podocytes were lyzed in the buffercontaining 1% Tritonx-100, 150 mM NaCl, 1 mM EDTA,50 mM Tris-HCl (pH 7.7), 1 mM NaF, 1 mM NaVO3, anda protease inhibitor cocktail (Sigma Chemical Co). Seventy-five micrograms of total protein was loaded to run 8%sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), and the gel was set up for transfer proteinto nitrocellulose membranes (Sigma Chemical Co.). Then,the membranes were rinsed in a Tris-buffered saline with

0.02% Tween-20 (TTBS), followed by immersing in 5% low-fat milk. Subsequently, the membranes were incubated withrabbit anti-TRPC6 antibody (Sigma Chemical Co); mouseanti-GAPDH antibody (Sigma Chemical Co). After rinsingthree times with TTBS, the membranes were incubatedwith HRP-conjugated goat anti-rabbit or mouse IgG (SigmaChemical Co.) for 45 min at room temperature and thendeveloped using ECL chemiluminescence reagent (SigmaChemical Co.). The specific protein bands were scanned andquantitated using densitometry in relation to the GAPDH,Western Blotting Detection System (GE Healthcare, ChalfontSt. Giles, UK). We repeated each Western Blot analysis usingprotein from three different and separate experiments.

2.5. Immunostaining. TRPC6 was fixed with 4% par-aformaldehyde, then permeabilized, and blocked with 0.3%TritonX-100 and 5% bovine serum albumin. The primaryantibody, rabbit anti-TRPC6 antibody (Sigma Chemical Co),was applied for overnight at 4◦C. FITC-conjugated goat anti-rabbit or mouse IgG (Sigma Chemical Co) and the nuclei dyeHoechst were used for 45 min at room temperature. Finally,the coverslips were mounted and images were taken byusing a immunofluorescence microscope (Zeiss, Germany).To determine the percentage of the cells in which RPC6 islocalized in nuclei, we counted at least 200 nuclei in triplicatein each experiment.

2.6. Statistical Analysis. Data were reported as mean ± SDwith n equal to the number of experiments. Statisticalevaluation was performed using a one-way ANOVA (two-sided test), followed by LSD (equal variances assumed) orDunnett’s T3 (equal variances not assumed) for post hoc testbetween two groups, and also using the nonparametric tests(Mann-Whitney U-test) as a posttest. Values of P < 0.05 wereconsidered as statistically significance.

3. Results

3.1. Effect of DEX on PAN-Induced Podocytes Changes.Podocytes were observed and photographed under invertedmicroscope. The relative area of podocytes was calculatedby Image J and SPSS 13.0. The cell bodies and nucleus ofPAN-induced podocytes were significantly decreased. Thecell bodies, which connected to each other between cells,stretched out like the branches. Foot processes appearedretraction, and the area of PAN-inducted podocytes wassignificantly reduced to 75% at 8 h (P < 0.05); foot processeswas obviously retracted, and the cell bodies were reducedto 46% at 24 h (P < 0.01); shrinked to 27% (P < 0.01),part of foot processes occurred disappearance or lost at48 h. However, after DEX treated, the area of podocytes wassignificantly greater at 8 h, 24 h, and 48 h, the difference wassignificant (P < 0.05). Based on these findings, we developedthe hypothesis that PAN-induced injury maybe prevented byDEX. Figure 1.

3.2. Effect of DEX on Pan-Induced TRPC6 mRNA Expression.TRPC6 mRNA results showed that the target bands can be


(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 1: PA-induced podocytes changes at different time points (inverted-phase contrast microscope ×200). Note: (a, d, g): the controlgroup, 8 h, 24 h, 48 h, resp., (foot processes and the connection between podocytes are intact) (b): PAN-induced 8 h; (c): DEX-treated 8 h;(e): PAN-induced 24 h; (f): DEX-treated 24 h; (h): PAN-induced 48 h (foot process retracted and lost, cell interconnected disappeared); (i):DEX-treated 48 h (foot processes and the connection between podocytes are still preserved).

seen in the 3rd and 4th lanes (150 bp). Our studies showedthat there was Trpc6 mRNA expression in the 3rd and 4thsamples, but the expression is low, and suggested that TRPC6mRNA expression is very low under normal circumstancesand cannot be shown by the agarose gel electrophoresis.TRPC6 mRNA expression did not change significantly atPAN-induced 8 h and 24 h. But TRPC6 mRNA expressionsignificantly increased at 48 h (P < 0.01); TRPC6 mRNAexpression did not show significantly changes at DEX-treated8 h; the TRPC6 mRNA expression was significantly decreasedat 24 h and 48 h (P < 0.01). Figure 2(a).

Under normal circumstances, TRPC6 expression is verylow in podocytes. GAPDH as internal control, Comparedwith the control group, found that TRPC6 mRNA expressionwas not significantly changed after PAN-induced 8 h. ButTRPC6 mRNA expression slightly increased at 24 h and 48 h

(P < 0.05); TRPC6 mRNA expression significantly decreasedafter DEX-treated 8 h, 24 h, and 48 h (P < 0.05), Figures 2(b)and 2(c).

3.3. Effect of DEX on PA-Induced TRPC6 Protein Expression.Western Blot analysis showed that TRPC6 and GAPDH,respectively, have specific band at 36 kd and 106 kd. Undernormal circumstances, podocytes have a certain amountof TRPC6 protein expression. Compared with the control,TRPC6 protein expression did not change significantly atPAN-induced 8 h but was higher at 24 h and 48 h (P < 0.01);TRPC6 protein expression did not significantly change atDEX-treated 8 h; decreased at 24 h; became normal (P <0.05). The protein expression significantly decreased at 48 h(P < 0.01), Figures 3(a) and 3(b).


PAN stimulation DEX intervention The control

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Figure 2: (a) TRPC6 mRNA expression changes at different time points. Note: 1, 4, 7-8 h; 2, 5, 8–24 h; 3, 6, 9–48 h. (b) GAPDH amplificationmap. (c) TRPC6 amplification map. Note: there was a slight peak in 3rd and 4th samples, ct value reached 34.77, 34.69, indicated that therewas Trpc6 mRNA expression in the 3rd and 4th samples, but the expression is low.

3.4. Effect of DEX on PAN-Induced TRPC6 DistributionChanges in Podocytes. TRPC6 was linear and evenly dis-tributed in the control, little in the cytoplasm; at PAN-inducted 24 h, TRPC6 was not continuity distributionalong the cell membrane, increased in cytoplasm; at 48 h,TRPC6 distribution increased in cell membrane, part ofthe cell membrane lost or gathered into granular, widelydistributed in cytoplasm. But, after DEX-treated, TRPC6,which significantly improved at different time points, is morehomogeneous distribution in the plasma membrane andbecame normal. Figure 4.

4. Discussion

TRPC6 is a member of the large transient receptor potentialsuperfamily of nonselective cation channels [4, 5]. Thissuperfamily consists of a group of six transmembranedomain-containing ion channels and has been subdividedinto six subfamilies; TRPC3; TRPC6, and TRPC7 have about75% homology, together constitute heteromeric polymer,and form the function channel [6]. Firstly, Winn et al. [7]and Reiser et al. [2], in 2005, determined one of thepathogenic genes of causing familial focal segmental glom-erulosclerosis (FSGS). Further studies showed that therewas interaction between TRPC6 protein and SD molecules,

together constituting the diaphragm hole complex. Thisfinding will make podocyte structural proteins and ionchannels linked. Recent studies have also shown that TRPC6is expressed in foot processes. TRPC6 within the cell bodyof podocytes and in primary processes in close vicinity toSD was associated with the nephrin, podocin, and CD2AP[8]. TRPC6 is a component of the SD multiprotein complex,involved in the physiologic and pathophysiologic role ofpodocytes [9].

Glucocorticoids are widely used for the treatment ofglomerular diseases characterized by podocyte injury andproteinuria [10]. It has been speculated that glucocorticoidsexert therapeutic effects through their immunosuppressiveand anti-inflammatory mechanisms [11]. Foot processes andSD contribute to the formation of the glomerular filter.It plays an important role in maintaining the podocytefunction. Podocyte injury may result in a leakage of proteinsinto the urine. So injury to podocytes and their SD typicallyleads to marked proteinuria. PAN, a podocyte toxin, is wildlyused to induce experimental nephrotic syndrome in rats [12–14], In in vitro study, PAN results in foot process effacementand variable dose-dependent apoptosis [15]. As a new SDmolecule, TRPC6 is an important molecule for keeping thestructure and function of podocytes as well as regulationof signaling transduction in podocytes and interacting with


1 2 3 1 2 3 1 2 3

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Figure 3: (a) The Western Blot band of TRPC6 and GAPDH atdifferent time points. Note: (1) the control; (2) PAN stimulation;(3) DEX intervention. (b) The Western Blot analysis of TRPC6 andGAPDH at each time point. Note: the Western Blot analysis showedthat the protein level of TRPC6 was increased at PAN-induced 24 hand 48 h, the difference was significant, P < 0.01; the proteinlevel of TRPC6 was decreased at DEX-treated 24 h and 48 h, thedifference was significant, P < 0.05.

other SD molecules, such as nephrin, podocin, and CD2AP[16–18]. Reiser et al. found that TRPC6 was expressed at thepodocyte membrane. In nongenetic forms of glomerular dis-ease, such as minimal change disease (NCD), membranousglomerulonephritis (MGN), and FSGS, TRPC6 overexpre-sion can directly affect cytoskeletal organization in podocytes[19]. Moller et al. also observed that TRPC6 overexpressioncorrelated with the development of podocyte injury. All ofthese suggest that TRPC6 overexpression is a pivotal factorresulting in podocyte injury. In the PAN-injected rats, theTRPC6 levels were increased markedly [19]. Knocking-downTRPC6 could effectively prevent the podocytes apoptosisinduced by PAN [12]. PAN results in foot process effacementand variable dose-dependent apoptosis [20, 21]. On thebasis of these findings, we hypothesized that TRPC6 wouldalso take part in the PAN-induced podocyte injury. DEXresisted podocyte injury via stabilizing TRPC6 expressionand distribution. The major purpose of our study was todetermine whether DEX mediated the specific cytoprotective

effect by blocking TRPC6-signaling pathway and blockingTRPC6 channels maybe beneficial to protect podocytes frominjury.

Our study found that, at PAN-induced and DEX-treated8 h, TRPC6 had no significant changes in protein expression.but was significantly higher at PAN-inducted 24 h and 48 h(P < 0.01); TRPC6 protein expression was significantlydecreased at DEX-treated 24 h and 48 h (P < 0.05).Immunofluorescence staining suggested that TRPC6 showeda linear uniformly distribution in the control group, onlya little in the cytoplasm; at PAN-inducted 24 h, TRPC6was mainly punctate distribution along the membraneand increased in cytoplasmic. At 48 h, TRPC6 distributionincreased in the cell membrane, part lost, gathered intoa granular, widely distributed in the cytoplasm; TRPC6was more homogeneous distribution after DEX treatment.The distribution was significantly weaker in cytoplasm.The difference, which is compared with other podocytemolecules, was that TRPC6 expression strengthened in PAN-inducted podocytes, the distribution increased but reducedafter DEX-treated. This is the special position of TRPC6 asa cation channel protein. But the specific mechanism is notvery clear, pending further study. Our results suggested thatTRPC6 might be a pharmacological target of maintainingthe function of podocytes in the future. So blocking TRPC6channels may be a new therapeutic strategy in proteinuricrenal diseases.

Our evidence also suggests that blocking TRPC6 chan-nels may be of therapeutic benefit in proteinuria. This createsthe exciting possibility that blocking TRPC6 channels withinthe podocyte may translate into long-lasting clinical benefitsin patients with FSGS. Our study suggested that TRPC6 filledthe damage signal, which was induced by PAN, and thenactivated the downstream molecules, caused the injury signaltransduction, so that foot process responds to retractedand lost. TRPC6 abnormal expression and distributionled to imbalance of TRPC6 channels and function. Thechanges of podocyte filtration rate caused the occurrenceof proteinuria; the DEX may have made the downstreammolecules, which have been activated, inactivated, podocytesrestored their original structure by blocking TRPC6 signalchannels, thus maintained SD structural and functionalintegrity by stabilizing TRPC6 expression, and played aprotective role in anti-proteinuria.

In summary, TRPC6 overexpression may be one of thefundamental changes in podocytes leading to proteinuriaand impairment of renal function. Perhaps, blocking theTRPC6 channel may be candidate for diagnosis and ther-apeutic target of renal diseases. Our study suggested thatTRPC6 signal pathway participated in the signal trans-duction mechanisms of DEX inhibiting podocyte injuryinduced by PAN. DEX stabilized TRPC6 expression throughbinding to its receptor. Our study will provide more clearlytheoretical basis for the molecular mechanism of DEXantiproteinuria and also provide new insights into the specialbeneficial effects of DEX on podocyte injury.


(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 4: Effects of DEX and PAN on the distribution and protein expression of TRPC6 at different time points (fluorescencemicroscope×400). Note: (a, d, g) were the control group, TRPC6 was linear and evenly distributed in the podocytes membrane surface, there aresome distribution in the cytoplasm; (b) PAN-inducted 8 h; (e) PAN-inducted 24 h, TRPC6 was not continuity distribution along the cellmembrane, increased in the cytoplasm; (h) after PAN-inducted 48 h, TRPC6 distribution increased in the cell membrane, part of the cellmembrane lost, gathered into a granular, widely distributed in the cytoplasm; (c) PAN-inducted + DEX-treated 8 h; (f) PAN-inducted +DEX-treated 24 h; (i) PAN-inducted + DEX-treated 48 h, TRPC6 was more homogeneous distribution in the membrane at each time point.

Disclosure

The authors declare that they are coauthors and contributedequally to this work.


The authors declare that they have no cinflict of interests.

Acknowledgments

This work was supported by the Natural Science FoundationProject of Guangdong Province (Project no. 5000424),the Technical Project of Guangdong Province (no.2005B36001019), the Science and Technology Research

Project of Guangzhou (no. 2006Z3-E0231). The authorsthank Professor Peter Mundel (America) and Professor JieDing, (Peking University First Hospital) for the PodocyteClone. They thank the Central Laboratory of GuangzhouFirst Municipal Hospital for Technique Helps.

References

[1] K. Zandi-Nejad, A. A. Eddy, R. J. Glassock, and B. M. Brenner,“Why is proteinuria an ominous biomarker of progressivekidney disease?” Kidney International, Supplement, vol. 66, no.92, pp. S76–S89, 2004.

[2] J. Reiser, K. R. Polu, C. C. Moller et al., “TRPC6 is a glomerularslit diaphragm-associated channel required for normal renalfunction,” Nature Genetics, vol. 37, no. 7, pp. 739–744, 2005.


[3] P. Mundel, J. Reiser, A. Z. M. Borja et al., “Rearrangements ofthe cytoskeleton and cell contacts induce process formationduring differentiation of conditionally immortalized mousepodocyte cell lines,” Experimental Cell Research, vol. 236, no.1, pp. 248–258, 1997.

[4] C. Montell, “The TRP superfamily of cation channels,”Science’s STKE, vol. 2005, no. 272, p. re3, 2005.

[5] I. S. Ramsey, M. Delling, and D. E. Clapham, “An introductionto TRP channels,” Annual Review of Physiology, vol. 68, pp.619–647, 2006.

[6] A. Dietrich, H. Kalwa, B. R. Rost, and T. Gudermann, “Thediacylgylcerol-sensitive TRPC3/6/7 subfamily of cation chan-nels: functional characterization and physiological relevance,”Pflugers Archiv European Journal of Physiology, vol. 451, no. 1,pp. 72–80, 2005.

[7] M. P. Winn, P. J. Conlon, K. L. Lynn et al., “Medicine: amutation in the TRPC6 cation channel causes familial focalsegmental glomerulosclerosis,” Science, vol. 308, no. 5729, pp.1801–1804, 2005.

[8] J. Reiser, K. R. Polu, C. C. Moller et al., “TRPC6 is a glomerularslit diaphragm-associated channel required for normal renalfunction,” Nature Genetics, vol. 37, no. 7, pp. 739–744, 2005.

[9] C. C. Moller, C. Wei, M. M. Altintas et al., “Inductionof TRPC6 channel in acquired forms of proteinuric kidneydisease,” Journal of the American Society of Nephrology, vol. 18,no. 1, pp. 29–36, 2007.

[10] T. Wada, J. W. Pippin, C. B. Marshall, S. V. Griffin, and S.J. Shankland, “Dexamethasone prevents podocyte apoptosisinduced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins,” Journal of the American Society ofNephrology, vol. 16, no. 9, pp. 2615–2625, 2005.

[11] T. Wada, J. W. Pippin, M. Nangaku, and S. J. Shankland, “Dex-amethasone’s prosurvival benefits in podocytes require extra-cellular signal-regulated kinase phosphorylation,” Nephron,Experimental Nephrology, vol. 109, no. 1, pp. e8–e19, 2008.

[12] X. Sun, Z. Fang, Z. Zhu, X. Yang, F. He, and C. Zhang,“Effect of down-regulation of TRPC6 on the puromycinaminonucleoside-induced apoptosis of mouse podocytes,”Journal of Huazhong University of Science and Technology -Medical Science, vol. 29, no. 4, pp. 417–422, 2009.

[13] Y. H. Kim, M. Goyal, D. Kurnit et al., “Podocyte depletionand glomerulosclerosis have a direct relationship in the PAN-treated rat,” Kidney International, vol. 60, no. 3, pp. 957–968,2001.

[14] C. X. Zheng, Z. H. Chen, C. H. Zeng, W. S. Qin, L. S. Li,and Z. H. Liu, “Triptolide protects podocytes from puromycinaminonucleoside induced injury in vivo and in vitro,” KidneyInternational, vol. 74, no. 5, pp. 596–612, 2008.

[15] S. F. Liu, J. Ding, Q. F. Fan, and H. Zhang, “Establishmentof a podocyte cell injury model induced by puromycinaminonucleoside,” Beijing Da Xue Xue Bao, vol. 40, no. 6, pp.586–589, 2008.

[16] W. Kriz, “TRPC6 - A new podocyte gene involved in focalsegmental glomerulosclerosis,” Trends in Molecular Medicine,vol. 11, no. 12, pp. 527–530, 2005.

[17] M. Estacion, W. G. Sinkins, S. W. Jones, M. A. B. Applegate,and W. P. Schilling, “Human TRPC6 expressed in HEK 293cells forms non-selective cation channels with limited Ca2+

permeability,” Journal of Physiology, vol. 572, no. 2, pp. 359–377, 2006.

[18] H. Zhang, J. Ding, Q. Fan, and S. Liu, “TRPC6 up-regulationin Ang II-induced podocyte apoptosis might result from ERKactivation and NF-κB translocation,” Experimental Biologyand Medicine, vol. 234, no. 9, pp. 1029–1036, 2009.

[19] C. C. Moller, C. Wei, M. M. Altintas et al., “Inductionof TRPC6 channel in acquired forms of proteinuric kidneydisease,” Journal of the American Society of Nephrology, vol. 18,no. 1, pp. 29–36, 2007.

[20] T. Wada, J. W. Pippin, C. B. Marshall, S. V. Griffin, and S.J. Shankland, “Dexamethasone prevents podocyte apoptosisinduced by puromycin aminonucleoside: role of p53 and Bcl-2-related family proteins,” Journal of the American Society ofNephrology, vol. 16, no. 9, pp. 2615–2625, 2005.

[21] S. F. Liu, J. Ding, Q. F. Fan, and H. Zhang, “Establishmentof a podocyte cell injury model induced by puromycinaminonucleoside,” Beijing Da Xue Xue Bao, vol. 40, no. 6, pp.586–589, 2008.


Research Article

A Study of the Wound Healing Mechanism of a TraditionalChinese Medicine, Angelica sinensis, Using a Proteomic Approach

Chia-Yen Hsiao,1 Ching-Yi Hung,1 Tung-Hu Tsai,2, 3 and Kin-Fu Chak1, 4

1 School of Life Sciences, Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan2 Institute of Traditional Medicine, National Yang-Ming University, Taipei 11221, Taiwan3 Department of Education and Research, Taipei City Hospital, Taipei 11221, Taiwan4 Department of Medicine, Mackay Medical College, Taipei 25245, Taiwan

Correspondence should be addressed to Kin-Fu Chak, [email protected]

Received 26 October 2011; Accepted 19 January 2012

Academic Editor: Saringat Hj. Baie

Copyright © 2012 Chia-Yen Hsiao et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Angelica sinensis (AS) is a traditional Chinese herbal medicine that has been formulated clinically to treat various form of skintrauma and to help wound healing. However, the mechanism by which it works remains a mystery. In this study we have establisheda new platform to evaluate the pharmacological effects of total AS herbal extracts as well as its major active component, ferulicacid (FA), using proteomic and biochemical analysis. Cytotoxic and proliferation-promoting concentrations of AS ethanol extracts(AS extract) and FA were tested, and then the cell extracts were subject to 2D PAGE analysis. We found 51 differentially expressedprotein spots, and these were identified by mass spectrometry. Furthermore, biomolecular assays, involving collagen secretion,migration, and ROS measurements, gave results that are consistent with the proteomic analysis. In this work, we have demonstrateda whole range of pharmacological effects associated with Angelica sinensis that might be beneficial when developing a woundhealing pharmaceutical formulation for the herbal medicine.

1. Introduction

Angelica sinensis (AS), which is called Dang-Gui in Chinese,has been used in medicine for more than two thousand yearsin East Asia, including China, Japan, Korea, and India. Inthe past, AS has been mostly used to treat gynecologicalconditions and anemia [1, 2] or formulated with Radixlithosperme as an aid to wound healing [3]. In recentstudies, AS has been shown to have multiple propertiesincluding the regulation of the immune system [4] and asantioxidant [5], antiinflammatory [6], anticancer [7] andothers. Components of AS have been identified and classifiedinto two groups; the essential oils and the water-solubleingredients [8]. Ferulic acid (FA) is one of the most abundantwater-soluble ingredients in AS and has been reported tobe the active component of AS [9]. FA is prominent as aROS scavenger because its structure is capable of stabilizingphenoxyl radical intermediates. Furthermore, FA is also able

to activate proteins like heme oxygenase-1 (HO-1), heatshock protein 70 (HSP70), Erk ½, and Akt, which help cellsto respond to environmental stress [10, 11].

Herbal drugs are difficult to analyze due to their com-plexity and the presence of toxicity due to alkaloids. To avoidthese complications, the effect of single active componentof herbs is often explored, and this single active componentrepresents the effects of the total herb; examples are shikoninfor Radix lithosperme [12], and Oridonin for Isodon rubescens[13]. However, the disadvantage of this single componentapproach is that the results can never be the same as thecomplete biochemical and pharmacological mechanisms ofthe total herb and may not reveal the real mechanism(s) ofthe formulated traditional Chinese medicine.

Proteomics is a powerful tool and has been widelyused to elucidate protein profile changes in response todrug treatment and to identify disease-relevant biomarkers.Using proteomics, Rhizoma paridis total saponin (RPTS)


was identified as contributing to the anti-hepatocellularcarcinoma effect (HCC) of this traditional medicine usingHepG2 cells [14]. Similarly, a proteomic analysis on Uncariarhynchophylla (Miq) Jack and its major component rhyn-chophylline was able to demonstrate an upregulation in theexpression of MIF and cyclophilin A in kainic acid-inducedepilepsy in rats [15].

Angelica sinensis (AS) is a basic component of many Chi-nese drugs that are used for wound healing, for example,shiunko [16]. Although AS has been applied in animalmodels and clinically, the mechanism by which AS helpswound healing remained to be clarified. Therefore thepurpose of this study was to explore the mechanisms bywhich an ethanol extract of AS exerts its protective effecton human skin fibroblasts using both biochemical andproteomic approaches. This approach also explored the effectof the drug’s active water-soluble component of FA. Basedon these findings, it should be possible to identify thepharmacological effects of AS and how these contributeto the process of wound healing when treated with someChinese traditional herbal medicines that contain AS.


2.1. HPLC Analysis. The HPLC system was equipped withBAS PM-80 pumps, a DGU-20A5 degasser, a CMA/170autosampler, and a Varian (model 340) photodiode arraydetector. Chromatographic separation was performed usinga Phenomenex Fusion RP-80 (2504.6 mm, 4 m). The mobilephases were acetonitrile (solvent A) and 2% acetic acid(solvent B). For the analysis of ferulic acid, the mobile phaseinvolved the following linear gradient: from 25% A to 75% Ain 0–15 min at a flow rate of 1.0 mL/min, and the detectionwavelength was set on 280 nm. The sample injection volumewas 20 μL.

2.2. Cell Culture. Human embryonic skin fibroblast was ob-tained from Bioresource Collection and Research Center(BCRC) no. 60118 (Detroit 551). Cells were cultured in10 cm culture dish (Corning) with minimum essentialmedium eagle (MEM) containing 2 mM L-glutamine, 1.5 g/Lsodium bicarbonate, 0.1 mM nonessential amino acids,and 1.0 mM sodium pyruvate (MEM alpha-modifications,Sigma), supplemented with 10% fetal bovine serum (SAFCBioscience), 50 units/mL penicillin, 0.05 mg/mL strepto-mycin, and 0.1 mg/mL neomycin (Sigma); the cells weregrown in a humidified atmosphere at 37◦C and 5% CO2.For passage, when fibroblasts had grown to a confluence ofapproximately 90% in the 10 cm culture dish, medium wasdiscarded and the cells washed with 5 mL PBS twice. Theywere then harvested using 2 mg/mL EDTA and 5 mg/mLtrypsin in phosphate-buffered saline (PBS). After washingwith 5 mL PBS twice, the cells were collected by centrifuga-tion at 1300 rpm for 5 min. The supernatant was discardedand the cells pipetted into fresh culture medium. They werethen aliquoted into several 10 cm culture dishes.

