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“EVALUATION OF ANTI-ARTHRITIC EFFECTS OF CURCUMA LONGA (TURMERIC) EXTRACT, OIL AND CURCUMIN IN COMBINATION WITH METHOTREXATE AND IBUPROFEN IN RATS”

 

By 

D. SONY B.Pharm

M.Pharm. Dissertation Protocol

Submitted to the

 

Rajiv Gandhi University of Health Sciences,

Bangalore-560041, Karnataka. 

In partial fulfillment

of the requirement for the Degree of 

MASTER OF PHARMACY

IN

PHARMACOLOGY 

Under the Guidance of

Dr. Benson Mathai K, M.Pharm, PhD.

Professor and Principal 

Dept. of Pharmacology and Toxicology

St. John’s Pharmacy College

St. John’s Educational Institutions

Bangalore –560104.

2011 - 13. 

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

BANGALORE, KARNATAKA.

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION


 
1.
 
Name of the candidate &

Address.

 
D. SONY

I M Pharm

ST. JOHN’S PHARMACY COLLEGE,

#6, 9TH CROSS, 2nd MAIN,

VIJAYANAGAR 2nd STAGE, BANGALORE-560104.

PHONE NO: 080 23300958/23300668.

 
  2. 
 
Name of the Institution.
 
St. John’s Pharmacy College

No 6, 9th cross, 2nd main, Vijayanagar,

2nd stage (Hampinagar),

Bangalore- 560104

Tel: 91-80-23300958/23300668

Email: admin@stjohns.edu.in

 
3.
 
Course of the study

& subject.

 
MASTER OF PHARMACY (M.PHARM)

Sub:- Pharmacology

 
4.
 
Date of admission.
 
18 JULY 2011
 
5.
 
Title of the Topic:

“Evaluation of anti-arthritic effects of curcuma longa (turmeric), oil and curcumin alone in combination with Methotrexate and Ibuprofen in rats.

 
6. 

6.1 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

6.2 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

6.3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

7. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

8. 
 

 
Brief resume of intended work

 

Need of the work 

            Globally, the incidence of degenerative and inflammatory joint diseases is very high and affects nearly 30% of the adults. [1] Osteoarthritis (OA) and Rheumatoid arthritis (RA) are the most common form of arthritis. RA affects about 1% of the population and is associated with significant morbidity and increased mortality.[2] Prevalence of OA after age 65 is about 60% in men and 70% in women.[3] 

               Arthritis shares the symptoms of pain, stiffness, and swelling of one or more joints. The most common form of arthritis, OA, involves a wear-and-tear degeneration of cartilage in   the joint space with only minimal inflammation. On the other end of the spectrum is RA, autoimmune disease, in which there is significant inflammation in the joints and other areas of the body as well. The heterogeneity of arthritic diseases creates significant challenges for effective medical treatment [4].  

               Pharmacotherapy serves as an important adjunctive role in OA treatment and a primary treatment in RA. In most cases people ailing with arthritis follow medication prescribed in the modern system of medicine. Ibuprofen, is the standard drug in the treatment of OA [5], while Methotrexate (MXT), is in RA [6]. However both these agents are known to cause undue side effects which compromise the ultimate therapeutic benefit. Reports suggest that the use of complementary and alternative medical therapies has been increasing among patients with arthritis.[7]

                   

                  Both turmeric extract and oil has been tested scientifically for the anti-arthritic effects and observed to be effective in preventing arthritis.[8] However the combination of turmeric with Ibuprofen, a standard drug in OA and with methotrexate, a standard in RA has never been performed. As turmeric is dietary and globally well accepted by people, the results from this study will help in advocating the combinative therapy of the two agents in RA and OA. The present study plans to see for the combinative effect as this will help in translating the results into humans in future. 

