Vishwrmn Mohn Prsd A.Thkurdesi

a Department of Pharmacology,Poona College of Pharmacy,Bharati Vidyapeeth Deemed University,Erandwane,Pune 411 038,India

b Indus Biotech Private Limited,1,Rahul Residency,Off Salunke Vihar Road,Kondhwa,Pune 411 048,India

Abstract Type-A procyanidine polyphenols(TAPP)are reported to have immunomodulatory and anti-inflammator potential in vitro.The objective of present work is to evaluate potential of TAPP extracted from Cinnamon(Cinnamomum zeylanicum)bark in animal models of inflammatio and rheumatoid arthritis in rats.Carrageenan-induced rat paw edema (CPE) and adjuvant induced established arthritis (AIA),in rats were used as the experimental models for inflammatio and arthritis respectively.Analgesic activity was evaluated in Randall-Selitto assay in AIA rats.TAPP showed significan anti-inflammator effect at dose of 4,8 and 25 mg/kg,p.o.but not at 2 mg/kg,p.o.dose in CPE model.The dose of 8 mg/kg,p.o.was selected for the evaluation of anti-arthritic activity in AIA model.TAPP(8 mg/kg,p.o.,daily from day-12 to day-21)treatment in established arthritic rats showed significan reversal of changes induced in AIA with respect to body weight drop(cachexia),ankle diameter,arthritic score,serum C-reactive protein(CRP)levels.Moreover,TAPP was found to be non-ulcerogenic as compared to AIA control rats.However,TAPP did not show analgesic effect on AIA-induced pain as seen in Randall-Selitto assay.In conclusion,TAPP showed disease-modifying potential in animal models of inflammatio and arthritis in rats.

Keywords: Type-A procyanidine polyphenols;Cinnamon bark;Anti-inflammatory Rheumatoid arthritis

1.Introduction

Rheumatoid arthritis (RA) is a chronic,inflammator,autoimmune disease,affecting freely movable joints,such as hand,knee and shoulder joints.The symptoms of active RA include pain,swelling,morning stiffness,warmth,redness,and limits in the functions of the joints.The systemic ramification of the disease,apart from morbidity and mortality,include cardiopathy,nephropathy,vasculopathy and pulmonary and cutaneous disorders[1].Although the cause of rheumatoid arthritis is unknown,autoimmunity plays a pivotal role in both its chronicity and progression,and RA is considered as a systemic autoimmune disease.

Existing treatment therapies for RA usually focused on anti-inflammator activity.They help to check the process of inflammatio and thus may also help in the repair process.Medications like non-steroidal anti-inflammator drugs (NSAIDs),corticosteroids and analgesics are used to suppress the symptoms while disease-modifying anti-rheumatic drugs(DMARDs)and biological response modifier often required to inhibit or halt the underlying immune process and prevent long-term damage.

Although NSAIDs,DMARDs and corticosteroids appear to be highly efficien drugs in the treatment of rheumatoid arthritis,they may cause side effects that can range in severity from mild to serious.The major adverse drug reactions (ADRs) associated with NSAIDs are gastrointestinal(ulceration or bleeding),and cardiovascular(myocardial infraction)with effects on other systems.Despite of its wide clinical use,gastrointestinal (GI)toxicity,upper GI adverse events such as perforation,ulceration and bleeding in about 20%of patients taking long-term NSAIDs is a major clinical limitation.Besides,some NSAIDs were withdrawn from the market because of the risk of heart attacks and stroke in some and some others had“Black box”warning in the package insert addressing risk of suffering from heart attacks and/or stroke [2].On the other hand,other therapies against arthritis such as DMARDs and biological agents had a risk of immunosuppression and serious infection respectively on longterm usage.Therefore,search for safer drugs for management of rheumatoid arthritis for chronic use is still going on.

Cinnamon(Cinnamomum zeylanicumSynC.verum,family:Lauraceae)bark is one of the oldest herbal medicines mentioned in many traditional text for inflammator [3],and pain related to disorders such as enteralgia(acute intestinal pain),bronchitis,and rheumatism [4].It is native to Sri Lanka,Mayanmar(Burma) and the southern coastal strip of India.In Chinese traditional medicine,cinnamon is indicated as an analgesic and antipyretic agent against cold,fever,headache,myalgia(mascular pain),arthralgia(arthritic pain)and amenorrhea(failure of menstruation).In Indian traditional literature including Ayurveda,many other valuable actions are attributed to cinnamon bark[5].