2.3. Cell Viability Assay (WST-1 Assay). The cell viabilityassay was performed according to the procedure describedin the manufacturer’s manual (Roche) with minor modifi-cations. This is a colorimetric assay for the quantification ofcell proliferation based on the cleavage of the water-solubletetrazolium salt (WST-1) by mitochondrial dehydrogenasesin viable cells. Drug-treated cells were seeded at a concentra-tion of 4 × 104 cells/well (about 40% confluence) in 1 mLculture medium into a 24-well culture plate and incubatedat 37◦C in an incubator containing 5% CO2. After cells hadattached, they were treated with 0.1% DMSO, 300 μg/mLAS extract, or 3.5 μM FA for 24 hr. The supernatant wasdiscarded and cell proliferation reagent WST-1 (Roche)was added to each well at a 1 : 50 ratio with fresh culturemedium. The cells were then incubated for an additional1 hr in dark. The absorbance was measured at 450 nm usingan ELISA reader (Thermo, Multiskan Spectrum), and theabsorbance of the background was measured at 690 nm.The difference in absorbance between 450 nm and 690 nmindicates the relative cell viability compared to 0.1% DMSO.The experiments were performed with three replicates foreach sample.

2.4. Sample Preparation for 2D PAGE. Fibroblasts werewashed twice in phosphate-buffered saline (PBS) and lysedwith 1 mL of NP-40 lysis buffer containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1% NP-40, 30 mM Na2P2O7,30 mM NaF, 1 mM Na3VO4, 1% protease inhibitor, and1% phosphatase inhibitor (Sigma). The whole cell lysatewas centrifuged at 14000 rpm for 20 min at 4◦C to removeinsoluble material. The supernatant was transferred to aconcentrator (GE Health, vivaspin 20, 100k MWCO) andcentrifuged at 6000×g, 4◦C, until volume less than 500 μL,then the lysis buffer was replaced with dH2O containing 1%protease inhibitor and 1% phosphatase inhibitor (Sigma).The protein concentration was quantified by the Bradfordprotein assay (Bio-Rad), and the samples were applieddirectly for 2D PAGE analysis.

2.5. Analysis of the Protein Profiles Using 2D PAGE. The 2DPAGE was performed according to the method describedin the manufacturer’s manual (Amersham Biosciences) withminor modifications. For the first-dimension IEF, pH 4–7,IPG strips (18 cm) were rehydrated with 400 μL rehydrationbuffer (0.5% IPG buffer, 8 M urea, 2% CHAPS, 50 mMdithiothreitol (DTT), and a trace of bromophenol blue) forat least 4 hr before 100 μg of protein sample was loadedby cup loading. IEF was then carried out under followingconditions: 150 V for 5 hr step and hold, 500 V for 3 hr stepand hold, 1000 V for 7 hr gradient, 8000 V for 3 hr gradientand 8000 V for 18 hr step-and-hold. Prior to the second-dimension SDS-PAGE, the IPG strips were equilibrated with4 mL of equilibration buffer, containing 50 mM Tris-HClpH 8.8, 6 M urea, 2% SDS, 30% glycerol, 50 mM DTT, and0.01% bromophenol blue at room temperature for 15 min,which was followed by equilibration in 50 mM Tris-HCl pH8.8, 6 M urea, 2% SDS, 30% glycerol, 5% iodoacetamide,and 0.01% bromophenol blue at room temperature for


15 min. The second-dimensional SDS-PAGE used a 12.5%separating gel and was performed without a stacking gel. Theequilibrated IPG gel strip was placed on top of the SDS-PAGEwith appropriate pressure to ensure a firm contact betweenthe strip and the gel slab. Electrophoresis was carried out at35 mA/gel until the tracking dye reached the bottom of thegel. The 2D PAGE was then silver stained.

2.6. Fast Silver Staining. After 2D PAGE electrophoresis, thegel was removed, immersed in fix solution (40% methanoland 10% acetic acid in dH2O) for 10 min, washed with dH2Ofor 10 min twice, and then immersed in solution A (0.25 mMsodium thiosulfate) for 30 min, which was then replaced bydH2O for 10 min. Next the gel was immersed in solution B(3.5 mM silver nitrate) for 30 min. After rinsing with dH2O,a mixture of solution C (0.357 M) and solution D (4.37 mM)in dH2O were used to develop the protein spots on the 2DPAGE gel. The reaction was stopped by 5% acetic acid.

2.7. Detection and Quantitative Analysis of the 2D PAGE Gels.2D PAGE images were obtained, and the amount of proteinin each spot was analyzed using ImageMaster 2D Elitesoftware version 5.0 (Amersham Biosciences). The volume ofa protein spot was defined as the sum of the intensities of thepixel units within the protein spot. To correct for quantitativevariations in the intensity of protein spots, spot volumes werenormalized as a percentage of the total volume of all the spotspresent in a given gel.

2.8. Protein Identification by LC-MS/MS. The process isdescribed in a document from the Institute of Biologi-cal Chemistry, Academia Sinica Institute of Biological Chem-istry (http://proteome.sinica.edu.tw/) and this was used withminor modification. Selected silver-stained protein spotswere excised from the 2D PAGE. They were then de-silver-stain using 1 : 1 Na2S2O (0.1 g in 1 mL H2O) and K6Fe(CN)6

(0.1 g in 1 mL H2O) in 25 mM ammonium bicarbonateuntil being transparent. Next the gel slices were added to25 mM ammonium bicarbonate pH 8.5 for 10 min, vacuum-dried, and rehydrated with 50% acetonitrile in 25 mMammonium bicarbonate pH 8.5 at room temperature for10 min. The supernatant was then discarded and the samplevacuum-dried. Subsequently, the protein within the spot wastrypsinized with 0.1% sequencing-grade modified trypsin(Promega, Madison, WI, USA) in 25 mM ammonium bicar-bonate, pH 8.5, at 37◦C for at least 16 hr. Each supernatantwas removed into a new Eppendorf tube, then 25 mMammonium bicarbonate pH 8.5 was added to the previousgel sample, and the mixture sonicated for 1 min, 10 times.The supernatant from this treatment was also removed intoa new Eppendorf tube. The process was repeated a thirdtime by adding 50% acetonitrile in 25 mM ammoniumbicarbonate pH 8.5 to the sample, which was followedby sonication for 1 min, 10 times. The three extractedsupernatants were pooled and evaporated to dryness undervacuum, and the dried pellet used to carry out integratednanoLC-MS/MS system (Micromass) (National ResearchProgram for Genomic Medicine, Academia Sinica). Eleven

rare differentially regulated protein spots were identifiedby LC-MS/MS (Orbitrap) mass spectrometry system (Pro-teomics Research Center, National Yang-Ming University) inthis way. The nanoLC-MS/MS data acquisition was carriedout by Micromass ProteinLynx Global Server (PGS) 2.0 dataprocessing software in a default mode and outputted as asingle Mascot-searchable peak list (.pkl) file. The LC-MS/MS(Orbitrap) data were processed by SWQUEST (ThermoFinnigan). The peak list files were used to query the Swiss-Port version 2010 05 database using Mascot program version2.2 (release date, 28-Feb-2007, Matrix Science, London, UK)with the following parameters: a taxonomy of Homo sapiens(20,400 sequences), peptide mass tolerance of 50 ppm,MS/MS ion mass tolerance of 0.25 Da, trypsin digestionwith one missed cleavage, no fixed modification, and thevariable modifications considered were methionine oxida-tion, cysteine carboxyamidomethylation, lysine acetylationand phosphorylation of tyrosine, serine, and threonine. Onlysignificant hits as defined by Mascot probability analysiswere considered. Protein identifications were accepted witha statistically significant Mascot protein search score ≥36 orSEQUEST score = 2.5 (critical), which corresponds to anerror probability of P < 0.05 using our data set. The proteinidentification with the highest score was selected to eliminateprotein redundancy within the database.

2.9. Cluster Analysis and Functional Classification of the Dif-ferentially Expressed Proteins. A plot of the calibrated inten-sity for the expression value of each protein as measuredby ImageMaster 2D Elite software version 5.0 (AmershamBiosciences, Sweden) among the different groups of sampleswas used in conjunction with an average linkage hierarchicalclustering algorithm (UPGMA, Unweighted Pair GroupMethod with Arithmetic Mean); this was done using Hierar-chical Clustering Explorer 3.5 [17]. The uncentered Pearson’scorrelation coefficient was determined as a measurement ofthe similarity metric, and the threshold value for the mini-mum similarity was set at 0.8. After clustering, each proteinwas allocated its place in a global temporal classification colorheat map. We used BGSSJ (Bulk Gene Search System for Java;http://bgssj.sourceforge.net/) [17] and the Swiss-Prot proteinknowledge database for the functional classification of theproteins.

2.10. Western Blotting. Proteins extracts from fibroblast cells(Detroit 551) were separated by 12.5% SDS-PAGE and thentransferred onto a nitrocellulose (NC) membrane. The NCmembrane was blocked with 5% nonfat milk in TBSTat room temperature for 1 hr and probed with variousprimary antibodies (anti-p-Erk1/2 (no. 9101), 1 : 2000; anti-Erk1/2 (no. 9102), 1 : 1000; anti-p-Akt (no. 9271), 1 : 1000and anti-Akt (no. 9272), 1 : 1000, Cell Signaling; anti-TGF-β (sc-52829), 1 : 1000, Santa Cruz; anti-β-tubulin (T4026),1 : 5000, Sigma Aldrich; anti-HSPB1 (ab39399), 1 : 1000and anti-STMN (cb1047), 1 : 1000, Abcam; anti-GSTP1(GTX112953), 1 : 3000 and anti-TPIS (GTX104618), 1 : 3000,GeneTax) in 5% BSA or non-fat milk in TBST. Afterwashing the NC membrane, the NC membrane was treated


with HRP-conjugated secondary antibody. The bands werevisualized by chemiluminescence using a chemiluminescenceimaging system (LAS-4000, Fujifilm) and performed den-sitometric analysis using Fujifilm MultiGauge software ver.2.0. The results are expressed as mean ± standard deviation.Student’s t-test was used to evaluate statistical significance,and a P value <0.05 was regarded as statistical significant(n = 4 for each experiment).

2.11. Intracellular ROS (Reactive Oxygen Species) Assay. Tomeasure the ROS content of the fibroblasts after DMSO,AS extract, or FA treatment, the intracellular content ofH2O2, which is in proportion to ROS, was determined usingthe redox-sensitive fluorescent dye 2′,7′-dichlorofluoresceindiacetate (DCF-DA) (Sigma). Briefly, cells were culturedto confluence and trypsinized. After centrifugation, discardsupernatant and the cells were resuspended and incubatedwith 10 μM DCF-DA (20 mM in DMSO for stock solutionand stored in −20◦C), which solved in 1 × PBS for 10minutes at 37◦C in the dark. Centrifuge again and cellswere then washed and resuspended in fresh culture medium.A total of 8 × 104 cells were added to a black flat 96-well ELISA plate with 200 μL of medium. ROS is able tooxidize DCF-DA to the fluorescent DCF, and therefore therelative concentration of intracellular ROS is determined by afluorescence reader using excitation at 485 nm and emissionat 538 nm. Student’s t-test was used to evaluate statisticalsignificance, and a P value < 0.05 was regarded as statisticalsignificant (n = 3 for each experiment).

2.12. Boyden Chamber Migration Assay. After cells werecultured to confluence, they were trypsinized, centrifuged,and resuspended in culture medium. A total of 2 × 104

fibroblasts were seeded in a Transwell (24 well, Corning) andtreated with DMSO, AS extract, or FA; the Transwell was theninserted in culture medium without bubbles. After 6 hr, theTranswell was removed from the culture medium and thecells fixed using methanol for 10 min. The methanol was thendiscarded and the cells air-dried. Finally the cells were stainedwith 5% Giemsa (solved in dH2O) in room temperatureovernight. The Transwell was washed with dH2O, and theinner cells on the Transwell were removed by scraping with acotton swab. The number of cells migrated down throughthe Transwell was manually counted under microscope.Student’s t-test was used to evaluate statistical significances,and a P value <0.05 was regarded as statistical significant(n = 3 for each experiment).

2.13. Wound Healing Assay. A total of 2.5 × 104 fibroblastswere seeded onto both sides of a culture insert (ibidi) in a24-well plate to generate 500 μm ± 50 μm gap between thecell layers before drug treatment. After the cells had attached,the culture insert was removed carefully. The cells were thentreated with the various drugs, which were added to theculture medium. The cells were then incubated for 24 hr.The medium was then discarded and the cells washed twicewith PBS to remove unattached cells. Photographs were thentaken by digital camera using microscopy (Olympus) and the

size of the gap measured. Student’s t-test was used to evaluatestatistical significances, and a P value < 0.05 was regarded asstatistical significant (n = 3 for each experiment).

2.14. Sircol Collagen Assay. The process is described in Sircolcollagen assay general protocol (Biocolor). In brief, 3 mL cellmedium was collected after drug treatment, and collagenwas precipitated by 4 M NaCl to avoid FBS interference. Thecollagen pellet was collected by centrifugation at 15000×g inroom temperature, and the pellet was dissolved in 0.5 mL of0.5 M acetic acid. 1 mL of Sircol dye reagent was mixed with100 μL resolved collagen sample and then gently inverted atroom temperature for 30 min. The resulting collagen wascollected then centrifugation at 10000×g for 10 min. Thepellet was dissolved in 1 mL alkali reagent by vortexing andthe collagen concentration was determined by a spectropho-tometer at OD540. Student’s t-test was used to evaluatestatistical significances, and a P value < 0.05 was regardedas statistical significant (n = 3 for each experiment).

3. Results

3.1. Safety and Efficacy Analysis of AS Ethanol Extracts and FATreatment. In order to establish a model system to analyzethe efficacy of total herbal extracts, we used Angelica sinensisethanol extracts (AS extract) and its active component,ferulic acid (FA), as samples. AS was extracted using absoluteethanol and then analyzed by HPLC to determine thequantity of FA in the AS extract.The concentration of FA inthe AS extract (see Figure 1 in the supplementary materialavailable at doi:10.1155/2012/467531) was 2.278 mg/mL.Traditionally, AS is a common basic component when for-mulating Chinese herbal medicines for wound healing, andtherefore we used human skin fibroblasts as an experimentalcell model to elucidate the underlying mechanisms by whichAS aids with the process of wound healing. AS extract andFA were dissolved in DMSO to give concentrations rangingfrom 10 to 500 μg/mL and 1 to 200 μM, respectively, inorder to determine the optimal drug concentrations forfibroblast cell growth. As shown in Figure 1, AS extracts wereable to significantly enhance cell viability by up to 125%in a dose-dependent manner (Figure 1(a)), but FA had noobvious effect on cell growth (Figure 1(b)). It was notedthat 500 μg/mL of AS extract was the highest concentrationthat was still able to help cell growth, and concentrationbeyond this point inhibited cell growth. Based on theseobservations, we chose 300 μg/mL of AS extract for this studyin order avoid any complicated pharmacological effects.Correspondingly, the appropriate concentration of FA basedon the level in the AS extract was 3.5 μM. Noticeably, ASextract significantly promotes cell growth, but the activecomponent FA is not able to induce a similar effect.This confirms that the pharmacological effect of a singlecomponent of an herb plant is not equivalent to that of awhole extract from the same plant in this case.


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Figure 1: Effects of AS extract or FA on cell viability after 24 hr treatment in human fibroblast. Graph represents the ratio of cell viabilitycompared with the DMSO control. (a) AS extract promotes cell growth in a dose dependent manner up to 400 μg/mL, but FA treatment (b)has no significant effect on cell proliferation. Fibroblasts were seeded in a 24 well plate with 4× 104 cells per well. After the cells were attached,different drug concentrations were added for 24 hr and cell proliferation was evaluated by WST-1 assay at OD450−OD690. The control wastreated with 0.1% DMSO only. The Student’s t test was used to evaluate the statistical significance of the results, which are presented as mean±SD (n = 5). ∗P < 0.05, ∗∗P < 0.01, compared with the control.

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Figure 2: Differential effect of AS and FA treatment on human fibroblasts as visualized by 2D PAGE. 300 μg/mL AS extract and 3.5 μM FAwere used to treat cells for 24 hr. Whole-cell lysates were extracted and analyzed by 2D PAGE (first dimension, 18 cm, pH 4–7, nonlineargradient of IPG strips; second dimension, 12.5% SDS-PAGE) and visualized by silver staining. There were 29 and 22 differentially expressedspots that could be identified from the AS extract and FA-treated cells, respectively, compared with the DMSO control. The numberedprotein spots were identified by LC-MS/MS mass spectrometry. The corresponding protein spot identities are shown in Table 1.

3.2. 2D PAGE Protein Profiling and the Identification of Differ-entially Expressed Protein Spots from Fibroblast Cells Treatedwith DMSO, AS Extract, or FA. To further investigate how ASextract and its active component FA can affect the processesof wound healing of the injured skin, two-dimensional (2D)PAGE was then carried out. In this study, 0.1% DMSO,300 μg/mL AS extract, or 3.5 μM FA-treated cell lysates wereseparated by 2D PAGE and visualized by silver staining. Thestained 2D PAGE gels were then analyzed by Image Master5.0. This software identified proteins showing quantitativelydifferent expression levels on the 2D PAGE gels using theDMSO treated cells as reference. As shown in Figure 2, we

were able to identify 29 and 22 protein spots for AS and FArespectively that were significantly increased or decreased bymore than 1.5- or 0.67-fold in intensity in response to ASextract, or FA treatment compared to DMSO, respectively.These protein spots were numerically labeled and are shownin Figure 2.

To further characterize these differentially expressed pro-teins, the concentration of total protein lysate was increasedto more than 1 mg, which was followed by 2D PAGE andstaining with Coomassie blue. Forty abundant differentiallyregulated protein spots were identifiable on this 2D PAGE gel,and these were excised and analyzed by LC-MS/MS (Q-TOF)


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mass spectrometry. In addition, eleven rare differentiallyregulated protein spots were excised from the earlier silverstained 2D PAGE gel and analyzed by the more sensitiveLC-MS/MS (Orbitrap) mass spectrometry. The resultingpeptides were screened using the Mascot database onlinesearch engine and SEQUEST to identify the proteins. Thedetailed results of the protein identification are presented inTable 1 using the DMSO treatment as reference. A Mascotscore �36 or a SEQUEST score = 2.5 (critical) indicatessignificant protein homology (P < 0.05). As shown inFigure 2, 29 and 22 differentially expressed protein spots werecharacterized from the cell extracts treated with AS extract orFA, respectively.

3.3. Functional Classification of the Differentially DisplayedProteins. Bioinformatic tools were used to categorize thedifferentially expressed proteins. All of the parameters ofthe proteins identified by mass spectrometry (Table 1), suchas protein ID and ratio of protein expression levels, wereintroduced into HCE 3.5 (Hierarchical Clustering Explorer),which has been widely used for genomic or proteomic datamining and pinpointing hidden meaningful results [17, 18].Based on the expression level ratios of the protein spotsafter the various treatments (AS extract/DMSO; FA/DMSO,Table 1), it was possible to categorized 34 protein spots intofive clusters (Figure 3). In cluster A, the 13 protein spots areall upregulated in fibroblasts on treatment with either ASextract or FA. In cluster B, the 8 protein spots are upregulatedin fibroblasts treated with AS extract, but the expressionlevels of these protein spots are not significantly changedupon treatment with FA. Five protein spots make up clusterC, and these are only overexpressed in fibroblasts treated withFA. In cluster D, 5 protein spots are downregulated whenthe fibroblasts was treated with AS extract, but there is nochange in the expression level on treatment with FA. Only3 protein spots form cluster E, and these are downregulatedupon treatment with either AS extract or FA. It is worthnoting that, except in clusters A and E, the protein expressionis different when cells are treated with either AS extract or FA.

After clustering, we use NCBI PubMed and BGSSJ (BulkGene Search System for Java) with the Swiss-Port databaseto classify the biological functions of these proteins. Thedelineated protein functions included protein transport,protein metabolism, calcium ion binding, oxidoreductaseactivity, antiapoptosis, protein binding, metabolism, signaltransduction, cell mobility, and cell growth.

Interestingly, seven oxidation-related proteins werefound among these differentially expressed proteins. Amongthem, five oxidation-related proteins were found to beupregulated in fibroblasts when treated with either ASextract or FA. These proteins were peroxiredoxin (PRDX)2, PRDX4, PRDX6, glutathione S-transferase Pi (GSTP1),and Parkinson’s disease protein 7 (PARK7) (Figure 3, clusterA). The other two oxidation related proteins, glutaredoxin-3 (GLRX3) and ferritin light chain (FRIL), were only upregulated in cells treated with FA (Figure 3, cluster C).The protein expression patterns of these proteins implythat FA alone might exert greater antioxidative ability onthe fibroblasts than AS extract. Furthermore, four of the

identified proteins were involved in the regulation of cellgrowth or antiapoptosis, namely, translationally controlledtumor protein (TCTP), heat shock protein beta-1 (HSPB1),GSTP1 (bifunctional protein), and calpain small subunit 1(CAPNS1) (Figure 3, clusters A and B). TCTP was upreg-ulated in fibroblasts treated with either AS extract or FA(Figure 3, cluster A). In contrast, HSPB1, GSTP1, andCAPNS1 were upregulated only in cells treated with ASextract (Figure 3, cluster B). This suggests that AS extracthas a greater effect on cell survival compared to FA. Thesebioinformatic findings are consistent with the results fromthe WST-1 cell growth test (Figure 1).

Calcium ions are very important in the regulation ofwound healing process, and its function is to modulate theexpression of genes involved in cell growth, differentiation,attachment, motility, and collagen secretion [19]. Someproteins related to the above functions were identified in thisstudy. Expression of annexin A2 (ANXA2) was upregulatedin fibroblasts when treated with either AS extract or FA(Figure 3, cluster A); however, annexin A5 (ANXA5) wasonly upregulated when the cells were treated with FA(Figure 3, cluster C). In contrast, synaptic vesicle membraneprotein VAT-1 homolog (VAT1) was only upregulated incells treated with AS extract (Figure 3, cluster B), whiletriosephosphate isomerase (TPIS) was upregulated in cellstreated with either AS extract or FA. Furthermore, phos-phoglycerate kinase 1 (PGK1) and L-lactate dehydrogenase Bchain (LDHB) were only upregulated in cells treated with ASextract. During the wound healing process, cells are hypoxic[20] and cell proliferation consumes a lot of energy in orderto repair the injured tissue. TPIS and PGK1 are important toglycolysis, and LDHB catalyzes the generation of lactate bythe reduction of pyruvate. Overexpression of these proteinswould seem to provide additional energy resource allowingthe cells to survive under stress.

A number of motility-associated proteins were also iden-tified. Actin-related protein 2/3 complex subunit 5 (ARPC5)and stathmin (STMN1) were only upregulated in cells treatedwith FA (Figure 3, cluster C), while vimentin (VIME) wasonly downregulated in cells treated with AS extract (Figure 3,cluster D). Surprisingly, NDKB and MARE1 were down-regulated in cells after both treatments (Figure 3, clusterE). These results suggest that treatment of fibroblasts witheither AS extract or FA improves the ability of the cell tosurvive under environmental stresses. With the exception ofVIME (for mobility), treatment of fibroblasts with AS extractmodulates the upregulation of proteins that are associatedwith cell growth (LEG1, CAPNS1), metabolism (PGK1,LDHB), calcium ion regulation (VAT1), and antiapoptosis(HSPB1). On the other hand, FA may have the uniqueability (Figure 3, cluster C) to modulate the upregulationof proteins associated with cell mobility (STMN1, ARPC5),antioxidative functions (GLRX3, FRIL), and calcium ion reg-ulation (ANXA5). Interestingly, we found some overlappingof proteins induced by either AS extract or FA treatment.Two proteins associated with cell mobility (MARE1, NDKB,Figure 3, cluster E) are down-regulated when fibroblasts aretreated with either AS extract or FA. In contrast, someproteins associated with metabolism (TPIS), antioxidative


Expression pattern Biological function

Protein transport

Calcium ion binding

Oxidoreductase activity



Protein binding

Metabolism

Signal transduction


Protein metabolism

Protein transport

Calcium ion binding

Protein binding

Protein metabolism

Proliferation

Metabolism

Metabolism


Cell motility

Calcium ion binding

Cell motility


Cell motility

Protein metabolism

Cell growth

Cell motility

Cell motility

Protein metabolism

Cell mobility

Cluster A

Cluster B

Cluster C

Cluster D

Cluster E

Spot numbers/protein ID

Anti apoptosis

Anti apoptosis

Anti apoptosis

Anti apoptosis

15-SEC13

2-ANXA2

1-PRDX6

5-PARK7

10-TCTP

9-PRDX2

17-HSP60

4-TPIS

16-STRAP

3-PRDX4

12-UBE2N

8-GSTP1

6-SAR1A

29-VAT1

21-HSPB1

27-HSP6

20-PSME1

26-CAPNS1

22-PGK1

19-LDHB

7-GSTP1

32-GLRX3

33-ARPC5

34-ANXA5

31-STMN1

30-FRIL

23-VIME

18-IF5A1

28-LEG1

24-VIME

14-MARE1

11-PDIA3

13-NDKB

Figure 3: Hierarchical clustering and functional classification of the changes in protein expression between the DMSO control cells andAS/FA-treated cells. The expression pattern for each protein was categorized by UPGMA using Hierarchical Clustering Explorer 3.5. Proteinswith a similar expression clustered into five different discrete groups (clusters A, B, C, D, and E) in a tree-like organization. Each row in thecolor heat map indicates a single protein and each column represents different groups of proteins from the DMSO, AS, and FA treatments. Ahigh protein expression value for a specific protein spot is represented by a bright red color, and a low protein expression value is representedby a bright green color. A black color indicates that the protein spot was expressed at an average level. A gray color represents protein spotsthat were undetectable. Further information about the differentially expressed protein spots that were identified is given in Table 1. Thefunctional classification of each protein was determined using BGSSJ and the Swiss-Prot protein sequence database.

functions (PRDXs, PARK7), antiapoptosis (GSTP1, TCTP),and calcium ion regulation (ANXA2) are upregulated whenfibroblasts are treated with either AS extract or FA (Figure 3,cluster A).

