Study of In-vivo Analgesic Activity | Experiment

Study of In-vivo Analgesic Activity ExperimentA) ANIMALSSwiss albino mice (20-25 g) of either sex were employ for study of in-vivo analgesic activity. Animals were unploughed under quantity examing ground conditions i.e. temprature is 24 2C and relative humidity is 60-70%. The study protocol was approved by the institutional animal moral philosophy 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 retained on balanced ration with free access to clean drinking irrigate system. 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. All of the animals were left for 2 sidereal days in the laboratory for acclimatization before the day of experiment and on the last day they were given water only. Minimum of 6 animals were used in each group.Wistar rats of either sex weighing (150-200 g) were used for studying in-vivo anti-inflammatory and antipyretic activity. Swiss albino mice of either sex weighing 20-25 g were used for in-vivo analgesic activity. Animals were maintained under prototype laboratory conditions (24 2C relative humidity 60-70%). Study protocol was approved by the institutional Animal Ethics Committee for the Purpose of Control and Supervision on Experiments on Animals (IAEC, Approval No. 1452/PO/a/11/CPCSEA) before experiment. Wiatar Rats and Albino-Swiss mice from Laboratory Animal House Section, Department of Pharmacology, Devsthali Vidyapeeth College of Pharmacy, Lalpur, Rudrapur (U. S. Nagar) were used in the study. The animals were procured from IVRI, Bareilly (U.P.). Minimum of 6 animals were used in each group.B) ACUTE TOXICITY STUDIESThe ac ute oral toxicity studies were performed to study the acute toxic effects and to determine minimum deadly dose of the synthesizingd compounds. Swiss albino mice of either sex weighing 20-25 g were used for the study. The aqueous solution of compounds were administered orally to different groups of over night fasted mice at the doses of 30, 100, 300, jet and 3000 mg/kg body weight. After administration of the compounds, animals were observed continuously for the first three hours for any toxic manifestation. There afterward, observations were made at regular intervals for 24 hrs. Further the animals were under investigation up to a period of one week. The dose calculated for the synthesized compounds are as following-I) ANALGESIC activityA) Method 1 Hot musical scale method Heat is used as a source of pain. Animals were individually placed on the hot plate maintain at constant temperature (55C) and the reaction of animals, such as paw licking or jump response was taken as the end response. Analgesic drugs/compounds increases the reaction time. The method was first described by Eddy Leimbach (A cut off period of 15 sec is observed to avoid damage to the paw). The compounds were fade away in the Carboxy methyl radical Cellulose (0.5% suspension). Control, commonplace and test compounds were given per orally to the animals and the reaction of time of animals at 15, 30, 60 120 min interval was noted on the hot plate after drug administration. The method of Eddy and Leimbach using techno heated plat analgesic apparatus was used. The standard drug Diclofenac Sodium (50 mg/kg) was used author drug for comparison. The chair was tabulated in Table. Results were expressed as means S.E.M. Statistical significance was analyzed using the one-way analysis of variance followed by Tukeys Multiple Comparison Test where p B) Method 2 Acetic Acid Induced Writhing MethodAnalgesic activity was determined by calculating total numeral of writhings, following intraperiton eal (I.P) administration of 0.6% (0.1 ml/10g) acetic acid in mice .7 Albino mice of either sex (25-30 g) were used. Synthesized compounds (QAA-04H-04S) were administered intraperitonealy (0.5 ml) as a suspension in unimpregnated 0.9% DMSO solution as vehicle. Diclofenac (10mg/kg) was used as the standard drug under same conditions. Acetic acid solution was administered intraperitonealy 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 muckle of vehicle. Results of percentage Analgesic activity of compounds were calculated using following formula and the results are shown in table.% Analgesic activity = No. of writhings for go out No. of writhings for test compound *100No. of writhings for controlII) ANTI-PYRETIC ACTIVITY STUDIESAlbino rats of Wistar strain of either sex weighing in the midst of 170-190g were used. For inductio n of fever in rats, 20% w/v of brewers yeast in distilled water was administered by subcutaneous injection. All animals were induced pyrexia by injection of 10 ml/kg of brewers yeast solution under the skin in between the shoulder blades. The site of the injection was massaged in order to lot 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 line up in rectal temperature was recorded 19 hours after yeast injection.The different groups of febrile rats were orally administered with the respective drugs and rectal temperature was recorded 30, 60, 120, 180 and 300 transactions post treatment. Decrease in rectal temperature post treatment indicated antipyretic effect. The difference in body temperature was recorded.III) ANTI-INFLAMMATORY ACTIVITYThe anti-inflammatory activity of compounds on carrageenan-induced rat paw oedema was determined according t o the method described by Winter et al. (1962). The experimental animals were divided into ten groups, each containing five animals. counterbalance group received sterile normal saline (0.85% NaCl) assigned as control and the second group received standard drug Ibuprofen (20 mg/kg b.w., p.o.). The 3rd to 10th groups were administered the test compounds (at a dose of 20 mg/kg b.w, suspended in 10 ml/kg of 2% gum acacia) orally. 