EVALUATION OF ANALGESIC ( IN VIVO ) ACTIVITY OF ARIFLEX TABLET IN COMPARISON WITH DICLOFENAC AND ACECLOFENAC USING ACETIC ACID INDUCED WRITHING MODEL IN MICE

Objective: The present study was conducted to evaluate analgesic activity of Ariflex Tablet which is a polyherbal formulation conceptualized and developed by Ari Healthcare Private in comparison to Aceclofenac and Diclofenac Tablet. Methods: Albino mice of either sex weighing 20–25 g were taken and divided into four groups with six animals in each group. Group 1 (Controlled Group) animals were starved overnight. Group 2 animals were orally administered with Diclofenac Tablet as Standard drug. Group 3 animals were orally administered with Aceclofenac Tablet as Standard drug and Group 4 Animals were orally administered with Ariflex Tablet. The test and standard drugs were orally administered with feeding needle after 1 h of injecting 1% acetic acid intraperitoneally in volume of 0.1 ml/10 g body weight. Writhing episodes were recorded for 30 min by counting the stretching. Results: All the tested formulations possess analgesic activity in acetic acid induced writhing model. Aceclofenac possesses strong analgesic activity compared to other formulations tested. In Ariflex Tablet Group, the number of writhes was 120.6±41.4. If compared to control group, the number of writhes was significantly less suggesting analgesic activity of Ariflex Tablet. Analgesic activity of Ariflex Tablet was close to that of Diclofenac Sodium. Conclusion: It can be concluded that Ariflex Tablet possesses significant analgesic activity. Ariflex Tablet can be used in the management of Osteoarthritis, Rheumatoid arthritis, Gouty arthritis, Lumbago, Sciatica, and Spondylitis.


INTRODUCTION
OA is a chronic degenerative joint disease characterized by loss of or injury to articular cartilage, sub-chondral thickening, and hypertrophy of bone and alterations of the synovial membrane and joint capsule [3]. In OA, bone rubbing causes pain, swelling, and restricted range of motion at the affected joint. The joint may also lose its normal shape. In the normal adult, articular cartilage consists of a delicate system of cells and matrix proteins, which have the function of creating a viscoelastic tissue with high biomechanical stability and low friction. Articular cartilage remains stable, if the process of degeneration and regeneration of cells and matrix proteins occurs in equilibrium. Chondrocytes are the cartilage cells, which produce and maintain the cartilaginous matrix, which consists mainly of collagen and proteoglycans. The alteration of chondrocyte transplantation and degeneration of cartilage due to various triggering factors causes OA [4].
At present, very few underlying factors are known to cause OA. However, some common factors such as age, sex, obesity, genetics, bone density, smoking, and local factors including trauma are main contributors to the pathogenesis of OA. OA with no known cause is termed as primary OA. It is mostly related to aging. Secondary OA results subsequent to another disease or condition. The above-mentioned factors initiate alterations in the equilibrium of cartilage formation and enhance degenerative cascade thus cause OA [1,3,4].
In general, OA is managed by symptomatic treatment methods such as use of pain and inflammation medications. Acetaminophen is considered to be the first-line therapy in the management of OA. Nonsteroidal anti-inflammatory drugs (NSAIDs) (selective and nonselective COX-2 inhibitors) are also commonly used for OA. Furthermore, the symptomatic slow acting drugs for OA such as diacerein, hyaluronic acid, and chondroitin sulfate are useful in OA management [5]. In OA, intra-articular corticosteroid injections are believed to be most effective in patients with evidence of inflammation, effusion, or both. Various other therapies such as transcutaneous nerve stimulation, thermal modalities, acupuncture, and surgery (including joint replacement) have also been used to treat OA. At present, though pharmacological, mechanical, and surgical interventions are used, there is no known cure for OA. Furthermore, above mentioned treatment options lead to many side effects and drawbacks. Thus, physicians and patients tend to move toward the use of alternative treatment methods [5][6][7].
Sandhigata Vata described under Vatavyadhi in Ayurveda can be correlated to OA. According to Ayurveda, Sandhi means joints and Vata has been considered the most important Dosha (humor) among the three Doshas. Thus, Sandhigata Vata means vitiated Vata residing at Sandhi. In Sandhigata Vata, vitiated Vata in joints causes severe pain, dryness, and obstructed joint movements. In Ayurveda, various local as well as oral treatment modalities have been used for the management of Sandhigata Vata. Various types of hot fomentations have been advocated as effective treatment measures. Local therapy includes massage with medicated oils such as Mahanarayan Taila, and Narayan Taila. followed by hot fomentation, whereas the oral therapy mainly includes the use
In the present study, an attempt has been made to evaluate analgesic (in vivo) activity of Ariflex Tablet in comparison with oral Diclofenac sodium and Aceclofenac using acetic acid induced writhing model.