Many scientifi pharmacological investigations are also reported on anti-inflammator potential of the bark of many species of cinnamon [4,6,7].The anti-inflammator action of the Japanese speciesCinnamomum seiboldii[8]andCinnamomi cortex[9]has been attributed to a series of tannins.The antinociceptive activity(analgesic)[10]and antipyretic(fever reducing)activityC.verumbark[8]were also been reported.

An interesting factor about cinnamon is that it can act both as an immunostimulant and a suppressant depending on species and doses[11].Another important activity is the immunomodulatory effects exerted by cinnamon bark.In vitroinhibitory activity against complement formation has been documented for cinnamon cortex and oil[11].The extract of cinnamon bark is reported to have anti-complementary[12]and immunosuppressive [13]activity.Cinnamon bark’s potential for inflammation pain and immune system makes it a good candidate as an antiarthritic agent[14].However,the component(s)responsible for these activities is not known.Cinnamon bark polyphenol extract had been shown anti-inflammator propertiesin vitroand therapeutic potential for prevention and treatment of inflammatio related diseases[15,16].

The oligomeric end products of the fl vonoid biosynthetic pathway in Cinnamon are procyanidins whose building blocks are (+)-catechin and (-)-epicatechin [17].Proanthocyanidin(procyanidin,oligomeric proanthocyanidins,leukocyanidin,leucoanthocyanin and condensed tannins)is a class of fl vanols,are now identifie and recognized for their favorable effects in human beings.

The anti-inflammator action has been attributed to polyphenolic components such as tannins[8]and procyanidins[17]from a variety of natural products.Based on the linkage between the successive monomeric units,procyanidins are classifie as Types A,B or C procyanidine polyphenols.Type-A procyanidine polyphenols (TAPP) from cinnamon bark has been reported to exhibit activities against microorganisms[18],suggesting a potential role of TAPP in regulating immune function with unknown mechanism.Further,TAPP has been shown anti-inflammator effects in cell culture studiesin vitro[16].Recently,we have demonstrated ameliorative effects of TAPP from cinnamon bark in animal model of allergic asthma [19].However,TAPP has not yet been evaluatedin vivousing animal models of rheumatoid arthritis (RA).The objective of present work is to evaluate effica y and safety of TAPP isolated fromC.zeylanicumin animal model of acute inflammatio and RA in rats.

2.Materials and methods

2.1.Materials

Wistar rats of either sex (130-200 g) were obtained from National Toxicology Center,Pune,India.The rats were divided into groups having six rats per group and housed in polypropylene cages at a temperature of (24±1)°C with 12 h:12 h dark-light cycle,with free access to standard pellet feed(Chakan Oil Mill,India)and filtere water.All experiments were carried out between 08:00 am and 17:00 pm in a quiet laboratory.The research protocol was approved(No.CPCSEA/48/08)by Institutional Animal Ethics Committee (IAEC) and as per Indian norms laid down by Committee for the Purpose of Control and Supervision of Experimental Animals (CPCSEA),New Delhi.

The test compound,TAPPwas provided by Indus Biotech Private Limited,Pune,India,after characterization as per reported procedures [19-21].The TAPP is a standardized extract ofC.zeylanicumbark with pentameric type-A procyanidine fl vonoid as a marker compound(75.9%purity)with presence of trimer and tetramer.The solutions of TAPP was freshly prepared daily by dissolving in mixture of propylene glycol and distilled water in ratio of 30:70 to make solution of 1 mg/mL concentration and administered orally.

