Analgesic and Antiinflammatory activity of Amukkarac curanam

Indian Journal of Pharmaceutical Sciences 442 July August 2009 Accepted: 05 August 2009 Revised: 24 March 2009 Received: 16 August 2007 Indian J. Pharm. Sci., 2009, 71 (4): 438-442 3. British Pharmacopoeia, British Pharmacopoeia Commission. Vol. 1, London: Her Majesty’s Stationary Office; 2009. p. 397. 4. European Pharmacopoeia, Council of Europe. 3rd ed, Strasbourg, France,: EDQM Publications; 2000. p. 504. 5. Nahata MC. Measurement of Cefixime in serum and cerebrospinal fluid by high performance liquid chromatography. J Liq Chromatogr 1991;14:3755-60. 6. Eric-Jovanovic S, Agbaba D, Zivanov-Stakic D, Vladimirov S. HPTLC Determination of Ceftriaxone, Cefixime and Ceftaxime in dosage forms. J Pharm Biomed Anal 1998;18:893-7. 7. Baertschi SW, Dorman DE, Occlowitz JL, Spangle LA, Collins MW, Wildfeuer ME et al. Isolation and structure elucidation of a novel product of acidic degradation of cefaclor. J Pharm Sci 1993;82:622-6. 8. Skibic MJ, Taylor KW, Occlowitz JL, Collins MW, Paschal JW, Lorenz LJ et al. Aqueous acidic degradation of carbacephalosporin in loracarbef. J Pharm Sci 1993;82:1010-4.

