ISSN 088 97S Asa Phamauol Sin POLAR DA Devs 22 (12), T— NO p/w. hina. com, su Anti-inflammatory effect and mechanism of proanthocyanidins from grape seeds! LI Wen-Guang”, ZHANG Xiao-Yu’, WU Yong-Jie, TIAN Xuan‘ (Department of Pharmacology , ont of Physiology, Lanchou Medical College; ‘National Laboratory of Organic Chemistry , Lanchou University , Lanchou 730000, China) KEY WORDS —proamthocyanidins; grape seeds: inflammation: interleukin-1; tumor necrosis factor; nittic ‘oxide: malondialdehyde: prostaglandins E; carrageenan ABSTRACT AIM: To investigate the antiinflammatory effect and mechanism of proanthocyanidins (PA) from grape seeds, METHODS; Croton oil-indced ear swelling in. mice and camageenan-induced hind paw edema in rats were prepared. The nitric oxide synthase (NOS) activity was measured by NADPH-disphoras stain assay, nitric oxide (NO) coment by Griess diazotiration assay, N-acetyl 3- D-glucosaminidase ( ®NAG ) activity by spectro: photography, malondialdehyde (MDA) content by thiobarbituric acid (TBA) fluorescence technique, and IL-1B, TNFa, and PGE, content by radioimmunoassay (RIA). RESULTS; PA 10 - 40 mg/kg ip inhibited camageenan induced paw edema in rats and croton oil- induced ear swelling in mice in a dose-dependent manner. PA J0 mg/kg reduced MDA content in inflamed paws, inhibited @-NAG and NOS activity, and lowered the content of NO, TL-I8, TNFe, and POE; in exudate from edema paws of rats induced by carrageenan. The inhibitory effect of PA on all above indices was more evident than that of dexamethasone 2 mg/kg. CONCLUSION: PA bas antiinflammatory effect on ‘experimental inflammation in rats and mice. Its mecha- nisms of antiinflammatory action are relevant (© oxygen free radical scavenging, anti-lipid peroxidation, and inhibition of the formation of inflammatory cytokines. | Project supported by the Natural Scjence Foundation of Gans Proving, No ZS W-AZHODY. £ Camespndece 10 Dr LI Wen-Guang. Pin 85-901 82-357. E-mail uyj@ lg. net Received 2001-46-01 Accepted 2001-10-12, INTRODUCTION Proandocyanidias, naturally occurring antioxidants widely available in fruits, vegetables, nus, seeds, flowers and bark, especially grape seeds, have een reported 10 possess a broad spectrum of biological, pharmacological, and therapeutic activities agninst foe radicals and oxidative stress. large number of reports demonstrated that proanthocyanidin were highly biosvail- able and provide potent protection against free radical- induced diseases, such as ischemia and reperfusion injury of many organs, advancing age, tumor promotion, and carcinogenesis!'~*), efc, However, fewer reports dem- onstrated their effects on arthritis. Proanthocyanidin from Polypodium feei rots, Hamamelis virginiana bark and avocado had been reported possess. antiphlogisic, antiviral, and analgesic activity evaluated. whether proanthocyani possessed ant-inflammatory activity and preliminarily explored its mechanisms of an-inflammatory action MATERIALS AND METHODS Chemicals and animals Proanthocyunidin (PA), brown-ted powder (purity: 96 % ), extracted from gripe seeds, provided by Laboratory of Applied Organic Chemistry, Lanzhou University. Dexametha- sone (Dex) was purchased from Lanwhou Pharmaceutical Factory (Batch number 950903). All above reagents were diluted with normal saline (NS). Carrageenan was the product of Liaoning Insite of Pharmacy, and ilwed (0 10 g/L with NS. Nitric oxide (NO) nitric oxide synthase (NOS) and N-aoetyl2-D-glucosamini- dase (8-NAG) detection kit were obiained from Jiancheng Instirute of Biotechnology (Nanjing, China). 1L-1 RIA kit was the product of Beijing Noth Biotechnology Company. 