Fitoterapia 81 (2010) 1109–1112

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Fitoterapia

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Comparative study on the anti-inflammatory and immune suppressive effectof Wilforlide A

Mei Xue a, Zhen-zhou Jiang b, Ji-ping Liu c, Lu-Yong Zhang a,⁎, Tao Wang a, Hao Wang a,Li Liu d, Zhi-xing Zhou a

a Jiangsu Center for Drug Screening Laboratory, China Pharmaceutical University No.24, Tongjiaxiang, Nanjing, Jiangsu Province, 210009, Chinab Key Laboratory of Drug Quality Control and Pharmacovigilance, China Pharmaceutical University No.24, Tongjiaxiang, Nanjing, Jiangsu Province, 210009, Chinac Department of Pharmacology, Shanxi University of Chinese Medicine, Xianyang, Shanxi Province, 712046, Chinad Department of Pharmacology, School of Medicine, Yangzhou University, Yangzhou 225001, China

a r t i c l e i n f o

⁎ Corresponding author. Tel./fax: 86 25 83271023.E-mail address: lyzhang@cpu.edu.cn (L.-Y. Zhang)

0367-326X/$ – see front matter © 2010 Elsevier B.V.doi:10.1016/j.fitote.2010.07.007

a b s t r a c t

Article history:Received 7 April 2010Accepted in revised form 2 July 2010Available online 16 July 2010

Tripterygium Glycosides (TG) is effective in the treatment of patients with a variety ofinflammatory and autoimmune diseases, especially rheumatoid arthritis (RA) in clinical.Wilforlide A (T1) serves as a quality control standard of TG that be listed in Drug Standard ofMinistry of Public Health of the People's Republic of China. The pharmacologic actions of T1remain to be unidentified. In this paper, we studied the anti-inflammatory and immunesuppressive effect of T1, and compared it with Triptolide (TP), which believed to be the majoractive component of TG. Carrageenan-induced rat pedal swelling, tampon-induced ratgranulation, and mice ear inhibition rate of swelling trail results show that high-dose T1 hasobvious anti-inflammatory effect. Colorimetric detection contents of hemolysin, carbonelimination from plasma of mice, mice delayed hypersensitivity immune, and organ indexwere measured. The results show that T1 has no significant immune suppressive activity, andthere has a significant difference with TP and TG. Therefore, we think the content of TP alsoshould be controlled as a supplement standard in order to ensure safe and effective medication.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Wilforlide ATripterygium glycosidesAnti-inflammatoryImmune suppressive

1. Introduction

TripterygiumwilfordiiHook f. (TWHF), a woody vine nativeto Eastern and southern China Korea, Japan, and Taiwan, has along history of use in traditional ChineseMedicine for treatingswelling, fever, chills, sores, joint pain, and inflammation, inaddition, having anticancer, antifertility and antibiosis activ-ities[1,2]. Tripterygium Glycosides (TG), an extract derivedfrom TWHF peeled root center and purified by columnchromatographic separation, is dominating pharmaceuticsin clinical application for treatment of patients with a varietyof inflammatory and autoimmune diseases such as rheuma-

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toid arthritis, systemic lupus erythematosus and skin diseases[3–5], and produced by 12 drug factories in China. Its mainconstituents have been reported to be diterpenes, triterpenes,and alkaloid compounds [6,7]. Triptolide (TP), believed to bethe major active component of TG [8]. Wilforlide A (T1) is atriterpene purified compound separated from TWHF [9–11]and serves as a quality control standard of TG. This standardbe official listed in Drug Standard of Ministry of Public Healthof the People's Republic of China (WS3-B-3350-98)[12]. Itschemical structure is shown in Fig. 1. However, T1 pharma-cologic actions remain to be unidentified. Does T1 also havestrong anti-inflammatory and immune suppressive effect andplay an important role in therapy of RA? A series ofexperiments is conducted in this study to examine itspharmacologic effect and compare it with TP and TG inorder to fully assess its rationality as a quality controlstandard and to study its further evaluation for RA.

Fig. 1. The chemical structure of Wilforlide A.

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2. Materials and methods

2.1. Materials

Wilforlide A was supplied by the medicinal chemistrylaboratory of China Pharmaceutical University. TPwas suppliedby the Institute of Dermatology of the Chinese Academy ofMedical Sciences. TG was supplied by Zhejiang DND drugfactory, batch number 0802702. Carrageenan, dinitrofluoro-benzene (DNFB) was purchased from Sigma (St. Louis, MO,USA). Sterile sheep blood, siderosis albumen reagent, andxylene were supplied by Nanjing Bianzhen biology company.

2.2. Animals

A total of 100male SD rats, bodyweight 160±10 g, and 200male ICR mice weighing 20±2 g were obtained from theCentral Animal Laboratory of Nantong Medical University. Therats were randomly divided into five groups, with ten rats foreach group, and were administered by oral gavage. The drugdosage was adjusted based on clinical equivalency dosageacting on the body surface area, and the dispensing solution is0.5% CMC-Na. The control group was treated with saline only.