3.4. Confirmation of the Differential Expression of the VariousProteins Detected by Proteomics Analysis. Western blot anal-

ysis was used to confirm the changes in protein expressiondetected by the proteomic approach. Human skin fibroblastswere seeded into plates and then treated with either ASextract or FA for 24 hr; then cell lysates were prepared asindicated in Materials and Methods. As shown in Figures 4(a)and 4(b), expression of GSTP1 and TPIS (Figure 3, clusterA) were upregulated in fibroblasts after treatment with either


GSTP1

DMSO AS FA

Fold 1 1.42 1.20

β-tubulin

(a)

TPIS

DMSO AS FA

Fold 1.34 1.21

β-tubulin

1

(b)

HSPB1

DMSO AS FA

Fold 1.43 1.14

β-tubulin

1

(c)

STMN1

DMSO AS FA

Fold 1.18 1.72

β-tubulin

1

(d)

RO

S co

nce

ntr

atio

n (

%)

DMSO AS FA0

80

100

120

∗∗#

(e)

Figure 4: Analysis of the results of 2D PAGE by immunoblotting and ROS assay. According to hierarchical clustering and functionalclassification, the expressions of GSTP1 and TPIS were both increased in 300 μg/mL AS extract and 3.5 μM FA-treated cells. In addition,STMN expression increased in AS-extract-treated cells, and HSPB1 expression increased in FA treated cells. GSTP1 expression was examinedby immunoblotting (a) using anti-GSTP1 antibody and these results were then further confirmed by functional ROS assay (e). The expressionof TPIS (b), HSBP1 (c), and STMN (d) were also confirmed by immunoblotting using specific antibodies. In all cases the results correlatedwith the clustering and functional classification of 2D PAGE. β-Tubulin was employed as the sample loading control. The relative expressionlevels were detected by Fujifilm Multigauge ver. 2.0 for densitometric analysis and normalized with internal control, and a Student’s t testwas performed to evaluate the statistical significance. Results are presented as mean ± SD (n = 3). ∗P < 0.01, #P < 0.1 but with a decreasedtrend compared with DMSO.

AS extract or FA, which is consistent with our proteomicanalysis (Figure 3 and Table 1). HSBP1 (Figure 3, cluster B)was significantly upregulated in fibroblasts after AS extracttreatment, but FA treatment did not significantly changeexpression of this gene (Figure 4(c)). Furthermore, STMN1was only significantly upregulated in cells treated with FAand no such change was detected when the cells were treatedwith AS (Figure 4(d)); this also agrees with the proteomicanalysis (Figure 3, cluster C). Thus western blot analysis ofselected proteins is in agreement with the expression patternof the proteins as detected by our 2D PAGE and massspectrometry analysis (Figure 3), which provides validationof our approaches.

3.5. Redox State of the Fibroblast after Treatment with ASExtract and FA. The PRDX family of proteins and GSTP1act as antioxidants [21], and these proteins show the greatestincrease in expression in response to treatment with ASextract or FA (Table 1). These results seem to indicate thatthe increase in these proteins may help to change the redoxstate of the cells after drug treatment. The redox-sensitivefluorescent dye 2′,7′-dichlorofluorescein diacetate (DCF-DA) was used to measure the redox state of cells after drugtreatment. As shown in Figure 4(e), the ROS level of cellstreated with AS extract was significantly reduced by 8%, butthis did not occur when the cells were treated with FA. It isworth noting that FA is an effective ROS scavenger due to


DMSO AS FA

Col

lage

n s

ecre

tion

(%

)

0

80

100

120

∗∗

(a)

∗∗ ∗

DMSO AS FA

Mig

rate

d ce

lls (

rela

tive

fold

)

0

0.5

1

1.5

2

(b)

Fold 1 1.52 0.81

β-tubulin

TGF-β

DMSO AS FA

(c)

1Fold 2.47 0.96

DMSO AS FA

p-Akt

Akt

β-tubulin

(d)

DMSO AS FA

Fold 1.59 0.91

p-Erk

Erk

β-tubulin

1

(e)

Figure 5: Functional confirmation of the effects of AS and FA treatment on human fibroblasts. (a) Collagen secretion was significantlyincreased after AS extract treatment. After cells were seeded in a 24 well plate and treated with 0.1% DMSO, 300 μg/mL AS extract or 3.5 μMFA for 24 hr, the medium were collected to analyze the collagen concentration. The graph represents the ratio of collagen secretion comparedto the DMSO control. (b) Fibroblast migration is promoted after treatment with AS extract or FA. A total of 2 × 104 cells were seeded intothe top of Boyden chamber with 0.1% DMSO, 300 μg/mL AS extract or 3.5 μM FA and added fresh culture medium into the bottom. After6 hr, the cells were stained by 5% Giemsa dissolved in water and the upper cells on Boyden chamber were scraped with cotton swabs then thecells counted using an inverted microscope. (c) TGF-β expression was increased after AS extract treatment and was correlated with collagensecretion (a). A total of 1 × 106 fibroblasts were seeded onto a 10 cm culture dish and then treated with DMSO, 300 μg/mL AS extractor 3.5 μM FA for 24 hr. After washing with 1 × PBS, the cells were lysed by NP-40 lysis buffer with protease and phosphatase inhibitor.TGF-β expression was determined by immunoblotting. The expression of phospho-Akt (d) and phospho-Erk (e) were also examined byimmunoblotting and it was found that AS extract was able to significantly increase expression of these proteins. The relative expression levelswere detected by Fujifilm Multigauge software and normalized with β-tubulin. The results are expressed as mean ± SD (n = 3). ∗P < 0.05,∗∗P < 0.01, compared with the control.

its structure [11]; nonetheless, it would seem that FA alone isnot completely effective in terms of antioxidant activity. Thussome component(s) from the AS extract may be necessary,together with FA, to obtain maximum anti-oxidative activity.

3.6. Functional Confirmation of the Effect of AS Extract onCollagen Secretion and Migration. Collagen secretion andcrosslinking are the most important step when repairingwounded tissue and collagen synthesis is controlled bytransforming growth factor beta (TGF-β), which is secretedby macrophages and fibroblasts [22]. Thus the effect of ASextract and FA on the secretion of collagen by fibroblasts wasexamined. As shown in Figure 5(a), AS extract was able tosignificantly promote collagen secretion by fibroblasts andthe increase was up to 5% compared to the control; incontrast, FA did not increase collagen secretion. Expressionof TGF-β was also analyzed by Western blotting, and it was

found that expression of TGF-β in response to treatmentwith either AS extract or FA correlated with the results of thecollagen secretion assay (Figure 5(c)).

The functions of STMN1, ARPC5, NDKB, and MARE1are known to be associated with cell mobility and woundhealing [23–26]; however, the expression levels of theseproteins, as detected by our proteomic analysis, varied, andthey grouped into different clusters (Figure 3, cluster C, D,E) in response to drug treatment. In these circumstances itis important to clarify the effects of each treatment on thefibroblasts, and therefore cell migration and wound healingassays were carried out on cells treated with either AS extractor FA. The Transwell assay was used for the cell migrationtest. As shown in Figure 5(b), we found that both AS extractand FA seemed to promote cell migration to the same degree.Furthermore, both AS extract and FA had the same effect onthe promotion of fibroblast wound healing (supplementaryFigure 2). These results suggest that FA might be the major


active component in AS extract that promotes cell migrationand wound healing.

Only CAPNS1 is involved in cell growth based on theproteomic classification (Figure 3, cluster B). Phosphory-lated Akt and Erk are known to be the factors that areinvolved in promoting cell growth, and therefore experi-ments were performed to measure the expression of thesetwo factors in fibroblasts after treatment with either ASextract or FA. As shown in Figures 5(d) and 5(e), theexpression levels of p-Akt and p-Erk were only upregulatedin fibroblasts treated with AS extract. These results areconsistent with our cell viability assay, which shows thatfibroblast cell growth is increased by about 25% whenfibroblasts are treated with AS extracts, but this did not occurwhen the cells are treated with FA (Figure 1). These findingsindicate that cell growth promotion is initiated by AS extracttreatment and this is likely to involve upregulation of the Aktand Erk pathway in the treated fibroblast cell.

4. Discussion

The cell viability test revealed that AS extract was able toeffectively promote human skin fibroblast proliferation withlow levels of cytotoxicity even at high concentrations. It iswell known that skin fibroblasts play an important role in theproliferation phase of wound healing [27, 28], and thereforetreatment with AS extract would seem to have beneficialeffects on fibroblast growth. It has been found previously thatfunctionally related proteins are often coordinately expressed[29]. In agreement with this hypothesis, five clusters ofproteins were identified using 2D PAGE analysis with similarchanges in cellular expression level in terms of AS/FAtreatments.

4.1. Proteins Involved in Antioxidative Activity. ROS plays animportant role in the early phase of wound healing [30],but excessive ROS in the wounded region is also harmfulto nearby normal cells. Peroxiredoxins (PRDXs), Parkinson’sdisease protein 7 (PARK7), and glutathione S-transferasePi (GSTP1) were categorized as cluster A proteins thatare upregulated in fibroblasts by either AS extract or FA(Figure 3, cluster A). PRDXs are a family of multifunctionalantioxidant thioredoxin-dependent peroxidases that protectcells from hydrogen peroxide [31, 32]. The PRDXs familyconsists of six members, and three of these (PRDx2, PRDX4,and PRDX6) were identified as upregulated in this study.PARK7 acts as a coordinator and induces the expressionof antioxidant genes such as glutamate cysteine ligase andglutathione [33]. GSTP1 is a multifunctional protein thatcan reduce hydroperoxides, carry out detoxification, andinhibit JNK phosphorylation; the latter results in a decreaseapoptotic cell numbers [34]. The mass spectrometry analysisindicated that PRDX-2, PDRX-4, PDRX-6, and PARK7 wereidentified in our sample (Table 1) with lower sequencecoverage. Nevertheless, the amount of hydrogen peroxidein the fibroblasts after AS extract treatment was substan-tially reduced (Figure 4(e)), which indicates the presenceof antioxidants in cells after drug treatment. Furthermore,

GSTP1 was significantly upregulated in fibroblasts that hadbeen treated with either AS extract or FA (Figure 4(a)).Together our results reveal that ROS was indeed significantlydecreased in cells after AS extract or FA treatment and that itis likely that PRDX-2, PDRX-4, PDRX-6, PARK7, and GSTP1are involved in this process.

4.2. Proteins Involved in the Regulation of Apoptosis. TCTP (atranslationally controlled tumor protein also named fortilin)and GSTP1 are upregulated by treatment with either ASextract or FA (Figure 3, cluster A); additionally, AS extractbut not FA is able to induce heat shock protein beta 1(HSPB1) expression (Figure 3, cluster B; Figure 4(c)). The N-terminus of TCTP is able to interact with Bcl-xL and helpthe cell to avoid apoptosis; in contrast, downregulation ofTCTP expression increases the number of apoptotic cells[35, 36]. It has also been reported that down-regulationof GSTP1 results in an increase of c-Jun NH2-terminalkinase-mediated apoptosis [37, 38]. HSPB1 (HSP27) worksas chaperone and helps protein folding; alternatively, itcan activate the proteasome allowing it to interact withdenatured proteins [39]. Phosphorylated HSPB1 is able toinhibit the interaction of Daxx with Fas or ASK1, whichdecreases the activation of downstream apoptosis-relatedproteins [40]. Furthermore, wound closure is delayed inknock-down SW480 cells indicating the involvement ofHSPB1 in regulating cell mobility [41]. Our results seemto demonstrate that AS extract and FA may activate anti-apoptosis in cells by upregulating the expression of TCTPand GSTP1. Moreover, AS extract but not FA may have theadditional function of inhibiting apoptosis and regulatingcell mobility during the wound healing process via upregula-tion of HSPB1.

4.3. Proteins Involved in the Regulation of Calcium Ion or Pro-tein Transportation. Calcium ions play an important role inwound healing, cell proliferation, differentiation, adhesion,migration, and collagen secretion [19]. Annexins are mem-brane proteins that contain the annexin core domain and aconserved calcium-lipid binding module that is involved incalcium ion regulation. In addition, expression of annexinA2 has been observed to be increased in migrating intestinalepithelial cells [42]. In this study it was found that annexinA2 was upregulated in fibroblasts treated with either ASextract or FA (Figure 3, cluster A). Moreover, a synapticvesicle membrane protein VAT-1 homolog (VAT-1) also isknown to participate in regulating calcium ion flux [43]. Wefound that treatment with AS extract but not with FA wasable to induce the upregulation of VAT-1 (Figure 3, clusterB). Interestingly, our biochemical assays further indicatedthat collagen secretion as well as the expression of TGF-β is substantially increased in fibroblasts after AS extracttreatment but not FA treatment (Figures 5(a) and 5(e)).Therefore we propose that AS extract has a greater capacitythan FA to affect calcium ion regulation as well as any relatedwound recovery effects.


AS extracts

FA

Cell mobility

Metabolism

Antioxidant

Calcium ion regulation

Cell mobility

Cell proliferation

Energy metabolism

Collagen secretion


TPIS

Erk and Akt pathways

Cell mobility

Antioxidant


LEG1, CAPNS1

LDHB, PGK1TGF-β

VAT1

HSPB1

VIME

Down regulation

Up regulation

MARE1, NDKB

PRDXs, PARK7

GSTP1, TCTPANXA2

STMN1, ARPC5

GLRX3, FRILANXA5

Anti apoptosis

Anti apoptosis

Figure 6: Summary of the effects of the AS and FA treatments on human skin fibroblasts.

4.4. Proteins Involved in Cell Mobility. Cell mobility is animportant part of the wound healing process [28]. Nucleo-side diphosphate kinases B (NDKB) and microtubule associ-ated protein RP/EB family member 1 (MARE1) were bothdown-regulated in cells treated with either AS extract orFA (Figure 3, cluster E). NDKB (also named nm23) seemsto be a metastasis suppressor gene and is thought to beinvolved in cell migration, proliferation, differentiation, anddevelopment [25]. MARE1 is also thought to promote poly-merization of microtubule and to stabilize microfilaments[26]. Downregulation of NDKB and MARE1 in fibroblaststhat are treated with either AS extract or FA would suggestthat there is a reduction in the suppression of migration,which might be beneficial to cells involved in the woundhealing process. Actin-related protein 2/3 complex subunit5 (ARPC5) and stathmin (STMN1) were both upregulatedby FA but not by AS extract (Figure 3, cluster C). ARPCs areable to regulate actin dynamics and function as a templatefor initiation of new actin filaments that branch off anexisting filament [24]. STMN1 is cytosolic phosphoproteinand is known to act as microtubule destabilizing proteinthat binds free form tubulin, which inhibit microtubulepolymerization [23]. Thus, our results indicate that both ASextract and FA are able to regulate cell mobility probably bycontrolling microtubule dynamics. Furthermore, it is worthnoting that ARPC5, STMN1, and NDKB have the highestsequence coverage and score in our mass spectrometricanalysis (Table 1). The migration and wound healing assays

(Figure 5(b)) show that AS extract and FA both promote cellmobility, and this agrees with the above findings.

4.5. Proteins Involved in Cell Growth or Metabolism. CAPNS1(calpain small subunit 1) is able to promote cell proliferation,migration, and adhesion [44]. In contrast, LEG1 (galectin-1) overexpression is thought to inhibit normal mousefibroblast growth and increase apoptosis [45]. Interestingly,we observed that CAPNS1 was upregulated (Figure 3, clusterB), while LEG1 was down-regulated (Figure 3, cluster D)when fibroblasts were treated with AS extract; such con-trasting changes in expression between CAPNS1 and LEG1should be beneficial to the fibroblast wound healing process.Furthermore, our results also show that, when fibroblastswere treated with AS extract there were activations of p-Akt and p-Erk expression (Figures 5(d) and 5(e)). Thus, inaddition to regulating the expression of CAPN1 and LEG1,AS extract seems to also modulate cell growth via the Erkand Akt pathways.

The main energy resource of fibroblasts is glycolysis, andan increase in LDHB expression in these cells would promotepropyl hydroxylase activity, which in turn would increaselactate production and collagen hydroxylation; these effects,in turn, would help cells tolerate an hypoxia environmentand accelerate the wound healing process [46]. We found thatboth AS extract and FA are able to upregulate the expressionof Triosephosphate isomerase (TPIS) in fibroblasts (Figure 3,cluster A), while only AS extract could activate the expression


of phosphoglycerate kinase (PGK1) and L-lactate dehydroge-nase B chain (LDHB) (Figure 3, cluster B). Thus we concludethat AS extract might have greater efficacy in providingextra energy for wound healing processes by upregulatingglycolysis, which would help to protect cells from hypoxia.

4.6. Potential Molecular Pharmaceutical Mechanism of ASExtract and FA in Regulating Wound Healing Processes ofFibroblasts. Taken together, our results revealed a possiblepharmacological wound healing processes induced by the ASextract or FA treatment of fibroblasts (Figure 6). Multiplefactors are likely to be involved in the wound healingprocesses induced by either AS extract or FA. Both AS extractand FA seem to activate the inhibition of ROS production(PRDXs and PARK7), enhance cell mobility (MARE1 andNDKB), promote glycolysis (TPIS), increase antiapoptosis(GSTP1 and TCTP), and regulate calcium ion (ANXA2),all of which ought to increase the viability of cells duringthe wound healing process. The unique pharmacologicalfunctions of AS extract that have been revealed in thisstudy include enhanced human skin fibroblast proliferationdue to suppression of LEG1 expression, and increasedCAPNS1 expression, and upregulation of PGK1 and LDHB,which promote glycolysis and activation of the Erk and Aktsignal transduction pathways. Fibroblasts treated with ASextract seem also to undergo a suppression of apoptosis dueto increased HSPB1 expression. Moreover, an increase incollagen secretion (TGF-β), calcium ion regulation (VAT1)and enhanced cell mobility (down-regulation of VIME) alsocontribute towards a more efficient wound healing process.In addition, a number of unique functions of FA have alsobeen revealed, which include ROS inhibition (GLRX3 andFRIL) and the promotion of cell mobility by upregulationof STMN1 and ARPCS and down-regulation of NDKB. Theextra functions of FA mentioned above should reinforce thehealing process associated with fibroblasts as a whole. Inthis work, we have described a technological platform that isable to reveal the underlying principles of a herbal medicine;namely, how an AS extract and its active component FAparticipate in the modulation of wound healing processassociated with fibroblasts. These results will help and benefitthe clinical applications of this herbal medicine when it isused to treat heat burns or traumatic injury to the skin.

Acknowledgments

This study was supported by grants from the NationalResearch Program for Genomic Medicine (NRPGM), NSC99-3112-B-010-006, and the National Science Council, NSC98-2320-B-010-008-MY3.

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Research Article

Fractionation of an Extract of Pluchea odorata Separatesa Property Indicative for the Induction of Cell Plasticity fromOne That Inhibits a Neoplastic Phenotype

Mareike Seelinger,1 Ruxandra Popescu,2 Prapairat Seephonkai,2 Judith Singhuber,2

Benedikt Giessrigl,1 Christine Unger,1 Sabine Bauer,1 Karl-Heinz Wagner,3

Monika Fritzer-Szekeres,4 Thomas Szekeres,4 Rene Diaz,5 Foster M. Tut,5 Richard Frisch,5

Bjorn Feistel,6 Brigitte Kopp,2 and Georg Krupitza1

1 Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18-20, 1090 Vienna, Austria2 Department of Pharmacognosy, Faculty of Life Sciences, University of Vienna, AlthanstraBe 14, 1090 Vienna, Austria3 Department of Nutritional Sciences, Faculty of Life Sciences, University of Vienna, AlthanstraBe 14, 1090 Vienna, Austria4 Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Vienna, Waehringer Guertel 18-20,1090 Vienna, Austria

5 Institute for Ethnobiology, Playa Diana, San Jose, Peten, Guatemala6 Finzelberg GmbH & Co. KG, Koblenzer Strasse 48-54, 56626 Andernach, Germany

Correspondence should be addressed to Georg Krupitza, [email protected]

Received 29 September 2011; Accepted 6 December 2011

Academic Editor: Ashok D. Taranalli

Copyright © 2012 Mareike Seelinger et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Introduction. Several studies demonstrated that anti-inflammatory remedies exhibit excellent anti-neoplastic properties. Anextract of Pluchea odorata (Asteraceae), which is used for wound healing and against inflammatory conditions, was fractionatedand properties correlating to anti-neoplastic and wound healing effects were separated. Methods. Up to six fractionation stepsusing silica gel, Sephadex columns, and distinct solvent systems were used, and eluted fractions were analysed by thin layerchromatography, apoptosis, and proliferation assays. The expression of oncogenes and proteins regulating cell migration wasinvestigated by immunoblotting after treating HL60 cells with the most active fractions. Results. Sequential fractionations enrichedanti-neoplastic activities which suppressed oncogene expression of JunB, c-Jun, c-Myc, and Stat3. Furthermore, a fraction (F4.6.3)inducing or keeping up expression of the mobility markers MYPT, ROCK1, and paxillin could be separated from another fraction(F4.3.7), which inhibited these markers. Conclusions. Wound healing builds up scar or specific tissue, and hence, compoundsenhancing cell migration support this process. In contrast, successful anti-neoplastic therapy combats tumour progression, andthus, suppression of cell migration is mandatory.

1. Introduction

Drug discovery is a constant need for biomedical researchand clinical progress. Particularly mega-biodiversity areassuch as the tropical rainforests of Central America are verypromising for the discovery of new pharmaceutical leadcompounds. Therefore, we chose an ethnomedical approachto drug discovery and focussed on traditional remedies of theMaya to preselect plants with proven health effects.

Traditional medicine plants are often used for hundredsof years [1], which is the reason why no or only littletoxic effects in humans can be expected. Especially in Africaand Central and South America, traditional medicine isadvised by shamans, curanderos, or herbalists who oftenkeep the use of the healing plants as a secret [2]. Althoughthese plants are used since ages, little is known about the“pinciples of activity” and/or the targeted cellular mech-anisms, and hence, these remedies have a great potential


for drug development. We aimed for the separation andisolation of distinct properties of the Maya healing plantPluchea odorata which are relevant for anticancer treatment.P. odorata grows in the USA, Mexico, Belize, Guatemala,Panama, Cayman Islands, Guadeloupe, Jamaica, Puerto Rico,St. Lucia, Venezuela, and Ecuador (Germplasm ResourcesInformation Network, United States Department of Agri-culture, 9 November 2004, http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl?104497) and is still used by the Maya totreat common cold, fever, flu, head colds, headache, hyper-tension, neuralgia, ophthalmia, palsy, pneumonia, snake bite,swellings, inflammation, and bruises of the skin [3]. Themedical solution is prepared by boiling two handfuls of leavesin one gallon of water, and then it is frequently applied on theaffected area until the inflammation subsides [4]. Further theplant is described as being antidote, astringent, diaphoretic,and haemostatic [5], as well as traditionally used by mothersafter giving birth to decrease the risk for infections andconveyance of tissue recovery [3]. Gridling et al. [6] andBauer et al. [7] describe a strong anti-neoplastic effect of thedichloromethane extract in HL60 and MCF-7 cells and ananti-inflammatory response in HUVECs (human umbilicalvein endothelial cells).

Pharmaceutical drugs are prepared under standardisedconditions and usually contain just one active principle. Incontrast, ethnopharmacologic remedies are mixtures of avast number of compounds, which are in many cases un-known, and vary in their composition and activity dependingon the growth area and the time of collection. This makesthem difficult for application and trading. Moreover, plantextracts can also contain compounds that are systemicallytoxic or counteract the desired effect of the active princi-ple(s). On the other hand, some of the different compoundsin a plant extract can synergise. The present work willgive examples of this phenomenon. A common reason toterminate a priori successful therapies of all kinds of cancerand other diseases is the acquisition of drug resistance.However, it has not been reported so far that complex plantmixtures induce treatment resistances in patients, most likelybecause several distinct active principles target various intra-and intercellular signalling pathways simultaneously therebypreventing that the organism develops an “escape mecha-nism.” Thus, the development of complex extracts shouldbe considered as a strategy to treat cancer. Standardisationprocedures for such mixtures must be individually developedto provide therapeutics which are effective and safe anddo not exhibit batch to batch variations. The fractionationand accompanying testing in bioassays and by appropriateanalyses are a feasible concept for standardisation andremedies emerging along such procedures can be approvedby national agencies (i.e., Avemar, WO 2004014406 (A1):“Use of a fermented wheat germ extract as anti-inflammatoryagent” by Hidvegi and Resetar).

Here we describe a fractionation approach of the dichlo-romethane extract of P. odorata, which was constantly con-trolled regarding its activity by bio-assays and analysesthat can be standardised. This resulted in fractions causingdistinct cellular responses indicative for wound healing oranti-neoplastic properties.

2. Material and Methods

2.1. Plant Material. The aerial parts (leaves, caulis, flore-scence) of Pluchea odorata (L.) Cass. were collected inGuatemala, Departamento Peten, near the north-westernshore of Lago Peten Itza, San Jose, within an area offour-year-old secondary vegetation ∼1 km north of theroad from San Jose to La Nueva San Jose (16◦59′30′′ N,89◦54′00′′ W). Voucher specimens (leg. G. Krupitza & R.O. Frisch, Nr. 1-2009, 08. 04. 2009, Herbarium W) werearchived at the Museum of Natural History, Vienna, Austria.6 kg of air-dried plant material has been extracted withdichloromethane by Bjorn Feistel (Finzelberg GmbH & Co.KG, Andernach, Germany), and the extract stored in anexsiccator in the dark at 4◦C until use.

