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Effects of an Orabase Formulation with Ethanolic Extractof Malva sylvestris L. in Oral Wound Healing in Rats

Ana Cristina Kovalik,1,2 Paula Bisetto,1 Marcia Thaıs Pochapski,1 Eduardo Baulm Campagnoli,1

Gibson Luiz Pilatti,1 and Fabio Andre Santos1

1Department of Dentistry, School of Dentistry, Ponta Grossa State University, Ponta Grossa, Brazil.2Department of Dentistry, School of Dentistry, Fundacao Universidade Regional de Blumenau, Blumenau, Brazil.

ABSTRACT Malva sylvestris L. is widely used in medicine for treatment of inflammatory processes. The plant has anti-

inflammatory properties due to substances such as mucilage, flavonoids, and tannins. A mouthwash with leaves from the plant

can be used for the treatment of wounds in the oral mucosa. The aim of this study was to assess the wound healing effect of

Malva sylvestris L. on a palate mucosa wound in rats. After intraperitoneal anesthesia, a 4-mm-diameter excisional wound was

made in the center of the palatal mucosa of 136 rats, using a punch-out biopsy tool. Eight animals were used as baseline

wound. The remaining rats were divided into four groups: CO, control; OB, orabase vehicle; CX, 2% chlorhexidine; and MA,

20% Malva in orabase. At 24 h postoperatively, the animals were immobilized without anesthetic to apply 25 mg of each

substance twice a day, totaling 50 mg daily. The wound areas were measured photographically and the reepithelialization rates

were determined histologically (%) after 0, 3, 7, 15, and 21 days. The data were analyzed by ANOVA and Tukey post hoc test.

Similar healing pattern was observed among the groups (P > .05; ANOVA). According to the methodology, Malva sylvestris

L. extract had no effect on wound healing in the palatal mucosa of rats.

KEY WORDS: � herbal medicine � medicinal food � mucosa � palate � phytotherapy � oral ulcer � rat � wound healing

INTRODUCTION

Herbal medicine is characterized by the therapeutic useof medicinal plants in their different dosage forms,

without the use of isolated active substances, and its ap-proach encourages community development, solidarity, andsocial participation.1–3 The indiscriminate use of plantswithout any phytochemical, pharmacological, and, aboveall, toxicological knowledge, is of great concern to health.The correct identification of these species, their manner ofuse, dosage, and quality control are also issues that need tobe resolved.4,5

Malva sylvestris L. (Malvaceae), usually known ascommon mallow, is native to Europe, North Africa, andAsia, and its traditional use has been documented for a longtime, although little evidence is available. Roots, stem,shoots, leaves, flowers, fruits, and seeds are applied ininfusions, decoctions, poultices, liniments, lotions, baths,and gargles.2,3,6 Young leaves are eaten raw in salads;leaves and shoots are consumed in soups and as boiledvegetables. Traditionally, the medicinal applications of thecommon mallow treat specified disorders of several sys-

tems of the body, such as the digestive, respiratory, geni-tourinary, muscular, and skeletal systems, as well as skinand mucosa injuries.5,6 This plant has anti-inflammatoryproperties due to substances such as mucilage, flavonoids,and tannins.

In dentistry, a mouthwash with stems and leaves fromthe mallow is used for the treatment of wounds in the oralmucosa.1,3 However, studies to scientifically prove theefficacy of herbal medicines are necessary because—aswell as encouraging patients and health care profes-sionals to incorporate medicinal plants into their thera-peutic regimen—they can guide people regarding theproper use of such substances, their effects, side-effects,and risks.2,4

Tissue repair or regeneration after intraoral surgicalprocedures represents a critical step for the patient and theprofessional because the extensive and constant coloni-zation by microorganisms could delay complete tissueregeneration, which can often compromise the outcome ofthe surgical procedure.2,7–9 In dentistry, the prescription ofherbal medicines for topical use after surgical proceduresto reduce the dental biofilm formation and to minimize theimpact of the mechanical trauma of mastication couldensure or accelerate tissue repair.7 Therefore, the objec-tive of this study was to evaluate the potential of Malvasylvestris L. to repair palate lesions created surgicallyin rats.

Manuscript received 3 January 2013. Revision accepted 6 November 2013.