'TNFe RIA kit was the product of Beijing Bangding Biotechnology Company. Prostuglandiny E, (PGE_) RIA kit was the product of Department ofISSN weston Acta Pamranal Sin FO} AR:PL 1 Deas 2121 his mal gps @ma ow ae. en Thrombus, Suzhou Medical College. China. All other reagents were of analytical purity. Kunming mice (weighing 22 ¢#2g. 2 #) and Wistar rats (weighing 218 g #26 g, ¢ $) were obtained from the Experimental Animal Center of Lanzhou Medical College (Grade ll . Certificate No 14-005 and. 14-006 respectively) Croton oil-induced ear edema in mice'”! Kunming mice (7 =50), were randomly divided imo 5 gioups (see Tab 1). PA and Dea were injected ip 30 min respectively before eroton oil inducing ear edema, Croion oil mixture was prepared"! ant applied (0.2 mt. each mouse) topically on the right ear pinna of mice. ‘The left pinna was used 2s control. The mice were killed 4h later. Disks from the pinnae were taken with a punch 7.0 mm in diameter. The edema extent was expressed in terms of the weight difference between the inflamed and the control Carrageenan-induced arthritis in rats'*” Fifty Wistar rats were randomly divided into controt (ip NS), PA 10, 20, 40 mg/kg and Dex 2 mg/kg groups to determine the anti-inflamimetory effect of PA. NS, PA. or Dex were injected ip 30) min before carrageenan (1% camageenan, 0.1 miL each rat) was given se in right hind pow plantar to induce the inflammation, Paw thickness ‘was measured prior to or 1, 2,3, 4. 6, 10, and 12 hy after challenge with carrageenan. The swelling degree wes caloulated according to the augment of paw thickness afer inducing onrits. Another 30 rats were randomly divided into control, PA 10 mg/kg, and Dex 2 mg/kg groups (Tab 8) treated as above experiment 0 assay the effect on formation of inflammatory factors, The other 10 rats, 0.) mL. normal saline sc in right hind paw plantar and 5 mL/kg NS ip. were used as blank control. AIT the rats were Killed 4h later after inducing inflammation, the skin of inflamed paw was cut open and the entire inflamed paw was ‘washed by extrusion with ice-cold PBS (50 mmol/L) 1 mL. Then the inflamed paw and exudate were harvested and stored at - 20 T before use. MDA determination in inflamed paws Inflamed rat paw was taken off skin and soaked in 2 mL. 15 % wichloroacetic acid for 3.h (4 °C). The super- natant (1.5 mL) was mixed with 2 mL TBA reagent [1 1 (vv) mixture of 0.57 % TBA and acetic acid]. The reaction mixture was boiled for 25 min. Then n-butanol (2 mL x2 times) was added. The mixture was shaken vigorously for } min, and centifged at 30K) x g for 10 min, The n-butanol layer was assayed by spectrofluora- Pan Fax AOL meter with 2, 520 am and Ay 350m. MDA content was calculated by reference to standards prepared from 1, 1,3. 3-tetraethoxypropane (Fluka) Determination of inflammatory factors in exudate IL-1, IL-8, and TNFe were measured by RIA method, NOS activity by NADPH-diaphorase stain assay, nitric oxide (NO) content by Griess diazotization assay, and BNAG activity by colorimetry, The detecting methods were according o the protocol enclosed in the detection kit Statistics assessed by Hest. Data were expressed as x + sand RESULTS Effect of PA on ear edema in mice PA 10~ 40 mg/kg reduced ear edema of mice induced by croton cil in a dose-dependent manner. The poterey of PA 40 mg/kg was obviously higher than that of Dex 2 mg/kg. (Tab 1). ‘Tab 1. Bifect of PA on ear edema induced by croton oil °P<0.05, ‘P<0.01 vs in mice, = 10. x +5. control. ema degree —_Tnhiition rate a omg 7% Contot - 8.9220 Dex 2 Lash a7 PA, 10 6822.9 Bo 2» 4s2L0 4 Basha Effect of PA on arthritis induced by carrageenan in rats PA 10 ~ 40 mg/kg obviously inhibited paw edema of rats induced by camageenan in