Review of Literature:

                  Curcuma longa (turmeric) has a long history of use in Ayurvedic medicine as a treatment for inflammatory conditions. It is one of nature's most powerful healers. Long known for its anti-inflammatory properties, research has revealed that turmeric is a natural wonder, proving beneficial in the treatment of many different health conditions from cancer to Alzheimer's disease.[9] Curcumin, the principal phytochemical turmeric is shown to prevent the ill effects of both methotraxate and Ibuprofen induced damage in rats.[10,11]  

                 Conventional medical treatment for rheumatoid arthritis and osteoarthritis includes the use of NSAIDs (traditional and selective inhibitors of cyclooxygenase [COX]-2), because they provide unmistakable and significant health benefits in the treatment of pain and inflammation. However, they are associated with an increased risk of serious gastrointestinal (GI) and cardiovascular (CV) adverse events.[12]  

               Generally various inflammatory mediators play a key role in arthritis mainly cytokines have emerged as crucial players in mediating synovial inflammation in the rheumatoid joint. Among the cytokines, Tumour Necrosis Factor (TNF-α) and Interleukin (IL-1) are considered to be of great importance in the pathogenesis of Arthritis.[13,14] You-Zhi Wang et al showed that EHCS significantly attenuates CIA in rats by decreasing the levels of IL-1β, IL-6, TNF-α and PGE2 in serum and the expression of COX-2 and transcription factor NF-κB in paw cartilage.[15]

          

                  Studies have shown that the administration of 110 mg/ml/kg turmeric for 28 days reduced the collagen-induced arthritis and arrested the degenerative changes in the bone and joints of collagen-induced arthritic rats.[8] The turmeric fraction depleted of essential oils is also shown to inhibit streptococcal cell wall -induced arthritis, joint inflammation and peri-articular joint destruction in a dose-dependent manner.[16] The authors also observed that turmeric oil was mildly joint-protective.[17] With regard to phytochemicals studies have shown that curcumin also shown to possess anti-arthritic effects.[18]

                 

                  Animal models of arthritis are used to study pathogenesis of disease and to evaluate potential anti-arthritic drugs for clinical use. Animal models for RA with a proven track record of 
 

predictability for efficacy in humans include: rat adjuvant arthritis, rat type II collagen arthritis[13,15,19], mouse type II collagen arthritis and antigen induced arthritis in several species.[20]  

                 Experimentally arthritis could be induced by various inflamogens of which Collagen-induced RA and monosodium iodoacetate-induced OA[21] are the most standard models of study in rats. The two agents induce OA and RA and the process of pathogenesis is observed to be biochemically, anatomically and clinically similar to that in humans. [22, 23]  
 

Aim of the study:

    To evaluate the anti-arthritic effects of turmeric powder, oil and curcumin in combination with Methotrexate and Ibuprofen.

    Objectives:

    The objectives of the present  study are as follows:

    1. To determine the anti-arthritic effects of turmeric powder, oil and curcumin against Collagen-induced rheumatoid arthritis.
    2. To determine the beneficial combinative anti-arthritic effects of turmeric powder, oil, curcumin with Methotrexate against Collagen-induced rheumatoid arthritis.
    3. To determine the anti-arthritic effects of turmeric powder, oil and curcumin against Monosodium iodoacetate-induced osteoarthritis.
    4. To determine the beneficial combinative anti arthritic effects of turmeric powder, oil, curcumin with Ibuprofen against Monosodium iodoacetate-induced osteoarthritis.
    5. To determine which one of turmeric powder, oil, and curcumin have better anti arthritic activity.
    6. To study whether the turmeric (powder, oil, curcumin) can overcome the side effects caused by the standard drugs (Methotrexate and Ibuprofen) used in RA and OA respectively.
 
 
 
 
 
 

Materials & Methods 

7.1 Source of data

       

             Whole work is planned to generate data from laboratory studies i.e.; experiments are performed as described in reference, experimental studies in journals and in textbooks available with college, Indian Institute of Science (IISc) library, Rajiv Gandhi University of Health Sciences (RGUHS) digital library (Helinet), websites like

              www.sciencedirect.com,

              www.ncbi.nlm.nih.gov/pubmed,

              www.google.com,

              www.ijp-online.com,

were used to obtain related information regarding this research protocol. 
 

7.2 Chemicals – All chemicals of standard quality and grade will be procured. 

7.3 Methods of collection of data

          The experiments will be conducted using laboratory animals and the data will be collected by analyzing appropriate parameters. All experiments are planned in accordance with Committee for the purpose of control and supervision of experiments on animals (CPCSEA), Chennai, India. The present study will be carried out after obtaining from Institutional Animal Ethical committee (IAEC).  