Conventional or synthetic drugs used in the treatment of diseases are sometimes inadequate and can have serious adverse effects. There is a world wide trend to search for traditional medicines. Siddha medicare is an ancient system of medicine popular amongst Tamil speaking world practiced for over several thousand years. In the present investigation Amukkarac curanam (AC), a polyherbal formulation [1] consisting of medicinal plants is taken for study. Its ingredients and formulation composition are tabulated in Table 1. One of the major ingredients is Withania somnifera reported to possess antistress [2] , antiinflammatory [3] and immunostimulant [4] properties. Other constituents Syzygium aromaticum, cinnamomum wightii, Elettaria cardamomum, Piper nigrum, Piper longum [5] , Zingiber officinale are also reported to be medicinally useful. However scientific data on analgesic and antiinflammatory activity of the formulation is not available. Hence in the present investigation analgesic and antiinflammatory potential of the formulation is explored.
All the ingredients of ammukkarac curanam were procured from Chennai local market and authenticated at Pharmacognosy department of Captain Srinivasa Murti Drug Research Institute of Ayurveda (CSMDRIA). Voucher sample of all Two different sets of mice were randomized with 3 different groups (n=6) for tail immersion and acetic acid induced writhing test respectively. The groups were: group-1 served as vehicle control and received 0.5% CMC solution, group 2 served as positive control and received either pentazocine 10 mg/kg, intraperitoneally (for tail immersion test) or aspirin 150 mg/kg (for acetic acid writhing test), group 3 served as test group and received the drug at dose of 500 mg/kg body weight suspended in 0.5% CMC.
The distal 5 cm of the tail was immersed in the water maintained at about 55° [ 6] . Time taken for withdrawal response was recorded at 0, 5, 15, 30, 60, 90 and 120 min after administering the drug. The cut off time was fixed at 15 s to prevent injury to the tail. Pentazocine was used as positive control.
Acetic acid 0.25 ml of 1% v/v [7] was given to all groups intraperitoneally after 30 min of the drug administration and onset of writhing was noted down and number of writhing (abdominal contraction, trunk twisting response and extension of hind limbs) were noted down for a period of 15 min. Number of writhings produced in the test groups were compared with standard one [8] and the analgesic activity (in terms of % maximum possible effect) was calculated as, dividing the difference between mean writhes of control group and mean writhes of drug treated group by mean writhes of control group and multiplying it by 100.
The Wistar rats were divided into four groups of 6 each. Pellets of surgical cotton weighing 10±1 mg were sterilized in hot air oven at 120° for 3 h. Four pellets each were aseptically implanted subcutaneously into both axillae and groin region, under light ether anesthesia [9] . The first group served as normal control and the second group served as vehicle control and received the vehicle, 0.5% CMC. The 3 rd group received AC suspended in 0.5% CMC, at the dose level of 500 mg/kg body weight, orally for 7 days. The 4 th group was treated with indomethacin at 10 mg/kg body weight orally. On 8 th day the animals were sacrificed, the pellets were dissected out, cleaned from extraneous tissues and dried in hot air oven over night at 70°. The weight of each pellet was recorded. The weight of the pellets taken out from AC administered rats was compared with the weight of pellets taken out from the control group and from the indomethacin administered rats. Blood samples and liver tissue were collected and biochemical studies were carried out.
Serum and 1% liver homogenate (prepared in double distilled water) were used for the estimation of acid phosphatase [10] , glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) [10] and total proteins [11] . Pellet weight was noted in both control and treated animals.
Data were statistically evaluated using ANOVA, expressed as mean±SD followed by Post Hoc Dunnett T3 multiple comparisons test using the 10 version of SPSS computer software. Data were considered significant at P < 0.05.
In tail immersion method (Table 2), Amukkarac curanam increased latency to thermal stimulation when compared to control. AC was found to exhibit very good analgesic activity. Its analgesic activity is significant when compared to pentazocine.
In order to distinguish between the central and peripheral analgesic action of AC, acetic acid-induced writhing response in mice was used. This method is simple, reliable and also affords rapid evaluation of peripheral type of analgesic action. AC showed 73.52% inhibition when compared to aspirin which is 98.32%. There is significant decrease in writhing response induced by acetic acid in AC treated group (Table 3), which proves its analgesic property. The abdominal contraction is related to the sensitization of nociceptive receptors to prostaglandins. It is therefore possible that AC exerts its analgesic effect probably by inhibiting synthesis or action of prostaglandins. Thus the analgesic property of the formulation was demonstrated using both the methods.
In the antiinflammatory study of AC using cotton pellet-induced granuloma formation, it reduced the activity of GPT, GOT and acid phosphatase activity in serum (Table 4). In liver GPT and GOT activity was reduced by AC (Table 5). AC reduced the activity of acid phosphatase. Protein in both liver and serum were reduced by AC.
The inhibition of GOT and GPT activity by this preparation may influence the formation of polypeptides like bradykinin and other kinin-like substances which are released during the inflammatory process. Acid phosphatase is frequently employed as a marker enzyme to assess the lysosomal change during inflammation. By stabilizing the lysosomal membrane, the antiinflammatory drug interferes in the synthesis of lysosomal enzymes which participate in the process of inflammation. Decrease in the levels of protein in serum is indicative of low activity of GPT and GOT by the drug, AC showed good antiinflammatory activity with 28% reduction in granuloma formation. This method described by Meier et al. [12] have showed that foreign body granuloma were provoked in rats by subcutaneous implantation of pellets of compressed cotton. This method has been useful for evaluation of steroidal and non-steroidal antiinflammatory drugs. Cotton pellet-induced granuloma is closely related to the formation of antibodies. AC is found to reduce the antibody formation and thus proves its antiinflammatory property. Based on these results it can be concluded that AC exerted potential analgesic activity, which could probably mediated through both central and peripheral mechanisms. It also has significant antiinflammatory property.

ACKNOWLEDGMENTS
Financial help by Project Director, Tamil Nadu State Aids Control Society and Director CCRAS, AYUSH for providing laboratory facility is greatly acknowledged.    Inflammatory control is compared with normal control and standard and AC is compared with inflammatory control. Values are significant when * P<0.05. Values are mean ± SD from 6 animals in each group.