Cytotoxic Effects of Conjugated Linoleic Acids on

0 downloads 0 Views 529KB Size Report
Dec 10, 2012 - Bendele A, McComb J, Gould T, McAbee T, Sennello G,. Chlipala E, Guy M. Animal models of arthritis: relevance to human disease. Toxicol ...
J Exp Integr Med 2013; 3(1):37-41

ISSN: 1309-4572

Journal of Experimental and Integrative Medicine available at www.scopemed.org

Original Research

The effect of two formulations of Entada phaseoloides seeds after topical application in ‘monoiodoacetate-induced osteoarthritis’ in rats Jayshree S. Dawane, Vijaya Pandit, Bhagyashree Rajopadhye, Manjiri Karandikar Bharati Vidyapeeth Deemed University (BVDU) Medical College, Pune, Maharashtra, India Received August 6, 2012 Accepted October 6, 2012 Published Online December 10, 2012 DOI 10.5455/jeim.061012.or.048 Corresponding Author Jayshree Shriram Dawane Department of Pharmacology, Bharati Vidyapeeth Deemed University Medical College, Pune-411043, Maharashtra, India. [email protected] Key Words Inflammation; Monoiodoacetate; Osteoarthritis

Abstract Objective: Arthritis is a very common clinical condition affecting both sexes and all ages. Most common forms of arthritis are osteoarthritis and rheumatoid arthritis. In all types of arthritis pain, inflammation and functional restriction are the presenting manifestation. Anti-inflammatory drugs like NSAIDs, corticosteroids and disease-modifying antirheumatic drugs etc are used for symptomatic relief but many times they are associated with adverse effects that can often be as difficult to manage as the disease itself. Therefore a need exists for new ways to treat these patients. In this study it was aimed to investigate the effect of topical application of two formulations of Entada phaseoloides (EP) seeds in the ‘monoiodoacetate (MIA)-induced osteoarthritis’ model in rats. Methods: Paste and ointment formulations of EP were tested. Thirty-two Wistar rats weighing 150-200 g were divided into four groups as (I) vehicle, (II) EP paste, (III), EP ointment and (IV) diclofenac ointment. Osteoarthritis was induced by intraarticular injection of 50 μl of MIA solution. Drug treatment was given topically according to groups for 14 days. Animals were observed for joint inflammation and gait. Joint histopathology was studied and scored. Results: Swelling and redness of left knee was seen in all rats within 24 h which subsided gradually. Lame gait and thickening of the joint capsule was seen only in control rats. Histopathologically, osteoarthritic changes were significantly less in drug-treated groups compared to control. Conclusion: Both the formulations of EP were effective in preventing the damage to the joint. © 2012 GESDAV

INTRODUCTION Osteoarthritis (OA) is the most common joint disease of humans. Among the elderly, knee OA is the leading cause of chronic disability in developed countries. OA has strong association with aging. This is the most common form of chronic disorder of synovial joints characterized by progressive degenerative changes in articular cartilage over the years, particularly in weightbearing joints [1]. Though the original insult generally remains unclear, the disease starts with the damage to the cartilaginous matrix. The repair process also gets initiated simultaneously which restricts the damage and maintains proper joint functioning. Once repair process fails, there is progressive damage to articular cartilage, bone and synovial membrane. Joint tissues like

http://www.jeim.org

cartilage, bone, synovium, capsule, ligament, muscle etc depends on each other for health and function. If one tissue is damaged, in due course, whole joint is affected leading to pain and disability. It is estimated that 80% of the population have radiographic evidence of OA by the age of 65 years [2]. Anti-inflammatory drugs like NSAIDs, corticosteroids and disease-modifying antirheumatic drugs etc are used for symptomatic relief but many times they are associated with adverse effects, that can often be as difficult to manage as the disease itself. Continuation of treatment with the same drugs leads to compromised quality of life. [3] Therefore a need exists for new ways to treat these patients.