Address correspondence to: Fabio Andre Santos, DDS, PhD, Department of Dentistry,School of Dentistry, Ponta Grossa State University, Av. Carlos Cavalcanti, n 4748,Uvaranas, Ponta Grossa CEP: 84030-900, Brazil, E-mail: fasantos11@gmail.com

JOURNAL OF MEDICINAL FOODJ Med Food 00 (0) 2014, 1–7# Mary Ann Liebert, Inc., and Korean Society of Food Science and NutritionDOI: 10.1089/jmf.2013.0001

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MATERIALS AND METHODS

Plant material

Dried stems and leaves of Malva sylvestris L., harvestedin the south of Brazil by Quimer Ervas e Especiarias (SaoPaulo, SP, Brazil), were triturated in a blender until a finelygranulated powder was obtained. To obtain the extract fromthis powder and ethanol 70% was added and the mixture(0,23 g/mL) was constantly shaken for 7 days at roomtemperature. The extracts were vacuum filtered and rotaryevaporated at 50�C under reduced pressure, and then frozen,lyophilized, and stored in the dark at 4�C. At the end of thisprocess it was possible to obtain around 7% of crude extract.

Orabase preparation

The 20% Malva sylvestris L. in orabase was preparedwith 5 mg of lyophilized crude extract added to 20 mg ofvehicle (gelatine, pectin, and sodium carboxymethylcellu-lose in a polyethylene and mineral oil base) with a totalvolume of 25 mg. The substances were prepared immedi-ately prior to the experiments.

Animals

The present study was approved by the Animal ResearchEthics Committee of the Ponta Grossa State University(Protocol No. 0787106) and followed the guidelines of theBrazilian Society for Laboratory Animal Science.

One hundred and thirty-six male Wistar rats (8–10 weeksold) weighing 270–300 g were divided into four groups (n = 8):Group 1, control (CO); Group 2, orabase vehicle (OB); Group3, 2% chlorhexidine (CX); and Group 4, 20% Malva in orabase(MA). Eight animals were baseline wound (0 day). Samplesize was determined after a pilot study (unpublished data).

The rats were anesthetized intraperitonealy (0.2 mL/100 g) with a mixture of ketamine hydrochloride (100 mg/mL, Vetalar�; Boehringer Ingelheim do Brasil Quımica eFarmaceutica, Sao Paulo, Brazil) and xylazine (100 mg/mL,Rompun�; Bayer HealthCare, SaoPaulo, Brazil). An exci-sional wound, 4 mm in diameter, was made in the center andbehind the second ridge of the palatal mucosa, using apunch-out biopsy tool (Silverline Leather Punch�, London,United Kingdom) and mucoperiosteal specimens were re-moved by sharp dissection exposing a circular bone area forsecondary healing.10

At 24 h postoperatively, the animals were immobilizedwithout anesthetic to apply 25 mg of each substance twicea day, totaling 50 mg daily. At the end of the periods 0, 3,7, 15, and 21 days, the animals were sacrificed by cervi-cal dislocation after previous sedation with ketamine andxylazine.

Photographic evaluation

To measure the wound healing area for each experimentaltime, digital photos were obtained of the palatal specimensusing a digital camera (Sony Cybershot DSC 707�; SonyBrasil Ltda, Sao Paulo, SP, Brazil) with opening, intensityflash, exposure time, and distance all standardized. To en-sure maximum accuracy, a millimeter rule was photo-graphed with the specimens as a scale reference. In eachphotograph, the wound edges were marked and the area ofdefect was measured.11 The digital photographs weretransferred to a computer and the wound surface area wascalculated in mm2 for each animal using the program ImagePro Plus� Version 4.5.0.29 (Media Cybernetics, SilverSpring, MD, USA).

Histologic evaluation

The specimens were immersed in 4% buffered formalinfor fixation for at least 48 h, decalcified in 5% EDTA for 40days, and processed for histological evaluation after thewounds were sectioned at the center. For each specimen,five serial sections, 6 lm apart, were cut perpendicular to thepalate midline and stained with hematoxylin and eosin. Thesections were examined using light microscopy (OlympusBX41�, DP-72, Tokyo, Japan) at a magnification of · 40and the distance between the epithelial margins in eachsection was measured in percentage using the softwareImage Pro Plus 4.5�. The percentage was obtained bymeasuring the interpalate distance between the first molars,taking as reference the junctional epithelium indicatingpalatal width. The second measurement was the distancebetween the epithelial edges of the healing wound. The ratiobetween the two measurements was obtained.

In both analyses the measurements were performed by asingle blinded, trained, and calibrated examiner in triplicate,and the arithmetic mean was used for statistical analysis ofthe data obtained.