2.2.Anti-inflammatory activity against CPE model

Anti-inflammator activity was evaluated on the basis of the inhibition of the CPE[22,23].Each rat in the experimental group(six rats each) was treated orally with 2,4,8 and 25 mg/kg body weight,1 h before an edematogenic agent,carrageenan(Sigma-Aldrich,USA)injection.Carrageenan(0.1 mL of a 1%suspension prepared in 0.9% NaCl) was injected into the subplanter aponeurosis of the left hind paw of rats with 26-gauge needle.One group of rats was treated orally with 5 mg/kg body weight of diclofenac sodium as a standard drug.Separate vehicle control group was also maintained.Paw volume was measured immediately after carrageenan injection (time 0) and at 1,3,6 and 24 h using a plethysmometer (Model 7150,Ugo Basile,Italy).Mean difference in paw volume (mL) was calculated with baseline (0 h) values for each rat.Percent (%) inhibition of paw volume at 3 h were calculated with a reported formula[24]from mean difference in mean paw volume(mL)in treated(Vt)and control(Vc)group according to the equation:%inhibition=(Vt/Vc)×100.

2.3.Effect on adjuvant-induced established polyarthritis(AIA)

Arthritis was induced according to method described earlier [25]and as modifie for therapeutic schedule.AIA was induced in 6-8 week-old rats by a single intradermal injection of 0.1 mL Freund’s complete adjuvant (FCA) into the footpad of the right hind paw.FCA contains 0.6 mg heat-inactivatedMycobacterium tuberculosisH37Ra(Difco Laboratory,Detroit,MI,USA)emulsifie in a sterile mixture of paraffi oil,saline and Tween-80.Arthritis was allowed to develop for next 12 d.On day 12,animals were randomly assigned to one of the following treatment groups:arthritic untreated controls(AIA controls),arthritic rats treated with diclofenac sodium (1 mg/(kg·d)) and arthritic rats treated TAPP (8 mg/(kg·d)).These groups of rats were orally treated once daily with vehicle (distilled water),diclofenac sodium or TAPP respectively from the day-12 to day-21 by oral feeding needle.Body weight and pain latency of inflame paw,ankle diameter,arthritis score and serum Creactive protein(CRP)levels were measured on day-0(baseline,before arthritis induction),day-12 and day-21 of the study.Total body weight was recorded every 3rd day from day 3 to day 21.The increase in body weight was calculated relative to that at day 0 allowing monitoring of the decrease in body weight gain associated with arthritis[26].The diameter of the inflame paw joint (ankle) was measured by Vernier Caliper [27].The joint was compressed by pressing the gauge till pain was elicited as indicated by squeaking or leg withdrawal.The distance moved by the screw gauge was recorded.

Swelling in hind paws were recorded by using a plethysmometer (UGO Basile Italy.) on day-0,day-12 and day-21 after the FCA injection [28].The rats were scored regularly for arthritis severity score based on swelling of paws [29]by two investigators who were blind to the treatment.Each paw was graded according to the severity and extent of erythema and swelling of periarticular soft tissues in the enlargement and distortion of the joints.The arthritis scores were ranged from 0(no sign)to 4(severe lesions),yielding a maximum score of 16 per animal.Pain stimulus was measured by the method reported earlier[30].

2.4.Biochemical estimations

The measurement of C-reactive protein was carried out according to earlier reported method[31].Blood samples were withdrawn retro orbital puncture under light anesthesia on day-0,day-12 and day-21 of the study.The serum was separated and samples were analyzed for CRP by commercially available enzyme-linked immunesorbent assay(ELISA)kit for CRP(R&D system,USA) according to the manufacturers’ recommendations.

2.5.Randall-Selitto assay

Pain latencies were measured using Randall-Selitto Analgesymeter(Ugo basile Model 7200)during AIA model on day 0(induction of AIA),day-12(start of treatments)and day-21(end of the study).Pain latencies were measured by the threshold stimulus for reaction(escape or paw withdrawal)using a weight of 500 g applied to the pads of hind paws.

2.6.Serum turbidity measurements

On day-21,rats were sacrifice by cervical dislocation after administration of light ether anesthesia.Blood was withdrawn by cardiac puncture.The serum turbidity of sacrifice arthritic rats on day-21 was measured by earlier reported method [32].To 0.1 mL of nonhaemolyzed serum,2.9 mL of 0.067 mol/L sorenson phosphate buffer composition:2 mL,0.067 mol/L monopotassium phosphate+98 mL,0.067 mol/L disodium phosphate in saline (pH 5.2) was added and gently agitated.The mixture was allowed to stabilize at room temperature for 15 min before being incubated in a constant temperature water bath at 69°C for 30 min.The samples were cooled in an ice bath and absorbance was read as serum turbidity in a spectrophotometer(Shimadzu,Japan)at the wavelength of 645 nm.