  1. Experimental animals
    • Species                                            Wistar rats
    • Age                                                  2-3months
    • Weight                                             150-230gm[24]
    • Gender                                             Both male and female

        Animals will be housed in polypropylene cages on clean paddy husk bedding till the completion of study. Animals will be maintained under controlled temperature at 25�C � 2�C with 12 hr light/dark cycle with food and water provided ad libitum. Animals which do not comply with above criteria and also which are found to be diseased will be excluded from the study. 

  1. Administration of drugs[]

    Collagen 200�l of the stock 2mg/ml (in RA)[25]

    Or Freund`s Adjuvant (0.1ml) (in RA)[24]

    Monosodium iodoacetate 30�l of the stock liquid (in OA)[21]

    Methotrexate (50mg/ kg body weight)

    Ibuprofen (Oral)

      Turmeric powder (5% mixed in diet)

      Turmeric oil (5% mixed in diet)

      Curcumin (5% mixed in diet) 

  1. Animal groups
 
    Experimental Model 1: Collagen-induced rheumatoid arthritis  
    Group 1: Normal animals                                                          06
    Group 2: RA controls                                                         06
    Group 3: RA + methotrexate (50 mg/kg b.wt) i.v. in 30 �l once a week for  four weeks    06
    Group 4: RA + turmeric powder (5% mixed in diet) 06
    Group 5: RA + turmeric oil (5% mixed in diet) 06
    Group 6: RA + curcumin (5% mixed in diet) 06
    Group 7: RA + turmeric oil (mixed in diet) + methotrexate (50 mg/kg b.wt) i.v. in 30 �l once a week for four weeks 06
    Group 8: RA + turmeric powder (mixed in diet) + methotrexate (50 mg/kg b.wt) i.v. in 30 �l once a week for four weeks 06
    Group 9: RA + curcumin powder (mixed in diet) + methotrexate (50 mg/kg b.wt) i.v. in 30 �l once a week for four weeks 06
                                                                          Total animals per experiment  54
     
     
     
    Model 2: Monosodium iodoacetate-induced osteoarthritis  
    Group 1: Normal animals                                                          06
    Group 2: OA controls                                                         06
    Group 3: OA + known drug (ibuprofen oral)                 06
    Group 4: OA + turmeric powder (5% mixed in diet) 06
    Group 5: OA + turmeric oil (5% mixed in diet) 06
    Group 6: OA + curcumin (5% mixed in diet) 06
    Group 7: OA + turmeric oil (mixed in diet) + ibuprofen oral 06
    Group 8: OA + turmeric powder (mixed in diet) + ibuprofen oral 06
    Group 9: OA + curcumin powder (mixed in diet) + ibuprofen oral 06
                                                                                          Total per experiment  54
    Two experiments (one with OA and other with RA) 54 x 2    108
  1. Methodology  
     
     

     Experimental Model 1:

          Collagen/Freunds Adjuvant-induced RA:

                         Collagen-Induced RA[22]:  Bovine type II collagen will be dissolved in 0.1% acetic acid at a concentration of 2mg/mL with constant mixing overnight at 40C. Then the solution will be emulsified with an equal volume (at a 1:1 ratio) of Freund incomplete adjuvant, and each rat will be injected  intradermally with 200 �L of the emulsion containing  200 mg of collagen in four sites on the back under isoflurane anesthesia. The day of induction will be designated as day 0 and will be evaluated till 28. On a daily basis, the swelling of both treated and control ankle joints will be monitored. The peri-articular swelling will be measured with a caliper starting on day zero and weekly thrice until the study will be completed on day 28 post initiation.

                        Freund`s Adjuvant-induced RA:[24] 

     Experimental Model 2:

          Monosodium iodoacetate-induced osteoarthritis:[23]

                            Osteoarthritis will be induced in all rats by a single intra-articular injection of monosodium iodoacetate dissolved in saline. Briefly, rats will be anesthetized with isoflurane, and monosodium iodoacetate (30μL/rat) will be injected into the left knee joint cavity using a 29-gauge needle inserted through the patellar tendon. To set up the osteoarthritis rat model in our lab, two groups of rats (n = 8 per group) will be used in the first experiment: one received monosodium iodoacetate and the other received saline. On a daily basis, the swelling of both treated and control ankle joints will be monitored. The peri-articular swelling will be measured with a caliper starting on day zero, and continued weekly thrice until the study will be completed on day 28 post initiation.  