2.2. Plant Extraction and Fractionation. Plant extractionwith dichloromethane was essentially as described earlier[6, 7]. 6000 g dried plant material was extracted with dichlo-romethane resulting in 220 g dry extract (extraction wasperformed by Finzelberg GmbH & Co. KG, Andernach,Germany).

Vacuum Liquid Chromatography (VLC) was used for theseparation of large amounts of extract. 36 g dichloromethaneextract was redissolved in dichloromethane, mixed with 70 gsilica gel and evaporated to dryness, and ground in a mortarto obtain a homogenous powder. A 12 × 40 cm column waspacked with 900 g silica gel, on top the silica gel-containingextract, and covered with sea sand to ballast the sample. Themobile phase was passed through by application of reducedpressure. After checking the collected fractions by TLC, thosewith similar bands were recombined, and this resulted inthree combined fractions (F1.1–F1.3; Table 1).

VLC Fractionation of F1.1. 10 g of F1.1, which was derivedfrom the crude extract, was dissolved in dichloromethane,mixed with 20 g silica gel, evaporated to dryness, refined in amortar, and applied on top of a 5 × 60 cm silica gel column.Compounds were eluted with the mobile phases shown inTable 2 by applying reduced pressure. Collected fractionswere checked by TLC, and similar fractions were recombinedyielding nine combined fractions (F2.1–F2.9).

Column Chromatography (CC) Fractionation of F2.6(Less Than 2 g of Residue was fractionated by CC). 1.6 gF2.6, which was gained by fractionation of F1.1 was dissolvedin dichloromethane, mixed with 3 g silica gel, evaporated todryness, placed on top of a 5 × 50 cm silicia gel column andeluted with mobile phases shown in Table 3. The collectedfractions were checked by TLC and those with similar bandpatterns were recombined to yield ten combined fractions(F3.1–F3.10).

CC Fractionation of F3.3. 1.08 g of F3.3, which wasderived from F2.6, was dissolved in dichloromethane andplaced on top of a 2× 30 cm silica gel column. Mobile phaseswere used as illustrated in Table 4. Fractions were checked byTLC, and those with similar band patterns were recombinedyielding twelve combined fractions (F4.3.1–F4.3.12).

CC Fractionation of F4.3.7. 146 mg F4.3.7 was dissolveddichloromethane and applied on a 80 × 1.5 cm silica gelcolumn and fractionated with one litre solvent (chloroform:


Table 1: Mobile phases used for VLC of the dichloromethane extract.

Mobile phase Relation Volume (l) Fractions Residue (g)

Petroleum ether 4

Petroleum ether : chloroform 9 : 1 5 F1.1 23.9

Chloroform 12

Chloroform : methanol 9 : 1 9 F1.2 10.8 g

Chloroform : methanol 7 : 3 9


Chloroform : methanol 3 : 7 9 F1.3 1.6 g


Methanol 9

Table 2: Mobile phases used for VLC of F1.1.


Dichloromethane : hexan 8 : 2 2 F2.1 1.63

Dichloromethane 2 F2.2 0.17

Dichloromethane : ethyl acetate 8 : 2 1 F2.3 1.28

Dichloromethane : ethyl acetate 6 : 4 1 F2.4



Ethyl acetate 1 F2.7 0.22

Ethyl acetate : methanol 8 : 2 1 F2.8 0.04

Ethyl acetate : methanol 6 : 4 1 F2.9 0.12

Table 3: Mobile phases used for CC of F2.6.


Dichloromethane 100% 1 F3.1 0.02

Dichloromethane : ethyl acetate 80 : 20 2F3.2 0.05

F3.3 1.14


F3.5 0.05


F3.7 0.02


F3.9 0.03

Ethyl acetate 100% 1 F3.10 0.04

Table 4: Mobile phases used for CC of F3.3.

Mobile phase Relation Volume (mL) Fractions Residue (mg)

Dichloromethane 100% 500 F4.3.1 0.4

Dichloromethane : ethyl acetate 90 : 10 500F4.3.2 3

F4.3.3 59

F4.3.4 91


F4.3.6 65


F4.3.8 8


F4.3.10 111

Dichloromethane : ethyl acetate 20 : 80 500 F4.3.11 63

Ethyl acetate 100% 500 F4.3.12 3


Table 5: Stationary phase, mobile phases, and detection methodsused for TLC.

Stationaryphase

Silica gel plates 60 F254 (Merck, Darmstadt, Germany)

Mobilephase

TLC system 1 : chloroform : methanol : water 90 : 22 : 3.5

TLC system 2 : chloroform : methanol : water90 : 3.5 : 0.2

TLC system 3 : dichloromethane : ethyl acetate 80 : 20

TLC system 4 : dichloromethane : ethyl acetate 85 : 15

TLC system 5 : chloroform : methanol : water 70 : 22 : 3.5

DetectionUV254,UV365, visible light

Anisaldehyde sulphuric acid reagent (ASR)

methanol: water, 95 : 1.5 : 0.1). Fractions were collected intubes, ten drops per minute, and every 30 minutes thetubes were changed. Afterwards the column was washed with300 mL methanol. Fractions were checked by TLC, and thosewith similar band patterns were recombined resulting intwelve fractions (F5.3.7.1–F5.3.7.12; see Figure 3(c)).

CC Fractionation of F3.6. 0.32 g F3.6, which was derivedfrom F2.6, was dissolved in methanol and applied on topof a 3.5 × 40 cm Sephadex column (2.5 × 40 cm for 3.6).Methanol was used as mobile phase, and fractions werecollected and checked by TLC, and those with similar bandpatterns were recombined to yield four combined fractions:F4.6.1 (9 mg), F4.6.2 (5 mg), F4.6.3 (207 mg), F4.6.4 (55 mg).

CC Fractionation of F4.6.3. 20 mg F4.6.3 (very oily),which was derived from F3.6, was dissolved in dichloro-methane, mixed with silica gel, and applied on top a2.5 × 15 cm dichloromethane conditioned silica gel col-umn. Successful elution was achieved with dichlorome-thane : ethylacetate (50 : 50). Fractions were checked byTLC and those with similar band patterns were recom-bined yielding six combined fractions (F5.6.3.1–F5.6.3.6; seeFigure 4(b)).

2.3. Thin Layer Chromatography (TLC). TLC was used forcontrol of fractionation. Stationary phase and mobile phasesare described in Table 5. The mobile phase varied betweenfive solvent systems. Plates were detected under UV254,UV366, and visible light, before and after spraying withanisaldehyde sulphuric acid reagent (ASR). ASR consistedof 0.5 mL anisaldehyde, 10 mL glacial acetic acid, 85 mLmethanol and 5 mL H2SO4 (sulfuric acid). The sprayed platewas heated at 100◦C for five minutes, and then compoundswere detected under UV254,365 and visible light. Unlessotherwise stated 8 μL extract or fraction solution was appliedto the plate.

2.4. Cell Culture. HL-60 (human promyelocytic leukaemiacell) cells were purchased from American Type CultureCollection (ATCC). The cells were grown in RPMI 1640medium which was supplemented with 10% heat-inactivatedfetal calf serum (FCS), 1% Glutamax, and 1% Penicillin-Streptomycin. Both medium and supplements were obtainedfrom Life Technologies (Carlsbad, CA, USA). The cells werekept in humidified atmosphere at 37◦C containing 5% CO2.

2.5. Proliferation Assay. Proliferation assays [8, 9] wereperformed to analyse the inhibition of proliferation of HL-60 cells treated with extract or fractions of P. odorata. Extractand fractions were dissolved in ethanol (final concentrationwas 0.2%). HL-60 cells were seeded in 24-well plates ata concentration of 1 × 105 cells per mL RPMI mediumallowing logarithmic growth within the time of treatmentwith plant extract or fractions. The control was treatedwith solvent. After 24 and 48 hours, the number of cellswas determined using the Sysmex Cell Counter (SysmexCorp., Kobe, Japan). Experiments were done in triplicate.Percentage of cell division progression compared to theuntreated control was calculated by applying the followingformula:

C48 or 72 h + drug− C24 h + drugC48 or 72 h − drug− C24 h + drug

× 100 = % cell division.

(1)

2.6. Apoptosis Assay. Determination of cell death by Hoechst33258 (HO) and propidium iodide (PI) double staining(both Sigma, St. Louis, MO, USA) allows identifying theamount and the type of cell death (early or late apoptosisor necrosis) [10–12]. Therefore, HL-60 cells were seeded ina 24-well plate at a concentration of 1 × 105 cells per mLRPMI medium. Cells were treated with fractions or extractof P. odorata. The cells were incubated for 8, 24, 48, and/or72 hours, depending on the experiment. At each time point,100 μL cell suspension of each well was transferred intoseparate wells of a 96-well plate, and Hoechst 33285 and pro-pidium iodide were added at final concentrations of 5 μg/mLand 2 μg/mL, respectively. After one hour of incubation at37◦C, stained cells were examined and photographed withan Axiovert fluorescence microscope (Zeiss, Jena, Germany)equipped with a DAPI filter. Type and number of cell deathswere evaluated by visual examination of the photographsaccording to the morphological characteristics revealed byHOPI staining. Experiments were done in triplicate.

2.7. Western Blotting. Preparation of lysates. HL60 cells wereseeded in a tissue culture flask at a concentration of 1 ×106 cells per mL RPMI medium. P. odorata fractions F1,F4.6.3, and F4.3.7 were analysed by western blots. HL60 cellswere either incubated with 40 μg/mL F1 or with 10 μg/mL ofone of the other two P. odorata fractions for the indicatedtimes. At each time point, 4 × 106 cells were harvested,placed on ice, and centrifuged (1000 rpm, 4◦C, 4 min). Then,the supernatant (medium) was discarded, and the pelletwas washed twice with cold phosphate buffered saline (PBS,pH 7.2) and centrifuged (1000 rpm, 4◦C, 4 min). The cellpellet was lysed in a buffer containing 150 mM NaCl, 50 mMTris (pH 8.0), 1% Triton-X-100, 1 mM phenylmethylsulfonylfluoride (PMSF), and 1 mM protease inhibitor cocktail (PIC)(Sigma, Schnelldorf, Germany). Afterwards the lysate wascentrifuged at 12000 rpm for 20 min at 4◦C. Supernatantwas transferred into a 1.5 mL tube and stored at −20◦C forfurther analyses.

Gel electrophoresis (SDS-PAGE) and blotting. Equalamounts of protein samples (lysate) were mixed with sodium


P. odorata

Dichloromethane crude extract

Co 5 15 40

0

20

40

60

80

100

120

∗

HL-

60pr

olif

erat

ion

(%)

(μg/mL)

(a)

P. odorata

1.1

Co 15 40

0

20

40

60

80

100

120

HL-

60pr

olif

erat

ion

(%)

∗

∗

(μg/mL)

F

(b)

Co 15 40

24 h 48 h 72 h

HL-

60ce

llde

ath

(%)

0

20

40

60

80

100

120

P. odorata

∗

∗ ∗

∗

∗

∗

Co 15 40 Co 15 40

(μg/mL)

1.1F

(c)

0 0.5 2 4 8 24

cl. caspase 3

ac. α-tubulin

α-tubulin

β-actin

1.1F

(d)

Figure 1: Antiproliferative effect of (a) dichloromethane crude extract, (b) fraction F1.1 in HL60 cells; 1 × 105 cells/mL were seeded into24-well plate, incubated with 5, 15, and 40 μg/mL extract or fraction F1.1, and the percentage of proliferation was calculated relative tosolvent treated control within a 24 h period, (c) Induction of apoptosis by F1.1: cells were grown as described and incubated with 15 and40 μg/mL of each fraction for 72 h. Then, cells were double stained with Hoechst 33258 and propidium iodide and examined under themicroscope with UV light connected to a DAPI filter. Nuclei with morphological changes which indicated cell death were counted, and thepercentages of dead cells were calculated. Experiments were performed in triplicate. Asterisks indicate significance compared to untreatedcontrol (P < 0.05), and error bars indicate ±SD. (d) 1 × 106 cells/mL were incubated with 40 μg/mL F1.1 and harvested after 0.5, 2, 4, 8, and24 h of treatment, lysed, and total protein applied to SDS-PAGE. Western blot analysis was conducted with the indicated antibodies. Equalsample loading was confirmed by Ponceau S staining and β-actin analysis.

dodecyl sulfate (SDS) sample buffer (1 : 1) and loadedonto a 10% polyacrylamide gel. Proteins were separatedby polyacrylamide gel electrophoresis (PAGE) at 120 Voltfor approximately one hour. To make proteins accessibleto antibody detection, they were electrotransferred fromthe gel onto a polyvinylidene difluoride (PVDF) Hybond

membrane (Amersham, Buckinghamshire, UK) at 95 Voltfor 80 minutes. Membranes were allowed to dry for 30minutes to provide fixing of the proteins on the membrane.Methanol was used to moist the membranes. Equal sampleloading was checked by staining the membrane with PonceauS (Sigma, Schnelldorf, Germany).


P. odorata

24 48 72

F2.1F2.2

F2.3 + 4F2.5

F2.6F2.7F2.8

F2.9

Co

(hours)

0

20

40

60

80

100

120

HL-

60ce

llde

ath

(%)

24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72 24 48 72

∗∗

∗

∗∗∗

∗∗∗

∗∗

∗

∗

∗∗

F2.1–F2.9

(a)

Co 2 10 2 10−20

0

20

40

60

80

100

120

HL-

60pr

olif

erat

ion

(%)

∗

∗

(μg/mL)

P. odorata

F3.3 F3.6

F3.3 and F3.6

(b)

Start

F2.

6

F3.

1

F3.

2

F3.

3

F3.

4

F3.

5

F3.

6

F3.

7

F3.

8

F3.

9

F3.

10(c)

Figure 2: (a) Induction of apoptosis of HL60 cells by F2.1–F2.9. 1× 105 cells/mL were seeded in 24-well plates and incubated with 10 μg/mLof each fraction for 72 h. Then, cells were double stained with Hoechst 33258 and propidium iodide and examined under the microscopewith UV light connected to a DAPI filter. Nuclei with morphological changes which indicated cell death were counted, and the percentagesof dead cells were calculated. Significance was calculated in comparison to control (Co). (b) Antiproliferative effect of F3.3 and F3.6: 1 × 105

cells/mL were seeded into 24-well plate, incubated with 2 and 10 μg/mL of each fraction, and the percentage of proliferation was calculatedrelative to solvent treated control within a 24 h period. Experiments were performed in triplicate. Asterisks indicate significance comparedto untreated control (P < 0.05) and error bars indicate ±SD. (c) Thin layer chromatography (TLC) of F2.6 and F3.1–F3.10; Mobile phase:TLC system 2. Detection: visible light with ASR.

Protein detection. After washing with PBS or TBS (Trisbuffered saline, pH 7.6), membranes were blocked in PBS-or TBS-milk (5% nonfat dry milk in PBS containing 0.5%Tween 20 or TBS containing 0.1% Tween 20) for onehour at room temperature. Then membranes were washedwith PBS/T (PBS containing 0.5% Tween 20) or TBS/T(TBS containing 0.1% Tween 20), changing the washingsolution four to five times every five minutes. Then everymembrane was incubated with a primary antibody (1 : 500)in blocking solution (according to the data sheet TBS-,PBS-milk or TBS-, PBS-BSA), at 4◦C over night gentlyshaking. Subsequently the membrane was again washed withPBS/T or TBS/T and incubated with the second antibody(peroxidase-conjugated goat anti-rabbit IgG or anti-mouseIgG) diluted 1 : 2000 for one hour at room temperature. Afterwashing the membranes, chemiluminescence was developedwith enhanced chemiluminescence (ECL) plus detectionkit (Amersham, UK) (two seconds to ten minutes), and

membranes were exposed to the Lumi-Imager F1 (Roche) forincreasing times.

2.8. Antibodies. Monoclonal mouse ascites fluid anti-acet-ylated α-tubulin (6-11B-1) and β actin (AC-15) antibod-ies were from Sigma (St. Louis, MO, USA). Monoclonalmouse α-tubulin (DM1A), β-tubulin (H-235), Cdc25A(F-6), and polyclonal rabbit paxillin (H-114), ROCK-1(C8F7), c-Jun (H-79), Jun b (210) were from Santa CruzBiotechnology, Inc. (Santa Cruz, CA, USA). Monoclonalrabbit cleaved caspase 8 (Asp391) (18C8) and phospho-Stat3 (Tyr705)(D3A7) antibodies and polyclonal rabbitcleaved caspase 3 (Asp175), human-specific cleaved caspase9 (Asp330), phospho-Chk2 (Thr68), Chk2, phospho-myosinlight chain 2 (MLC2-Ser19), myosin light chain 2, Stat3,and MYPT1 were from Cell Signaling (Danvers, MA, USA).Polyclonal rabbit phospho-Cdc25A (Ser177) antibody wasfrom Abgent (San Diego, CA, USA), polyclonal rabbit


F4.3.1–F4.3.12

Co 5 10

0

20

40

60

80

100

120

F4.3.2F4.3.3

F4.3.5F4.3.6

F4.3.7F4.3.8F4.3.9F4.3.10F4.3.11

F4.3.1

F4.3.4

F4.3.12

Co

P. odorata

HL-

60pr

olif

erat

ion

(%)

(μg/mL)

5 10 5 10 5 10 5 10 5 10 5 10 5 10 5 10 5 10 5 10 5 10

(a)

0

20

40

60

80

100

120

P. odorataF4.3.7

Co 5 10 Co 5 10

(μg/mL)

48 h24 h

HL-

60ce

llde

ath

(%)

(b)P. odorata

0

20

40

60

80

100

120

HL-

60ce

llde

ath

(%)

Co 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5 2 5

(μg/mL)

F5.3.7.1–F5.3.7.12

5.3.7.15.3.7.2

Co

5.3.7.35.3.7.45.3.7.55.3.7.6

5.3.7.75.3.7.85.3.7.95.3.7.105.3.7.115.3.7.12

∗

(c)

Figure 3: (a) Antiproliferative effect of F4.3.1–F4.3.12 in HL60 cells; 1 × 105 cells/mL were seeded into 24-well plates, incubated with 5and 10 μg/mL of each fraction, and the percentage of proliferation was calculated relative to solvent treated control within a 24 h period. (b)Induction of apoptosis by F4.3.7 and (c) by F5.3.7.1–F5.3.7.12 in HL60 cells; 1 × 105 cells/mL were seeded in 24-well plates and incubatedwith (b) 5 and 10 μg/mL of F4.3.7 for 24 and 48 h and (c) with 2 and 5 μg/mL of each indicated fraction for 24 h. Then, cells were doublestained with Hoechst 33258 and propidium iodide and examined under the microscope with UV light connected to a DAPI filter. Nucleiwith morphological changes which indicated cell death were counted, and the percentages of dead cells were calculated. Experiments wereperformed in triplicate. Asterisks indicate significance compared to untreated control (P < 0.05), and error bars indicate ±SD.

phospho-MYPT1 (Thr696) from Upstate (NY, USA), mousemonoclonal γH2AX (pSer139) (DR 1017) from Calbiochem(San Diego, CA, USA), and mouse monoclonal c-Myc Ab-2(9E10.3) from Thermo Fisher Scientific, Inc. (Fremont, CA,USA). The secondary antibodies peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Dako(Glostrup, Denmark).

2.9. Statistical Analysis. For statistical analyses, Excel 2003software and Prism 5 software package (GraphPad, SanDiego, CA, USA) were used. The values were expressed

as mean ± standard deviation, and Student’s t-test wasapplied to compare differences between control samples andtreatment groups. Statistical significance level was set to P <0.05.

3. Results and Discussion

The dichloromethane extract of P. odorata exhibits stronganti-neoplastic activity (Figure 1(a)). Therefore, we fraction-ated this extract and constantly monitored the activities bybio-assays measuring apoptosis and/or proliferation rates to


0

20

40

60

80

100

120

HL-

60ce

llde

ath

(%)

P. odorata

(μg/mL)

∗

∗

∗

∗

∗

F4.6.3

Co 10 Co 2 5 10 Co 2 5 10

48 h24 h8 h

(a)

0

20

40

60

80

100

120

HL-

60ce

llde

ath

(%)

P. odorata

(μg/mL)

Co 2 5 Co 2 5 Co 2 5 Co 2 5 Co 2 5 Co 2 5

5.6.3.15.6.3.25.6.3.3

5.6.3.45.6.3.55.6.3.6

∗

F5.6.3.1–F5.6.3.6

(b)

Figure 4: (a) Induction of apoptosis; 1× 105 HL60 cells/mL were seeded in 24-well plates and incubated with 2 and/or 10 μg/mL of F4.6.3 for8, 24, and 48 h or (b) 2 and 5 μg/mL of F5.6.3.1–F5.6.3.6 for 24 h. Then, cells were double stained with Hoechst 33258 and propidium iodideand examined under the microscope with UV light connected to a DAPI filter. Nuclei with morphological changes which indicated cell deathwere counted, and the percentages of dead cells were calculated. Experiments were performed in triplicate. Asterisks indicate significancecompared to untreated control (P < 0.05), and error bars indicate ±SD.

get closer to the active principles. In the first round thecrude extract was split in three distinct fractions of whichfraction F1.1 exhibited the strongest anti-proliferative andproapoptotic activity (Figures 1(b) and 1(c)). 40 μg F1.1/mLinduced caspase 3 and decreased β-actin and α-tubulin levels,and therefore also reduced acetylation levels of α-tubulinwere detected within 4 h of treatment (Figure 1(d)). Thissuggested that the overall protein decrease was due to theearly activation of caspase 3 and subsequent cell death.

F1.1 was further processed yielding fractions F2.1–F2.9.F2.6 was nearly as active F2.7 (Figure 2(a)) but contained∼10 times more fraction material (1.9 g versus 0.2 g, resp.).Therefore, F2.6 was further processed yielding fractionsF3.1–F3.10 (Figures 2(b) and 2(c)).

10 μg/mL of F3.4–F3.6 exhibited potent anti-proliferativeproperties in HL60 cells suppressing cell growth by 100%.The TLC analysis showed that both, F3.3 and F3.6, containeda distinct main compound, and therefore F3.3 and F3.6 werefurther fractionated. Also F3.2 was processed, however, thisyielded only several low-activity fractions (data not shown).

(i) The subfractionation of F3.3 on a silica gel columnyielded the potent antiproliferative and proapoptoticfraction F4.3.7 (Figures 3(a) and 3(b)). Furtherfractionation of F4.3.7 caused the decomposition ofthe strong cytotoxic activity into many low activefractions (F5.3.7.1–F5.3.7.12; Figure 3(c)), which insum approximated the activity of the precursor.

(ii) The subprocessing of F3.6 yielded fractions F4.6.1–F4.6.4. F4.6.3, which was very oily, was the most

pro-apoptotic one (Figure 4(a)). Upon further frac-tionation, the activity of F4.6.3 also decomposedinto several low-activity fractions (F5.6.3.1–F5.6.3.6;Figure 4(b)).

Therefore, we went back to F4.3.7 and F4.6.3 andcontinued analyses with these two distinct high-activityfractions. The TLC patterns of F4.3.7 and F4.6.3 were clearlydifferent from each other evidencing that they containdifferent compounds (Figure 5(a)). To characterise the twodistinct fraction types, posttranslational modifications andexpression levels of proteins, which are relevant for apop-tosis and cell cycle arrest, were investigated. F4.3.7 slightlyinduced Chk2 phosphorylation and hence its activation,whereas F4.6.3 did not (Figure 5(b)). Chk2 was shown tophosphorylate Cdc25A at Ser177 and tags it for proteasomaldegradation [13, 14]. However, treatment of HL60 cells withF4.3.7 caused the dephosphorylation of Ser177 and proteinstabilisation. Also F4.6.3 stabilised Cdc25A despite Ser177phosphorylation. This implicated that Cdc25A was notregulated by Chk2 activity in this scenario. Moreover Cdc25Astabilisation suggested an increase in cell cycle activity [15].Nevertheless, cell proliferation was inhibited.

In search of molecular causes for reduced proliferation,we found that the fractionation steps enriched a spindletoxin or an indirect microfilament-targeting activity. Thiswas reflected by induced α-tubulin acetylation, which is anindicator of the polymerisation status of tubulin microfil-aments [16, 17], within 2 h of treatment with F4.3.7 andF4.6.3 (Figure 5(c)). Thereafter, caspase 3 became activated.Also the phosphorylation of H2AX occurred after treatment


Start

F4.

6.1

F4.

6.2

F4.

6.3

F4.

6.4

F4.

3.1

F4.

3.12

F4.

3.11

F4.

3.10

F4.

3.2

F4.

3.3

F4.

3.4

F4.

3.5

F4.

3.6

F4.

3.7

F4.

3.8

F4.