2.7.Microscopic study

Microscopic evaluation was done by analyzing the histology of stomach sections [60].Freshly excised stomach of one animal from each group was washed with saline and preserved in 10% formaldehyde solution for histopathological studies.They were processed for 12 h using isopropyl alcohol,xylene and paraffin Then sections of stomach were cut (5 μm thickness)with microtome.Sections were deparaffinatio and stained using hematoxylin and eosin (H&E) stain.The sections were then observed and photomicrographs were taken for histology assessment.

2.8.Statistical analysis

Data were obtained for each set of parameters separately for experiments namely CPE and AIA model and expressed as means±SEM.Data were analyzed by two-way repeated measures ANOVA followed by Bonferroni posttests separately for each parameter.Data for serum turbidity measurement were analyzed by one-way ANOVA followed by Dunnett’s test.Level of significanc was set atP＜0.05.All statistical tests were carried out using Prism 5.0(Graph Pad,San Diego CA,USA)statistical software.

3.Results

3.1.Effect of TAPP in CPE model

Subplanter injection of carrageenan produced significan amount of edema(increase in paw volume)in all the groups of animals.Increase of paw volume(edema)was observed within an hour (paw volume increases by 0.69 mL).This edema was sustained up to 6 h(increase by 1.65 mL)(Table 1).The standard NSAID,diclofenac sodium (5 mg/kg,p.o.) caused significan reduction in edema (anti-inflammator effect) compared with vehicle treated rats (P＜0.001) at 1 h of treatment until 6 h.Oral treatment of the test compound,TAPP showed significan(P＜0.001)anti-inflammator effects at 4 mg/kg,8 and 25 mg/kg dose levels.However,at 2 mg/kg,p.o.dose,TAPP did not show anti-inflammator effect.The onset of effect for TAPP was at 3 h,3 and 1 h for dose levels of 4,8 and 25 mg/kg respectively.At 3 h,TAPP (8 and 25 mg/kg) showed 27.47%,35.21% and 63.38% inhibition of edema respectively compared to 88.02%inhibition showed by diclofenac sodium(5 mg/kg)treatment.

Table 1 Effect of various doses of TAPP against carrageenan-induced rat paw edema(CPE)in rats.

3.2.Effect of TAPP on AIA in rats

3.2.1.Effect on body weight reduction(Cachexia)in AIA rats

Single intradermal injection of FCA into the footpad of the rat hind paw brought significan (P＜0.001) reduction in body weight (cachexia) in all the rats on day-12 of the study.In AIA control rats treated with vehicle,the weights were further reduced (from 202.50 g to 184.67 g) during day-12 to day-21(Table 2).In the same period (day-12 to day-21),subacute oral administration of diclofenac sodium (1 mg/kg) halted the cachaxia of AIA (P＜0.01).The body weights were increased from 197.17 g to 206.5 g after diclofenac sodium treatment.Similar cachexia reversal effect was found in rats after subacute oral treatment (day-12 to day-21) with TAPP in AIA.TAPP(8 mg/kg)showed significan (P＜0.05)increase in body weight from 196.50 g to 208.33 g.

3.2.2.Effect on ankle diameter in AIA rats

Ankle diameter of inflame paw in AIA rats were found to increase significantl (P＜0.001)in all rats on day-12 compared with day-0 of the study(Table 2).On day-21 of the study,mean ankle diameters of rats with AIA rats treated with vehicle did not have significan change(Table 2)compared with day-12.On the other hand,significan (P＜0.001) reduction in ankle diameter of AIA rats was found on day-21 compared with day-12 with treatment of diclofenac sodium(from 7.64 mm to 6.82 mm)and TAPP(from 8.30 mm to 7.30 mm)(Table 2).

3.2.3.Effect on serum C-reactive protein levels in AIA in rats

The C-reactive protein (CRP) level in serum samples were found to increase on day-12 of the study in all rats (Table 2).The increase in serum CRP levels was sustained on day-21 in AIA control rats.On day-21,significan reduction of CRP levels were found in serum samples of AIA rats treated with diclofenac sodium (from 10.24 mg/mL to 7.47 mg/mL) and TAPP (from 10.28 mg/mL to 8.15 mg/mL)(Table 2).