dose-dependent manner. The potency of PA 20 and 40 mg/kg arived peak at 6 h after carageena and admini- stration with inhibition rate (IR) of 71.3 % and 853.6%, respectively, which were much higher than that ‘of Dex. The lasting time of action was longer in groups treated with PA than that tated with Dex 2 mg/kg (Tab 2). The plateau of inhibitory rate of PA 10 mg/kg on inflammation was seen at 3— 4h with TR 48.1% and 39.3 %, respectively: and then faded. So we adopted 4 has final point and BA 10 mg/kg as the dose for the evaluation of the anti-inflammatory mechanism of PA in the following tes. Effects of PA on formation of MDA, NO, andISSWORSR-OTS Avs Phamacol Sin POLAK 20M Dee: 22 12), E-aul s@ aul. shee. c.en Phe Fae -2-04742109, 1s Tob 2. Effect of PA on paw edema degree (PED, mm) induced by carrageenan in rats. m=10. +5. *P>0.05, °P<0.05, P<0.01 vs control. IR: inhibition rate {%). Drgs th 2h 3h 4h ob Won ah mek? PED OR OPED OR PEDRO PED PDR DRED OR Cooml 1620.5 - 22410 = Bist = 37a10 - 43206 - 47207 - 3 PAIO Lie03" BA 14204 %9 1530.65 WL 22206 W3 Z420H" 2412067 I gaan 89 D— Os204 500.9204 9.6 1020.6 06.6 L020 2413216 U3 20208 522207 514 W960. G2 0620.3 AZ 08204 TF OB20K MOTOS BH 16206 7 13206 OT DA? jweor 0 11205 HS Lte08 HE ZO RSs 1203 MO 42008 MT ateog o inflammatory cytokines, NOS activity, and BNAG release in carrageenan-induced rat arthritis PA 10 mg/kg markedly suppressed the generation of MDA in inflamed paws, inhibited the NOS activity and B-NAG relewe, and reduced content of NO and cytokines such as IL-1, and TNFa, PGE, in exudates from rat paw stimulated by carrageenan. The inhibitory potency of PA on all above indices was higher than that of Dex 2 mg/kg (Tab 3, 4). Tab 3. Effects of PA on formation of MDA and NO, NOS activity, and P-NAG release in currageenan-induced rat arthritis, 2 = 10. 2 5. "P<0.05, P<0.01 ws contr Drs MDA) NO’ NAG (NOS mmgrka? pad"! moh! UL O.2520.0F 94% oe esr 072018 S22) a3 585204 oasz02" Wes ose weer 0.3520.0% ele 1132. 4296+ 1D? DISCUSSION Proanthocyanidin, contained in 2 large number of plants. is very important in human health protection and Tab4. Effect of PA on content of TI-1f, TNFa, and PGE; in carrageenan-induced rat arthritis. "P< 0.05, *P<0.01 2s control. diseases prevention. Our results showed that PA. from grape seeds dose-dependently inkibited the plantar edema of rats induced by carrageenan and ear edema of mice induced by croion oil, The effective dosige of PA fromgrape seeds was 10 mg/kg, which was lower than that of PA from Polypodium fee’ roots, 200 mg/kg! ‘The antiphlogistic mechanism of PA from grape seeds was partly due t© the inhibition of prostaglandin. biosynthesis same as PA from Polypodiua fei roots. PANAG was released from Tysosome, its change of activity in the inflammatory exudate reflected sability of lysosome membrane. PA matked lessened fsNAG activity demonstrating that PA had protective effect on lysosome membrane Free radicals-induced lipoperoxidation has been im- plicated in over @ fundred diseases including arthritis Recent studies showed that nitric oxide (NO), which showed free radical feanure, was also a key inflammatory rmediatoz”), In our experiment, PA from grape seeds significantly suppressed the content. of fipoperoxidaion product MDA in carrageenan-induced inflamed paws of rats similar (o that in vitro'!"