ANTI-INFLAMMATORY AND ANTI-ARTHRITIC ACTIVITY OF A POLYHERBAL FORMULATION, BAIDYANATH MAHARASNADI KADHA IN EXPERIMENTAL RATS

M.Pharm. Dissertation Protocol

Submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore.

By

DAYANAND

B.Pharm.

Under the guidance of

Mr. SHIV KUMAR

M.Pharm

Assistant Professor,

Department of Pharmacology,

NET Pharmacy College, Raichur

DEPARTMENT OF PHARMACOLOGY

N.E.T PHARMACY COLLEGE

RAICHUR

2010-2011.

Rajiv Gandhi University of Health Sciences, Karnataka

Bangalore

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1 / Name of candidate and address (In Block Letters) / DAYANAND.B.CHAWARE
R/S - HULSOOR
TALUKA-BASAVAKALYAN.
DISTRICT- BIDAR.
KARNATAKA.
2 / Name of the Institute / N.E.T. PHARMACY COLLEGE,
RAICHUR.
3 / Course of study and subject: / M.PHARM. PHARMACOLOGY.
4 / Date of admission of course: / 03-07-2010
5 / Title of the topic:ANTI-INFLAMMATORY AND ANTI-ARTHRITIC ACTIVITY OF A POLYHERBAL FORMULATION,BAIDYANATH MAHARASNADI KADHA IN RATS
6 / Brief Resume of the intended work:
6.1: Need for the study Enclosure-I
6.2: Review of Literature Enclosure-II
6.3: Main objectives of study Enclosure-III
7 / Materials and Methods:
7.1 Source of data Enclosure-IV
7.2 Method of collection of data Enclosure-V
7.3 Does the study require any investigation or interventions to be conducted on patients of humans or animals? If so, please describe briefly.
YES (Rats)
7.4 Has ethical clearance been obtained from your institution in case of 7.3?
YES: IAEC NO.:576/2002/bc/IAEC/CPCSEA
8 / List of References Enclosure-VI
9 / Signature of the candidate / (DAYANAND)
10 / Remarks of the Guide / The work proposed in this synopsis can be carried out at our institute.
11 / Name and designation of
(In block letters)
11.1 Guide
11.2 Signature / Mr. SHIV KUMAR
M.Pharm
Assistant Professor,
Department of Pharmacology,
NET Pharmacy College, Raichur
11.3 Co-Guide (if any)
11.4 Signature / ------
------
11.5 Head of Department
11.6 Signature / Dr. BHEEMACHARI
M. Pharm.,Ph.D
PROFESSOR & HOD,
Department Of Pharmacology,
N.E.T. Pharmacy College,
Raichur.
12 / 12.1Remarks of the Chairman and Principal
Signature / The work proposed in this synopsis can be carried out at our institute.
Dr. H. DODDAYA
M.Pharm., Ph.D
Principal,
N.E.T. Pharmacy college,
Raichur 584103.

Enclosure-I

6) Brief resume of the intended work.

6.1: Need for the study:

Rheumatoid arthritis (RA) is a common autoimmune disease characterized by chronic inflammation of the synovial membrane with concomitant destruction of cartilage and bone. Although the etiology and pathogenesis of Rheumatoid arthritis is not yet understood, it has been suggested that abnormalities of cytokines play an important role in pathogenesis1.

In the Version 2 estimates for the Global Burden of Disease 2000 study, published in the World Health Report 2002 (3), RA is the 31st leading cause of YLD (Years lived with Disability) at global level, accounting for 0.8% of total global YLDs2. About 1% of the world's population is afflicted by rheumatoid arthritis, women three times more often than men. Onset is most frequent between the ages of 40 and 50, but people of any age can be affected3.

The management of Rheumatoid arthritis was revolutionized with the advent of corticosteroids and Disease Modifying Anti-Rheumatic Drugs (DMARD’S) like Methotrexate, Sulphasalazine and Leflunomide. Conventional DMARD’S however have several limitations like slow onset of action, induction of partial remission and modest 5 year retention rates4.