FIG. 1. Percentage of weight gain (meanand standard error) in each experimental pe-riod among four experimental groups (CO,control; OB, orabase; CX, 2% chlorhexidine;and MA, 20% Malva in orabase). *Significantdifference (P < .05) at 3 days with all groups.**Significant difference (P < .001) at 7 dayswith OB groups (n = 8). ANOVA and Turkeypost hoc test.

2 KOVALIK ET AL.

Statistical analysis

Intra-examiner reproducibility was assessed twicewithin 48 h to check the reproducibility of the measure-ments related to the residual wound area and the per-centage of wound healing using the Bland and Altmanprocedure.12 Comparisons among groups were tested byone-way ANOVA and Tukey post hoc test. The normalityof the distribution of the data was confirmed using theShapiro–Wilks test. An alpha value of £ .05 was used toindicate statistically significant differences among thegroups.

RESULTS

The animals of CO group had a lower weight gain per-centage than other groups after 3 days. However, in 7 days,statistically significant difference was observed only for the OBgroup. After 15 and 21 days all groups did not show statisticallysignificant difference in weight gain percentage (Fig. 1).

The intra-examiner reproducibility for wound area andpercentage of histological wound healing was within thelimits of agreement.

The differences between macroscopic wound area andpercentage of wound histological healing in the experi-mental groups compared with the control group did notreach statistical significance at any time during each ex-perimental period. The mean area of the circumscribed de-fects decreased significantly with time.

Macroscopic evaluation

The mean and standard error of wound area is shown inFigure 2. Statistically significant difference was found be-tween the baseline wound (0 day) and the orabase vehiclegroup at 3 days. Statistically significant difference was alsofound between the baseline wound and the group with 20%Malva in orabase at 1 week. At 3 days the fibrin layer cov-ering the bone tissue was rarely viewed and during the firstpostoperative week the healing was very slow. At 15 days, thewound was largely covered by epithelium with only a smallresidual wound. By day 21, the wound was almost fullyhealed with a minimal or nonexistent central depression ofthe 4-mm defect.

Histomorphometric evaluation

The mean and standard error of percentage of histologicalwound healing is shown in Figure 3. Similarly to the mea-surement of the macroscopic wound area, the wound widthdecreased significantly with time. However, there were nosignificant differences among the groups. Further, at 15days, the group of 2% chlorhexidine presented a delay in thepattern of repair compared with baseline wound.

Histological qualitative evaluation

The normal palatal mucoperiosteum was observed anddivided into epithelium, lamina propria, submucosa, andperiosteum. The epithelium was stratified squamous

FIG. 2. Mean and standard error of woundsurface areas in square millimeters evaluatedat four time points (n = 8). Groups: BW,baseline wound; CO, control; OB, orabase;CX, 2% chlorhexidine; and MA, 20% Malvain orabase. *Significant difference (P < .05) at3 days with OB group, at 7 days with MAgroup, and at 15 and 21 days with all groups.ANOVA and Tukey post hoc test.

FIG. 3. Mean and standard error of wound-healing percentage evaluated by histologicalanalysis at four time points (n = 8). Groups:BW, baseline wound; CO, control; OB, or-abase; CX, 2% chlorhexidine; and MA, 20%Malva in orabase. *Significant difference(P < .05) at 15 days with CO, OB, and MAgroups and at 21 days with all groups. AN-OVA and Tukey post hoc test.

MALVA SYLVESTRIS ON ORAL WOUND HEALING 3

orthokeratinized. In the midpalatal suture, cartilage wasobserved, which was surrounded by two sublayers of peri-osteum: osteogenic and fibrous layers.

At 3 days there was a slight proliferation of epithelialmargins. The clot was rarely observed, but was present inisolated sites and most often near the edges of the remainingepithelial tissue. The bone was exposed to the oral cavity andthe beginning of the necrotic area was seen. Some granulationtissue was observed near the lesion edge. The presence ofsome osteoclasts was viewed and, in all groups, a mild layerof bacterial colonization was observed that increased withtime (Fig. 4). Epithelialization moderately increased on day 7(Fig. 5) and faster reepithelialization occurred on days 15 and21 with only a small residual wound (Figs. 6 and 7). In allgroups, bone necrosis was observed in the area of the woundand this necrosis increased at day 15 (Fig. 6). Bone necrosissubsequently increased so that it was possible to view thesequestral bone underneath the marginal epithelial cells thatwere migrating. The sequestral bone was more visible in somesamples on day 21 (Fig. 7). In this case, reepithelialization

was incomplete and inflammatory infiltrate was higher.Granulation tissue was more pronounced in the second week.Collagenous fiber was viewed with time. The Figures 4–7have shown wound histologic reepithelialization pattern indifferent time points.