!, and markedly lessened the activity of NOS and the content of NO in existe of carrageenan-induced paw edema in rats. These rewults demonstrated that inhibition of lipoperoxidation and NO formation was one of ant-inflammatory mechanisms of n Fo8,/ Eton Ny iviton way Initia eb? ate % mel res ag Lt re/% Blank 0.122007 - oe - 0.520. - Consol 62210 - 51194 - Mes : Pato zestor we R50 obo ont w. Dex s2eh0 48.9 Tpaae 2.3 weit 3ISSN 8.9750 sa + E-mail sma shene2-cn ct Pratl Sig “PRP AEF 200 Dees 22 12) Finan Mt IO Pa. His well Known that IL-1 and TNFa were proinflammatory cytokines. They can interact with other inflammatory factors such as PGE>, NO, and five radicals, and take part in the formation and development ‘of rheumatoid arthritis") This experiment demon strated that PA obviously inhibited formation of IL-1 and TNFa. suggesting that the major ant-inflammatory mechanism of PA is related to its suppressive effect on the fommation of inflammatory cytokines, Because it also inhibits the lipoperoxidation and the formation of PGE, and NO, © ils setion range is quite extensive. But what the pivotal role of ant-inftammation of PA is and a what Point, PA affects inflammatory network remained t0 be clarified ‘REFERENCES 1 Shao ZH, Qn YM, Becker LB. Hoek TL. Li €Q, Schuacker PT, etal. Grape seed prunhocyanins reduce oxidant sess in canliomyocytes. Acad Emerg Med 2001 8: 562 YeX. Knit RLs Liv W, Joshi $$, Kuszyasti CA, MeGinn TR, eal. The eytonc effects of «novel THO arape seed rowuthacyaiin exact on cultura! huran cancer cells. Ml Cell Biccher 1999; 196: 99— 106 3. Sao My Mauik G, Roy 5. Baxhi D. Das DK. Cantioprtctive effets of grape seed proantocvanitin aginst fschemic reperfusion injury. 3 Mol Cell Carbo! 1999: 31 9-97 4 Subarus A. Wagner H. Analgesic and ant iflamestory setiviy of the proamboeyandin shelieguein A fram Pobpodin fei MET. Phytomedicine 2000; 7: 401~ 5, 3 Bhktneier CA, Cinatl J Je, Rabenau H, Doerr HW, Biber As Koch E, Anvil and antiphlogisic scivies of Hemant virginiana tak, Pata Me 196: 62; 241 ~5 (6 De Ofiveiam MIM, Santos M, Cari CA. Anglzsic activity of cdinerc preathocyanitins preliminary expeiments, Ang Ins Biol Sao Palo) 1975; 42; 14530. 7 Confort A, Cacti Py Saree L, Schisvon O. Veronese Fy Velo GP. Ani-nfanmaiory avity of mmometoxypaly cibylene glycol syperonide dismutase o advent rtrts in rats. Pharmacol Res 191; 23: 51-6. 8 Hu YE, Wa YI. Blfect of ecombjnan human taxi fro tau growth factor on acute inflation in mice and rs ‘Acta Pharmacel Sin 2005 22: 375-9. 9 Salvemini D, Wang ZQ. Wyatt PS, Bourdon DM, Marino MH, Manning PT. etal. Nisic oxide: key mediator inthe early and Ive phase of camigrenuindvedl rit paw inflammation, Br J Prarnaodl 199; 118; @9~ 38, 10 Zhang XY. Gao MT. 14 WG, Tian X. nw JH. The anu Tipoperoniative effect af GSE x vito In; Zheng RL. editor, Adkanccs in five radical lie sciences: v7. Beljngs Avoric Energy Publishing Howse: 1999, pl10—20 1 Chios GCY. Liu SXL, Systemic ai-inftammation by synthe imerleukin-I Whockers. Acta Pharmacol Sin 1999s, 29, 05-8. 12 Hii H Shin WS, Koizumi T, Takato T. Susaeni T, Koiauni ¥. et al. Peroxyitite production by TNF alps and Tetheta; implication foe suppesion of ostesblasic dferetaion, Am J Physiol Endocrinol Meub 2000; 270; Fast ~7 13) Brady TC, Cheng LY. Day BY, Capo JD. Exineelllar superoxide dismuase is upregulated with inducible isc oxide abuse ater NF-kappa B activation. Am J Physiol 1997; Lone —6. METH RES ROE A BLP, RDA, RPA, OB AME Fhe B OHS RRR ORE NASER AMT BRE ERE, LH 730000, TH) FRG MEHR: MOH, RE, APRA, ee NF: MAEM: ME: MRR ES, AR SRE Bil: He REP TEAR (PAIL RAE RAL W. Bik: ECS Mh BE A LR SMR RUE BOY, OTE PA WRAL BH. NOS iATEH] NADPH SOB MEI GIL, NO & TH! Gress & BUG IR BLU, BANAG FLAP AEM) iL. MDA TH TB: INGE, IL-LB, TNFa AI PGE, & BI a UAMae. SS: PA 10-40 me/ke ip HR TEI SW Fh RM EY FC BA EP AE SAY TEAR. PA 10 mg/kg Bik BL AL MDA ¢ ak, #4 UR aE 16 HRP NOS fi BNAG ith, MEME IL-1B, TNF #1 PGE, fi. SEH RE TO EAM 2 mg/kg. ie, PAK MRE RAE TH RE WH. TESLA OLALARER FLA ALE, MBER te An RRA FB Chie Bt)