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 beneficial paw served as a mention 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 oed ema and the percentage proscription by the compounds was calculated using the formula (Kouadio et al., 2000)% curtailment = 1- 100where dt is the difference in paw volume in the test compound-treated group and dc the difference in paw volume in the control group.IV) ANTIMICROBIAL ACTIVITYAntimicrobial chemotherapy plays an important role in the treatment of many infectious diseases. However repeated and irrational use of some antibiotics result in resistance i.e., ineffectiveness of drug against the microorganisms. In the recent past, the emergence of drug resistance to antibiotics is more. This situation stimulated us to prepare new serial of antimicrobials.The principle use of antibiotics is to help the body fight bacteriuml and/or fungous infections. The course of an infection is often linked to a race between the pathogens ability to grow in the host tissue and the tissues ability to capture and destroy the invading pathogen. Antibiotics are given to develop or kill some of the invading Pathogens hopefully, the bodys tissue can then destroy the rest.The effectiveness of an antibiotic is preliminarily determined by the size of the zone of inhibition, but zone size varies according to how easily the antibiotic diffuses through the agar, the type of medium used and many other factors. If a sporty zone appears in which there is No microbial growth around the disk, it is called as the zone of inhibition, even though killing may have occurred in this zone.(A) Antibacterial ActivityIn our current study, antibacterial activity was carried out by the agar diffusion method. Here the responses of the organisms to the synthesized compounds were measured and compared with the responses of the standard drugs. The standard reference drugs used in the antibacterial screening were Norfloxacin and Gatifloxacin. For antibacterial activity 2 gram positive bacteria i.e. Enterococci, Staphylococcus aureus and two gram negative bacteria i.e. Escherichia coli Shigella speci es were taken. Petridishes, cork borer, beakers, glass syringes and test tubes were sterilized by dry heat sterilization at 160C for 1hr in hot air oven.All the synthesized compounds were dissolved in DMF to make the dumbnesss of 40g/ml.Preparation of nutrient agar mediaPreparation of the bacteriological media involves the following steps-All ingredients were dissolved in distilled water by boiling.The pH of the medium was determined with a pH meter and adjusted if necessary.The medium so prepared was sterilized by autoclaving at a temperature of 121C for 15mins.Preparation of agar platesThe sterilized nutrient media was cooled to 45-46C and inoculated with respective suspension of micro-organisms. They were mixed well and 200ml each of inoculated media were transferred into separate petridishes. They were allowed to cool at means temp. Until the agar medium completely solidified. Bores were made using cork borer and 0.1ml solution of test drug and control solutions were separatel y added to each bores. The sterile discs of standard reference drugs were placed on the surface. The petridishes were kept for 2hrs to allow the drug to diffuse into the agar media. A sterile atmosphere was maintained during the entire work on by carrying out the work under Laminar Air Flow bench. All the plates were incubated for 24hrs at 37C. At the end of incubation period, diameters of the zone of inhibition were measured and recorded.(B) Antifungal ActivityThe antifungal activity was carried out by agar diffusion method. The responses of the fungal microorganisms to the synthesized compounds were recorded and compared with the standard reference drugs. Two fungal strains namely Aspergillus niger and Aspergillus flavus were taken for the study. Petridishes, cork borer, beakers, glass syringes and test tubes were sterilized by dry heat sterilization at 160C for 1hr in hot air oven. Each sample compound was dissolved in DMF to make the concentrations of 40g/ml. Clotrimazole and A mphotericin B were used as standard dugs.Media for fungiSabouraud Dextrose Agar 65g procured from Himedia, MumbaiDistilled water 1000mlPreparation of agar mediaThe preparation of the media involves the following steps-Sabouraud Dextrose Agar was dissolved in 1000ml of sterile distilled water by boiling.The pH of the medium was determined with a pH meter and adjusted to if necessary.The medium so prepared was sterilized by autoclaving at a temp. of 121C for 15mins.The sterilized nutrient media was cooled to 45-46C and inoculated with respective suspension of fungal organisms. They were mixed well and 200ml each of inoculated media were transferred into separate petridishes. They were allowed to cool at room temp. Until the agar medium completely solidified. Bores were made using cork borer and 0.1ml solution of test drug and control solutions were separately added to each bores. The sterile discs of standard reference drugs were placed on the surface. The petridishes were kept for 2hrs to allow the drug to diffuse into the agar media. A sterile atmosphere was maintained during the entire process by carrying out the work under Laminar Air Flow bench. Then the plates were incubated at 25C for 48hrs. The zone of inhibition was measured and recorded.V) IN-VITRO ANTI-INFLAMMATORY ACTIVITYMethod followed In vitro inhibition of white denaturationDenaturation of proteins is one of the causes of inflammation. Production of auto- antigens in certain rheumatic diseases may be due to in vivo denaturation of proteins. A number of anti-inflammatory drugs are known to inhibit the denaturation of proteins. Mizushima and other have employed protein denaturation as in vitro screening model for anti-inflammatory compounds.MaterialsBovine serum albumin (sigma)Buffer tablets (7.4 pH)DMFIbuprofen (standard)Distilled water (q.s.)METHODThe test compounds were dissolved in minimum amount of dimethyl formamide (DMF) and diluted with phosphate airplane pilot (0.2M, pH 7.4). The final concentration of DMF in all solutions was less than 2.5%. Test solution (1ml) containing different concentration of drug was mixed with 1ml of 1mg/ml albumin solution in phosphate buffer and incubated at 271C for 15 min. Denaturation was induced by keeping the reaction mixture at 601C in water bath for 10 min. after cooling, the turbidity was measured at 660nm in spectrophotometer. The percentage inhibition of denaturation was calculated from control where no drug was added. And compared against standard (Ibuprofen).