DISCUSSION

The pattern of repair occurred over time; however, therewas no significant difference among groups in the times.The groups that received some form of covering of the le-sion demonstrate the importance for animal welfare. Thiswas observed because the CO group had no weight gainafter 3 and 7 days. Further, the wound protection can reducethe impact of physical injury caused by food and chemicalsdirectly on the site of injury, because good outcomes werealso observed in relation to the OB and MA groups. Asorabase forms a protective film over the injury, it may haveprovided similar repair in the periods of 15 and 21 days inthe OB and MA groups, to exert protective mechanical

FIG. 4. Photomicrographs of wound-healing pattern after 3 days. (A) CO,control; (B) OB, orabase; (C) CX, 2%chlorhexidine; (D) MA, 20% Malva inorabase. There was a slight prolifera-tion of epithelial margins. The bonewas exposed to the oral cavity and thebeginning of the necrotic area wasseen. Some granulation tissue was ob-served near the lesion edge. The pres-ence of some osteoclasts was viewedand, in all groups, a mild layer ofbacterial colonization was observed.NC, nasal cavity; T, teeth; PB, palatalbone; E, epithelium; black arrow,bacterial colonization; outline arrow,clot. Hematoxylin and eosin stain,original magnification · 40.

FIG. 5. Photomicrographs of wound-healing pattern after 7 days. (A) CO,control; (B) OB, orabase; (C) CX, 2%chlorhexidine; (D) MA, 20% Malva inorabase. Epithelialization process hasshowed moderate increase. In allgroups, bone necrosis was observed inthe area of the wound. Palatal bonewas exposed to the oral cavity andnecrotic area was seen. Some granu-lation tissue was observed near the le-sion edge. All groups had a layer ofbacterial colonization. NC, nasal cavi-ty; T, teeth; PB, palatal bone; E, epi-thelium; black arrow, bacterialcolonization; outline arrow, clot; NB,necrotic bone.

4 KOVALIK ET AL.

action.13 As the results between the OB and MA groupswere similar, and were not significantly different from CXand CO, it is not possible to state that mallow had a positiveeffect in reducing the extent of the injury. This probablyoccurred because of the protection exerted by orabase andnot by the pharmacological properties exerted by the activemetabolites in the stems, leaves, and flowers of the plant.However, further studies should be conducted to test thiseffect with other vehicles and with extracts at differentconcentrations.

The vehicle incorporated into mallow could have been asubstance in the form of gel, such as commercially avail-able chlorhexidine. However, orabase is not toxic, does notharm tissue with which it is in contact, and is more ad-herent than gel; all of which maintains the concentration ofthe substance active for as long a period of time as possibleon the wound, ranging from 15 min to 2 h depending on themobility of the affected region and the saliva, which re-moves the substance more easily, depending on the rateof flow.14,15

In this present study, we included 2% chlorhexidine di-gluconate in natrosol; besides being considered the goldstandard in the control of bacterial colonization and prolif-eration, it is the substance of choice for use after oral sur-gical procedures because of its broad spectrum of action.16

In this group, we did not use orabase because it does notpromote the dissociation and release of chlorhexidine andinterferes with its action. Chlorhexidine in a concentrationof 2% was employed, as it is used in topical form aftersurgical procedures in the oral cavity.17 In this study, after15 days, chlorhexidine showed a negative effect comparedwith baseline wound (0 day), with no significant differencefrom the other groups during the same period. This reduc-tion of injury repair pattern reflects the harmful effectscaused by the substance on the proliferation of fibroblasts,and consequently of collagen and noncollagen substances,as well as keratinocytes.18–22 The inhibition of the prolif-eration and function of fibroblasts in vitro depends on timeand the concentration of chlorhexidine on the injured tis-sue.18,20 As the concentration used in this study was high,

FIG. 6. Photomicrographs of wound-healing pattern after 15 days. (A) CO,control; (B) OB, orabase; (C) CX, 2%chlorhexidine; (D) MA, 20% Malva inorabase. The reepithelialization wasincomplete and inflammatory infiltratewas higher. Necrotic bone area andbacterial colonization was seen in allof experimental groups. Granulationtissue was more pronounced in thesecond week near the lesion edge. Thesequestral bone was observed. NC,nasal cavity; T, teeth; PB, palatal bone;E, epithelium; black arrow, bacterialcolonization; NB, necrotic bone; GT,granulation tissue; SB, sequestral bone.Hematoxylin and eosin stain, originalmagnification · 40.