37

Dawane et al: Entada phaseoloids in monoiodoacetate-induced osteoarthritis Entada phaseoloides (Mimosaceae) is a gigantic climber tree with twisted and angled stem. Seeds of this plant commonly called as Gilla (Sanskrit), Hathibij (Hindi), Garambi (Marathi) and Gogo are traditionally used worldwide for medicinal purpose [4]. Paste of the seed pulp is used as an herbal medicine to reduce inflammation and pain of joints and lymph nodes. In addition seeds are reported to have emetic, antihelmintic and antimalignant activities [5]. No reference is available where this preparation is tested for its topical anti-inflammatory activity using modern medicine parameters. So, the purpose of present study was to find efficacy of Entada phaseoloides seed pulp after topical application in an animal model of arthritis.

Animals Albino wistar rats of either sex weighing 150-200 g were used for the study. Animals were procured from Central Animal House recognized by CPCSEA (Regd.No.258; Committee for the Purpose of Control and Supervision on Experiments on Animals, Tamil Nadu, India) of BVDU Medical College. 12 h day and night cycle was maintained during housing. The rats were fed with commercial rat diet and Aquaguard water ad libitum. The experiments were designed and conducted in accordance with the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India, and Institutional Animal Ethical Committee Guidelines. Groups

MATERIALS AND METHODS The study was started after obtaining approval from Institutional Animal Ethics Committee (approval Letter No. IAEC/BVDUMC/01/2008-2009). Collection and authentication of plant material Entada phaseoloides seeds were obtained from M/s Gopal Govind Lokhande, 764, Pune, India, known vender of ayurvedic and unani medicine. Authenticated in Agharkar Research Institute, Pune. Drugs and chemicals Mono-iodoacetate was obtained from Sigma Chemical Co. (USA), diclofenac from Novartis India Ltd (Mumbai), polyethylene glycol (PEG) 400 and 3350 of analytical grade were obtained from from local supplier Sharad Chemical Agencies (Pune), and sodium pentobarbital from Loba Chemie Industries (Mumbai). Formulations used -Paste of Entada phaseoloides seeds powder [4, 5]: EP seed pulp was powdered and sieved through mill No.80. Fresh paste was prepared by addition of water in the powder (5 g/5 ml) and mixed to make uniform paste. -Ointment of Entada phaseoloides seeds powder [6]: PEG ointment base was prepared by mixing PEG 400 (60%) and 3350 (40%). 5 g PEG mixture was warmed to 65ºC, stirred while cooling until congealed. 5 g EP powder was incorporated in 5 g ointment base using tile and spatula. This preparation was divided into ten equal parts on the tile and one part is applied topically to each rat every day. -Standard drug: market preparation of diclofenac ointment as Voveran® Emulgel was used as positive control. One hundred gram of this ointment contains 1.16 g of diclofenac diethyl ammonium (equivalent to 1 g diclofenac sodium), isopropyl alcohol, propylene glycol, perfume, Cream 45, and other additives.

38

Rats were randomly divided into four groups of 8 animals in each: group-I, control treated with ointment base; group-II, Entada phaseoloides paste; group-III, Entada phaseoloides ointment; and group-IV, diclofenac sodium ointment. All topical formulations were gently applied to the left knee joint surface. Ointment base was applied to control rats. Procedure Monoiodoacetate (MIA) was dissolved in saline 90 mg/ml. Under pentobarbital anaesthesia, rat was positioned on the back and left leg flexed 90º at the knee. The patellar ligament was palpated below the patella and 50 μl (4.5 mg) of MIA solution was given by intraarticular injection through the patellar ligament of knee using 26G needle. Care was taken not to advance the needle too far into the cruciate ligament. Topical treatment was started, as per groups on the same day and continued up to day 14. On day 15 rats were sacrificed; skin removed and knee joints observed for gross changes [7]. Knee joints removed and fixed in 10% formalin. All joints were decalcified by 10% formic acid for 15 days and embedded in paraffin. The blocks were cut into thin sections and were stained by haematoxylin and eosin and histopathology studied. Parameters observed Gross changes in the knee joint like color and thickness of joint capsule, and surface irregularity were observed. Microscopically, following parameters were observed [7]: -Chondrocyte changes; structural change in the joint surface, ulceration, fibrillation of cartilage surface, disorganization of chondrocytes, exposure of subchondral bones, and cellular changes in hypertrophied chondrocytes. -Inflammatory changes; degeneration and necrosis in the joint, inflammatory cell infiltration in synovial tissue, synovial cell proliferation, and uptake of Safranin-O staining.