Further, for the management as well as treatment of various inflammatory disorders, drugs like Ibuprofen, Diclofenac and other NSAIDS are quite often prescribed. These drugs act by inhibiting the synthesis of prostaglandins. At the same time they also cause lot of adverse effects ranging from gastric ulcer to renal and vascular complications5. Although a panel of drugs has been developed for the treatment of Rheumatoid arthritis, therapies are not satisfied with these marketed drugs. An ideal drug for treatment of Rheumatoid arthritis still awaits discovery.

The rich floral diversity of India has provided herbal health practitioners and other traditional healers in the country with an impressive pool of natural pharmacy from which plants are selected as ingredients to prepare herbal remedies and medicines (phyto medicine) for the treatment, management and control of a variety of human ailments. Furthermore there is a growing evidence that several classes of compounds found in plants exhibits anti-inflammatory and anti- arthritic with fewer side effects.

Considering the available literature in ayurveda, we propose to study the Anti-inflammatory & Anti-arthritic activity of a marketed poly herbal formulation for inflammatory disorders, “Baidyanath Maharasnadi kadha”, manufactured from Shree Baidyanath Ayurved Pvt. Ltd. Kolkata. It is a preparation of Prawahi Kwath variety in which the decoction of the herbs with additives is fermented like in Asav preparation.6

However, there are not adequate experimental evidences about the effectiveness of Baidyanath Maharasnadi kadha as anti-inflammatory and anti-arthritic drug.

In this present context, an attempt of study is proposed to evaluate the effect of Baidyanath Maharasnadi kadha on inflammation and arthritis in rats.

Enclosure-II

6.2) Review of literature:

  1. Through survey of literature (National and International Journals, HELINET RGUHS Bangalore) reveals that, inflammation and arthritis are increasing day by day disappointingly.Various reasons have been cited for such inflammation and arthritis, due to some abnormalities of cytokines play an important role in pathogenesis1. However, the inflammation and arthritis protection is unsatisfactory with modern medicine.
  2. Upon literature, survey several plants and their formulations displayed claiming to be effective and their result is unsatisfactory.
  3. In search for a good plant or polyherbal formulation, which treats inflammation and arthritis, we found it would be much useful and worthful to evaluate a formulation rather than a medicinal drug. Since,formulation will be readily available in the market and used by the community.
  4. Baidyanath Maharasnadi kadha is proprietary Ayurvedic medicine manufactured by Shree Baidyanath Ayurved Bhavan PVT.Ltd Kolkata.6
  5. Maharasnadi kadha, a herbal formulation contains fourteen plants namely Rasna (Alpinia galanga), Amaltas (Cassia fistula Linn), Bala (Hibiscus tiliaceus), Erand (Ricinus communis), Javas (Syzygium aromaticum),Sontha (Zingiber officinale), Gokshurak (Tribulus terristris), Ashwagangdha (Withania somnifera), Devdar (Cedrus deodara), Brihati (Solanum indicum), Vidariknd (Ipomoea digitata), Kachura (Curcuma zedoaria), Guggulu (Commiphora wightii) and Punarmotha (Boerhavia diffusa).6
  6. Baidyanath Maharasnadi kadha (prawahi quath) is decoction obtained after boiling the herbs in water. Thus it contain water , soluble substances and is more powerful than churnas.It is easily assimilated in the system.
  7. Rasna (Alpinia galanga) is oneactive ingredient of Baidyanath Maharasnadi kadha,rhizome used as carminative(in dyspepsia), stomachic, circulatorystimulant, diaphoretic, anti inflammatory.7
  8. Amalatas (Cassia fistula Linn.).Its The whole plant possesses medicinal properties useful in the treatment of skin diseases, inflammatory diseases, rheumatism, anorexia and jaundice.8
  9. Bala (Hibiscus tiliaceus) belonging to family Malvaceae. Roots, flowers, bark used as anti rheumatic.9
  10. Erand (Ricinus communis)10 is usedOilfromseedsandyoungleaf—purgative.Oilisusedin dermatosis andeczema.Root— anti inflammatory,11adecoctionis administered forlumbagoandallied complaints. Bark—purgative.
  11. Javas (Syzygium aromaticum)12 belonging to family Myrtaceae. Leaves and roots used as vermifuge. Plant—stimulant, antirheumatic, anti inflammatory.13Berries—white, thesizeofapea; edible.
  12. Sontha (Zingiber officinale)14belonging to the family Zingiberaceae.Rhizome used as antiemetic, antiflatulent, hypocholesterolaemic, anti-inflammatory, antispasmodic.15expectorant, circulatorystimulant, diaphoretic, increasesbioavailabil-ity of prescription drugs. Used for irritable bowel and diarrhoea, colds and influenza.
  13. Gokshurak (Tribulus terristris)16belonging to the family Zygophyllaceae. Fruits used as diuretic, demulcent, anti-inflammatory,17 anabolic, spasmolytic, musclerelaxant,hypotensive, hypoglycaemic.
  14. Ashwagangdha (Withania somnifera)18 belonging to the family Solanaceae. Root—used as an anti inflammatory19 drug for swellings, tumours, scrofula and rheumatism; and as a sedative and hypnotic in anxiety neurosis. Leaf—anti-inflammatory, hepatoprotective, antibacterial. Fruits and seeds—diuretic. Withanine—sedative, hypnotic. Withaferin A—major component of biologically active steroids; as effective as hydrocortisonedose for dose.
  15. Devdar (Cedrus deodara)20 belonging to the family Pinaceaae Bark used as decoction is used internally as astringent,antidiarrhoeal and febrifuge. Essentialoil—antiseptic (used in skin diseases). Anti inflammatory.21
  16. (Solanum indicum)22. Plantand root used as stimulant,digestive,carminative, astringent,expectorant,diaphoretic,anthelmintic.Usedforcatarrhalaffections,asthma,drycough;dysuria;intestinalworms;colic,flatulence,vomiting.Berries—usedinasthmaandrheumatism.
  17. Vidarikand (Ipomoea digitata)23 belonging to the family Convolvulaceae used as cholagogue,galactagogue,alterative,demulcent, purgative. Resinfromroot—usessimilartoJalap.Flourofrawrhizomeisgiven in enlargementofliverandspleen,alsoformenorrhagia,debility and fataccumulation.
  18. Kachura(Curcuma zedoaria)24 belonging to family Zingiberaceae is used as carminative, stomachic, gastrointestinal stimulant, diuretic, expectorant, demulcent, rubefacient. Used inflatulence and dyspepsia. Fresh root is used for checking lecorrhoeal discharge; also for blood purification. Zedoary’s effect on digestive organs is similar to ginger but milder. Anti inflammatory.25
  19. Guggulu(Commiphora wightii)26 belonging to family Burseraceae Oleo-gum-resin used for reducing obesity and in rheumatoid arthritis, osteoarthritis, sciatica in inflammatory condition.27
  20. Punarmotha (Boerhavia diffusa)28 belonging to family Nictaginaceae is used as diuretics, anti inflammatory, anti bacterial, anti spasmodic.