3.2.4.Effect on total arthritis score in AIA rats

The total arthritis score in inflame paw was increased significantl in 12 d after induction of arthritis(Table 3).Further,increase in total arthritis score was found in AIA control rats on day-21 of the study(Table 3).However,significan reductionin arthritis score of AIA was brought by subacute treatment of diclofenac sodium (from 6.83 to 2.67) or TAPP (from 6.17 to 2.00)as observed on day-21 of the study(Table 3).

Table 2 Effect of TAPP(8 mg/kg,p.o.)on body weight,ankle diameter and serum C-reactive protein(CRP)levels against FCA-induced established polyarthritis(AIA)in rats.

Table 3 Effect of TAPP(8 mg/kg,p.o.)on total arthritis score and pain latency against FCA-induced established polyarthritis(AIA)in rats.

3.2.5.Effect on pain latency of inflamed paw(Randall and Selitto assay)in AIA rats

The pain latency tested in Randall and Selitto analgesiometer was found to decrease significantl in all rats.The reduction was found to be as high as 19.50 to as low as 3.83 (Table 3).The pain latencies were found to further decrease to 2.25 sec in AIA control rats.Subacute administration of diclofenac sodium or TAPP in AIA rats did not change pain latencies on day-21 compared to day-12(Table 2).

3.2.6.Effect on change in serum turbidity in AIA rats

Absorbance of various serum samples on day-21 in AIA control rats are presented in Fig.1.The serum samples of nonarthritic rats showed absorbance of 0.3493 whereas AIA control rats(vehicle treated)reduced absorbance to 0.1295(P＜0.001).The subacute treatment with diclofenac sodium in AIA control rats from day-12 to day-21 did not cause any change in absorbance compared with serum samples of AIA control rats.The serum samples of AIA rats treated with TAPP showed mild increase(from 0.1295 to 0.1621,P＜0.05)in absorbance compared with AIA control(Fig.1).

Fig.1.Effect of TAPP(8 mg/kg)on serum turbidity of FCA-induced arthritic rats on day-21.Data are represented as absorbance at 645 nm±SEM and was analyzed by one-way ANOVA followed by Dunnett’s “t” test.Absorbance of serum samples was measured on day-21 of the study. n=6. ###P ＜0.001 as compared with non-arthritic rats;*P ＜0.05 as compared with arthritic control group.

3.2.7.Histology of gastric mucosa in AIA rats

Gastric mucosal superficia layer of stomach samples of rats removed from normal non-arthritic (Fig.2A) rats were found to be smooth.The mucosal epithelial layer of stomach sections of AIA control rats treated with vehicle (Fig.2B) or TAPP at oral dose of 8 mg/kg(Fig.2D)were also found to be normal and smooth with mild capillary dilation.However,superficia layer of gastric mucosa of AIA rat treated with subacute treatment of diclofenac sodium(1 mg/kg,p.o.)had multiple areas of erosions and hemorrhage(Fig.2C).

4.Discussion

The cinnamon bark polyphenol extract is water-soluble and can be standardized to type-A proanthocyanidins [33].In the present study,we are reporting anti-inflammator and antiarthritic potential of type-A proanthocyanidins from cinnamon bark,TAPP,in animal models.

The recognition of different mediators for different phases of CPE has important implications for interpreting the antiinflammator effect of the drugs.The development of such edema in the rat paw is a biphasic event.The initial phase of the edema has been attributed to the release of histamine and serotonin which is then maintained during the plateau phase because of kinin-like substances [34].Further,involvement of histamine and serotonin with other mediators in the pathogenesis of rheumatoid arthritis was also reported by many researchers[35-37].Effectiveness of the test compounds in the 1st hour after carrageenan injection is indicative of their antihistaminic and/or anti-serotonin action.The 2nd accelerating phase of carrageenan edema is attributed to release of prostaglandins[38].Our test compound,TAPP was not effective in the 1st hour of treatment but was found effective in the 2nd phase at 8 mg/kg and higher dose(observed at 3 h)thus suggesting prostaglandins inhibition effect of TAPP(Table 1).However,possibility of other mechanism such as inhibition of leukocyte emigration by TAPP cannot be ruled out.