3.9

(a)

0 0.5 2 4 8 24

F4.6.3 F4.3.7

0 0.5 2 4 8 24

p(Thr68)Chk2

Chk2

p(Ser177)Cdc25A

Cdc25A

β-actin

(b)

0 0.5 2 4 8 24

cl. caspase 9

ac. α-tubulin

α-tubulin

F4.6.3 F4.3.7

0 0.5 2 4 8 24

γH2AX

cl. caspase 8

cl. caspase 3

β-tubulin

(c)

0 0.5 2 4 8 24

F4.6.3 F4.3.7

0 0.5 2 4 8 24

β-actin

c-Myc

JunB

c-Jun

Stat3

p(Tyr705)Stat3

(d)

0 0.5 2 4 8 24

F4.6.3 F4.3.7

0 0.5 2 4 8 24

Paxillin

ROCK1

p(Ser19)MLC2

MLC2

p(Thr696)MYPT1

MYPT1

β-actin

(e)

Figure 5: (a) Thin layer chromatography (TLC) of F4.6.3 (left panel) and F4.3.7 (right panel); mobile phase: TLC system 2; detection: UV254.Analysis of (b) cell cycle and checkpoint regulators, (c) apoptosis-related proteins, (d) oncogenes, (e) proteins required for mobility; 1 ×106 HL60 cells/mL were incubated with 10 μg/mL F4.6.3 and F4.3.7, respectively, and harvested after 0.5, 2, 4, 8, and 24 h of treatment. Cellswere lysed and obtained proteins samples applied to SDS-PAGE. Western blot analysis was performed with the indicated antibodies. Equalsample loading was confirmed by Ponceau S staining β-actin or β-tubulin analysis.


with both fractions indicating DNA damage presumably dueto caspase-3-induced DNase activity of DNA fragmentationfactor (DFF; [18]), because caspase 3 activation and H2AXphosphorylation appeared simultaneously [19]. Evidently,the molecular onset of apoptosis occurred faster than theorchestrated expression of the investigated cell cycle regu-lators. Interestingly, the activation of caspase 3 concurredwith the activation of caspase 8, but not with activation ofcaspase 9, indicating that the extrinsic apoptosis pathwaybecame activated and not the intrinsic one. The signature-type processing of caspase 3 into the active fragment wasmore prominent by F1.1 than by F4.3.7 and F4.6.3. This wasmost likely due to the four times higher F1.1 concentrationused (40 μg/mL), which caused also the degradation of α-tubulin and β-actin as consequence of swift onset of celldeath.

The low or absent activation of Chk2 (resp.) suggestedthat a genotoxic activity, which was formerly present inthe crude extract [6] and in other subfractions [7], waseliminated throughout the described fractionations. Avoid-ing genotoxicity can be beneficial, because it reduces DNAdamage and subsequent mutations that may cause sec-ondary malignancies. Cdc25A, a classified oncogene [20,21] was upregulated by both fraction types and thereforewe investigated also the expression of other oncogenes,which are related to tumour growth and progression. F4.6.3had a substantial effect on the repression of c-Myc andonly a minor effect on c-Jun. Even more effective F4.3.7suppressed also the phosphorylation of Tyr705 of Stat3 andthus inhibited its function, which is known to play a rolein tumour progression [22]. This fraction further repressedJunB, c-Jun, and c-Myc more effectively than F4.6.3.

Since P. odorata is used also as a wound healing remedy,this property also involves tissue regeneration and theregulation of a process called “epithelial to mesenchymaltransition” (EMT) [23, 24]. The most prominent featureof EMT is the acquisition of a mobile phenotype [25, 26].If transient and tightly regulated, EMT is beneficial forthe organism, because it contributes to acute inflammationand tissue repair [27–29]. In contrast, the chronic statusoccurrence of EMT causes pathologies such as progressionof cancer [30]. The mobility of cancer cells is a prerequisitefor the intra- and extravasation of the vasculature, tissueinvasion, and metastatic spread [23, 31], and it is mediated byproteins that allow cell plasticity and movement. Therefore,the alteration of motility-related gene products in cancerouscells, such as paxillin [32, 33], ROCK1 [34], MLC2 for theformation of stress fibres [35] and MYPT [36], are indicatorsfor increased mobility and hence cancer progression. Alsoleukaemia cells such as differentiated HL60 attach to theECM and vascular cells and transmigrate through vesselwalls, invade tissues [37–39], and add to the progressionof the disease. Hence, HL60 cells possess a repertoire ofproteins facilitating cell movement although they normallygrow in suspension. Treatment with F4.3.7 caused ROCK1repression below constitutive and detectable levels, whichwas not the case with F4.6.3 (Figure 5(d)). Also MYPTexpression decreased upon treatment with F4.3.7. Paxillinbecame upregulated by F4.6.3 treatment and marginally by

F4.3.7, and this was also the case for the phosphorylation ofMLC2. Since P. odorata is successfully used for tissue recoveryand healing of skin bruises, the increased mobility of cells(fibroblasts, epithelial cells, macrophages, etc.) is required toclose the tissue disruption. This very property, however, isdetrimental throughout cancer treatment, and elimination ofthis activity might be beneficial for this purpose. Therefore,future research has to address the question whether F4.3.7is advantageous for cancer cell treatment, whereas F4.6.3exhibits advanced wound healing properties.

4. Conclusions

A spindle-damaging activity became enriched by fractiona-tions, which was reflected by increased α-tubulin acetylationfollowed by the activation of caspases 8 and 3 and the typicalnuclear morphology of apoptosis. The impact on apoptosisand the orchestration of apoptosis regulator activation weresimilar for both fractions. In comparison to earlier work,genotoxic components activating Chk2 were eliminated.

Importantly, F4.6.3 induced mobility markers, whereasF4.3.7 inhibited the expression of mobility markers ordid not interfere with their constitutive expression. Thisproperty implicates an inhibitory effect of F4.3.7 on tumourprogression which was also reflected by the downregulationof the Jun family of oncogenes and the suppression of Stat3activity.

These findings can provide a basis for the development ofremedies (1) supporting wound healing in case of F4.6.3 or(2) interfering with tumour progression in case of F4.3.7.

Acknowledgments

The authors wish to thank Toni Jager for preparing thefigures. This work was supported by the Funds for Innovativeand Interdisciplinary Cancer Research to G. Krupitza.

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Research Article

Inhibitory Effect of Nelumbo nucifera (Gaertn.) onthe Development of Atopic Dermatitis-Like Skin Lesions inNC/Nga Mice

Rajendra Karki,1 Myung-A Jung,1, 2 Keuk-Jun Kim,3 and Dong-Wook Kim1

1 Department of Oriental Medicine Resources, College of Natural Science, Mokpo National University, 61 Muan-gun,Jeollanam-do 534-729, Republic of Korea

2 Jeollanamdo Institute of Natural Resources Research, Jangheung-gun, Jeollanam-do 529-851, Republic of Korea3 Department of Clinical Pathology, Taekyeung College, Gyeungsan, Gyeongsangbuk-do 712-719, Republic of Korea

Correspondence should be addressed to Dong-Wook Kim, [email protected]

Received 5 October 2011; Revised 8 November 2011; Accepted 15 November 2011


Copyright © 2012 Rajendra Karki et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Atopic dermatitis (AD) is a chronic inflammatory skin disease which has a complex etiology that encompasses immunologicresponses. The study was carried out to examine the effect of Nelumbo nucifera (Gaertn.) leaf (NL) on the AD-like skin lesioninduced by repeated epicutaneous application of 2,4-dinitrochlorobenzene (DNCB) on the dorsal skin of NC/Nga mice. Threedifferent doses of NL (5, 25, and 50 mg/mice/day) were administered orally from the day of sensitization with DNCB for 4 weeks.The efficacy of NL was judged by histopathological examination, blood IgE level, measurement of transepidermal water loss(TEWL), scratching behavior, and skin severity score. NL resulted in the suppression of clinical severity score, TEWL, scratchingbehavior, and blood IgE level. Histopathologic analyses revealed that thickening of the epidermis and mast cell degranulation wassignificantly reduced in NL group. These results suggest that NL may be a useful natural resource for the management of AD.

1. Introduction

Atopic dermatitis (AD) is a chronic and relapsing inflamma-tory skin disease relying on the interplay of environmental,immunological, and genetic factors [1]. The prevalence ofAD has increased dramatically in the past 3 decades and theincrease in recent years can be attributed to environmentalinfluences including industrialized and urban settings [2].Epidemiological reports suggest that AD affects up to 10–20% of children worldwide and can persist into adulthood. Ithas been estimated that AD symptoms are developed among65% of patients in the first year of life and among 90% ofthe patients before the age of five [3]. The lesional skin inAD is characterized by edema, hemorrhage, erosion, dryness,and alopecia typically localized on the ears, back, and neckand in the facial region [4]. Pathological examination ofthe lesional skin in AD reveals spongiosis, hyperkeratosis,and parakeratosis in acute lesions and marked epidermalhyperplasia, acanthosis, and perivascular accumulation oflymphocytes and mast cells in chronic lesions [5]. Although

the complex interrelationships between genetic, environ-mental, skin barrier, pharmacological, psychological, andimmunological factors contribute to the pathogenesis of AD,the immunological basis of the disease is of considerableimportance and has been extensively studied [6]. The allergicdiseases like AD, asthma, and rhinitis are characterizedby Th2-dominated responses, which are mediated by IL4,IL-5, and IL-13 and induce B-cell class switching to IgE[7]. NC/Nga mice were established as an inbred strainfrom Japanese fancy mice in 1957 and have recently beenshown to spontaneously develop AD-like dermatitis with IgEhyperproduction in air-uncontrolled, conventional circum-stance [8, 9]. NC/Nga mice, however, show no skin lesionwhen raised in specific-pathogen-free (SPF) condition. Anepicutaneous application with chemical antigens such aspicryl chloride evokes contact hypersensitivity reaction inmice that had previously been sensitized with same agents.Interestingly, repetition of the topical application with suchagents not only induces a shift in kinetics of the skinreaction from delayed-type to immediate-type response but


Control

(a)

NL5

(b)

NL25

(c)

NL50

(d)

Figure 1: Clinical skin symptoms. Three different doses of NL (5, 25, and 50 mg/mice/day) were administered orally from the day of DNCBsensitization. The photographs were taken on day 28 before the sacrifice of the mice.

also changes the cytokine milieu from Th1 to Th2 profile[10, 11].

Nelumbo nucifera Gaertn. (Nymphaeaceae), a largeaquatic herb widely found in India, China, Japan, and Korea,not only possesses ornamental and dietary value but alsohas been using as a medicinal herb in eastern Asia. Almostevery part of N. nucifera including leaves, flowers, seeds, andrhizomes has been reported to possess different therapeuticeffects [12]. The leaves have been mentioned to showantiobesity activity by increasing lipolysis in the adiposetissue of mice [12, 13]. There are reports on N. nuciferarhizome possessing acetylcholinesterase activity and semenpossessing antidiarrheal and tranquilizing activities [14]. N.nucifera is rich in alkaloids and flavonoids. Moreover, N.nucifera leaves contain abundant amount of flavonoids likeluteolin, quercetrin, and isoquercitrin [15]. In this paper, theeffects of water extract of N. nucifera leaves (NLs) on theslink skin symptoms of NC/Nga mice caused by the repeatedtopical application of DNCB (2, 4-dinitrochlorobenzene)were evaluated.


2.1. Animals. Male 5-week-old NC/Nga mice (20–23 gramsbody weight) were purchased from SLC Inc. (Tokyo, Japan).The mice were kept in standard plastic cages under controlled

temperature (25 ± 5◦C), humidity (50 ± 10%), 100% freshhepa-filtered air, and 24 hours light-dark cycle (lights onfrom 06:00 to 18:00) in the Animal Research Center ofMokpo National University. The mice were supplied withbasal diet and sterilized water without any restriction duringthe experiment. All mice were acclimated for a week priorto the initiation of experiments. All animal studies wereapproved by the Animal Care Committee of the GraduateSchool of Natural Sciences, Mokpo National University, andall husbandry practices and animal care were in accordancewith the guidelines of Korean Council on Animal Care.

2.2. Reagents. DNCB was purchased from Sigma-Aldrich,Inc. (St. Louis, MO, USA). Olive oil and acetone werepurchased from Kanto Chemical Co., Inc. (Tokyo, Japan) andCarlo erba Reagents SA. (France), respectively. DNCB wasdissolved in olive oil/acetone (3 : 1) as 1% (w/v) and 0.4%(w/v) solution and used for the sensitization and elicitation.

2.3. Application of DNCB. Dorsal hair of NC/Nga mice wasremoved by using electric shaver and hair removing creamcontaining 80% of thioglycolate. On the next day, the dorsalskin was sensitized with 200 μL of 1% DNCB (w/v) in oliveoil/acetone (3 : 1). Four days later, the mice were challengedby applying 200 μL of 0.4% DNCB (w/v) on the dorsal skin


repeatedly 3 times per week (Monday, Thursday, and Sat-urday) for 4 weeks. Each group contained 7 mice.

2.4. Preparation of Nelumbo nucifera Leaf (NL) Extract.Leaves of N. nucifera were obtained from Muan, Korea. Theleaves (1 Kg) were extracted with 3000 mL of distilled waterby using soxhlet extractor for 3 hours, and the extraction wasrepeated for 3 times. The residue was removed by filtrationand then the filtrate was evaporated and freeze-dried to givethe powder of NL. The yield of the dried extract was about12% of the starting material.

2.5. Administration of NL. NL was dissolved in distilled waterand fed to the mice per orally using gastric sonde from theday of sensitization until 4 weeks. The control was fed withdistilled water. NL was administered at 3 different doses (5,25, and 50 mg/mice/day).

2.6. Transepidermal Water Loss (TEWL). TEWL on thedorsal skin of mice was measured using a skin evaporativewater recorder (Tewameter TM300; Courage and Khazawa,Cologne, Germany). The resulting data were analyzed bythe microprocessor and were expressed in g/m2/h. TEWLwas measured in each week for 4 weeks. Measurements wererecorded when TEWL readings were stabilized at approxi-mately 30 seconds after the probe was placed on the skin.

2.7. Evaluation of Scratching Behavior and Blood IgE Level.Mice were placed individually in a clear plastic cage andallowed to acclimatize for 15 minutes. Thereafter, behaviorwas videotaped for 10 minutes. Scratching of the nose,ears, and dorsal skin with the hind paws was observedduring playback. Licking of the belly and dorsal skin duringgrooming was disregarded. Each occurrence of scratchingof the head, neck, dorsal skin, ears, and nose was scoredto obtain the maximum score. The IgE level in blood wasmeasured using IgE kit (Shibayagi Co. Ltd., Gunma, Japan)according to the manufacturer’s instructions.

2.8. Evaluation of Skin Severity. The severity of dermatitison the face, ears, and dorsal part of the body was assessedblindly. The evaluated symptoms consisting of (i) ery-thema/hemorrhage, (ii) pruritus and dry skin, (iii) edema,(iv) excoriation/erosion, and (v) lichenification were scoredas follows: none = 0; mild = 1; moderate = 2; severe = 3. Thesum of the scores for each evaluated symptom (maximumscore: 15) was considered as the skin severity score. The skinseverity was evaluated every week for 4 weeks.

2.9. Histopathological Examination. After 4 weeks, all themice were euthanatized under diethyl ether anesthesia andthe dorsal skin was excised, fixed in 10% phosphate-bufferedformalin (pH 7.2), and embedded in paraffin. Sections of4 μm were microtomed. The sections were deparaffinizedin xylene, dehydrated in graded alcohol bath, and stainedwith hematoxylin and eosin or acidified toluidine blue.Finally, they were examined through optical microscope(Olympus, Tokyo, Japan), and thickness of epidermal layer

Time (weeks)

0 1 2 3 4 5

Skin

seve

rity

scor

e

0

2

4

6

8

10

12

14

16

ControlNL5

NL25NL50

aa

Figure 2: Skin severity score. The skin severity of mice was assessedmacroscopically in a blinded fashion as mentioned in Section 2.The symptoms considered were (i) erythema/hemorrhage, (ii)pruritus and dry skin, (iii) edema, (iv) excoriation/erosion, and (v)lichenification. aP < 0.05 versus control.

was calculated using Aperio ImageScope image analysis. Thenumber of mast cells per square millimeter of dermis infour sites chosen at random was counted from the toluidinestained sections.

2.10. Statistical Analysis. All the experimental data were ex-pressed as mean ± standard deviation. The significance ofvariation among different groups was determined by one-way ANOVA analysis. P value ≤ 0.05 was considered to besignificantly different.

3. Results

3.1. Effect of NL on Skin Severity. NC/Nga mice have beenshown to develop AD-like skin lesions by repeated applica-tion of picrylchloride. The skin severity in control was in-creased gradually with the number of DNCB challenges.Oral administration of NL for 4 weeks suppressed thedevelopment of AD-like skin lesions. The skin severity ofeach group on day 28 is as shown in Figure 1. Skin severityin each group was more or less similar up to 14 daysfrom the day of sensitization. But after 14 days, there wasdrastic change in the skin symptoms of control than NL-administered groups. The skin severity score of each groupis as shown in Figure 2.

3.2. Histopathological Analyses. Hematoxylin and eosinstaining of the dorsal skin sections revealed hyperkeratosis,parakeratosis, acanthosis with varying degrees of spongiosis,exocytosis of mononuclear cells in the epidermis, andinfiltration of inflammatory cells into the upper dermis ofcontrol group while all those parameters were suppressedin NL-administered groups as shown in Figure 3(a). Thequantitative data of epidermal thickness are as shown in


NL25 NL50

NL5Control

(a)

Control NL-25 NL-50

(μm

)

0

20

40

60

80

100

120

a

NL5

(b)

Figure 3: Histopathological changes. The dorsal skin excised at the end of the experiment was fixed in formalin, microtomed in 4 μmsections, and stained with hematoxylin and eosin (magnification 400x). The thickness of epidermal layer was calculated using AperioImageScope V9.1.19.1571 image analysis. aP < 0.05 versus control.

Figure 3(b). The epidermal thickness of control was 88.668±15.2μm while that of NL50 was 61.288 ± 21 μm (P < 0.05).Toluidine blue staining of the dorsal skin sections revealed ofmore infiltration and degranulation of mast cells in the upperdermis of control than NL-administered groups as shown inFigure 4(a). The quantitative data on mast cell infiltrationand degranulation are as shown in Figure 4(b). The numberof mast cells per millimeter square in control, NL5, NL25,and NL50 was found to be 67±20, 41±18, 44±13, and 41±9, respectively.

3.3. Effect of NL on Blood IgE Level. The epicutaneous sensi-tization and challenge of dorsal skin of NC/Nga mice withDNCB elevated the Ig E level from 1.36 ± 0.435μg/mL(normal, nontreated group) to 6.87 ± 0.286μg/mL. Theadministration of NL50 suppressed the IgE level to 3.5 ±0.93μg/mL, which was the suppression approximately by2 folds against the control. Similarly, NL25 suppressed theelevation of IgE to 4.7 ± 0.8μg/mL which was statisticallysignificant (P < 0.05). Figure 5 shows the effect of NL onthe blood IgE level on day 28.


Control NL5

NL25 NL50

(a)

Deg

ran

ula

ted

mas

tce

lls/a

rea

0

20

40

60

80

100

Control NL5 NL25 NL50

(b)

Figure 4: Mast cell degranulation. The dorsal skin excised at the end of the experiment was fixed in formalin, microtomed in 4 μmsections, and stained with acidified toluidine blue. The number of mast cells/unit area of dermis in 4 sites chosen at random was counted.(magnification, 400x), arrow heads showing degranulated mast cells.

3.4. Effect of NL on Scratching Behavior and TEWL. Therewas no difference in the average number of scratchingbehavior among the groups up to 14 days. However, therewere changes in the scratching behavior among the groupsfrom the 3rd week. The scratching behavior evaluated onday 25 is as shown in Figure 6. The number of scratchingwas decreased by more than threefold in NL50. Consistentwith effect of NL on mast cell degranulation, NL decreasedthe scratching behavior. TEWL of NL-administered groupswas not different from control up to 14 days. However,administration of NL25 and NL50 significantly inhibitedTEWL on days 21 and 28 (Figure 7).

4. Discussion

AD is a chronic inflammatory skin disease which has a com-plex etiology, and it encompasses immunologic responses,susceptibility genes, environmental triggers, and compro-mised skin-barrier function [1]. The skin lesions of ADpatients are characterized by the presence of inflammatoryinfiltrates consisting of T lymphocytes, monocytes/macro-phages, eosinophils, and mast cells [5]. Steroidal drugs likecorticosteroids are commonly prescribed for the alleviationof the symptoms of AD. However, the repeated use ofcorticosteroids can cause severe skin atrophy, susceptibility to


Normal Control NL5 NL25 NL500

2

4

6

8

a

b

(μg/

mL)

Figure 5: Blood IgE level. Blood was taken from each mouse atthe end of the experiment, and the blood IgE level was measuredusing mouse IgE-specific ELISA kit. aP < 0.05 and bP < 0.01 versuscontrol.

Control NL5 NL25 NL50

Nu

mbe

rs

0

10

20

30

40

50

60

70

b

a

Figure 6: Effect of NL on scratching behavior. The scratchingbehavior of each mouse was videotaped for 10 minutes on day25 after sensitization. Scratching of the nose, ears, and dorsal skinwith the hind paws was observed during play back. aP < 0.05 andbP < 0.01 versus control.

infection, and adrenal suppression [16]. NC/Nga mice, orig-inated from Japanese fancy mice as an inbred line, wheneverraised in conventional circumstances spontaneously developAD-like skin lesions characterized by increased numberof eosinophils, mast cells, lymphocytes, and macrophages[4]. Although SPF NC/Nga mice showed neither clinicalsigns nor IgE hyperproduction, application of haptens ormite antigens is necessary to evoke the eczema stably [17].Consistent with these reports, epicutaneous sensitizationand challenge of dorsal skin of NC/Nga mice with DNCBevoked the incidence of AD like skin lesions. Histologicalexamination of skin lesion stained by hematoxylin and eosinand toluidine blue revealed the thickening of epidermis anddermis because of leukocyte infiltration, hyperplasia, dermisulcer, and infiltration of immunocytes on the skin such as Tcell, mast cell, and eosinophils. In NL-administered groups,

Time (weeks)

0 1 2 3 40

10

20

30

40

50

60

70

80

ControlNL5

NL25NL50

bb

bb

(g/m

2/h

r)

Figure 7: Effect of NL on TEWL. TEWL was measured everyweek for 4 weeks after sensitization using Tewameter (TM300).Measurements were recorded when TEWL readings were stabilizedat approximately 30 seconds after the probe was placed on the skin.bP < 0.01 versus control.

the thickness of epidermis and dermis was reduced and theinfiltration of immunocytes was significantly decreased ascompared to control. Excessive production of chemokinesuch as thymus and activated-regulation chemokine (TARC)by activated keratinocytes attracts the immunocyte intothe epidermis resulting in acanthosis [4]. In our in vitrostudy, treatment of NL (250 and 500 μg/mL) for 24 hourssignificantly inhibited the production of TARC by activatedkeratinocyte (data not shown) suggesting that inhibitionof TARC inhibited the infiltration of immunocytes therebyinhibiting epidermal thickening. Moreover, Th2-specificchemokines, TARC, and monocyte-derived chemotacticcytokine, and their receptor, CCR4, have been reported tobe highly expressed in the lesions of the NC/Nga mice[4]. Haptens such as picryl chloride are commonly usedto induce allergic dermatitis and have been thought toevoke primarily a Th1-dominated response. However, it hasbeen recently reported that multiple challenges with picrylchloride to the skin of mice over an extended period causethe skin inflammation to shift to a chronic Th2-dominatedinflammatory response that is similar to human AD [18].It is well established that the elevation of serum IgE inAD may be due to the Th1/Th2 imbalance skewed to Th2,which plays important roles in the pathology of AD [19].Mast cells, a critical participant in the various biologi-cal processes including allergic diseases and inflammatoryreaction, store biologically potent inflammatory mediatorsuch as histamine and express on their surface membranereceptors with high affinity and specificity for IgE [20]. Theinteraction of antigen with surface-bound IgE initiates aseries of biochemical events that culminates in the release ofhistamine and the production of cytokines [21]. Histologicalexaminations revealed that NL possessed the significantinhibitory effect on mast cell degranulation possibly due toinhibition of IgE hyperproduction. Pruritus, an unpleasant


sensation provoking the desire to scratch in AD, might bedue to histamine, proteases, and cytokines from various cellsincluding degranulated mast cells [22]. In our study, NLinhibited the episode of scratching which might be due toinhibition of degranulation of mast cells. Xeroderma andskin barrier dysfunction, which are associated with AD, aredue to lower level of ceramides that accelerate TEWL anddecrease water capacitance resulting in atopic dry skin [23].In our study, administration of NL inhibited TEWL therebyimproving the skin condition. Although the hapten-repeatedsensitization model is not a genetically driven model, manyof its aspects may be applicable to extrinsic allergen-drivenAD. In conclusion, we demonstrated that the oral admin-istration of NL inhibited the development of AD like skinlesions in NC/Nga mice induced by repeated epicutaneoussensitization of the dorsal skin by DNCB. NL containsabundant amount of flavonoids like luteolin, quercetrin, andisoquercitrin [15] as pharmacologically active components.The anti-inflammatory effect of those flavonoids suggests thepossibility of their therapeutic efficacy in various inflamma-tory diseases [24]. Furthermore, oral administration of NLfor 4 weeks had no remarkable toxic effects such as reductionof food intake and body weight. Thus, the results fromour experiment suggest that NL can be a potential naturalresource for the management of AD although the mechanismof action involved in the treatment remains to be explored.

5. Conclusions

The present study provides evidence that oral administrationof N. nucifera leaf extract attenuated the DNCB-inducedatopic dermatitis like skin lesions in NC/Nga mice. Althoughwe studied the effect of NL in chemical antigen-inducedmodel, many of its aspects may be applicable to extrinsicallergen-driven AD.

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[18] M. Q. Man, Y. Hatano, S. H. Lee et al., “Characterization ofa hapten-induced, murine model with multiple features ofatopic dermatitis: structural, immunologic, and biochemicalchanges following single versus multiple oxazolone challen-ges,” The Journal of Investigative Dermatology, vol. 128, no. 1,pp. 79–86, 2008.

[19] S. Romagnani, “Immunologic influences on allergy and theTH1/TH2 balance,” The Journal of Allergy and Clinical Immu-nology, vol. 113, no. 3, pp. 395–400, 2004.