Enclosure-III

6.3) Main objectives of the study:

The main objective of the proposed work is to evaluate the anti-inflammatory and anti-arthritic activity of Baidyanath Maharasnadi kadha, a poly herbal formulation. The whole study is divided into two phases:

Phase I:

1. Determination of LD50 and dose selection for the study (i.e. selection of two doses 1/20, and 1/5 from the LD50 value and considered as low and high doses respectively).

Phase II:

To evaluate anti inflammatory activity of the extracts in experimental animal models like:

  1. Carrageenan induced paw oedema in rats.
  2. Cotton pellet granuloma method in rats.
  3. Formalin induced inflammation model in rats.

To evaluate anti-arthritic activity of the extracts in experimental animal models like:

  1. Adjuvant induced arthritis like inflammation in rats

Enclosure-IV

7)MATERIALS AND METHODS:

7.1) Source of data:

Whole work is planned to generate data from laboratory based studies as described in objectives of the study and experimental studies in journals and text books available with college and other institutions, national/international journals (IJP, IJPP, Ethanopharmacol and Indian J Exp Biol.).

Enclosure-V

7.2: Methods of collection of data:

Data will be generated from experimental animal studies and results will be subjected for statistical analysis by ANOVA followed by Dunnett’s‘t’ test. P values less than 0.05 will be considered as statistically significant.