DOI 10.5455/jeim.061012.or.048

Journal of Experimental and Integrative Medicine 2013; 3(1):37-41 Histopathological change of each knee was quantitatively expressed simply by the summation of individual grades [slight: 1(+); moderate: 2(++); severe: 3(+++)] and calculating the average pathology scores, for each finding [7]. Statistical analysis The statistical package Graph Pad Prism 5 was used to analyse all results. Values are expressed as mean ± SEM. One way ANOVA followed by post hoc Dunnett’s test was used for analysis of data and for comparisons between treated and control groups; p < 0.05 was considered significant.

RESULTS AND OBSERVATIONS Gross observations of the knee joint were done after removal of the skin over the joint. The capsule of the knee joint in EP paste treated group appeared white and glistening, while the capsule of the knee joint in control group was slightly thickened with irregular surface and lost normal shine.

Total histopathological score of all drug treated groups was significantly (p < 0.001) less in comparison with control (Table 1). It was seen that both EP formulations and diclofenac sodium reduced inflammation significantly (p < 0.001; Fig.1). All formulations to some extent, also had shown chondroprotective effect (p < 0.05, Fig.2). After injection of MIA in left knee joint, swelling appeared on the knee joint in all the animals which was gradually resolved by the 5th day. Gait of all animals was not affected initially but in the 2 nd week there was a change in the gait of control rats. On gross observation, knee joint capsule of the control group animals was thick, irregular and dull-looking whereas in drug treated rats it was white, smooth and glistening (Fig.3). On microscopic observation, it was seen that both EP formulations and diclofenac sodium reduced inflammation significantly (Fig.4&5). All formulations, to some extent, also had shown chondroprotective effect and significantly reduced total pathology score.

Figure 1. Inflammatory changes according to histopathological score

Figure 3. Gross observation of knee joint

Figure 2. Chondrocyte damage score of all study groups

Figure 4. Histopathologic changes in knee joint of a control rat

http://www.jeim.org

39

Dawane et al: Entada phaseoloids in monoiodoacetate-induced osteoarthritis noninvasive nature of the treatment [10, 11]. So, in present study, paste and ointment formulations of E.phaseoloids were studied. Monoiodoacetate-induced arthritis model has been used to induce degenerative joint disease closely resembling osteoarthritis. This is a rapid, reproducible animal model that mimics both the histological changes and symptoms of OA associated joint discomfort allowing both the rapid analysis of the mechanisms involved with OA pain and the evaluation of therapeutic interventions to treat the joint discomfort associated with the disease [12].

Figure 5. Histopathologic changes in knee joint of a drug-treated rat

DISCUSSION At present, no treatment is available which can halt the degradation of the cartilage or stimulate its fresh syhthesis or reverse the pathological changes of OA. All treatment is aimed at pain relief and increasing the range of joint mobility [8]. The present study was planned to evaluate the efficacy of topical application of Entada phaseoloids formulations in animal models of osteoarthritis. Oral methanolic extract of EP seeds was effective in subacute inflammation [9]; however, oral administration of E.phaseoloids is difficult because of its emetic action. E.phaseoloids is used topically as freshly prepared paste by practitioners of traditional and ayurvedic medicine for rheumatoid arthritis and osteoarthritis [4, 5]. Topical anti-inflammatory agents used commonly are available in ointment forms, i.e. ready to use formulations. The delivery of drug through the skin has long been a promising concept because of the ease of access, large surface area, vast exposure to the circulatory and lymphatic networks, and