[20] D. D. Metcalfe, D. Baram, and Y. A. Mekori, “Mast cells,” Phys-iological Reviews, vol. 77, no. 4, pp. 1033–1079, 1997.

[21] J. R. Gordon, P. R. Burd, and S. J. Galli, “Mast cells as a sourceof multifunctional cytokines,” Immunology Today, vol. 11, no.12, pp. 458–464, 1990.

[22] S. Stander and M. Steinhoff, “Pathophysiology of pruritus inatopic dermatitis: an overview,” Experimental Dermatology,vol. 11, no. 1, pp. 12–24, 2002.

[23] C. W. Blichmann and J. Serup, “Assessment of skin mois-ture. Measurement of electrical conductance, capacitance andtransepidermal water loss,” Acta Dermato-Venereologica, vol.68, no. 4, pp. 284–290, 1988.

[24] K. Morikawa, M. Nonaka, M. Narahara et al., “Inhibitoryeffect of quercetin on carrageenan-induced inflammation inrats,” Life Sciences, vol. 74, no. 6, pp. 709–721, 2003.


Research Article

Topical Application of Chrysanthemum Indicum L.Attenuates the Development of Atopic Dermatitis-LikeSkin Lesions by Suppressing Serum IgE Levels, IFN-γ, andIL-4 in Nc/Nga Mice

Sunmin Park,1, 2 Jung Bok Lee,1 and Suna Kang1

1 Department of Food and Nutrition, Hoseo University, 165 Sechul-Ri, BaeBang-Yup, Asan-Si,ChungNam-Do 336-795, Republic of Korea

2 Department of Mechatronics Engineering, Hoseo University, Asan 336-795, Republic of Korea

Correspondence should be addressed to Sunmin Park, [email protected]

Received 22 September 2011; Revised 25 October 2011; Accepted 1 November 2011


Copyright © 2012 Sunmin Park et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chrysanthemum indicum L. (CIL) is widely used as an anti-inflammatory agent in Asia and our preliminary study revealed thatCIL reduced interleukin (IL)-4 and IL-13 in 2,4-dinitrochlorobenzene (DNCB)-treated HaCaT cells, a human keratinocyte cellline. We investigated the atopic dermatitis (AD) effect of topically applied CIL in mice with AD-like symptoms. After topicalapplication of 1,3-butylen glycol (control), CIL-Low (5%), CIL-High (30%), or 0.1% hydrocortisone (HC) on the AD-like skinlesions in DNCB-treated NC/Nga mice for 5 weeks, the ear thickness, mast cell infiltration, and serum immunoglobulin E (IgE),IgG1, IL-4 and interferon (IFN)-γ were measured. The gene expressions of IL-4, IL-13, and IFN-γ in the dorsal skin were assayed.CIL treatment dosedependently reduced severity of clinical symptoms of dorsal skin, ear thickness, and the number of mast cellsand eosinophils. CIL-High significantly decreased serum IgE, IgG1, IL-4, and IFN-γ levels and reduced mRNA levels of IFN-γ,IL-4, and IL-13 in dorsal skin lesion. The improvement by CIL-High was similar to HC, but without its adverse effects such as skinatrophy maceration, and secondary infection. In conclusion, CIL may be an effective alternative substance for the management ofAD.

1. Introduction

Atopic dermatitis (AD) is a common skin disease charac-terized by chronic and relapsing inflammatory dermatitiswith immunological disturbances [1]. The incidence of ADis continuously increasing worldwide with a prevalence rateof approximately 10–20% and is more common amonginfants and children. Most patients with AD have increasedcirculating eosinophils and immunoglobulin (Ig) E due toelevated interleukin (IL)-4, IL-5, and IL-13 produced by T-helper (Th) 2 cells [2, 3]. Th2 immune responses are knownto play an important role in the pathogenic mechanism ofAD. In addition, Th1 immune response related to interferon-gamma (IFN-γ) is also associated with the pathophysiologyof AD. Therefore, AD is known to be a biphasic inflammatoryskin disease, provoked by Th1/Th2 immune responses [4].

Acute AD skin lesions exhibit Th2 type inflammatorycytokine profiles, whereas the chronic phase is characterizedby Th1 type immune responses releasing Th1 cytokines suchas IFN-γ and IL-12 and delayed-type hypersensitivity reac-tions [4, 5]. The imbalance of Th1 and Th2 immune respons-es plays important roles in the development of AD [6, 7].

NC/Nga mice are the most extensively studied animalmodel of AD since the inbred strain was established in 1957[8]. These mice spontaneously develop AD-like eczematousskin lesions when kept in air-uncontrolled conventionalhousing, but not when maintained under specific pathogen-free (SPF) conditions [8]. However, when these mice aremaintained under conventional conditions, the low inci-dence of AD-like lesions is a drawback [8]. In contrast, chem-ical hapten, 2,4-dinitrofluorobenzene (DNFB), or 2,4-dinitrochlorobenzene (DNCB) application produces 100%


reproducible AD-like lesions in NC/Nga mice [8–10]. BothDNCB- and DNFB-induced contact hypersensitivity is a T-cell-mediated inflammatory skin reaction that is believed tobe associated with Th1 activation [11, 12]. Since the clin-ical symptoms displayed in the NC/Nga mice treated withchemicals and humans with AD are similar, the mice areconsidered to be AD animal model suitable for understand-ing and evaluating effective therapeutic strategies for AD[8]. Since topical hydrocortisone exerts anti-inflammatory,antipruritic, and vasoconstrictive actions, it is widely ac-cepted that topical hydrocortisone therapy is crucial for themanagement of AD. However, it cannot be used for longperiods, and adverse side effects are frequently observedlike burning, itching, irritation, dryness, folliculitis, hypertri-chosis, acneiform eruptions, hypopigmentation, perioraldermatitis, allergic contact dermatitis, maceration of theskin, secondary infection, skin atrophy, and striae.

Accordingly, a wide variety of natural products are cur-rently being evaluated for alternative AD therapies, with theideal agent possessing potent efficacy with a minimal sideeffect profile [10, 13, 14]. Chrysanthemum indicum L. is wide-ly used for anti-inflammatory agents in Southeast Asian folkmedicine. It was also found to have anti-microbial, anti-oxidant, and anti-inflammatory action [15, 16]. Chrysanthe-mum indicum L. inhibits inflammatory mediators, such asNO, PGE2, TNF-α, and IL-1β, via suppression of MAPKsand NF-κB-dependent pathways [15]. In addition, our pre-liminary study revealed that the 1,3-butylene glycol extractof dried flowers of Chrysanthemum indicum L. decreasesthe expression of IL-4 and IL-13 the most in DNCB-treatedHaCaT cells, human keratinocytes, when the extracts of23 herbs including Chrysanthemum indicum L. were com-pared. These effects may be due to the sesquiterpene com-pounds such as kikkanol A, B, and C and flavone gly-cones such as eriodictyol 7-O-β-D-glucopyranosiduronicacid, acaciin, and luteolin 7-O-β-D-glucopyranoside [17,18]. Thus, Chrysanthemum indicum L. may relieve AD-likesymptoms. However, it has not yet been determined whetherChrysanthemum indicum L. suppresses the development andprogression of AD. In this study, we used the DNCB-treatedNC/Nga murine model to examine the inhibitory effectof Chrysanthemum indicum L. on the development andprogression of AD-like skin lesions.

2. Materials and Method

2.1. Preparation of Chrysanthemum indicum L. Extracts.Dried flowers of Chrysanthemum indicum L. were purchasedin Kyung-Dong Herb market (Seoul, Korea) in 2008, iden-tified by Dr. Joo YS (Department of Herbology, WoosukUniversity, Wanju-gun, Korea), and a voucher specimen(No. 2008-05 and 2008-06) deposited at the herbarium ofDepartment of Food & Nutrition, Hoseo University. Since1,3-butylene glycol is a good solvent for making skin lotion,dried flowers of Chrysanthemum indicum L. (1 kg) wereextracted at room temperature for 12 hours with 20 or 3.3L of 1,3-butylene glycol, filtered, and the filtrates centrifugedat 450×g to make 5 or 30% extracts. When more than 30%Chrysanthemum indicum L. was extracted with 1,3-butylene

glycol, the extract formed a precipitate. The supernatantswere used for topical application.

2.2. Animals. Twenty female 6-week-old NC/Nga mice werepurchased from Charles River Japan (Yokohama, Japan) andmaintained under conventional conditions: a 12 h light/12 hdark cycle, room temperature of 22-23◦C, and humidity of55 ± 15%. The mice had free access to food and water.All surgical and experimental procedures were performedaccording to the guidelines of the Animal Care and UseReview Committee at Hoseo University, Korea.

2.3. Induction of AD-Like Skin Lesion. Mice were anes-thetized with a mixture of ketamine and xylazine (100 and10 mg/kg body weight), after which the animal’s back hairand right ear were shaved 1 day prior to sensitization. In thefirst day, 1% DNCB in acetone/olive oil (3 : 1) was applied tothe dorsal skin and right ear then 0.2% DNCB was appliedevery other day [19]. The same volume of acetone/olive oilvehicle was applied instead of DNCB solution to controls.

2.4. Topical Application of 1,3-Butylene Glycol Extracts ofChrysanthemum indicum L. To determine the AD effectof Chrysanthemum indicum L., two dosages were assignedbased on the preliminary cell-based study and the maximumdosage to extract. A preliminary study showed that 50 μg/mLof Chrysanthemum indicum L. was effective against an ADmodel in HaCaT cells, a human keratinocyte cell line, butnot 5 μg/mL. After the induction of the AD-like skin lesions,animals were divided into four groups of 10 mice each.These groups were then treated topically in the dorsal skinand right ear for 5 weeks with one of four agents: 1,3-butylene glycol (BG; control), 5 or 30% dried flower ofChrysanthemum indicum L. (CIL-Low or CIL-High), or 0.1%hydrocortisone butyrate (HC; positive control) twice a day.Mice with no induction of AD-like skin lesion were treatedwith 1,3-butylene glycol as a normal control.

2.5. Evaluation of Skin Lesion Severity. The relative dermatitisseverity was assessed macroscopically using the followingscoring procedure. The total skin severity score was definedas the sum of the individual scores for each of the followingfour signs: (1) erythema and hemorrhage, (2) edema, (3)erosion (excoriation), and (4) scaling (dryness) [20]. Inthis system, 0 was defined as exhibiting no symptoms, 1 asmild symptoms, 2 as moderate symptoms, and 3 as severesymptoms. Additionally, the mice were photographed onceper week.

Ear thickness was measured before and after induction ofthe inflammatory response using a digital micrometer (MSI-Viking, Duncan, SC). The micrometer was applied near thetip of the ear just distal to the cartilaginous ridges, andthe thickness was recorded in micrometers. To minimizetechnique variations, a single investigator performed themeasurements throughout each experiment.

2.6. Measurement of Serum IgG1 and IgE Levels andIterleukin-4 (IL-4) and Interferon-γ (IFN-γ) Cytokine Levels.The total serum IgG1 and IgE was quantified by sandwich


enzyme-linked immunosorbent assay (ELISA) QuantitationKit (R&D Systems, Minneapolis, MN, USA) according tothe manufacturer’s protocol. The serum concentrations ofthe cytokines (IL-4 and IFN-γ) were also quantified using amouse cytokine enzyme immunoassay kit (R&D Systems).

2.7. Real-Time Quantitative Reverse Transcriptase-PolymeraseChain Reaction (RT-PCR). The dorsal skin tissues from fivemice from each group were collected at the end of treat-ment. Total RNA was isolated from the skin tissues usinga monophasic solution of phenol and guanidine isothiocy-anate (Trizol reagent, Gibco-BRL, Rockville, MD), followedby extraction and precipitation with isopropyl alcohol. ThecDNA was synthesized from equal amounts of total RNAwith superscript III reverse transcriptase, and polymerasechain reaction (PCR) was performed with high-fidelity TaqDNA polymerase. Equal amounts of cDNA were mixedwith SYBR Green supermix (Bio-Rad) mix and were ana-lyzed using a realtime PCR machine (BioRad Laboratories,Hercules, CA). The expression level of the gene of interestwas corrected for that of the house keeping gene, β-actin.The following primers were used for PCR reactions (5′-3′),mouse IFN-γ, sense 5′-CGGCACAGTCATTGAAAGCCTA-3′, and antisense 5′-GTTGCTGATGGCCTGATTGTC-3′; IL-4, sense 5′-TCTCGAATGTACCAGGAGCCATATC-3′, and antisense 5′-AGCACCTTGGAAGCCCTACAGA-3′; mouse IL-13, sense 5′-cagctccctggttctctcac-3′, and anti-sense 5′-ccacactccataccatgctg-3′; mouse β-actin, sense 5′-CATCCGTAAAGACCTCTATGCCAAC-3′, and antisense 5′-ATGGAGCCACCGATCCACA-3′. The primers were de-signed to sandwich at least one intron in order to distinguishbetween the products derived from mRNA and genomicDNA.

2.8. Histological Analysis. Dorsal samples were taken 24 hafter final DNCB administration on day 35 and they werefixed in 10% buffered neutral formaldehyde and embeddedin paraffin wax. Histological sections were 6 μm thick andwere stained with haematoxylin and eosin for countingnumber of eosinophils. The sections were also stained with0.5% toluidine blue for investigating the number of mastcells. The cell counts were performed in six consecutivemicroscopic fields at 400x magnification.

2.9. Statistical Analysis. Statistical analysis was performedusing SAS software and all results expressed asmean ± standard deviation. The biological and metaboliceffects of CIL-Low, CIL-High, HC (positive control), andvehicle (a negative control) were compared by one-wayANOVA. Significant differences in the main effects amongthe groups were identified by Tukey’s test at P < 0.05. The sig-nificance of differences between the mice with and withoutAD-like skin lesion was determined by two-sample t test.

3. Results

3.1. Severity of Skin Lesion. The NC/Nga mice developedAD-like skin lesions following repeated application of DNCBas evidenced by skin lesion scores, whereas normal controls

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Figure 1: Changes in severity scores and ear thickness of atopicdermatitis (AD) in NC/Nga mice. AD was induced in Nc/Ngamice by topical application of 2,4-dinitrochlorobenzene (DNCB)in the dorsal skin, and a right ear was topically treated with 1,3-butylen glycol (BG; control), 5% Chrysanthemum indicum L. (CIL-Low), 30% Chrysanthemum indicum L. (CIL-High), and 0.1%hydrocortisone (HC) on the lesions for 5 weeks. Normal controlsdid not have DCNB-induced AD. CIL decreased clinical severity ofatopic dermatitis symptoms and ear thickness in a dose-dependentmanner, and CIL-High had a statistically similar effect on theseverity as HC. (a) Changes in severity scores. (b) Changes of earthickness. Each value represents the mean ± SD of 10 mice in eachgroup. ∗Significantly different among the different treatments inNc/Nga mice at P < 0.05. a, b, c Values with different superscriptswere significantly different among Nc/Nga mice by Tukey test atP < 0.05. †Significantly different between control (BG) and normalcontrol at P < 0.05.

did not exhibit any skin lesions. The total scores werecalculated from sums of the scores for erythema, edema,erosion, and dryness; with a score of 12 indicating the mostsevere state. The normal controls exhibited no changes inskin lesion scores over time (Figure 1(a)). In accordance withthe previous finding, the clinical severity of skin lesions in thecontrol group increased gradually depending on the times ofchallenge with DNCB. All mice in the control group treatedwith BG (control) exhibited AD-like skin lesions includingerythema, edema, erosion, and dryness in the dorsal skin(Figure 1(a)). However, topical application of CIL dose-dependently lowered the skin lesion scores below that of thecontrols: CIL-Low significantly decreased the clinical scores


at 4 and 5 weeks from the DNCB sensitization in comparisonto the control, and CIL-High lowered the scores more thanCIL-Low (Figure 1(a)). The decreased clinical scores in CIL-High were similar to those observed with HC treatment.These results indicated that CIL-Low and CIL-High reducedthe progression of AD-like skin lesions in comparison to thecontrol, and CIL-High exhibited the similar improvementto HC. The scores increased after topical application andbegan to be lowered from day 21 in CIL-Low and CIL-High,whereas they reached a maximum at day 28 in the BG group(Figure 1(a)).

3.2. Ear Thickness. Ear thickness gradually increased aftertopical application of DNCB in BG and CIL-Low groups upto the 4th week but in CIL-High and HC groups, only up tothe 3rd week (Figure 1(b)). This indicated that the AD symp-toms were alleviated after the peak of ear thickness. Earthickness in the CIL-Low group significantly lowered onlyat the 4th week while CIL-High suppressed its increase incomparison to the BG group from the 2nd week of sen-sitization (Figure 1(b)). Ear thickness of the CIL-Low andCIL-High groups was significantly different at the 4th and5th weeks. The decrease in the CIL-High group was not sig-nificantly different from HC (Figure 1(b)). Ear thickness didnot change in the normal control group during the exper-imental periods.

3.3. Serum IgG1, IgE, IL-4, and INF-γ Levels. At day 35 afterthe sensitization, serum IgG1 and IgE levels were higher inthe control group than in the normal control group, but thelevels were dosedependently reduced by CIL treatments inDNCB-treated mice (Figure 2(a)). CIL-Low and CIL-Highdecreased circulating levels of IgG1 and IgE, but it wasstatistically significant only for CIL-High which had levelsclose to those of HC. IL-4 produced by Th2 cells was muchgreater in the control group than the normal control groupwhereas only CIL-High significantly suppressed the increasein DNCB-treated mice, and the suppression was as much asin HC, a positive control (Figure 2(b)). The control groupalso exhibited higher levels of INF-γ produced by Th1 cellsthan the normal control group, but CIL-High lowered thelevels in DNCB-treated mice, but it did not reach levels ofthe normal control group (Figure 2(b)). The reduction ofserum INF-γ levels was statistically similar to HC. The ratioof IL-4 and IFN-γ was higher in control group than normalcontrol, whereas the ratio was dosedependently lowered byCIL in DNCB-treated mice, but only CIL-High significantlydecreased the ratio. The ratio was statistically similar betweenCIL-High and HC (data not shown).

3.4. Histological Findings and Mast Cell Counts in the InflamedSkin. The dorsal skin of the mice in the control groupsexhibited hypertrophy, hyperkeratosis, intercellular edema,the liquefaction degeneration of the basal layer, and infiltra-tion of inflammatory cells such as mast cells and eosinophilsin contrast to the normal control group (Figure 3). Thesymptoms seen in dorsal skin such as hypertrophy, hyper-keratosis, intercellular edema, and liquefaction degenerationof the basal layer were relieved by the treatments with CIL

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Figure 2: Serum levels of IgG1, IgE, IL-4, and IFN-γ levels atthe end of experimental periods. AD was induced in Nc/Ngamice by topical application of 2,4-dinitrochlorobenzene (DNCB)in the dorsal skin, and a right ear was topically treated with1,3-butylen glycol (BG; control), 5% Chrysanthemum indicum L.(CIL-Low), 30% Chrysanthemum indicum L. (CIL-High), and 0.1%hydrocortisone (HC) on the lesions for 5 weeks. Normal controlsdid not have DCNB-induced AD. After 5 weeks of treatments,serum was separated to measure immunoglobulins and cytokines.CIL-High significantly reduced circulating levels of cytokines andimmunoglobulins in comparison to the control, and CIL-Highexhibited a statistically similar amelioration to HC. (a) Serum levelsof IgG1 and IgE. (b) Serum levels of IL-4 and INF-γ. Each valuerepresents the mean ± SD of 10 mice in each group. ∗Significantlydifferent among the different treatments in Nc/Nga mice at P <0.05. a, b, c Means on the bars with different superscripts weresignificantly different among Nc/Nga mice by Tukey test at P < 0.05.†Significantly different between control (BG) and normal control atP < 0.05.

in a dose-dependent manner. As seen in Figure 3(a), hyper-trophy, hyperkeratosis, intercellular edema, and liquefactiondegeneration of the basal layer were ameliorated with CIL-Low and CIL-High treatment in comparison to the controlgroup (Figure 3(a)). The improvement of the symptoms inCIL-High was similar to the improvements in the HC group.

An increased number of mast cells and eosinophils wereobserved in the skin lesions of the control group comparedto the normal control group (Figures 3(a) and 3(b)).The increase in mast cell and eosinophil counts was dosedependently suppressed by CIL treatment in DNCB-treated


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Figure 3: The number of mast cells and eosinophils of the dorsal skin with histopathological analysis. AD induced in Nc/Nga mice bytopical application of 2,4-dinitrochlorobenzene (DNCB) in the dorsal skin, and a right ear was topically treated with 1,3-butylen glycol(BG; control), 5% Chrysanthemum indicum L. (CIL-Low), 30% Chrysanthemum indicum L. (CIL-High), and 0.1% hydrocortisone (HC) onthe lesions for 5 weeks. Normal controls did not have DCNB-induced AD. After 5 weeks of treatments, the dorsal skin was fixed with 10%formaldehyde, embedded in paraffin, and then sections were made. The skin sections were stained with hematoxylin and eosin and toluidineblue staining. CIL-High significantly elevated mast cells and eosinophils in the lesion of the dorsal skin in comparison to the control andexhibited a statistically similar increment to HC. Each value represents the mean± SD of 5 mice in each group. (a) The number of eosinophilsas determined by hematoxylin and eosin staining. Eosinophils exhibited as blue dots. (b) The number of mast cells determined by toluidineblue staining. Mast cells appear as blue dots. ∗Significantly different among the different treatments in Nc/Nga mice at P < 0.05. a, b, cMeans on the bars with different superscripts were significantly different by Tukey test at P < 0.05. †Significantly different between control(BG) and normal control at P < 0.05.

mice. CIL-Low decreased the mast cells and eosinophilsin comparison to the control, but it was not significantlydifferent. CIL-High significantly lowered them, and thedecrease was similar to the HC group (Figures 3(a) and 3(b)).

3.5. Cytokine mRNA Expression Levels. To determine cytok-ine production in the inflamed dorsal skin lesions, mRNAexpression of IL-4, IL-13, and INF-γ cytokines was analyzed.The dorsal skin tissues of DNCB-treated control miceexhibited much higher expression levels of IL-4, IL-13,and IFN-γ than those of the normal controls (Figure 4).CIL suppressed the mRNA expression of IL-4 and IL-13produced by Th2 cells in a dose-dependent manner, whilethe expression of IFN-γ produced by Th1 was also reduced byCIL treatment (Figure 4). However, the decreases in mRNAlevels were significantly different only in CIL-High, not inCIL-Low, and the reduction of INF-γ was less than thatof IL-4 and IL-13 in CIL treatment. The decreased expres-sions of IL-4, IL-13, and IFN-γ by CIL-High were similarto those following treatment with HC, a positive control(Figure 4).

4. Discussion

Atopic dermatitis is a biphasic inflammatory skin disease,provoked by an imbalance between Th1 and Th2 immuneresponses [4]. Th2 immune responses are mediated by

interleukin (IL)-4, IL-5, and IL-13 while Th1 immuneresponses are modulated by IFN-γ [2]. In particular, Th2responses are key elements to the pathogenesis of atopicdisorders. NC/Nga mice were the first mouse model of ADreported by Matsuda et al. [21], and the mice treated withDFBN, DNCB, or picryl chloride have also been used as ananimal model for human AD [22]. Elevated levels of serumtotal IgE have been reported to correlate with the appearanceof the AD-like lesions in NC/Nga mice, with massiveinfiltration of IL-4- and IL-13-producing Th2 cells and thedegranulation of mast cells and eosinophils [4]. HC is apotent topical corticosteroid used to alleviate rash, eczema,and dermatitis. However, HC is a steroidal agent with adverseeffects such as facial hypertrichosis, folliculitis, miliaria, andgenital ulcers [23], which has inspired to investigations ofnonsteroidal agents for relieving AD. Recently, several studieshave reported that Herbal Oriental Medicine therapy maybe effective in AD patients [24]. The present study showedthat CIL dose dependently reduced the severity of AD-likeskin lesions by decreasing serum IgE and IgG1 levels andinfiltration of mast cells and eosinophils in DNCB-treatedNC/Nga mice. The results also indicate that the effects of CILwere probably due to a decrease in IFN-γ, IL-4, and IL-13production by activated Th1 and Th2 cells. CIL-High wasfound to be as effective as HC for alleviating AD.

Mast cells are known as key effector cells in IgE-mediatedallergic disorders and are activated by cross-linking of a high


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Figure 4: IL-4, IL-13, and IFN-γ expression in the dorsal skin.AD induced in Nc/Nga mice by topical application of 2,4-dinitrochlorobenzene (DNCB) in the dorsal skin, and a rightear was topically treated with 1,3-butylen glycol (BG; control),5% Chrysanthemum indicum L. (CIL-Low), 30% Chrysanthemumindicum L. (CIL-High), and 0.1% hydrocortisone (HC) on thelesions for 5 weeks. Normal controls did not have DCNB-inducedAD. After 5 weeks of treatments, total RNA was extracted from thedorsal skin, and cDNA was generated. The mRNA expression ofIL-4, IL-13, and INF-γ was measured by realtime PCR, and theirrelative expression was standardized according to respective β-actinmRNA levels. CIL-High significantly decreased the expression of IL-4, IL-13, and INF-γ in the lesion of the dorsal skin in comparisonto the control and exhibited a statistically similar decrease as HC.Each value represents the mean ± SD of 5 mice in each group.∗Significantly different among the different treatments in Nc/Ngamice at P < 0.05. a, b, c Means on the bars with different super-scripts were significantly different by Tukey test at P < 0.05. †Sig-nificantly different between control (BG) and normal control atP < 0.05.

affinity IgE receptor [3]. Upon activation, mast cells undergodegranulation and release a variety of biologically activesubstances, which play an important role in host defenseand allergic reactions including AD. Infiltration of mast cellsinto the dermis is a necessary characteristic for defining anappropriate animal model for AD [25]. We investigated theefficacy of CIL for preventing AD-like skin lesions in DNCB

challenged NC/Nga mice and its mechanism for preventingand alleviating AD was explored since CIL has been widelyused as an anti-inflammatory agent in Southeast Asian folkmedicine. We found that CIL-High significantly suppressedthe numbers of mast cells infiltrating in the skin lesions ofthe atopic dermatitis mice, suggesting that the activation andmigration of mast cells may be an immunopharmacologicaltarget of CIL-High.