Treatments: Animals were treated p.o. with turmeric oil in 0.01% Tween 80 and dosed at 5 ml/kg daily after the injection of inflamogens. The positive control drug ibuprofen 200 mg/kg b. wt will be administered through the oral route, while methotrexate (50 mg/kg b. wt stock 30 �l) will be administered once a week through the intravenous route. The experiments will be terminated on day 28 post injection of the inflamogen. The animals will be euthanized by administering an over dose of ketamine or halothane. Blood and the inflamed bones will be collected and the carcasses will be immediately sent to incinerator.  
 

Statistical analysis:-

       The data obtained from experimentation will be subjected to one way Analysis Of Variance (ANOVA) followed by suitable post hoc test.

Total No. of animals required:

    • No of the rats for Collagen-induced RA                                       = 54

      (9gps � 6 animals)              

    • No of rats for Monosodium Iodoacetate-induced OA                 = 54

      (9gps � 6 animals)                  

    • Total No of rats                                                                            = 108
 

7.4  Does the study require any investigation or intervention to be conducted on patients or other humans or animals? If so. Please describe briefly?

Yes, the study requires investigation on wistar rats. 

7.5  Has ethical clearance been obtained from your institution in case of 7.3?

 Institutional Animals Ethical Committee (IAEC) meeting will be held in our Instituition in February and the approval letter will be sent to university after obtaining the approval. 
 
 
 

LIST OF REFERENCES 

  1. Woolf AD, Pfleger B. Burden of major musculoskeletal conditions. Bull World Health Organ 2003, 81:646-56.
  1. McInnes, I.B., Scheet, G. Cytokines in the pathogenesis of Rheumatoid arthritis. Nat Rev Immunol 2007;7:429-42. 
  1. Sarzi-Puttini P, Cimmino MA, Scarpa R et al. Osteoarthritis: an overview of the disease and its treatment strategies. Semin Arthritis Rheum 2005; 35:1-10. 
  1. An Ayurvedic Approach to Arthritis by Micheal K.Farley, M.D. Chopra Center ACT Project 1, 2010. 
     
  1. Farkouh ME, Greenberg JD, Jeger RV, Ramanathan K, Verheugt FW, Chesebro JH, Kirshner H, Hochman JS, Lay CL, Ruland S, Mellein B, Matchaba PT, Fuster V, Abramson SB. Cardiovascular outcomes in high risk patients with osteoarthritis treated with ibuprofen, naproxen or lumiracoxib. Ann Rheum Dis 2007; 66:764-70. 
  1. Agarwal SK. Biologic agents in rheumatoid arthritis: an update for managed care professionals. J Manag Care Pharm 2011; 17:S14-8. 
  1. Setty AR, Sigal LH. Herbal medications commonly used in the practice of rheumatology: mechanisms of action, efficacy, and side effects. Semin Arthritis Rheum 2005; 34:773-84. 
  1. Taty Anna K, Elvy Suhana MR, Das S, Faizah O, Hamzaini AH. Anti-inflammatory effect of Curcuma longa (turmeric) on collagen-induced arthritis. An anatomico-radiological study Clin Ter 2011a; 162:201-7. 
  1. Jurenka JS. Anti-inflammatory properties of curcumin, a major constituent of Curcuma longa: a review of preclinical and clinical research. Altern Med Rev 2009; 14:141-53. 
  1. Hemeida RA, Mohafez OM. Curcumin attenuates methotraxate-induced hepatic oxidative damage in rats. J Egypt Natl Canc Inst 2008; 20:141-8. 
  1. Banerjee M, Tripathi LM, Srivastava VM, Puri A, Shukla R. Modulation of inflammatory mediators by ibuprofen and curcumin treatment during chronic inflammation in rat. Immunopharmacol Immunotoxicol 2003; 25:213-24.  
  1. Patrignani P, Tacconelli S, Bruno A, Sostres C, Lanas A. Managing the adverse effects of nonsteroidal anti-inflammatory drugs. Expert Rev Clin Pharmacol 2011; 4:605-21. 
  1. Zhe-Ming Wang, Shi-gong Zhu, Zai-Wang Wu, Yue Lu, Hong-Zheng Fu, Rui-Qin Qian. Kirenol upregulates nuclear Annexin-1 which interacts with NF-κB to attenuate synovial inflammation of collagen-induced arthritis in rats. J Ethnopharmacol 2011; 137:774-82. 
     