MIA causes degenerative changes in articular cartilage by inhibiting glyceraldehyde-3-phosphate leading to disruption of chondrocyte metabolism [13]. It has also been demonstrated that the injection of MIA into the knees of rats resulted in increase in matrix metalloproteinases (MMP) activity which are implicated in the cartilage destruction, ultimately resulting in disruption of chondrocyte metabolism and eventual cell death. So, initial injury caused by injection of MIA is cartilage damage. After initial insult, there is accumulation of inflammatory cells which release inflammatory cytokines [14]. Cytokines set up vicious cycle of inflammation and damage to joint. In this model, chondroprotective and antiinflammatory agents are effective. Phytochemical studies of E.phaseoloides showed the presence of amorphous saponins (entadamide A and entadamide B), levorotatory glycosides, raffnose, traces of alkaloids [15, 16]. The membrane-permeabilizing activity showed by a large number of saponins, whether triterpenoid or steroid. Strong foaming of aqueous solutions is the characteristic of saponins. This could be the factor responsible for increasing availability of EP in paste formulation leading to better efficacy [17, 18].

Table:1 Histopathological score after topical application of Entada phaseoloides on MIA-iInduced osteoarthritis Microscopic Findings

Group I

Group II

Group III

Group IV

Structural change in the joint (surface irregularities)

0.88 ± 0.84

0.38 ± 0.52

0.38 ± 0.52

0.25 ± 0.46

Ulceration

0.25 ± 0.46

0±0

0.13 ± 0.35

0±0

Fibrillation of cartilage surface

0.13 ± 0.35

0±0

0.13 ± 0.35

0.13 ± 0.35

Disorganization of chondrocytes

0.88 ± 0.36

0.13 ± 0.35

0.13 ± 0.35

0±0

Exposure of subchondral bones

0±0

0±0

0±0

0±0

Cellular changes of chondrocytes hypertrophy

1±0

0±0

0±0

0.25 ± 0.46

Degeneration Necrosis

1 ± 0.93

0.25 ± 0.46

0.25 ± 0.46

0±0

Inflammatory cell infiltration in synovial tissue

0.38 ± 0.52

0.25 ± 0.46

0±0

0.13 ± 0.35

Synovial cell proliferation

0.75 ± 0.46

0.13 ± 0.35

0.25 ± 0.46

0±0

Safranin-O staining (reduction of staining in cartilage)

1 ± 0.93

0±0

0±0

0±0

Total Pathology Score

6.25 ± 0.66

1.13 ± 0.32***

1.38 ± 0.38***

0.75 ± 0.27***

***Statistically different compared to group I.

40

DOI 10.5455/jeim.061012.or.048

Journal of Experimental and Integrative Medicine 2013; 3(1):37-41 3.

Flower RJ, Moncada S, Vane JR. Analgesic, antipyretics and anti-inflammatory agents-drugs employed in the treatment of gout. In: Goodman LS, Gilman A (eds), The Pharmacological Basis of Therapeutics, Macmillan, New York, NY, USA, pp 682709, 1980.

4.

Kirtikar KR, Basu BD, Blatter E. Indian Medicinal Plants. Lalit Mohan Basu Publishers, Allahabad, Uttar Pradesh, India, pp 906907, 1935.

5.

The wealth of India, A Dictionary of On Indian Raw Materials and Products-Industrial National Institute of Science Communication and Information Resources, New Delhi, India, pp 174-175, 1948.

6.

Reynolds J, Parfitt K, Parsons A, Sweetman S. Martindale: The Extra Pharmacopoeia, 29th edition, The Pharmaceutical Press, London, UK, pp 1129-1130, 1989.

Figure 6. Hypothetical schema showing the possible effects of Entada phaseoloides in MIA-induced osteoarthritis

7.

Kobayashi K, Imaizum R, Sumichika H, Tanaka H, Goda M, Fukunari A, Komatsu H. Sodium iodoacetate-induced experimental osteoarthritis and associated pain model in rats. J Vet Med Sci 2003; 65:1195-9.