It is known that mast cell activation is tightly modulatedby IgE from B cells, and increased total serum IgE levels are ahallmark of AD [24]. AD induced by DNFB application acti-vates B cells in NC/Nga mice by a Th2 reaction through IL-4that elevates IgE production [25]. IgE expression is knownto cause both acute and chronic phase skin symptoms.Consistent with these reports, we found that serum IgE levelswere significantly increased by repeated DNCB application inNC/Nga mice, as was IL-4 and IL-13 production by activatedTh2 cells. These cytokines are known to play important rolesin the inflammation and hypertrophy of the skin in AD [25].We also found that higher IgE, IL-4, and IL-13 levels wereassociated with more severe skin lesions. Topical applicationof CIL-High significantly improved the severity of AD-likeskin lesions by suppressing serum IgE, IL-4, and IL-13 levels.The improvement by CIL-High was as effective as with HC.

In addition to cytokines released from Th2 cells, Th1cytokines such as INF-γ are involved in AD development[4]. Under normal conditions, the differentiation of naiveT cells to Th1 and Th2 lineages is regulated by cytokinesthat are secreted from various cells, including themselves,and the Th1/Th2 balance is maintained [6, 7]. However, inatopic dermatitis, the balance shifts to Th2 dominance; thiseventually leads to excessive Th2 cytokine production [6].Some studies have found that increased INF-γ levels alleviateAD-like symptoms, but those results remain controversial.In the present study, repeated DNCB application increasedboth IFN-γ and IL-4 production from activated CD4+ Tcells of draining lymph nodes in NC/Nga mice. CIL dosedependently decreased both Th1 (INF-γ) and Th2 cytokines(IL-4 and IL-13) as effectively as HC. Furthermore, the ratioof Th1 to Th2 cytokines was increased by CIL treatment.

In conclusion, CIL-High reduced the development ofAD-like skin lesions resulting from repeated DNCB applica-tion in NC/Nga mice by suppressing total serum IgE levelsand IFN-γ and IL-4 production by activated CD4+ T cells.CIL-High ameliorated AD symptoms as effectively as HC.Sesquiterpenes and flavonoids in CIL may be the activecomponents exerting anti-AD-like effects; further study isneeded to identify the primary active components.

Acknowledgment

This work was supported by Business for Cooperative R&Dbetween Industry, Academy, and Research Institute fundedKorea Small and Medium Business Administration in 2009.All authors have no conflict of interests. The data in thispaper, or parts thereof, has not been submitted or publishedelsewhere for publication. All authors listed in the papersubmitted to this journal have contributed substantially to


the work, participated in the writing of the paper and seenand approved the submitted version.

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Research Article

Wound Healing and Anti-Inflammatory Effect in Animal Modelsof Calendula officinalis L. Growing in Brazil

Leila Maria Leal Parente,1 Ruy de Souza Lino Junior,2

Leonice Manrique Faustino Tresvenzol,1 Marina Clare Vinaud,2

Jose Realino de Paula,1 and Neusa Margarida Paulo3

1 Laboratory of Natural Products, Pharmacy Faculty, Federal University of Goias, 74605-220 Goiania, GO, Brazil2 Department of General Pathology, Institute of Tropical Pathology and Public Health, Federal University of Goias,74605-050 Goiania, GO, Brazil

3 Veterinary School, Federal University of Goias, 74001-970, Goiania, GO, Brazil

Correspondence should be addressed to Marina Clare Vinaud, [email protected]

Received 1 October 2011; Accepted 14 October 2011


Copyright © 2012 Leila Maria Leal Parente et al. This is an open access article distributed under the Creative CommonsAttribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Calendula officinalis is an annual herb from Mediterranean origin which is popularly used in wound healing and as an anti-inflammatory agent. In this study, the ethanolic extract, the dichloromethane, and hexanic fractions of the flowers from plantsgrowing in Brazil were produced. The angiogenic activity of the extract and fractions was evaluated through the chorioallantoicmembrane and cutaneous wounds in rat models. The healing activity of the extract was evaluated by the same cutaneous woundsmodel through macroscopic, morphometric, histopathologic, and immunohistochemical analysis. The antibacterial activity ofthe extract and fractions was also evaluated. This experimental study revealed that C. officinalis presented anti-inflammatoryand antibacterial activities as well as angiogenic and fibroplastic properties acting in a positive way on the inflammatory andproliferative phases of the healing process.

1. Introduction

Calendula officinalis L. (Compositae) is an annual herbfrom Mediterranean origin. It is believed that it has beenintroduced in England during the 13th century [1] andfound in Europe as a cultured plant. Wild plants are rare[2]. Its medicinal use seemed to be most diffused from the13th century and especially in the wound healing aspect.It was used as balm and creams, as antiseptic and anti-inflammatory agents during the north-American civil war aswell as in the first World War [2, 3].

The flowers of C. officinalis are found in chapters whichmay distinguish the varieties through its color and size.They have medicinal use especially as an anti-inflammatoryagent, for the treatment of wound, first-degree burns,contusions, and skin rashes. The German sanitary authoritiesrecommended its topic use in leg ulcers and its internal useonly against inflammatory lesions in the oral and pharynx

mucosae [4–6]. The main chemical components found in theflowers are saponins, triterpenes, alcohol triterpenes, fattyacid esters, carotenoids, flavonoids, coumarines, essentialoils, hydrocarbons, and fatty acids [7–9].

The anti-inflammatory activity of C. officinalis flowerscultured in Europe and Asia has been evaluated and evi-denced through the model of edema induction of the earthrough croton oil and the model of edema induction of thepaw through carrageenan [2, 3, 7, 10–12]. The angiogenicactivity of the aqueous extract of C. officinalis cultured inEngland was also evidenced [13].

Most of the literatures that report the medicinal activityof C. officinalis were performed in European or Asian coun-tries from plants cultured in those locations. The differentconditions of culturing a plant may alter some specificpatterns of the vegetal metabolism which may activate orinactivate some metabolic pathways [14]. This study aimedat the evaluation of the wound healing and antibacterial


activities of the ethanolic extract and its fractions from theflowers of C. officinalis cultured in Brazil (Parana estate).

2. Material and Methods

2.1. Plant Material. Dried and pulverized flowers of C.officinalis collected in Parana estate, Brazil, during the monthof February, 2006, were commercially obtained from thecompany Empresa Clorofila (Goiania, GO, Brazil). To thequality control of the plant material, a pharmacognosticevaluation was performed which analyzed the organolepticproperties, ashes microscopy, and the ashes contents. Alsothe total flavonoids content dosages and thin-layer chro-matography analysis were performed [3, 15].

To obtain the ethanolic extract (CEE), the pulverizedflowers were extracted with 96◦ GL PA ethanol by coldmaceration and concentrated by rotary evaporator at 40◦C.To obtain of the hexanic and dichloromethane fractions, theCEE was soluble into a MeOH/H2O (7 : 1) mix followed byan extraction with hexane and afterwards with dichloro-methane. The resulting extracts which were hexanic (HCF)and dichloromethane (DCF) were concentrated by rotaryevaporator at 40◦C.

As the ethanolic extract, the hexanic and dichlorometh-ane fractions were insoluble in distilled water, solutions of1% of the extract and fractions previously diluted in 70%ethanol were prepared and then used to test the angiogenicactivity of the CAM. To test the angiogenic activity of theethanolic extract on cutaneous wounds in rats, an aqueoussolution of 1% of the extract was daily prepared.

2.2. Experimental Animals. 36 Wistar female rats, 60 daysold, weighing between 160 and 190 g-interval were used.The animals were adapted during a ten-day period andmaintained in individual polyethylene cages covered withcoarse saw dust under controlled environmental conditionssuch as room temperature of 23 ± 2◦C, relative air humiditybetween 50 and 70%, and photoperiod light/dark of 12 h.Water and ration were supplied ad libitum.

75 fertilized eggs from Cobb chickens, two days old, wereused and obtained from matrixes with age varying from 34to 35 weeks old. We followed the ethical principles in animalexperimentation recommended by the National Council forControl of Animal for Experiments (CONCEA).

The experimental protocol of the animals used in thisstudy was evaluated and approved by the Ethics in ResearchCommittee of the Federal University of Goias (protocolnumber 019/2007).

2.3. Chorioallantoic Membrane Model (CAM)

2.3.1. Preparation of the CAM. The CAM was prepared aim-ing the angiogenic activity evaluation according to anadapted methodology [16]. The eggs were randomly dis-tributed into four groups with 15 eggs each and submittedto the following treatments: SC1: solvent control 1 (70%ethanol); PC: positive control at 1% (17 β-estradiol);SC2: solvent control 2 (distilled water); CEE: ethanolic

extract at 1%; DCF: dichloromethane fraction at 1%; HCF:hexanic fraction at 1%.

According to previously established protocols [16], theopening of the egg shell, the removal of the albumen, and thetreatments were performed inside a type II security biologi-cal chamber. On the third day of incubation, through a smallopening of the egg shell, 2 to 3 mL of albumen were removedfollowed by the sealing of the orifice with histological paraffinin fusion point. The incubation continued until the 10thday when the eggs were treated with 0.1 mL of the solutionsdescribed above which were instilled above the CAM surface.The eggs were sealed again and then incubated for two days.

After the euthanasia of the embryos, through medullarsection of the atlantoocciptal region, fragments of the CAMfrom each egg were collected to morphometric analysis andquantification of the blood vessels.

2.3.2. Morphometric Evaluation of the CAM. To the morpho-metric evaluation of the CAM, a fresh fragment was collectedfrom 10 eggs from each group and distended on a glass slideand microscopically analyzed.

The microscopical analysis was performed with a digitalimage analysis system. The percentage of the vascular areaper field was calculated through the Image J 1.3.1 (NIH,USA) software. 30 random fields were photographed andanalyzed per sample, and this number was determined byaccumulated mean [17].

2.3.3. Evaluation of the Inflammatory Cells and the Numberof Blood Vessels in the CAM. The membranes were fixedin tamponated formalin, processed and blocked into paraf-fin, and then sectioned into 5 micrometer sections andstained with hematoxylin and eosin (HE). 20 randomlychosen fields were photographed, number determined byaccumulated mean. The inflammatory cells found in theCAM were classified in a qualitative form into absent(0/3), discrete (1/3), moderate (2/3), and accentuated(3/3).

The counting of the blood vessels in mesoderm fromthe CAM was performed through planimetry by countingof the points by using the GIM; P 2.4.3 software. A squarereticulum composed by 25 points was overlapped on top ofthe histologic image [18, 19].

2.4. Cutaneous Wounds in Rats Model. The animals wereweighted and randomly divided in two groups (n = 18)and subdivided in three subgroups (n = 6). The groupswere evaluated in determined moments of the postoperatory(PO) period according to the protocol previously establishedby Lopes et al. [20] and Garros et al. [21] aiming ofthe evaluation of the initial phases of the inflammatoryprocess (4 and 7 PO days) and the proliferative phase of theinflammatory process (14 PO days) resulting in a generalevaluation of the healing. The groups are namely:

Group 1. Control (C), animals were treated with distilledwater. C1 was evaluated in the 4th PO day, C2 in the 7th POday, and C3 in the 14th PO day.


Group 2. CEE, animals were treated with aqueous solutionof the ethanolic extract at 1%. CEE1 evaluated in the 4th POday, CEE2 in the 7th PO day and CEE3 in the 14th PO day.

To the wound induction, a circular methalic punch of1 cm of diameter was used on the dorsocervical region ofeach animal. The anesthetics consisted of the associationbetween ketamin (70 mg/Kg, IM) and xylazine (10 mg/Kg,IM). Right after the surgery and daily afterwards on the samehour, 100 μL of the CEE solution and of the solvent (distilledwater) were instilled on the animals wounds.

Each animal was daily examined as to their generalaspect, and the macroscopic evaluation of the wound wasperformed. The animals were euthanized in a CO2 chamberat 4, 7, and 14 PO days accordingly to previous protocolsby Lopes et al. [20] and Garros et al. [21]. Fragments of thecutaneous wounds were collected to the histopathologic andimmunohistochemical analysis.

2.4.1. Histopathologic and Morphometric Evaluation. Themorphometric analysis of the wounds was performedthrough the photographed images of the wounds at dayszero, 4, 7, and 14 PO aiming of the determination of thecontraction of the wound area [22].

The histopathologic analysis was performed after thefragments of the wounds were fixed with 10% bufferedformalin, processed, and blocked with paraffin, and thensectioned into 5 micrometer sections and stained withhematoxylin and eosin (HE). The sections were alsostained with Picrossirius to the collagen quantificationunder polarized light [23] through the calculus of thepercentage of the marked area in green or reddish-yellowby field by using the IMAGE J 1.3.1 (NIH, USA) soft-ware.

The histopathologic analysis at 4, 7, and 14 PO daysdetermined the healing phases of the wound through histo-logic variables such as the presence of fibrin, hemorrhage,hyperemia, inflammatory infiltration, reepithelialization,and epithelial hyperplasia [24].

2.4.2. Angiogenic Activity Evaluation through Immunohis-tochemistry. Sections were incubated with bovine serumalbumin (BSA), the blockage of endogenous peroxidase wasperformed, and the sections were incubated with the vascularendothelial growth factor (VEGF) primary antibody (147)(Santa Cruz Biotechnology-507), 1 : 500 dilution in a wetchamber, overnight at 4◦C.

After this step, the streptavidin-biotin-peroxidase com-plex (kit LSAB-Dako K0690) was instilled over the sectionsfor 20 minutes each reaction in a wet chamber at room tem-perature. The reaction was revealed with diaminobenzidine(DAB) solution, for 1 minute. A PBS solution was used forwashing between the steps. The sections were counterstainedwith Mayer’s hematoxylin, for 30 seconds, and the slideswere mounted with synthetic resin (Sigma Aldrich, USA) andhistological coverslips.

The counting of the blood vessels was performed throughplanimetry, and the intensity of the VEGF expression into theendothelial cells was evaluated.

2.5. In Vitro Antibacterial Activity. To determine theminimum inhibitory concentration (MIC), the cultures ofStaphylococcus aureus (ATCC 6532), Staphylococcus aureus(ATCC 13048), Micrococcus roseus (ATCC 1740), Micrococcusluteus (ATCC 9341), Bacillus cereus (ATCC 14576), Bacillusstearothermophylus (ATCC 1262), Enterobacter cloacae(HMA/FTA 502), Enterobacter aerogenes (ATCC 13048), Es-cherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC9027), and Serratia marcescens (ATCC 14756) were usedthrough the plaque assay method. The microorganisms werereactivated and processed according to NCCLS [25].

The MIC was determined through the plaque assaymethod when 1000 mg of the CEE, 500 mg of the HCF,and 350 mg of the DHC were incubated in essay tubes with1.0 mL of DMSO.

2.6. Statistical Analysis. The results were submitted to thestatistical analysis through the GraphPad InStat programme(Version 3.05 for Windows). From the Kolmogorov-Smirnovnormality test, the morphometric data from the CAM wereevaluated through the Kruskal-Wallis test followed by theDunn posttest. The counting of the blood vessels was ana-lyzed through ANOVA and the Tukey test. The histopatologicand immunohistochemical variables were analyzed throughthe Mann-Whitney test. The significance level was of P <0.05.


The pulverized flowers of C. officinalis presented a yellowishcoloring and soft and mildly aromatic odor. The microscopicanalysis of the dust through the Steinmetz and Etzold stain-ing evidence its structures: biseriate multicellular trichomeof the corolla tube from the ligulate flower, ligule epidermalwith striated cuticle and ligule parenchyma containingdroplets of oil and pollen grains.

The total content of the ashes was of 8.47%. The thinlayer chromatography evidenced the presence of rutin (Rf-0.47), flavonoids (Rf-0.29), and aglycone flavonoids (Rf-0.92). The contents of total flavonoids from the pulverizedflowers were of 0.77% and in the CEE of 1.29%. WHOmonographs [3] describe that C. officinalis should notpresent less than 0.4% of total flavonoids calculated ashyperoside up until 10% of the total ashes.

All data obtained from the pharmacognostic analysis arein accordance to what is described in the literature [3, 15]which indicates that the plant material tested correspondedto the pulverized flowers of C. officinalis.

The evaluation of the angiogenic activity in the CAMshowed through the morphometric analysis an increase inthe vascular area in the positive control, ethanolic extract,dichloromethane fraction, and hexane fraction groups whencompared to the solvent control 1 (Table 1).

The quantification of the blood vessels performedthrough planimetry of the CAM stained with H&E andtreated with positive control, ethanolic extract, dichlo-ro-meth-ane fraction, and hexane fraction evidenced anincrease in the number of blood vessels when compared tothe solvent control 1 group (Table 2).


Table 1: Morphometry (median, minimum/maximum) of theblood vessels from the fresh chorioallantoic membrane fromfertilized chicken eggs.

Treatment Marked area of the CAM

CEE 10.47 (1.43/57.49)∗

HCF 9.33 (1.61/71.22)∗

DCF 9.11 (2.26/54.35)∗

PC 8.11 (3.56/20.64)∗

SC1 5.10 (0.61/49.83)

SC2 5.78 (1.20/40.37)

CAM: chorioallantoic membrane, CEE: ethanolic extract of flowersfrom Calendula officinalis at 1%, HCF: hexanic fraction at 1%, DCF:dichloromethane fraction at 1%, PC: positive control at 1% (17 β-estradiol),SC1: solvent control with 70% ethanol, SC2: solvent control with distilledwater. ∗P < 0.05 when compared to SC1.

When substances are administered on the surface of theCAM, they may induce nonspecific inflammatory reactionsleading to a secondary vessel proliferative response [26]. Inthis study, the CAM treated with C. officinalis in its extract orfraction forms presented a discrete inflammatory infiltration.We may infer that the increase in the neovascularizationobserved in the CAM is due to the direct acting of theC. officinalis and its fractions and is not related to theinflammatory reaction which is in accordance to similarfindings by Patrick et al. [13]. Although there are reports inthe literature [13] concerning the angiogenic activity of C.officinalis, there were not found descriptions of which of itscomponent is responsible for this specific activity.

In the evaluation of the wound healing activity ofC. officinalis, only the ethanolic extract was used becausethe extract and the fractions presented similar angiogenicproperties.

The macroscopic evaluation of the wounds did not showpurulent exudate in any of the wounded animals, but it waspossible to observe a serous exudation in the animals fromthe CEE group up until the 4th PO day and in the controlgroup up until the 7th PO day. The crusts started to formfrom the 3rd day, and, in the CEE group, they presentedthinner and wetter when compared to the control groupwhich were thicker and drier. At the 7th PO day, the crustsbegan to detach showing signs of epithelialization of thewounds, and, in the 14th PO day, the complete healing wasverified on both groups.

The microscopic evaluation of the CEE group at 4 and 7PO days showed a significant decrease as to the presence offibrin (median = 1.0 and 2.0, resp.)and hyperemia (median= 1.0 and 1.5, resp.) when compared to the control group atthe same PO days (median = 3.0 and 2.5, resp.) (Figure 1). Asto the evaluation of the other variables such as hemorrhage,inflammatory infiltration, reepithelialization, and epithelialhyperplasia, there was not a significant difference foundbetween the analyzed groups.

Previous studies evaluated and evidenced the anti-inflammatory activity of C. officinalis and related this activityto the presence of triterpenes, especially to faradiol estersand taraxasterol [2, 3, 7, 10–12]. In this study, the sameeffect was evidenced due to the observation of the significant

Table 2: Influence of the Calendula officinalis extract and fractionson the number of blood vessels (median, minimum/maximum) ofthe chorioallantoic membrane from fertilized chicken eggs.

Treatment Number of blood vessels

CEE 1 (0/4)∗

HCF 1 (0/4)∗

DCF 2 (0/13)∗

PC 2 (0/13)∗

SC1 1 (0/3)

SC2 1 (0/3)

CEE: ethanolic extract of flowers from Calendula officinalis at 1%, HCF:hexanic fraction at 1%, DCF: dichloromethane fraction at 1%, PC: positivecontrol at 1% (17 β-estradiol), SC1: solvent control with 70% ethanol, SC2:solvent control with distilled water. ∗P < 0.05 when compared to SC1.

difference of the presence of fibrin and hyperemia foundwhich represents the circulatory alterations directly relatedto the inflammatory phase of the healing process.

It was possible to observe a significant increase inthe collagen amount in the CEE group at 4 and 7 POdays (median = 8.27 and 6.33, resp.) when compared tothe control group (median = 5.33 and 4.31, resp.) whichindicates fibroplasia (Figure 2). On the 4th PO day, it waspossible to observe the brilliant green coloration of thecollagen on both groups, and, on the 7th PO day, the reddish-yellow one especially on the CEE group.

There are no reports found in the literature on the colla-gen mensuration through Picrossirius staining of cutaneouswounds treated with C. officinalis. In this study, the statisticalconfirmation of the increase in the collagen concentrationin the wounds treated with the CEE at 4 and 7 PO daysindicates that the extract contributed to the greater synthesisof this fiber. This indicates a positive role of the extract on thecutaneous wounds healing process. The triterpene faradiolpalmitic ester stimulated the proliferation and migrationof fibroblasts in previous in vitro studies [27]; we believethat this may have occurred in our study, stimulating thefibroplasias evidenced here.

The immunohistochemical evaluation showed anincrease in the number of blood vessels in the dermis of therats treated with CEE as it had a positive marking for VEGF(Figure 3). This finding confirms the intense activity of theextract on the neovascularization also observed on the CAMmodel.

The immunohistochemistry technique is a viable toolto angiogenesis evaluation through VEGF marking in thisexperimental model. Our data indicated that the CEEangiogenic property is not directly related to the increaseof the intensity of expression of this marker. Other pro-aniogenic factors such as fibroblast growth factor (FGF),angiogenic cytokines as interleukin 8 (IL-8), tumor necrosisfactor-alfa (TNF-α), and transformation growth factor-beta(TGF-β) [28, 29] may be related to the angiogenic activityevidenced in the C. officinalis extract by this study.

The antibacterial activity of the CEE and the hexanicfraction was observed against Gram-positive bacteria, andthis effect was not observed on the dichloromethane fraction.


Fb

30 μm

(a1)

Fb

30 μm

(a2)

Fb

(b1)

30 μm 30 μm

Fb

(b2)

Figure 1: Photomicrograph of cutaneous wounds in rats at 4 (a1, a2) and 7 (b1, b2) PO days highlighting the presence of fibrin (Fb). (a1)and (b1) refer to the control group, treated with distilled water. (a2) and (b2) refer to CEE group. HE staining.

(a1)

30 μm

(a2)

30 μm

(b1)

30 μm

(b2)

30 μm

Figure 2: Photomicrograph of the cutaneous wound in rats (n = 6) at 4 (a1, a2) and 7 (b1, b2) PO days. (a1) Control group, treatedwith distilled water, type III collagen production evidenced through green polarized light; (a2) CEE group, type III collagen intenseproduction; (b1) control group, type I collagen production evidenced by orange-yellow polarized light; (b2) CEE group, intense type Icollagen production. Picrossirius staining.


20 μm

(a)

(b)

Figure 3: Photomicrograph from the dermis of rats at 7th PO days(n = 6). (a) Solvent control with distilled water. (b) EEC at 1%.Arrows indicate the VEGF positive marking on the dermis fromboth groups. Immunohistochemistry, 400x.

The lowest concentrations of the extracts that inhibit micro-organism’s growth were CEE, MIC of 0.39 mg/mL againstS. aureus 13048 and B. stearo thermophylus; FDC, MIC of4.37 mg/mL against S. aureus 6532 and S. aureus 13048,MIC of 1.08 mg/mL against B. stearo thermophylus and B.cereus, MIC of 0.5 mg/mL against M. roseus; FHC, MIC of0.19 mg/mL against S. aureus 13048. According to Alonso[2], the antibacterial activity is due to the presence offlavonoids and essential oils in C. officinalis.

In the case of wound contaminations, there is the for-mation of a great quantity of exudate and toxin productionswhich may retard the healing process [30] in addition to therisk of presenting an entrance pathway to systemic infections.The antibacterial activity of C. officinalis evidenced in thisstudy corroborates with the healing activity detected by thisplant in this study as it prevents secondary infections of thewounds that can aggravate the injury.

4. Conclusions

Calendula officinalis growing in Brazil presented anti-inflam-matory and antibacterial activities as well as the capabilityof stimulating fibroplasia and angiogenesis. Therefore, the C.officinalis extracts act in a positive form on the inflammatoryand proliferative phases of the healing process of cutaneouswounds.

Acknowledgments

This study was performed with the collaboration of theAnimal Pathology and Preventive Veterinary Medicine Sec-tors of the Veterinary School of the Federal University ofGoias; also of the Natural Products Research Laboratoryfrom the Pharmacy Faculty and the Pathology Sector ofthe Tropical Pathology and Public Health Institute from theFederal University of Goias; also of the company EmpresaPerdigao S/A (Rio Verde-GO). The authors would like tothank CAPES (Coordenacao de aperfeicoamento de pessoalde nıvel superior) for the financial support.