PHASE-1:

1. Determination of LD50 of Baidyanath Maharasnadi kadha:29

The acute toxicity of Baidyanath Maharasnadi kadha will be determined by using albino rats. At the commencement of its dosing, each animal should be
between 8 - 12 weeks old and its weight should fall in an interval within ± 20 % of the mean initial weight of any previously dosed animal. The temperature in the experimental animal room should be 22°C (± 3°C). Although, the relative humidity should be maintained 50-60%. Lighting should be artificial, the sequence being 12 hours light and 12 hours dark. The animals are housed individually. For feeding, conventional rodent laboratory diets may be used with an unlimited supply of drinking water.

Overnight fasted animals will be administered with single dose of Baidyanath Maharasnadi kadha and observed for its mortality up to 48hours study period (short term toxicity). Based on the short-term toxicity profile, the text dose will be determined as per OECD guidelines No 425. From the LD50 dose, 1/20 and 1/5 will be selected and considered as low and high doses respectively.

PHASE-II

7.2.1: Determination of anti-inflammatory activity:

1) Carrageenan induced paw oedema in rats:30

Animals of either sex weighing between (150-200g) will be divided into following 5 groups of 6 animals each.

Group A: Negative control (receives distilled water) p.o.

Group B: Positive control group (Carrageenan)

Group C: Carrageenan +Standard (Indomethacin 10mg/kg) p.o

Group D: Carrageenan + low dose of Baidyanath Maharasnadi kadha. p.o.

Group E: Carrageenan + high dose of Baidyanath Maharasnadi kadha p.o.

Acute inflammation will be produced by injecting 0.1ml of 1% Carrageenan into sub plantar surface of rat hind paw. The Baidyanath Maharasnadi kadha decoction, reference, standard drug and control distilled vehicle has to be administered 60 min before Carrageenan injection. The paw volume will be measured at 0, 0.5, 1, 2, 3, 4 and 5 hrs interval using plethysmograph apparatus.

2) Cotton pellet-induced granuloma:31

Animals of either sex weighing between (150-200 g) will be divided into following 5 groups of 6 animals each.

Group A: Negative control (receives distilled water) p.o

Group B: Positive control group (Cotton pellets)

Group C: Cotton pellets + Standard (Indomethacin 10mg/kg) p.o.

Group D: Cotton pellets + Low dose of Baidyanath Maharasnadi kadha. p.o.

Group E: Cotton pellets + High dose of Baidyanath Maharasnadi kadha p.o.

The rats will be anaesthetized with anesthetic ether and sterile cotton pellets weighing 10 ± 1 mg will be implanted subcutaneously into both sides of the groin region of each rat and drugs will be administered orally to groups A, C, D, E respectively for seven consecutive days from the day of cotton pellets implantation. On 8th day, the animals will be anaesthetized by anesthetic ether and the pellets together with granuloma tissues will be carefully removed and made free from extraneous tissues. The wet pellets will be weighed and dried in an oven at 60°C for 24 hours to constant weight, after that the dried pellets were weighed again. Increment in the dry weight of the pellets will be taken as a measure of granuloma formation.

3) Formalin-induced acute inflammatory model:32

Animals of either sex weighing between (150-200 g) will be divided into following 5 groups of 6 animals each.

Group A: Negative control (receives distilled water) p.o.

Group B: Positive control group (formalin)

Group C: Standard (formalin + Indomethacin 10mg/kg) p.o.

Group D: Low dose of Baidyanath Maharasnadi kadha. p.o.

Group E: High dose of Baidyanath Maharasnadi kadha p.o.

Rats will be injected with 0.1ml of formalin (2% in distilled water) in plantar aponeurosis of the left foot; on the first and third day of the test, rat paw volume will be measured for alternate days for 10 days. The percent inhibition of mean increase in paw edema of each group will be measured on the 10th day and compared with control.

7.2.2: Determination of anti-arthritic activity33

1) Adjuvant induced arthritis like inflammation in rats:

Animals of either sex weighing between (150-200g) will be divided into following 5 groups of 6 animals each.

Group A: Negative control (receives vehicle) p.o.

Group B: Positive control (Complete Freunds adjuvant)

Group C: Standard Indomethacin (10 mg/kg) p.o.

Group D: Low dose of Baidyanath Maharasnadi kadha. p.o.

Group E: High dose of Baidyanath Maharasnadi kadha p.o.