From the results of the present work, we can say that E.phaseoloides has good penetrability through the skin due to the presence of saponins in it. It inhibits the release of chemical mediators and breaks the vicious circle of the joint damage as seen from the less inflammatory score in the drug-treated groups. It also presents some chondroprotective effect. Thus, it is apparent that EP is effective in the present animal model of osteoarthritis (Fig.6).

8.

Satoskar RS, Bhandarkar SD, Rege NN. Pharmacology and Pharmacotherapeutics. 21st edition, Churchill-Livingstone, New York, NY, USA, pp 1039-1040, 2009.

9.

Gupta R, Rathi BS, Thakurdesai PA, Bodhankar SL. Antiinflammatory and analgesic effect of methanolic extract of Entada phaseoloides seeds. J Cell Tissue Res 2006; 6:609-13.

In conclusion, both paste and ointment topical formulations of Entada phaseoloides show protective effect in osteoarthritis in this experimental model. Further studies are required to evaluate the exact mechanism of action of anti-inflammatory and chondroprotective effect. We have planned experiments to derive exact mechanism of action, to find out optimum duration of treatment and to study effectiveness of other formulations.

10. Kyuki K, Shibuya T, Tsurumi K, Fujimura H. Anti-inflammatory effect of diclofenac sodium ointment (cream) in topical application. Jpn J Pharmacol 1983; 33:121-32. 11. Aukunuru J, Bonepally C, Guduri V. Preparation, characterization and optimization of ibuprofen ointment intended for topical and systemic delivery. Trop J Pharm Res 2007; 6:85560. 12. Bendele A, McComb J, Gould T, McAbee T, Sennello G, Chlipala E, Guy M. Animal models of arthritis: relevance to human disease. Toxicol Pathol 1999; 27; 134-42. 13. Guzman RE, Evans MG, Bove S, Morenko B, Kilgore K. Monoiodoacetate-induced histological changes in subchondral bone andarticular cartilage of rat femorotibial joints: an animal model of osteoarthritis. Toxicol Pathol 2003; 6:619-24. 14. Janusz MJ, Hookfin EB, Heitmeyer SA, Woessner JF, Freemon AJ, Hoyland JA, Brown KK, Hsieh LC, Almstead NG, De B, Natchus MG, Pikul S, Taiwo YO. Moderation of iodioacetateinduced experimental osteoarthritis in rats by matrix metalloproteinase inhibitors. Osteoarthritis Cartilage 2001; 9:751-60. 15. Okada Y, Shibata S, Ikekawa I, Javellana AMJ, Kamu O. Entada saponin-III, a saponin isolated from the bark of Entada phaseoloides. Pytochemistry 1987; 26:2789-96.

REFERENCES 1.

Brandt KD. Osteoarthritis. In: Kasper DL, Braunwald E, Hauser S, Longo D, Jameson JL, Fauci AS (eds) Harrison’s Principle of Internal Medicine, 16th edition, McGraw Hill, New York, NY, USA; pp 2036-2045, 2005.

2.

Mohan H, Textbook of Pathology. Jaypee Brothers Medical Publishers, 5th edition, New Delhi, India, pp 850-851, 2005.

16. Ikegami F, Sekine T, Aburada M, Fujji Y, Komatsu Y, Murakoshi I. Synthesis of entadamide A and entadamide B isolated from Entada phaseoloides and their inhibitory effects on 5-lipoxygenase. Chem Pharm Bull (Tokyo) 1989; 37:1932-33. 17. Francis G, Kerem Z, Makkar HP, Becker K. The biological action of saponins in animal systems: a review. Br J Nutr 2002, 88, 587-605. 18. Bangham AD, Horne RW, Glauert AM, Dingle JT, Lucy JA. Action of saponins on biological cell membranes. Nature 1962; 196:952-3.

This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided that the work is properly cited.

http://www.jeim.org

41