     
  1. Inoue A, Matsumoto I, Tanaka Y, Iwanami K, Kanamori A, Ochiai N, Goto D, Ito S, Sumida T. Tumor necrosis factor alpha-induced adipose-related protein expression in experimental arthritis and in rheumatoid arthritis. Arthritis Res Ther 2009; 11:R118.  
  1. You-Zhi Wang, Shi-Qin Sun, Ya-Bin Zhou. Extract of the dried heartwood of Caesalpinia sappan L. attenuates collagen-induced arthritis. J Ethnopharmacol 2011; 136:271-78. 
  1. Funk JL, Frye JB, Oyarzo JN, Kuscuoglu N, Wilson J, McCaffrey G, et al. Efficacy and mechanism of action of turmeric supplements in the treatment of experimental arthritis. Arthritis Rheum 2006; 54:3452-64. 
  1. Funk JL, Frye JB, Oyarzo JN, Zhang H, Timmermann BN. Anti-arthritic effects and toxicity of the essential oils of turmeric (Curcuma longa L.). J Agric Food Chem 2010; 58:842-9. 
  1. Joe B, Rao UJ, Lokesh BR. Presence of an acidic glycoprotein in the serum of arthritic rats: modulation by capsaicin and curcumin. Mol Cell Biochem 1997; 169:125-34. 
  1. Yago T, Nanke Y, Kawamoto M, Furuya T, Kobashigawa T, Kamatani et al IL-23 induces human osteoclastogenesis via IL-17 in vitro, and anti-IL-23 antibody attenuates collagen-induced arthritis in rats. Arthritis Res Ther 2007; 9:R96. 
  1. Alison M. Bendele. Animal models of rheumatoid arthritis. J Musculoskel Neuron Interact 2001;1:377-85. 
  1. Beyreuther B, Callizot N, St�hr T. Antinociceptive efficacy of lacosamide in the monosodium iodoacetate rat model for osteoarthritis pain. Arthritis Res Ther 2007; 9:R14. 
  1. Tsubata M, Takagaki K, Hirano S, Iwatani K, Chiyuki ABE. Effects of Flavangenol, an Extract of French Maritime Pine Bark on Collagen-Induced Arthritis in Rats. J Nutr Sci Vitaminol 57, 251-57.  
     
  1. Pomonis JD, Boulet JM, Gottshall SL, Phillips S, Sellers R, Bunton T, Walker K. Development and pharmacological characterization of a rat model of osteoarthritis pain. 2005; 114:339-46.  
  1. Banji D, Pinnapureddy J, Banji OJ, Kumar AR, Reddy KN. Evaluation of the concomitant use of methotrexate and curcumin on Freund's complete adjuvant-induced arthritis and hematological indices in rats. Indian J Pharmacol 2011; 43:546-50.  
  1. Daans M, Lories RJ, Luyten FP. Dynamic activation of bone morphogenetic protein signaling in collagen-induced arthritis supports their role in joint homeostasis  and disease. Arthritis Res Ther 2008; 10:R115.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
9.
 
Signature of the candidate
 

(D.Sony)

 
10.
 
Remarks of the guide
 
Recommended for approval
11. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

12.

Name & Designation of

11.1 Guide 
 
 

11.2 Signature of Guide 
 
 

11.3 Head of the Department  
 
 
 
 

11.4 Signature of HOD 
 

12.1 Remarks of the Chairman & Principal 
 
 

12.2 Signature

Dr. Benson Mathai, M.Pharm, Ph.D.

Professor &  Principal

Department Of Pharmacology,

St. John’s Pharmacy College,

#6, 9th Cross, 2nd Main,

Vijaynagar 2nd Stage, Bangalore-560104. 
 
 
 
 

Dr. Benson Mathai, M.Pharm, Ph.D.

Professor & Head,

Department Of Pharmacology,

St. John’s Pharmacy College,

#6, 9th Cross, 2nd Main,

Vijaynagar 2nd Stage, Bangalore-560104.


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