2.3. Suppression of carrageenan-induced rat pedal swelling

Before the rats were administered, the foot volume of therats was measured by the displacement technique using acalibrated glass tube. Each group received different drugsafter 30 min; 0.1 mL of 10 g/L carrageenan was injected intothe right hind foot of each rat under the plantar aponeurosis.Measurements of foot volume were performed immediately1, 2, 4, and 6 h after injection of carrageenan. The edema rateand inhibition rate of each group were calculated as follows:

Edema rate ðEÞ %ð Þ = ðVt−V0ÞInhibition rate ðIÞ %ð Þ = ðEc−EtÞ= Ec

ð1Þ

where V0 is the mean paw volume before carrageenaninjection (mL), Vt is the mean paw volume after carrageenaninjection (mL), Ec is the edema rate of the control group, andEt is the edema rate of the treated group.

2.4. Suppression of xylene-induced mice ear swelling

Each group received different drugs after 30 min. On theright ear of each mouse (15 μL, two sides each), 30 μL xylenewas smeared, while the left ear did not receive treatment.After 1 h, the mice were sacrificed by breaking their cervicalvertebrae, and both ears were cut. The middle of the ears wasperforated with a hole puncher and weighed. The edema rateand inhibition rate of each group were calculated as follows:

Edema rate ðEÞ %ð Þ = ðWt−W0ÞInhibition rate ðIÞ %ð Þ = ðEc−EtÞ= Ec

ð2Þ

where W0 is the weight of the left ear (mg), Wt is the weightof the right ear (mg), Ec is the edema rate of the controlgroup, and Et is the edema rate of the treated group.

2.5. Suppression of tampon-induced rat granulation swelling

All animals were oral gavage once daily for 7 days. Afterthe first administration, a highly compressed steam-sterilizedtampon in etherization condition was inserted into the rats'bilateral axillary fossa. The rats were sacrificed 24 h after thelast administration, the tampons were taken out, baked(40 °C, 12 h), and weighed. The weights of the granulationswelling were calculated as follows:

The weight of the granulation swelling = Wt−W0 ð3Þ

where W0 is the weight of the tampon before it was inserted(mg), and Wt is the weight of the tampon 7 days after it wasinserted.

2.6. Formation reaction of blood serum hemolysin

Mice were oral gavage once daily for 7 days. After thesecond administration, each mouse was injected by perito-neal 0.2 mL SRBC suspension to immunize. After 8 days,before the animals were sacrificed, blood was drawn from apicked eyeball and centrifuged at 3000 rpm for 10 min within1 h after collection. Then 1 mL blood serum, diluted 800times, was taken, and 0.5 mL SRBC suspension was addedbefore cooling in an ice bank. This was followed by addition of1 mL complement (guinea pig serum, diluted 10 times),reaction for 10 min in 37 °Cwater, termination of the reactionin the ice bank, and centrifugation at 4000 rpm for 10 min.Then 1 mL supernatant was taken, 3 mL Siderosis albumenreagent was added, and the solution was antigraded andmeasured by ultraviolet-visible spectrophotometer at540 nm. Further, 0.25 mL SRBC suspension was diluted to4 mL with Siderosis albumen reagent. Half hemolysis absor-bance can be obtained using the same measurement method.The sample HC50 was calculated as follows:

Sample HC50 = Sample absorbance= half hemolysis absorbanceð Þ× Dilution times ð4Þ

Table 1Effect of WilforlideA on mice ear swelling induced by xylene (x± s).

Groups N Dosage Edema rate (mg) Inhibition rate (%)

Control group 10 – 2.62±0.92 0TP 9 60 μg/kg 1.23±0.35★★ 53.28TG 10 13.8 mg/kg 1.58±0.21 39.46High-dose T1 10 300 μg/kg 1.28±0.28★ 50.90Low-dose T1 10 60 μg/kg 2.30±0.56◆◆ 12.29

★Significant difference at Pb0.05 levels compared with the Control group,★★Significant difference at Pb0.05 levels compared with the Control group.◆◆Significant difference at Pb0.01 levels compared with the TP group.

Table 2Effect of WilforlideA on rat granulation induced by tampon (x± s).

Groups N Dosage Mgranulation (mg)

Control group 9 – 60.66±9.51TP 10 30 μg/kg 39.53±8.45★★

TG 9 7.5 mg/kg 43.99±7.38★★

High-dose T1 8 150 μg/kg 47.68±11.53★★◆

Low-dose T1 10 30 μg/kg 51.15±9.04★◆

★Significant difference at Pb0.05 levels compared with the Control group,★★Significant difference at Pb0.05 levels compared with the Control group.◆Significant difference at Pb0.05 levels compared with the TP group.

