2.1 Reagents Used All reagents and chemicals that were used in the experiments were of analytical grade. Distilled water, centrally circulated around the laboratory, was used for any dilution, washing, or control purpose. PNR were purchased from University Ayurvedic Research Centre, Jahangirnagar University, Savar, Bangladesh. Pharmaceutical grade Tramadol, and Diclofenac Sodium were purchased from Square Pharmaceuticals Bangladesh Ltd. Ketamine for anesthesia was purchased from Advanced Chemical Industries (ACI) limited. All other reagents were purchased from Sigma Aldrich (USA) unless mentioned otherwise.
2.2 Dose and Route of Administration 0.9% NaCl was administered to the animals Per Oral (p.o.) at a volume that would not cause any additional psychological or physiological stress to the animals. For experimental purpose 10mL/kg, 20mL/kg, and 40mL/kg doses of PNR were used.
2.3 Maintenance and Use of Test Animals Healthy Swiss Albino mice (5-6 weeks old, only males) weighing 20-25g and Sprague- Dawley rats weighing 130-160g were procured from Jahangir Nagar University Animal House. All test subjects were kept in the air-conditioned animal house of the Pharmacy Department North South University at a temperature of 25±2ºC with a 12h light/dark cycle. The rats were kept in white plastic cages of dimension 30×20×13 cm. Soft sterilized wood shavings were used as bedding. The test subjects were provided with standard rat pellet diet and filtered drinking water ad libitum. This study was approved by an ethics committee of North South University which gave its consent in absolute accordance with the recommendations of the international association for the study of pain.
2.4 Grouping and Drug Administration The animals were randomly divided into several groups of 8 mice or rats per group for the planned analgesic and anti-inflammatory tests. Control groups were treated with 0.9% NaCl p.o. at a volume that would not cause any additional psychological or physiological stress to the animals. Positive controls were treated with Tramadol and Diclofenac Na. Treatment groups were treated with three doses (10mL/kg, 20mL/Kg, and 40mL/Kg) of PNR (p.o.).
2.5 Determination of CNS Modulation in Analgesic Activity 2.5.1 Hot plate test The hot plate test was performed on the test subjects in a slightly modified version from the one described earlier. The animals were placed on hot plate apparatus (Model-35100, manufacturer-UGO Basile of Italy) maintained at a temperature of 54±0.5ºC for a maximum time of 20s per exposure to prevent blister formation and skin damage, both of which might affect the results. The mice were screened for initial nociceptive effect; only those which showed an initial response (jumping or paw-licking) within 8-13 seconds were retained to carry out the experiment. The control group was administered with 0.9% NaCl. The treatment groups were treated with PNR (10mL/kg, 20mL/Kg, and 40mL/kg, p.o.) and Tramadol (10mg/kg p.o.).Naloxone (5mg/kg i.p.) was administered with PNR (10mlL/kg, 20mL/Kg, and 40mL/Kg) and Tramadol to four different groups, other than the treatment groups, to ensure that the observed results were not caused due to activity of endogenous opoids. It would also reconfirm the role of opioid agonism of the PNR, if any, as Naloxone is an antagonist of opioid receptor and would inhibit any activity, if shown, by PNR. Reaction time was recorded as latency period, when the animals licked their fore and hind paws and jumped, at 0, 30, 60, 120, 180, 240 and 300 minutes after the treatment.
2.5.2 Tail immersion test The tail immersion test was performed according to the procedures used by Wang et al., with minor modifications. Briefly, the lower two-third of mouse’s tail was immersed in a constant temperature water bath at 50±0.20C. The reaction time, i.e. the amount of time it takes the animal to withdraw its tail, was measured at 0, 30, 60, 90, and 120 min after drug treatment. PNR (10mL/kg, 20mL/Kg, and 40mL/Kg p.o.), Tramadol (10mg/Kg p.o.), and 0.9% NaCl (p.o.) were administered to treatment groups. Naloxone (5mg/kg i.p.) was administered with PNR (10mL/kg, 20mL/Kg, and 40mL/Kg) and Tramadol to four different groups, other than the treatment groups, to ensure that the observed results were not caused due to activity of endogenous opoids. It would also reconfirm the role of opioid agonism of the PNR, if any, as Naloxone is an antagonist of opioid receptor and would inhibit any activity, if shown, by PNR. To avert any sort of tissue injury, the cut-off time for tail immersion was fixed at 20s.