Rats will be injected with Complete Freunds adjuvant containing 10 mg of heat killed Mycobacterium tuberculosis in 1ml paraffin oil (0.1ml) into the left paw intra dermally. Groups will be treated with the Baidyanath Maharasnadi kadha, distilled water and standard respectively until 14 days from the day of injection of Complete Freunds Adjuvant.

Parameters to be studied

1. Hematological examination:34

Rats will be anaesthetized with anesthetic ether and blood samples will be collected by retro-orbital route using EDTA as anti-coagulant and subjected to estimation of RBC, WBC (Total and Differential leucocytes count) and mean hemoglobin concentration.

2. Serum bio-chemical examination:35

For biochemical determination, blood will be collected from the retro-orbital route without any anti-coagulant. After one hour serum will be separated by centrifugation and maintained at -4oc until further use. The serum will be subjected for determination of Blood Urea Nitrogen (BUN), Total protein, Albumin, Alanine amino transferases (ALT), Asparatate amino transferases (AST) and Alkaline Phosphate (ALP) by using Semi auto-analyzer.

3. Body weight:36

Body weight for each group of rats will be recorded for alternative days during the period of arthritis. The difference between mean body weights in each group will be calculated to determine the change in the body weight between the first day and fourteenth day.

4. Ankle diameter: 36

Changes in the ankle diameter of both injected and non injected paws from day 0 to day 14 on alternative days will be assessed using a vernier scale for all the animals.

5. Arthritis Index and hind paw volume: 36

The paw volume will be measured at 10:00 am alternative days from day 0 to day 14 using plethysmograph apparatus for all the animals.

Arthritis index will be calculated by the following equation:

AI = (hind paw volume on day x)-(hind paw volume on day 0)

(hind paw volume on day 0)

6. Body temperature: 36

Body temperature as an index of inflammation was monitored for rats from day 0 to day 14 using rectal thermometer at 11:00 am on every alternative days and compared with that of the day 0 to all the animals.

7. Radiological studies:33

Before sacrificing the animals, X-ray analysis of hind paw joints of the animals will be carried out for evaluating the extent of bone damage.

8. Histo-pathological studies:36

The animals will be sacrificed by cervical displacement method and the ankle joint tissues will be excised and fixed in 10% buffered formalin, decalcified in 10% EDTA, embedded in paraffin, sectioned and stained with haemotoxylin and eosin and evaluated under light microscope

STATISTICAL ANALYSIS

All values will be expressed as mean ± SEM for 6 animals in a group. Results will be subjected to statistical analysis using one-way ANOVA (analysis of variance) followed by Dunnet‘t’ test. p< 0.05 will be considered as statistically significant

7.3: Does the study require any investigations or interventions to be conducted on patients other human or animals? If so, please describe briefly:

Study requires investigation on albino rats.

7.4: Has ethical clearance been obtained from your institute in case of 7.3:

YES: IAEC NO.:576/2002/bc/IAEC/CPCSEA

ENCLOSURE –VI

List of References:

  1. Manfredsdottir VF, Vikingsdottir T, JonssonT, Geirsson AJ, Kjartansson O, Heimisdottir M et al. The effects of tobacco smoking and rheumatoid factor seropositivity on disease activity and joint damage in early rheumatoid arthritis. Rheumatology 2006; 45: 734-740.
  2. Deborah S, Colin M, Bruce P. The global burden of rheumatoid arthritis in the year 2000.Draft 15-08-2006.
  3. arthritis. Accessed on 06-12-2010.
  4. Shanker S, Handa R. Biological agents in rheumatoid arthritis.
  5. Neis AS. Principles of Therapeutics Goodman and Gillman’s The Pharmacological Basis of Therapeutics. 10thed. McGraw-Hill, Medical Publishing Division.2001; New Delhi: 687.
  6. acessed on 01/12/2010
  7. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 37-38.
  8. Ilavarasana R, Mallik M, Venkataraman S. Anti-Inflammatory And Antioxidant Activitiesof Cassia Fistula Linn Bark Extracts. Afr. J. Trad. CAM (2005) 2 (1): 70 - 85
  9. Joy PP. Thomas J. Mathew S., Skaria BP. Medicinal Plants. Kerala Agricultural University of Aromatic Medicinal Plants Research Station. Kerala, India. 1998; 192.
  10. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 551.
  11. Ilavarasana R, Mallik M, Venkataraman S.Anti-inflammatoryandfreeradicalscavengingactivity of Ricinuscommunis rootextract. Journalof Ethnopharmacology. 2006; 103: 478–480
  12. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 639-40.
  13. Jain A, Sharma S, Goyal M, Dubey S, Jain S, Sahu J, Sharma A, Kaushik A. Anti-inflammatory activity of Syzygium Cumini leaves. International Journal of Phytomedicine 2010; 2: 124-126.
  14. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 733-34.
  15. VendruscoloA., Takaki I., Bersani-Amado LE., Dantas JA., Bersani-Amado CA., Cuman RKN. Antiinflammatory and antinociceptive activities ofZingiber officinale Roscoe essential oil in experimental animal models. Indian J Pharmacol February 2006; 38 (1):58-9.
  16. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 670.
  17. Hong CH., Hur SK..Oh OJ.,Kim SS.,Nam KA, Lee SK. Evaluationofnaturalproductsoninhibitionofinducible cyclooxygenase(COX-2)andnitricoxidesynthase(iNOS)incultured mousemacrophagecells. Journal of Ethnopharmacology. 2002; 83: 153-59.
  18. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 717-18.
  19. SinghG., Sharma PK., Dudhe R., Singh S.Biological activities of Withania somnifera. Scholars Research Library Annals of Biological Research, 2010, 1 (3) : 56-63.
  20. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 133.
  21. ShindeUA., Phadke AS., Nair AM.,.Mungantiwar AA,Dikshit VJ., Saraf MN.. Studiesontheanti-inflammatory and analgesic activity of Cedrusdeodara (Roxb.)Loud.Woodoil. JournalofEthnopharmacology. 1999; 65: 21–27
  22. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 612.
  23. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 331.
  24. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 188-89.
  25. Navarro1DF., De Souza MM., NetoRA., GolinV., Niero R., Yunes RA. Monache FD., and V. Filho C. Phytochemical analysis and analgesic propertiesof Curcuma zedoaria grown in Brazil. Phytomedicine2002; 9: 427–432.
  26. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 88.
  27. Bagul MS., Srinivasa H., Kanaki NS., Rajani M. Anti inflammatory activity of two ayurvedic formulation containing guggul. Indian J pharmacol 2005; 37 (6): 399-400.
  28. Khare CP. Indian Medicinal plant- An Illustrated dictionary, Spingler Science, New Dehili, India; 1938: 96.
  29. OECD 2001 guidelines on acute oral toxicity. Environmental health and safety monograph series on testing and adjustment no.425.
  30. Winter CA, Risley EA, Nuss. Carrageenin Induced Oedema in hind paw of the rat as assay for anti-inflammatory drugs. Proceedings of society of Experi Biolo Medi 1962; 111: 544- 47.
  31. Carey WM, Dasi JMB, Nimmagadda RV, Gottumukkala KM. Anti-inflammatory activity of methonolic extract of Bambusa vulgaris leaves. International Journal of Green Pharmacy 2009: 234-238.
  32. Perez RM, Perez S, Zavala MA, Salazar M. Anti-inflammatory activity of the bark of Hippocratea excelsa. Journal of Ethnopharmacology 1995; 47: 85-90.
  33. Ramprasath VR, Shanthi P, Sachdanandam P. Curative effect of Semicarpus anacardium Linn. nut milk extract against adjuvant arthritis with special reference to bone metabolism. Chemico-Biological Interactions 2006; 160: 183-192.
  34. Richard PC, Louis JD, Lynn O, Eileen MB, Frank D, Robert B and Alan JL. Comparison of inflammatory changes in established type II collagen and adjuvant induced arthritis using out bred wistar rats.hit. J. lmmunopharmac1985; 7(6): 811-826.
  35. Pinto MG, Marques PR, Gayer CRM. Sub-acute toxicity evaluation of a hydro alcoholic extract of Pterodon pubescens seeds in mice with collagen –induced arthritis. Journal of Ethnopharmacology 2001; 77: 159-164.
  36. Gumaa AA, Elshenaway MM, Affi NA, Mohammed EA, Thabit RH. Dual effect of nitric oxide donar on adjuant arthritis. International Immunopharamacology 2009; 9: 439- 447.

1