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After the animals were sacrificed, the thymus gland andspleen were dislodged, weighed, and calculated as follows:

Thymus gland spleenð Þindex %ð Þ = weight of the thymus gland spleenð Þ= body weight × 100% ð5Þ

2.7. Mice delayed hypersensitivity immune

Mice were oral gavage once daily for 7 days. After the firstadministration, the middle abdomen of the mice wasdepilated and smeared with 50 g/20 μL LDNFB. In 6 days,10 g/20 μL DNFB was smeared on the right ear of each mice(10 μL two sides each), while the left ear was smeared with20 μL acetone as control. After 30 h, the mice were sacrificedby breaking their cervical vertebrae, and both ears were cut.The middle of the ears was perforated with a hole puncherand weighed. The edema rate and inhibition rate of eachgroup were calculated as in Eq. (2).

2.8. Carbon elimination from the plasma of mice

Mice were oral gavage once daily for 7 days. After 1 h inthe last administration, injecting 0.1 mL/10 g Indian ink intothe caudal vein, 20 μL blood was drawn from a picked eyeballafter 1 min (t1) and 5 min (t2); after addition of 2 mL 0.1%Na2CO3, absorbance (OD1, OD2) was measured by anultraviolet–visible spectrophotometer at 280 nm. After theanimals were sacrificed, their liver and spleen were dislodgedandweighed. The clearance index (K) and phagotrophy index(a) were calculated as follows:

K = lgOD1−lgOD2ð Þ= t1−t2ð Þa = body weight = liver weight + spleen weightð Þ × K1=3 ð6Þ

2.9. Statistical analysis

Data were expressed as mean, standard deviation (S.D.).Statistical significance was tested using Student's t-test forcomparison between means. The difference was consideredsignificant at a P value less than 0.05.

Fig. 2. Effect of Wilforlide A on rat pedal swelling induced by carrageenan.

3. Results

3.1. Anti-inflammatory effect on WilforlideA

From Fig. 2, each experimental group reached edema peakat 4 h and obvious inhibition at 6 h. None of the groupsshowed any significant difference, while the control groupdid not have significant difference. Tables 1 and 2 show thathigh-dose T1, TP, and TG markedly inhibited mice auricularswelling induced by dimethylbenzene and rat granulationinduced by tampon (Pb0.05). As compared to the controlgroup, low-dose T1 slightly decreased, but there was nosignificant difference. The above results show that T1 hasobvious anti-inflammatory effect, but it is inferior to that of TPand TG.

3.2. Immune suppressive effect on WilforlideA

From Table 3, TP and TGmarkedly reduced mice hemolysinresponse and the index of carbon elimination from plasma(Pb0.05). High-dose T1 and low-dose T1 did not have asignificant difference with the control group, but comparisonwith TP showed a significant difference (Pb0.05). Every trailgroup had a slightly decreasedmice auricular swelling induced

Table 3Effect of WilforlideA on mouse hemolysin response and carbon eliminationfrom plasma of mice (x± s).

Groups N Dosage HC50 a phagotrophy index

Control group 10 – 149.85 4.45±0.53TP 10 60 μg/kg 82.23★★ 3.52±0.78★

TG 10 13.8 mg/kg 78.68★★ 3.81±0.61★

High-dose T1 10 300 μg/kg 115.51◆◆ 4.38±0.53◆

Low-dose T1 10 60 μg/kg 124.18◆◆ 4.21±0.87◆

In the table, comparison with the control group: ★Pb0.05, ★★Pb0.01;comparison with TP: ◆Pb0.05, ◆◆Pb0.01.

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by DTH and the weight of mice immune organ, but none of thegroups showed a significant difference (PN0.05). Therefore,detaileddata arenot listed in thepaper. The results showthatT1has no significant immune suppressive effect, but that of TP andTG is obvious significant.

4. Conclusion

The colorimetric detection contents of hemolysin andcarbon elimination from plasma of mice immune respectivelyrepresent different processes of immune modulation (non-specificity immune; specificity immune). Engine block im-mune function has multiplicity. The reason for thenonsignificant difference on DTH andweight of mice immuneorgan may be related to the administration period andactivation of T lymphocytes or drug insensitivity for cellularimmunity, but the three experimental results are also similar.Therefore, we conclude that T1 has obvious anti-inflamma-tory effect, while its immune suppressive effect is very weak.The anti-inflammatory mechanisms and other pharmacolog-ical properties of T1 require further examination. Ourexperimental results show that using the content of T1alone cannot evaluate the overall quality of TG and thecurrent standards remain to be insufficient. This problem notshould be to evade that the anti-inflammatory and immunesuppressive effect of TP is very stronger. Safety range of TP isnarrow, activity and toxicity are close relationship andcouldn't be separated [13–15]. Therefore, we think thecontent of TP also should be controlled as a supplementstandard in order to ensure safe and effective medication.

Acknowledgements

The study was supported by the following grants: SpecificFund for Public Interest Research of Traditional ChineseMedicine, Ministry of Finance (No. 200707008) and Megapro-jects of Science Research for the 11th Five-Year Plan: Standard-

ized Platform Construction and Scientific Application in NewTechnologies for New Drug Screening (No. 2009ZX09302-002).

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