Study site
The study was conducted at Padmashree Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune-411018.

Ethical consideration and approval
All laboratory animal handling and experimental procedures were performed in accordance with the CPCSEA guidelines (198/99/ CPCSEA) and study protocol. The Institutional Animal Ethics Committee (IAEC) of Padm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune -18 has approved the study in the meeting held on December 31, 2014. The approved protocol number was DYPIPSR/ IAEC/14-15/P-04.

Study drugs
Study drugs used in the study are Diclofenac sodium, Aceclofenac, and Ariflex tablet. Drugs including Diclofenac sodium and Aceclofenac were purchased from market whereas Ariflex tablet was supplied by Ari Healthcare Private Limited Pune. The compositions of Ariflex tablet are mentioned in Table 1.

Drug material
The material used for the study is shown in following Table 2.

Study animals
Male Albino Mice of 3-4 weeks age and having weight between 20 g and 25 g were used in the study. The details are given in Table 3.

Procurement of study animals
All animals were obtained from National Institute of Biosciences Pune. Animals were housed standard laboratory conditions of temperature and 12 h light and 12 h dark cycle with free access to standard pellet diet and water.

Study procedure
Albino mice of either sex weighing 20-25 g were taken and divided into four groups with six animals in each group. Group 1 (Controlled Group) animals were starved overnight. Group 2 animals were orally administered with Diclofenac Tablet as Standard drug. Group 3 animals were orally administered with of Aceclofenac Tablet as Standard drug and Group 4 Animals were orally administered with Ariflex Tablet. The test and standard drugs were orally administered with feeding needle after 1 h of injecting 1% acetic acid intraperitoneally in volume of 0.1 ml/10g body weight. Writhing episodes were recorded for 30 min by counting the stretching.

RESULTS
• Animals in control group experienced more pain as evident from the results. The number of writhes in control group was 261±28.99 (Mean±SEM) • In Diclofenac group, the number of writhes was 95.4±11.81 (Mean±SEM). If compared to control group, the number of writhes was significantly less suggesting analgesic activity of Diclofenac • The number of writhes in Aceclofenac group was 33.8±11.53 (Mean±SEM). If compared to control group, the number of writhes was significantly less suggesting strong analgesic activity of Aceclofenac • In Ariflex Tablet Group, the number of writhes was 120.6±41.4. If compared to control group, the number of writhes was significantly less suggesting analgesic activity of Ariflex Tablet. The details are presented in following Table 4 and Fig. 1.