FIG. 7. Photomicrographs of wound-healing pattern after 21 days. (A) CO,control; (B) OB, orabase; (C) CX, 2%chlorhexidine; (D) MA, 20% Malva inorabase. The reepithelialization wascompleted in the most of the experi-mental groups. The sequestral bonewas observed in few groups. Most ofthe palatal bone was resorbed. NC,nasal cavity; T, teeth; PB, palatal bone;E, epithelium; black arrow, bacterialcolonization; SB, sequestral bone. He-matoxylin and eosin stain, originalmagnification · 40.

MALVA SYLVESTRIS ON ORAL WOUND HEALING 5

the result was inconsistent compared with the study thatcompared 5-mm palatine lesions with treatment using0.12% and 1% chlorhexidine, where the effect on the repairwas beneficial.11,23

Applying the substances in each group was initiated onlyafter 24 h. During this period, fibrin layer and clot formationon the palatal bone occurs—which is considered funda-mental for the new connective and epithelial tissue at the siteof injury—which preserves the epithelial margins and thecells of the tissue and also releases growth factors nearthe edges.24,25 Further, this interval of time is important forthe recovery of the animals after surgical procedure.11

Another important detail was the choice not to anesthetizethe animals prior to each daily application of substances.When topically administrating any medication intraorally, itwill rarely remain on the site of the injury for long. There isa dynamic movement and removal of the substance by salivaand through the articulation of the teeth, muscles, and ton-gue during mastication and swallowing. The action exertedby solutions such as chlorhexidine mouthwash occurs byresidual effect on the epithelial surface exerted by itsheightened substantivity.16 Besides being able to replicateeven more closely a real clinical situation, there was no riskof loss of animals during the experimental periods.

The mechanical and physical trauma caused by mastica-tion was possibly a factor that delayed the repair process. Itis believed that the absence of coagulum in the 3-day groupwas due to the friction caused by feeding against the palateinjury. The absence of epithelial edges that were also de-tected in some animals affected the rate of formation of newconnective and epithelial tissue, and prolonged the inflam-matory phase, thereby reducing the number of cells thatinduce the formation of new tissue and growth factors.24,26

Another possible consequence of the trauma of mastication,this time associated with bacterial colonization, was thenecrosis of the palatal bone plate exposed to the oral envi-ronment. This was a common finding in another similarexperiment.11 Although not measured quantitatively, thepresence of osteoclasts was observed, particularly in theperiods of 7 and 15 days. These factors cause a prolongedinflammatory phase that perpetuates the continuous flow ofinflammatory cells to the site of injury.25

The palate injury model adopted in this research wasbased on the simple standardization technique, consideringlesion extension (circular shape) and depth (removal ofthe mucoperiosteum and exposure of the palatal bone). Insome studies using the same methodology, researchers haveadopted a 3-, 4-, or 5-mm circular scalpel.11,23,24,27,28 In thispresent study, the choice of the 4-mm circular scalpel wasbased on a pilot study, where it was observed that the closingof the 3-mm lesion occurred up until day 9, in accordancewith the results of the other studies.11,23,24 In this presentresearch, the observation of repair over a longer period wasthe criterion for choosing the size of the lesion. Further, thesurgical use of rats was due to the ease of handling andrelatively low maintenance costs.28

This research is only preliminary study about Malvasylvestris in oral wound healing. Although the results have

shown that the extract of mallow did not promote the ac-celerated healing of the lesion, there were no negative orharmful effects on tissues of the animals, but further studiesshould be carried out with different vehicles and concen-trations to test their effectiveness, not only regarding heal-ing, but also in terms of its anti-inflammatory andantimicrobial potential.

We concluded that there was a significant reduction in thewound over a period of time. Extract of 20% Malva syl-vestris L. in orabase did not present an effect on the healingof wound in the palatal mucosa of rats. Further studies canbe necessary for comparing these results.

ACKNOWLEDGMENTS

This work was supported by CAPES (Coordenacao deAperfeicoamento de Pessoal de Nıvel Superior) and Ara-ucaria Foundation (Fundacao Araucaria de Apoio ao De-senvolvimento Cientıfico e Tecnologico do Parana amparaa formacao de recursos humanos do Estado do Parana),Brazil.

The authors would like to express our gratitude to PontaGrossa State University and Fundacao Universidade Re-gional de Blumenau, Blumenau, Brazil.

The authors wish to thank Dr. Sean Stroud for reading thisarticle and offering his valuable comments.

AUTHOR DISCLOSURE STATEMENT

None of the authors have conflict of interests.

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