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Research Article

A Therapeutic Approach for Wound Healing by Using EssentialOils of Cupressus and Juniperus Species Growing in Turkey

Ibrahim Tumen,1 Ipek Suntar,2 Hikmet Keles,3 and Esra Kupeli Akkol2

1 Department of Forest Products Chemistry, Faculty of Forestry, Bartin University, 74100 Bartin, Turkey2 Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler, 06330 Ankara, Turkey3 Department of Pathology, Faculty of Veterinary Medicine, Afyon Kocatepe University, 03200 Afyonkarahisar, Turkey

Correspondence should be addressed to Esra Kupeli Akkol, [email protected]

Received 8 June 2011; Accepted 16 July 2011

Academic Editor: Saringat Hj. Baie

Copyright © 2012 Ibrahim Tumen et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Juniperus and Cupressus genera are mainly used as diuretic, stimulant, and antiseptic, for common cold and wound healing inTurkish folk medicine. In the present study, essential oils obtained from cones of Cupressus and berries of Juniperus were evaluatedfor their wound healing and anti-inflammatory effects. In vivo wound healing activity was evaluated by linear incision and circularexcision experimental wound models, assessment of hydroxyproline content, and subsequently histopathological analysis. Thehealing potential was comparatively assessed with a reference ointment Madecassol. Additionally acetic-acid-induced capillarypermeability test was used for the oils’ anti-inflammatory activity. The essential oils of J. oxycedrus subsp. oxycedrus and J.phoenicea demonstrated the highest activities, while the rest of the species did not show any significant wound healing effect.The experimental study revealed that J. oxycedrus subsp. oxycedrus and J. phoenicea display remarkable wound healing and anti-inflammatory activities, which support the folkloric use of the plants.

1. Introduction

Juniperus L. (Cupressaceae) has almost 70 species throughoutthe world and are mostly distributed in the Northern Hemi-sphere [1]. The widespread areas extend from Japan and EastAsia to Europe, as well as from North and East Africa toNorth America. In Turkey, the Juniperus genus is representedby 10 taxa under seven speciess and has also been used byAnatolian people since ancient times. Fruits of Juniperus spe-cies are used to produce pekmez (a traditional Turkish fruitconcentrate), which is rich in nutritional constituents [2].Juniper berries are used in Northern European countries toimpart sharp and clear flavour to the meat dishes [3, 4].The coniferous parts and leaves of Juniperus are utilizedin medicine as an antihelmintic, diuretic, stimulant, andantiseptic and for wound healing. J. excelsa is used againsttuberculosis and jaundice [5]. The literature survey indicatesthat the major phenolic constituents in extracts of Juniperusspecies are lignans, coumarins, sesquiterpenes, abietane,

labdane and pimarane diterpenes, flavonoids, biflavonols,flavone glycosides, and tannins [1].

The genus Cupressus is one of several genera within thefamily Cupressaceae. Based on genetic and morphologicalanalysis, the Cupressus is found in the Cupressoideae sub-family [6]. They are widespread in the northern hemisphere,including western North America, Central America, north-west Africa, the Middle East, the Himalaya, southern China,and north Vietnam. They are evergreen trees or large shrubs,growing to 5–40 m tall. In Turkish folk medicine, fruits ofCupressus sempervirens are used to treat common cold andcough [7]. In previous studies, the major constituents havebeen identified in Cupressus species as α-pinene and Δ-3-carene [8]. The leaves and fruits of this plant are quite rich intannins and flavonoids but they are free from alkaloids andlow in saponins [9].

The aim of the present study is to evaluate the woundhealing activity of the oils from cones of Cupressus semper-virens var. horizontalis and C. sempervirens var. pyramidalis


Table 1: Sampling site, local name, date, and altitude of the tree species.

Species Sampling site Local name Collection date Altitude

Cupressus sempervirens var. horizontalis Antalya-Turkey Dallı Servi October, 2010 670 m

Cupressus sempervirens var. pyramidalis Antalya-Turkey Piramid Servi October, 2010 650 m

Juniperus communis L. subsp. nana Syme. Keltepe-Karabuk Bodur Ardıc September, 2010 1,210 m

Juniperus excelsa Bieb. Silifke-Mersin Boylu Ardıc October, 2010 220 m

Juniperus foetidissima Willd. Beypazarı-Ankara Kokulu Ardıc October, 2010 240 m

Juniperus oxycedrus L. subsp. oxycedrus Keltepe-Karabuk Katran Ardıcı September, 2010 1,200 m

Juniperus phoenicea L. Bodrum-Yokusbasi Finike Ardıcı September, 2010 170 m

Table 2: Effects of the essential oils from C. sempervirens var. hori-zontalis, C. sempervirens var. pyramidalis, J. communis, J. excelsa, J.foetidissima, J. oxycedrus, and J. phoenicea on linear incision woundmodel.

MaterialStatistical mean ± SEM

(tensile strength %)

Vehicle13.24 ± 2.25

(9.1)

Negative control12.14 ± 2.54

—

Cupressus sempervirens var. horizontalis14.51 ± 2.17

(9.6)

Cupressus sempervirens var. pyramidalis14.27 ± 2.09

(7.8)

J. communis subsp. nana13.82 ± 2.04

(4.4)

Juniperus excelsa15.27 ± 2.43

(15.3)

Juniperus foetidissima14.66 ± 1.86

(10.7)

Juniperus oxycedrus subsp. oxycedrus17.41 ± 2.19

(31.5)∗∗

Juniperus phoenicea18.05 ± 2.26

(36.3)∗∗

Madecassol19.87 ± 1.41

(50.1)∗∗∗

∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; SEM: Standard error of themean.Percentage of tensile strength values: vehicle group was compared to negativecontrol group; the oils and the reference material were compared to vehiclegroup.

and berries of J. communis, J. excelsa, J. foetidissima, J. oxyce-drus, and J. phoenicea by means of in vivo circular excisionand linear incision wound models and anti-inflammatoryactivity.


2.1. Plant Material. Berries and cones of 5 different Juniperspecies and 2 different varieties of Cypress belonging to Cup-ressaceae family natively growing in Turkey were chosen asthe study material. Specimens were taken from each speciesas 1 kg weight from their growth sites and stored at −24◦Cuntil analysis [10]. Species names, sampling site, local name,

collection date, and altitude of all specimens are listed inTable 1.

2.2. Hydrodistillation. The essential oils of each sample wereobtained by hydrodistillation with a Clevenger apparatus us-ing 250 g (partially crushed) of fresh berries and cones. Theessential oils were collected for 3 h and dried over anhydroussodium sulphate and under refrigeration in a sealed vial untilrequired [11, 12].

2.3. Biological Activity Tests

2.3.1. Animals. Male, Sprague-Dawley rats (160–180 g) andSwiss albino mice (20–25 g) were purchased from the animalbreeding laboratory of Saki Yenilli (Ankara, Turkey).

The animals were left for 3 days at room conditions foracclimatization. They were maintained on standard pelletdiet and water ad libitum throughout the experiment. A min-imum of six animals were used in each group. Throughoutthe experiments, animals were processed according to thesuggested European ethical guidelines for the care of labor-atory animals.

2.3.2. Preparation of Test Samples for Bioassay. Incision andexcision wound models were used to evaluate the woundhealing activity. For the in vivo wound models, test sampleswere prepared in an ointment base (vehicle) consisting ofglycol stearate, 1, 2 propylene glycol, liquid paraffin (3 : 6 : 1)in 1% concentration. 0.5 g of each test ointment was appliedtopically on the wounded site immediately after wound wascreated by a surgical blade.

The animals of the vehicle group were treated with theointment base only, whereas the animals of the referencedrug group were treated with 0.5 g of Madecassol (Bayer,00001199). Madecassol contains 1% extract of Centellaasiatica.

For the assessment of anti-inflammatory activity, testsamples were given orally to test animals after suspending ina mixture of distilled H2O and 1% Tween 80. The controlgroup animals received the same experimental handling asthose of the test groups except that the drug treatment wasreplaced with appropriate volumes of dosing vehicle. Indo-methacin (10 mg/kg) in 1% Tween 80 was used as referencedrug.


Table 3: Effects of the essential oils from C. sempervirens var. horizontalis, C. sempervirens var. pyramidalis, J. communis, J. excelsa, J. foetid-issima, J. oxycedrus, and J. phoenicea on circular excision wound model.

MaterialWound area ± SEM (contraction %)

0 2 4 6 8 10 12

Vehicle 19.55 ± 2.0917.98 ± 2.20

(2.55)16.54 ± 2.11

—14.89 ± 1.95

—9.55 ± 1.56

(9.51)6.31± 1.16

(8.42)2.97 ± 0.87

(5.41)

Negative Control 19.43 ± 2.15 18.45 ± 2.23 16.42 ± 2.17 14.72 ± 1.91 10.51 ± 1.75 6.89 ± 1.23 3.14 ± 1.49

C. sempervirens var.horizontalis

19.40 ± 2.1317.56 ± 1.40

(2.34)16.02 ± 1.50

(3.14)14.91 ± 1.62

—9.57 ± 1.40

—5.86 ± 1.32

(7.13)3.56 ± 0.69

—

C. sempervirens var.pyramidalis

20.03 ± 2.1217.90 ± 1.91

(0.44)16.69 ± 1.92

—14.92 ± 1.60

—8.84 ± 1.67

(7.43)7.08 ± 1.12

—2.59 ± 0.84

(12.79)

J. communis subsp. nanaSyme.

19.55 ± 2.2217.81 ± 1.81

(5.56)16.75 ± 1.80

—15.16 ± 1.87

—9.57 ± 1.74

—6.02 ± 0.99

(4.60)3.04 ± 0.89

—

J. excelsa 19.50 ± 2.1614.99 ± 1.78

(16.63)13.24 ± 2.09

(19.95)11.26 ± 1.70

(24.38)7.39 ± 1.58

(22.62)5.47 ± 1.07

(13.31)2.44 ± 0.90

(17.85)

J. foetidissima 19.96 ± 2.5116.16 ± 1.89

(10.12)14.02 ± 1.78

(15.24)13.49 ± 1.71

(9.40)9.09 ± 1.54

(4.82)6.10 ± 1.27

(3.33)2.49 ± 1.05

(16.16)

J. oxycedrus subsp.oxycedrus

19.41 ± 2.0817.51 ± 1.92

(2.61)16.02 ± 1.70

(6.05)12.62 ± 1.63

(15.25)6.03 ± 1.03

(36.86)∗3.85 ± 0.96

(38.99)∗1.69 ± 0.60

(43.10)∗

J. phoenicea 19.59 ± 2.1916.46 ± 2.54

(8.45)14.02 ± 1.96

(15.24)11.97 ± 1.86

(19.61)7.44 ± 1.39

(22.09)4.21 ± 1.28

(33.28)∗1.76 ± 0.61

(40.74)∗

Madecassol 19.68 ± 2.0314.46 ± 1.83

(19.58)11.77 ± 1.45

(28.84)8.76 ± 1.21(41.17)∗∗

4.74 ± 0.89(50.37)∗∗

2.12 ± 0.31(66.40)∗∗∗

0.00 ± 0.00(100.00)∗∗∗

∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; SEM: standard error of the mean.Percentage of contraction values: vehicle group was compared to negative control group; the oils and the reference material were compared to vehicle group.

Table 4: Effect of topical treatment of test ointments for 7 days onhydroxyproline content.

MaterialHydroxyproline(mg/g) ± SEM

Vehicle 20.21 ± 2.43

Negative Control 19.25 ± 2.67

C. sempervirens var. horizontalis 18.3 ± 2.41

C. sempervirens var. pyramidalis 25.9 ± 3.17

J. communis subsp. nana 24.3 ± 3.52

J. excelsa 29.5 ± 3.01

J. foetidissima 21.8 ± 2.54

J. oxycedrus subsp. oxycedrus 50.8 ± 2.44∗∗

J. phoenicea 44.3 ± 2.81∗

Madecassol 75.6 ± 2.95∗∗∗

∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; SEM: standard error of the mean.

2.3.3. Wound Healing Activity

Linear Incision Wound Model. Animals, seven rats in eachgroup, were anaesthetized with 0.15 cc Ketalar [13], and thehairs on the dorsal part of the rats were shaved and cleanedwith 70% alcohol. Two 5 cm length linear-paravertebral in-cisions were made with a sterile blade through the shavedskin at the distance of 1.5 cm from the dorsal midline on eachside. Three surgical sutures were placed each 1 cm apart.

The ointments prepared with test samples, the referencedrug (Madecassol) or ointment base (glycol stearate: propy-lene glycol: liquid paraffin (3 : 6 : 1)) were topically applied

on the dorsal wounds in each group of animals once dailythroughout 9 days. All the sutures were removed on the lastday, and tensile strength of previously wounded and treatedskin was measured by using a tensiometer (Zwick/Roell Z0.5,Germany) [14, 15].

Circular Excision Wound Model. This model was used tomonitor wound contraction and wound closure time. Eachgroup of animals (seven animals in each) was anaesthetizedby 0.01 cc Ketalar. The back hairs of the mice were depilatedby shaving. The circular wound was created on the dorsalinterscapular region of each animal by excising the skin witha 5 mm biopsy punch; wounds were left open (Tramontina,Machado, Nogueira Filho Gda, Kim, Vizzioli & Toledo,2002). Test samples, the reference drug (Madecassol, Bayer)and the vehicle ointments were applied topically once aday till the wound was completely healed. The progressivechanges in wound area were monitored by a camera (Fuji,S20 Pro, Japan) every other day. Later on, wound area wasevaluated by using AutoCAD program. Wound contractionwas calculated as percentage of the reduction in woundedarea. A specimen sample of tissue was isolated from thetreated skin of each group of mice for the histopathologicalexamination [16].

2.3.4. Hydroxyproline Estimation. Tissues were dried in hotair oven at 60–70◦C till consistent weight was achieved. After-wards, samples were hydrolyzed with 6 N HCl for 4 hours at130◦C. The hydrolyzed samples were adjusted to pH 7 andsubjected to chloramine T oxidation. The coloured adductformed with Ehrlich reagent at 60◦C was read at 557 nm [17].


Table 5: Wound healing processes and healing phases of the vehicle, negative control, test ointments, and Madecassol-administered animals.

Groups Wound healing processes

Vehicle S U RE FP CD MNC PMN NV

Negative control ++/+++ +++ — ++/+++ ++ +++ ++/+++ ++

C. sempervirens var. horizontalis ++/+++ ++/+++ — ++/+++ ++ ++ ++/+++ ++/+++

C. sempervirens var. pyramidalis ++ ++/+++ — ++ ++ +/++ ++ ++

J. communis subsp. nana Syme. ++/+++ ++/+++ — ++/+++ ++ +/++ ++ +++

J. excelsa ++/+++ ++/+++ — ++ +/++ ++ ++ +/++

J. foetidissima +/++ +/++ −/+ ++ +/++ +/++ +/++ ++

J. oxycedrus subsp. oxycedrus ++ +/++ −/+ ++ +/++ +/++ +/++ +/++

J. phoenicea +/++ +/++ −/+ ++ +/++ +/++ +/++ ++

Madecassol +/++ −/+ + ++ +/++ + +/++ +/++

HE- and VG-stained sections were scored as mild (+), moderate (++), and severe (+++) for epidermal and/or dermal remodeling. S: scab, U: ulcer, RE: re-epithelization, FP: fibroblast proliferation, CD: collagen depositions, MNC: mononuclear cells, PMN: polymorphonuclear cells, NV: neovascularization, I:inflammation phase, P: proliferation phase, and R: remodeling phase.

Table 6: Inhibitory effect of the essential oils from C. sempervirens var. horizontalis, C. sempervirens var. pyramidalis, J. communis, J. excelsa,J. foetidissima, J. oxycedrus, and J. phoenicea on acetic-acid-induced increased capillary permeability.

Material Dose (mg/kg) Evans blue concentration (μg/mL) ± SEM Inhibition (%)

Control 10.69 ± 0.86

Cupressus sempervirens var. horizontalis100 10.40 ± 0.95 2.7

200 10.12 ± 0.74 5.3

Cupressus sempervirens var. pyramidalis100 10.75 ± 0.87 —

200 11.16 ± 0.91 —

Juniperus communis ssp. nana100 10.13 ± 0.79 5.2

200 10.01 ± 0.38 6.4

Juniperus excelsa100 10.57 ± 0.88 1.1

200 9.04 ± 0.85 15.4

Juniperus foetidissima100 12.27 ± 0.95 —

200 11.25 ± 0.99 —

Juniperus oxycedrus ssp. oxycedrus100 8.82 ± 0.76 17.5

200 7.15 ± 0.32 33.1∗∗

Juniperus phoenicea100 9.04 ± 0.89 15.4

200 7.77 ± 0.44 27.3∗

Indomethacin 10 5.28 ± 0.26 50.6∗∗∗

∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 significant from the control; SEM: standard error of the mean.

Standard hydroxyproline was also run and values reported asmg/g dry weight of tissue [18].

2.3.5. Histopathology. The skin specimens from each groupwere collected at the end of the experiment. Samples werefixed in 10% buffered formalin, processed, and blocked withparaffin and then sectioned into 5 micrometer sections andstained with hematoxylin & eosin (HE) and Van Gieson(VG) stains. The tissues were examined by light microscope(Olympus CX41 attached Kameram Digital Image AnalyzeSystem) and graded as mild (+), moderate (++), andsevere (+++) for epidermal or dermal remodeling. Re-epi-thelization or ulcer in epidermis, fibroblast proliferation,mononuclear and/or polymorphonuclear cells, neovascular-ization, and collagen depositions in dermis were analyzedto score the epidermal or dermal remodeling. At the endof the examination, wound healing phases—inflammation,

proliferation, and remodeling—were determined for allgroups.

2.3.6. Anti-Inflammatory Activity

Acetic-Acid-Induced Increase in Capillary Permeability. Effectof the test samples on the increased vascular permeabilityinduced by acetic acid in mice was determined accordingto Whittle method [19] with some modifications [20].Each test sample was administered orally to a group of 10mice in 0.2 mL/20 g body weight. Thirty minutes after theadministration, the tail of each animal was injected with0.1 mL of 4% Evans blue in saline solution (intravenous) andthen we waited for 10 min. Then, 0.4 mL of 0.5% (v/v) AcOHwas injected intraperitoneally. After 20 min incubation, themice were killed by dislocation of the neck, and the viscerawere exposed and irrigated with distilled water, which wasthen poured into 10 mL volumetric flasks through glass wool.


Figure 1: Histopathological view of wound healing and epidermal/dermal remodeling in the vehicle, negative control, oils, and referenceointment Madecassol-administered animals. Skin sections show the hematoxylin & eosin- (HE-) stained epidermis and dermis in A and thedermis stained with Van Gieson (VG) in B. The original magnification was×100 and the scale bars represent 120 μm for figures in A, and theoriginal magnification was ×400 and the scale bars represent 40 μm for B. Data are representative of 6 animals per group. (1) Vehicle group:10-day-old wound tissue treated with only vehicle, (2) negative control group: 10-day-old wound tissue, untreated group, (3) C. sempervirensvar. horizontalis group: 10-day-old wound tissue treated with the essential oil of C. sempervirens var. horizontalis, (4) C. sempervirens var.pyramidalis group, 10 day old wound tissue treated with essential oil of C. sempervirens var. pyramidalis, (5) J. communis group: 10-day-oldwound tissue treated with essential oil of J. communis, (6) J. excelsa group: 10-day-old wound tissue treated with essential oil of J. excelsa,(7) J. foetidissima group: 10-day-old wound tissue treated with essential oil of J. foetidissima, (8) J. oxycedrus group: 10-day-old woundtissue treated with essential oil of J. oxycedrus, (9) J. phoenicea group: 10-day-old wound tissue treated with essential oil of J. phoenicea, and(10) reference group: 10-day-old wound tissue treated with Madecassol. Arrows pointing events during wound healing; s: scab, u: ulcer, re:re-epithelization, f: fibroblast, c: collagen, mnc: mononuclear cells, pmn: polymorphonuclear cells, and nv: neovascularization.

Each flask was made up to 10 mL with distilled water, 0.1 mLof 0.1 N NaOH solution was added to the flask, and theabsorption of the final solution was measured at 590 nm(Beckmann Dual Spectrometer; Beckman, Fullerton, Calif,USA). A mixture of distilled water and 0.5% CMC was givenorally to control animals, and they were treated in the samemanner as described above.

2.3.7. Statistical Analysis of the Data. The data on percent-age anti-inflammatory and wound healing was statisticallyanalyzed using one-way analysis of variance (ANOVA). Thevalues of P ≤ 0.05 were considered statistically significant.

Histopathologic data were considered to be nonparamet-ric; therefore, no statistical tests were performed.


In the present study, wound healing and anti-inflammatoryeffects of the oils obtained from Cupressus sempervirens var.horizontalis, C. sempervirens var. pyramidalis J. communis,J. excelsa, J. foetidissima, J. oxycedrus, and J. phoenicea were

evaluated. The oils were mixed with glycol stearate, 1,2 pro-pylene glycol, liquid paraffin (3 : 6 : 1) in 1% concentrationfor the assessment of the wound healing activity. Linearincision and circular excision experimental wound modelswere employed. Moreover, skin samples were evaluatedhistopathologically. The experimental results are given inTables 2–6.

As shown in Table 2, topical application of the ointmentprepared with the oils of aerial parts of J. oxycedrus subsp.oxycedrus and J. phoenicea onto the incised wounds demon-strated the best wound healing activity. On day 10 the ten-sile strength values were 31.5% and 36.3%, respectively. Therest of the oils did not show any remarkable wound healingactivity.

The contraction values of vehicle, negative control, oilsand reference drug treated groups were shown in Table 3. Theoils obtained from the berries of J. oxycedrus subsp. oxycedrusand J. phoenicea were found to have wound healing potential,while the vehicle and negative control groups and the otheroil ointments showed no statistically significant wound heal-ing activity. The wound contractions were determined as


Re-epithelizationProliferationInflammation

Veh

icle

Neg

ativ

eco

ntr

ol

Mad

ecas

sol

(%)

0

20

40

60

80

100

C.s

empe

rvir

ens

var.

hori

zont

alis

C.s

empe

rvir

ens

var.

pyra

mid

alis

J.co

mm

unis

subs

p.na

na

J.ex

cels

a

J.fo

etid

issi

ma

J.ox

yced

rus

subs

p.ox

yced

rus

J.ph

oeni

cea

Figure 2: Healing phases of the vehicle, negative control, testointments, and Madecassol-administered animals.

38.99% and 43.10% for J. oxycedrus subsp. oxycedrus and33.28% and 40.74% for J. phoenicea when compared toreference drug Madecassol (90.78%–100%).

For the assessment of collagen synthesis hydroxyprolinelevels of treated tissues were evaluated. As shown in Table 4significant increase in hydroxyproline content was observedfor the tissues treated with the essential oils of J. oxycedrusssp. oxycedrus and J. phoenicea.

Phases in wound healing processes (inflammation, proliferation, and remodeling) were observed and withinthe experimental groups with different degree (Table 5,Figures 1 and 2). The reference drug, essential oils of J.oxycedrus subsp. oxycedrus and J. phoenicea treated groupsdemonstrated faster remodeling respectively compared tothe other groups which showed delayed wound healingprocesses. As an evidence of delay, wound-associated tissuedebris still remained in the dermal tissues.

The effect of the extracts on the inflammatory phaseof wound healing was examined by using the methodof Whittle, based on the inhibition of acetic-acid-inducedcapi-llary permeability. As shown in Table 6, the inhibitoryactivity was observed for the oils from Juniperus oxycedrusssp. oxycedrus and J. phoenicea at the dose of 200 mg/kgwith the highest inhibitory values of 33.1% and 27.3%,respectively, while the essential oils from both species showedno significant effect at the dose of 100 mg/kg. On the otherhand the rest of the oils did not show any inhibitory activity.

Previous studies revealed that the essential oils ofJuniperus and Cupressus are rich in α-pinene. Particularly J.phoenicea possesses the highest α-pinene content comparedto the other species in comparative phytochemical analysis;β-Pinene, sabinene, and limonene are the constituents whichare also present in the essential oils of both genera [8, 10].In a previous study, α-pinene was found to have moderate

anti-inflammatory effect at 500 mg/kg dose on carrageenan-induced hind paw edema test [21]. Anti-inflammatoryactivity is essential for wound healing, since a long durationin the inflammatory phase causes a delay in healing process.In order to shorten the healing period as well as for minimalpain and scar, anti-inflammatory activity is required [22].Presence of α-pinene in Juniperus species could probablycontribute to wound healing activity by providing anti-inflammatory effect. Moreover, limonene is an importantmonoterpene which has a role in wound healing [23]. In amodel of chronic skin inflammation, the researchers havealso shown a significant effect of limonene and perillyl alco-hol on skin repair and proinflammatory cytokines levels [24].Furthermore, J. oxycedrus ssp. oxycedrus and J. phoeniceawere found to have high flavonoid and phenolic acid contentwhich provide them to show remarkable antioxidant activity[5]. As in many of the diseases, antioxidant activity also helpsto promote wound healing process [25].

Collagen is an important extracellular matrix pro-tein which provides strength and integrity to the tissueand plays an important role in haemostasis and epithelisa-tion. High levels of hydroxyproline in regenerated tissue sug-gest enhanced collagen synthesis. Hence enhanced collagensynthesis by J. oxycedrus ssp. oxycedrus and J. phoenicea maysignificantly contribute to healing and provide strength to re-paired tissue [18].

4. Conclusion

According to the experimental results, the oils from berriesof J. oxycedrus ssp. oxycedrus and J. phoenicea were foundto have better activity on the wound healing compared tothe other essential oils and control groups. This might bedue to the synergistic effect of the constituents present in theoils. This study provides a scientific evidence for the folkloricutilization of Juniperus for wound healing activity.

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Ethnopharmacological Approaches to Wound Repair · 2019. 8. 7. · Ethnopharmacological Approaches to Wound Repair Guest Editors: Esra Küpeli Akkol, Fatma U. Aﬁﬁ, Saringat Hj