DISCUSSION
OA is a chronic degenerative joint disease characterized by loss or injury of articular cartilage, sub-chondral thickening, hypertrophy of bone, and alterations in the synovial membrane and joint capsule [3].
Chondrocytes are the cartilage cells, which produce and maintain the cartilaginous matrix that is mainly composed of collagen and proteoglycans. The alteration of chondrocyte transplantation and degeneration of cartilage due to various triggering factors causes OA [3].
Goals of treatment for OA include pain alleviation and improving the function of the joint to mitigate the reduction in activity. Various pharmacological and non-pharmacological treatments are used in OA. But being unable to modify the natural course or progression of disease, no treatment has proven curative in nature. Role of both pharmacological and non-pharmacological modalities of treatment has been emphasized in guidelines provided for non-surgical management of OA [20].    Acetaminophen is considered as first-line drug for pain relief. If pain relief is inadequate with acetaminophen, NSAIDs especially, selective COX-II inhibitors are recommended in appropriate dosage. If NSAIDs fail to provide sufficient pain relief and risk of adverse effects of NSAIDs such as gastric bleeding is anticipated, use of intra-articular corticosteroid is advised. Use of the steroidal injection is limited to 2-3 times a year owing to greater chances of progressive cartilage damage through repeated injections in weight-bearing joints [20].
Invasive treatments such as arthroscopic lavage, cartilage debridement, osteotomy, and lastly total joint replacement are considered only when all above conservative treatments fail to provide symptom relief [20].
Various other therapies such as transcutaneous nerve stimulation, thermal modalities, acupuncture, and surgery (including joint replacement) have also been used to treat OA. Although pharmacological, mechanical and surgical interventions are used, there is no known cure for OA. Furthermore, above mentioned treatment options lead to many side effects and drawbacks on their long-term irrational use.
Acetaminophen overdose leads to hepatotoxicity which is common and potentially fatal incident [21]. Numerous prospective studies have identified prevalence of 15-20% for gastric ulcers and 5-8% for duodenal ulcers after 12 weeks of therapy of NSAIDs [22]. Numerous epidemiologic studies have demonstrated that the risk of a significant GI complication is increased in patients on chronic NSAID therapy to a rate of 1-4% per year [22]. Corticosteroid drugs do not independently cause ulcer disease. The use of these drugs in conjunction with NSAIDs, however, increases the risk of a gastrointestinal (GI) event approximately two-fold [22]. NSAIDs are also associated with adverse effects such as renal toxicity, cardiovascular events, increased blood pressure, and deterioration of congestive heart disease [23]. Skin atrophy, Hyperglycemia, Electrolyte abnormalities, Weight gain/moon facies, Purpura, Acne/hirsutism, GI bleed, ulcer, Fatty liver, Osteoporosis, etc., are side effects associated with the use of corticosteroids [24].
To establish analgesic activity of Ariflex tablet, before the clinical trials, this experimental study was conducted. In this study, evaluation of analgesic activity (in vivo) of Ariflex tablet in comparison with Diclofenac Tablet and Aceclofenac Tablet using Acetic acid induced writhing model was done. From the results, it is clear that all the formulations tested possess analgesic activity in acetic acid induced writing model. Aceclofenac being selective COX II inhibitor possesses strong analgesic activity among tested formulations. After Aceclofenac, the less number of writes was found in Diclofenac sodium group. The observed results are in line with the published literature. Ariflex tablet being herbal formulation, showed less analgesic activity as compared to Diclofenac Sodium and Aceclofenac. When compared to control group Ariflex Tablet possesses significant analgesic activity. The analgesic activity of Ariflex Tablet was close to that of Diclofenac Sodium.
Many ingredients of Ariflex Tablet such as Shallaki (B. serrata) [26], Nirgundi (V. negundo) [34,35], and Shunthi (Z. officinale) [38,39] possess analgesic activity. Few ingredients of formulation have central as well as peripheral analgesic activity [34]. Few ingredients reduce the secretion of prostaglandins and also provide relief from the symptoms of pain by inhibition of COX enzyme [38]. Few ingredients have pain suppressing activity possibly mediated through PG synthesis inhibition and through the opioid receptors [34,35,38]. Thus, the observed significant analgesic activity of Ariflex Tablet could be the results of synergetic activities of various analgesic herbs present in the formulation.

CONCLUSION
It can be concluded that Ariflex Tablet possesses significant analgesic activity. Ariflex Tablet can be used in the management of OA, Rheumatoid arthritis, Gouty arthritis, Lumbago, Sciatica, and Spondylitis.