The ability of anti-inflammator test compounds to offer benefit in clinical management of rheumatic diseases and so modificatio of arthritic syndromes in laboratory animals is an important criterion in anti-inflammator screening procedure.In 1963,AIA in rats was used as primary animal model for RA using intradermal administration of Freund adjuvant in oil emulsion [39].AIA is a rather aggressive and monophasic form of arthritis.Usually,the AIA is severe and finall leads to complete ankylosis and permanent joint deformations similar to progressive symptoms of RA patients.AIA has been used extensively as an experimental model for studying immune-inflammator processes of arthritic diseases in humans,in particular RA,as well as for screening and testing novel anti-arthritic agents[40].Therefore,we tested TAPP against established polyarthritis model of AIA rats using therapeutic schedule of treatment [25,41].We induced AIA in rats,allowed it to develop for 12 d and evaluated effects of TAPP on day-21 of the study.The progression of arthritis was confirme in our study by scoring total arthritis lesions.The diseases progressed till day-12 and then became quiescent.TAPP halted the progression which was evident by significan decrease shown in the total arthritic score(Table 3).

The inflammatio associated with AIA is mainly dependent on prostaglandin E2(PGE2)generated by cyclooxygenases(COXs)[40,42].Besides,the role of cytokines like TNF-α and IL-1 has also been implicated in this model[43].TAPP treatment to AIA rats reduced ankle diameters of arthritic paw.Therefore,the anti-inflammator action of TAPP may be mediated by prostaglandin and/or cytokine inhibition.These results are also in line with reports that anti-inflammator action of cinnamon bark has been attributed to polyphenolic component such as tannins [8]and procyanidines [17].The inhibition of lipid peroxidation,capillary permeability and fragility,and enzymes such as phospholipase A2,cyclooxygenase,and lipoxygenase were reported as alleged mechanism for cinnamon tannins and polyphenols[8,17].

Fig.2.Photomicrograph of stomach on day-21 of the study.The figur shows sections of stomach of (A) normal rats,(B) AIA control rats with treatment from day-12 to day-21 of vehicle(distilled water),(C)diclofenac sodium(1 mg/kg,p.o.)treated arthritic rats shows erosion(arrow)with hemorrhage at the superficia part of the mucosa and(D)TAPP(8 mg/kg,p.o.)treated arthritic rats;M.mucosa,MM.muscularis mucosa,and L.lumen.Stained with hematoxylin and eosin(H&E)at 40×.

Analgesia is an important ancillary property of all antiinflammator agents.Most of the anti-inflammator drugs increased pain threshold in various animal models [44].This is natural because many endogenous chemical mediators of inflammatio play a part in generating pain impulses(for example,histamine,serotonin and prostaglandins) and some other mediators such as bradykinin and cytokines,are involved in prolongation of the sensation of the pain[45].

RA is an inflammator condition of the joint that is associated with hyperalgesia and functional impairment[46].Joint inflam mation induced by AIA is associated with hyperalgesia and slight compression of the joint which caused leg withdrawal and squeaking.The hyperalgesia associated with arthritis has been reported to involve prostaglandin synthesis[47].Increased levels of substance P,calcitonin gene-related peptide and up-regulation of neurokinin-1 receptor in the dorsal horn of the rat spinal cord have been reported to play an important role in modulating both inflammatio and hyperalgesia in the FCA model[48].However,in the present study,TAPP did not prevent reduction pain latency in Randall-Selitto assay.Therefore,nociceptionrelated mediators(substance-P,calcitonin gene-related peptide and neurokinin-1) are not involved in mechanism of action of TAPP.This also pointed toward the possibility of disease modificatio as possible mechanism of action of TAPP against RA.

AIA has been generally believed to be the result of a delayedtype hypersensitivity(DTH)response to a disseminated antigen probably derived from the injected bacterial cell wall[49].AIA in rats is a useful model of inflammator cachexia that mimics the human pathophysiology in many important ways [50].AIA induces inflammatio as primary lesion which reached to peak after 3-5 d with secondary lesions occur after a delay of about 11-12 d.Secondary reactions in AIA are characterized by immune responses,inflammatio and decrease of weight(cachexia).In our study,the test compound,TAPP significantl protected rats from cachexia because of arthritis induction.

