Study of In-vivo Analgesic Activity in Animals

Study of In-vivo Analgesic Activity in Animals A) ANIMALS Swiss albino mice (20-25 g) and wistar rats (150-200 g) of either sex were used for study of in-vivo analgesic activity. Animals were kept under standard laboratory conditions i.e. temprature is 24  ± 2 °C and relative humidity is 60-70%. The study protocol was approved by the institutional animal ethics committee (IAEC) before experiment (Approval No. 1452/PO/a/11/CPCSEA). Albino-Swiss mice were taken from Laboratory Animal House, Devsthali Vidyapeeth College of Pharmacy, Lalpur, Rudrapur (U. S. Nagar) and used for the study. The animals were procured from IVRI, Bareilly (U.P.) The animals were kept in polypropylene cages and maintained on balanced ration with free access to clean drinking water. All experimental procedures were conducted in accordance with the guide for Care and use of laboratory animals and in accordance with the Local animal care and use committee. Paddy husk was provided as bedding material, which was cleaned every day. The cages were maintained clean. All o f the animals were left for 2 days in the laboratory for getting used to before the day of experiment and on the last day they were given water only. Minimum of 6 animals were used in each group. B) ACUTE TOXICITY STUDIES The acute oral toxicity studies were carried out to study the acute toxic effects and to determine minimum toxic dose of the synthesized compounds. For the study swiss albino mice of either sex weighing 20-25 g were used. The aqueous solution of compounds were administered orally to different groups of over night fasted mice at the doses of 30, 100, 300, 1000 and 3000 mg/kg body weight. After administration of the compounds, animals were observed continuously for any toxic manifestation for the first three hours. There after, observations were made at regular intervals for 24 hrs. Further the animals were under investigation up to a period of one week. I) ANALGESIC ACTIVITY For the study of analgesisc activity two methods were used. (A) Hot Plate method (B) Acetic caid induced writhing method A) Method 1: Hot plate method186,187,188,189 By applying heat pain is inced to animals. All the animals one by one are kept in the hot plate maintain at constant temperature (55 °C) and there reactions was noted i.e. paw licking or jumping response. Work plan Albino rats of either sex (150-200 g) were selected and divided into four groups of six animals each. All the animals were fasted for 24 hrs. before the start of the experiment and water was given adlibitum. The animals were treated as follows : Group 1 : Control group received 0.5% sodium CMC (1mg/kg) orally. Group 2 : Diclofenac sodium 50mg/kg were administered orally. Group 3 : Novel benzimidazole substituted pyrazolidine 3,5 dione derivative in dose level of 50mg/kg was administered orally. Group 4 : Novel 2-quinolone substituted pyrazolidine 3,5 dione derivative in dose level of 50mg/kg was administered orally. Here Group 1 is the control, group 2 is active standard and group 3 and group 4 are test. Experimental Details The hot plate method is based on the fact that analgesic compounds increases the response time. This method was first described by Eddy Leimbach, where a cut off period of 15 sec is observed to avoid damage to the paw. All the synthesized compounds were dissolved in the CMC (0.5% suspension). After administration of control, standard and test compounds the animals were kept at the hot plate and their reaction time were note at 15, 30, 60 120 min interval. All the doses were given orally to animals. Diclofenac Sodium at dose of 50 mg/kg was used standard drug for comparison. The results so obtained were tabulated in Table 10, 12, 14 and 16 and figure 07, 09, 11 and 13. Results were expressed as means  ± S.E.M. Statistical significance was analyzed using the two-way anova analysis of variance followed by Tukey’s Multiple Comparison Test where p B) Method 2: Acetic Acid Induced Writhing Method186,187,188,189 In this method pain is induced by intraperitoneal (I.P) administration of 0.6% (0.1 ml/10g) acetic acid in mice. Analgesic activity was determined by calculating total number of writhings. Work plan Albino mice of either sex (25-30 g) were used for the study. All the animals were fasted for 24 hrs. before the start of the experiment and water was given adlibitum. The animals were treated as follows : Group 1 : Control group received 0.5% sodium CMC (1mg/kg) orally. Group 2 : Diclofenac sodium 20mg/kg were administered orally. Group 3 : Novel benzimidazole substituted pyrazolidine 3,5 dione derivative in dose level of 20mg/kg was administered orally. Group 4 : Novel 2-quinolone substituted pyrazolidine 3,5 dione derivative in dose level of 20mg/kg was administered orally. Here Group 1 is the control, group 2 is active standard and group 3 and group 4 are test. Experimental Details All the synthesized compounds were administered intraperitonealy (0.5 ml) as a suspension in sterile 0.9% DMSO solution as vehicle. Diclofenac Sodium at dose of 20 mg/kg was used standard drug for comparison. Acetic acid solution was intraperitonealy administered 30 min after administration of the compounds. 