In the past,many plant-derived procyanidines reported to have cytokine inhibitory effectin vitro[51,52].Similar effects were also attributed to Cinnamon bark extract and polyphenols[16,53].In vitropre-challenge with cinnamon extract is reported to suppress lipopolysaccharide(LPS)-induced cytokines expression[54].Recently,immune regulatory action of TAPP in mouse 3T3-L1 adipocytes is attributed to TTP (tristetraprolin) [55].Besides,TAPP is capable of affecting immune responses by regulating anti-inflammator mediators as well as the TTP gene expression in macrophages[16].Taken together,cytokine inhibition emerges as a probable mechanism of TAPP responsible for cachexia reducing effects in AIA-induced established polyarthritis in rats.This probability is also supported by our result that TAPP reduces serum C-reactive protein (CRP) levels of rats on day-21 compared to day-12 levels (Table 2) whereas AIA control rats showed sustained increase in CRP till day-21(Table 2).

CRP is an acute-phase protein and has been identifie as an important biomarker for various inflammator,degenerative,and neoplastic diseases [56,57].Elevated levels of CRP have been found in the blood during almost all diseases associated with active inflammatio or tissue destruction,particularly in RA patients [57,58].Sustained increase in serum CRP levels suggests a lasting production and stimulation of acute-phase proteins during disease progression.

In our study,serum turbidity (showed by absorbance of serum samples) of AIA control rats was found to be signifi cantly reduced compared with normal rats (Fig.1).Treatment from day-12 to day-21 with TAPP but not diclofenac sodium(an NSAID) prevented AIA-induced fall in turbidity (Fig.1).The decrease in serum turbidity in AIA control rats,is known to be caused by denatured products and was shown to be correlated with severity [59]and immune response seen in AIA[60].Further,increased levels of serum lysozyme levels were reported to be strongly correlated with decreases in serum turbidity in AIA [59].Therapeutically useful anti-inflammator drugs were shown to reverse this decrease in serum turbidity[32].Taken together,our results strongly pointed to diseasemodifying properties of TAPP and its potential to slow down the RA progression.

One of the most prominent and serious side effects of NSAIDs is occurrence of gastrointestinal damage(ulcerogenicity).Hence,it was necessary to test our compounds for their ulcerogenicity potential.In the present study,subacute treatment of TAPP(8 mg/kg,p.o.)did not cause severe ulcerogenicity as compared to significan ulcerogenicity shown by diclofenac sodium(1 mg/kg,p.o.)in established AIA in rats.

The absence of the analgesic and the ulcerogenicity potential with reduction in serum CRP levels in AIA rats qualifie TAPP as a disease-modifying anti-rheumatic drug (DMARD).DMARDs suppress the rheumatoid process and bring about a remission,but do not have nonspecifi anti-inflammator or analgesic action.TAPP offer added advantage of moderate antiinflammator effects at daily dose of 8 mg/kg,p.o.in arthritic rats.The DMARD effects shown by TAPP can also be substantiated by earlier reports on cinnamon bark extracts.For example,cinnamon bark was shown to inhibit gastric secretions[11]and eradicateH.pylori(a bacteria known to exacerbate gastric acidity and ulcers)[61,62].TAPP is also expected to be protective on the cardiovascular system as cinnamon polyphenols especially TAPP by virtue of its antioxidant activity[63,64].

5.Conclusion

In conclusion,TAPP,type-A procyanidine polyphenols isolated from the bark ofC.zeylanicumshowed anti-inflammor and anti-arthritic effects in animal models without ulcerogenicity potential.Lack of analgesic activity in present study and reports of immunomodulatory potential suggested TAPP as potential DMARD.Further studies are required to explain the mechanism of action of TAPP toward autoimmune and inflam matory disease processes.

Acknowledgements

The authors would like to acknowledge Dr.K.R.Mahadik,Principal,Poona College of Pharmacy,Bharati Vidyapeeth Deemed University,Pune,India for providing necessary facilities to carry out the study.Research support was provided by Indus Biotech Private Limited but played no role in collection,analysis and interpretation of data.