10 min after intraperitoneal injection of acetic acid solution, the number of writhings per animal was recorded for 20 min. Control animals received an equal volume of vehicle. Results were expressed as means  ± S.E.M. Statistical significance was analyzed using the two-way anova analysis of variance followed by Tukey’s Multiple Comparison Test where p II) ANTI-PYRETIC ACTIVITY STUDIES:190 For antipyretic activity yeast induced pyrexia model was used for the study. Work plan Albino rats of either sex (150-200 g) were selected and divided into four groups of six animals each. All the animals were fasted for 24 hrs. before the start of the experiment and water was given adlibitum. The animals were treated as follows : Group 1 : Control group received 0.5% sodium CMC (1mg/kg) orally. Group 2 : Peracetamol 100mg/kg were administered orally. Group 3 : Novel benzimidazole substituted pyrazolidine 3,5 dione derivative in dose level of 100mg/kg was administered orally. Group 4 : Novel 2-quinolone substituted pyrazolidine 3,5 dione derivative in dose level of 100mg/kg was administered orally. Here Group 1 is the control, group 2 is active standard and group 3 and group 4 are test. Experimental Details For induction of fever in rats, 20% w/v of brewer’s yeast in distilled water was administered by subcutaneous injection. All animals which were used for study, were induced pyrexia by injection of 10 ml/kg of brewer’s yeast solution under the skin in between the shoulder blades. The place of the injection was massaged in order to spread the suspension beneath the skin. Basal rectal temperature was measured before the injection of yeast, by inserting digital clinical thermometer to a depth of 2 cm into the rectum. The rise in rectal temperature was recorded after 19 hours of yeast injection. The rectal temperature was taken after 30, 60, 120, 180 and 300 minutes post treatment. If a drug is having antipyretic effect then there is a fall in the rectal temprature. Results were expressed as means  ± S.E.M. Statistical significance was analyzed using the two-way anova analysis of variance followed by Tukey’s Multiple Comparison Test where p III) ANTI-INFLAMMATORY ACTIVITY: 186,187,188,189 For anti-inflammatory activity carrageenin-induced rat paw oedema method was used. Work plan Albino rats of either sex (150-200 g) were selected and divided into four groups of six animals each. All the animals were fasted for 24 hrs. before the start of the experiment and water was given adlibitum. The animals were treated as follows : Group 1 : Control group received sterile normal saline (0.85% NaCl) orally. Group 2 : Ibuprofen 20mg/kg were administered orally. Group 3 : Novel benzimidazole substituted pyrazolidine 3,5 dione derivative in dose level of 50mg/kg was administered orally. Group 4 : Novel 2-quinolone substituted pyrazolidine 3,5 dione derivative in dose level of 50mg/kg was administered orally. Here Group 1 is the control, group 2 is active standard and group 3 and group 4 are test. Experimental Details This method was described by Winter et al. in 1962. The experimental animals were divided into ten groups, each containing five animals. After 30 min of administration of test compounds, 0.1 ml of 1% (w/v) carrageenin was injected subcutaneously in the subplantar region of the left hind paw. The right paw served as a reference to non inflammed paw for comparison. The initial paw volume was measured within 30 sec of the carrageenin injection by plethysmometer. The relative increase in paw volume was measured in control, standard and test compounds at 1, 2, 3, 4, 5, 6, 7 and 8 h after the carrageenin injection. The difference between initial and final readings was taken as the volume of oedema and the percentage inhibition by the compounds was calculated using the formula- where dt is the difference in paw volume in the test compound-treated group and dc the difference in paw volume in the control group. Results were expressed as means  ± S.E.M. Statistical significance was analyzed using the two-way anova analysis of variance followed by Tukey’s Multiple Comparison Test where p Uttarakhand Technical University, Dehradun 1