Study of In-vivo Analgesic Activity | Experiment

Study of In-vivo Analgesic bodily function ExperimentA) ANIMALSSwiss albino mice (20-25 g) of either get glowering were employ for study of in-vivo painkiller activity. Animals were unploughed chthonic standard laboratory conditions i.e. temprature is 24 2C and relative humidity is 60-70%. The study protocol was approved by the institutional animal ethics committee (IAEC) before experiment (Approval no(prenominal) 1452/PO/a/11/CPCSEA). Albino-Swiss mice were taken from Laboratory Animal Ho lend oneself, Devsthali Vidyapeeth College of Pharmacy, Lalpur, Rudrapur (U. S. N agar-agar) 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 stop access to clean drinking weewee. All experimental procedures were conducted in concord 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 le ft for 2 old age 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 apiece group.Wistar rats of either sex weighing (150-200 g) were used for poring over in-vivo anti-inflammatory do drugs and antipyretic activity. Swiss albino mice of either sex weighing 20-25 g were used for in-vivo analgesic activity. Animals were maintained under standard laboratory conditions (24 2C relative humidity 60-70%). Study protocol was approved by the institutional Animal Ethics Committee for the Purpose of Control and comptroller 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 g roup.B) ACUTE perniciousness STUDIESThe acute oral toxicity studies were performed to study the acute toxic effect and to deter bite minimum lethal dose of the synthesized 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 diametrical groups of over night fasted mice at the doses of 30, 100, ccc, 1000 and 3000 mg/kg embody weight. later on administration of the compounds, animals were observed continuously for the first three hours for some(prenominal) toxic manifestation. Thereafter, observations were make at regular intervals for 24 hrs. Further the animals were under investigation up to a period of one week. The dose mensurable for the synthesized compounds are as following-I) ANALGESIC activenessA) Method 1 heated up plate method Heat is used as a reference book of pain. Animals were individually displace on the hot plate maintain at constant temperature (55C) and the recept ion of animals, such as mitt licking or jump response was taken as the end response. Analgesic doses/compounds increases the reaction time. The method was first described by plait Leimbach (A cut off period of 15 sec is observed to avoid damage to the script). The compounds were change state in the Carboxy Methyl Cellulose (0.5% happy chance). Control, standard and tally compounds were given per orally to the animals and the reaction of time of animals at 15, 30, 60 120 min interval was illustrious on the hot plate after do drugs administration. The method of Eddy and Leimbach employ techno heated plat analgesic apparatus was used. The standard drug Diclofenac Sodium (50 mg/kg) was used reference drug for comparison. The result was tabulated in Table. Results were expressed as means S.E.M. Statistical significance was analyzed employ the one-way analysis of variance followed by Tukeys Multiple par visitation where p B) Method 2 Acetic Acid generate Writhing MethodAn algesic activity was determined by calculating total number of writhings, following intraperitoneal (I.P) administration of 0.6% (0.1 ml/10g) acetic caustic 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 uninventive 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 certain an equal brashness of vehicle. Results of serving Analgesic activity of compounds were calculated exploitation following formula and the results are shown in table.% Analgesic activity = No. of writhings for delay No. of writhings for test compound *100No. of writhings for controlII) ANTI-PYRETIC ACTIVITY S TUDIESAlbino rats of Wistar strain of either sex weighing amid 170-190g were used. For installment of fever in rats, 20% w/v of brewers yeast in distilled water was administered by subcutaneous injection. All animals were bring forth 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 spread the suspension beneath the skin. Basal rectal temperature was measured before the injection of yeast, by inserting digital clinical thermo bill to a depth of 2 cm into the rectum. The revive in rectal temperature was recorded 19 hours after yeast injection.The variant groups of febrile rats were orally administered with the respective drugs and rectal temperature was recorded 30, 60, 120, 180 and 300 minutes post discussion. 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 carrageenin-induced rat paw oedema was determined harmonize to the method described by Winter et al. (1962). The experimental animals were divided into x groups, each containing five animals. First group true sterile expression 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 tenth groups were administered the test compounds (at a dose of 20 mg/kg b.w, hang 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 right paw served as a reference to non inflammed paw for comparison. The initial paw tidy sum 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 inject ion. 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 (Kouadio et al., 2000)% Inhibition = 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 ACTIVITYAntimicrobic chem differentapy plays an primary(prenominal) role in the treatment of many infectious diseases. However repeated and irrational use of some antibiotics result in resistance i.e., ineffectuality of drug against the microorganisms. In the recent past, the emergence of drug resistance to antibiotics is more. This stake stimulated us to prepare new series of antimicrobials.The principle use of antibiotics is to help the body fight bacteriuml and/or fungal infections. The of course of an infection is often linked to a race between the pathogens ability to grow in the host tissue paper and the tissues abi lity to capture and destroy the invading pathogen. Antibiotics are given to break 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 soft the antibiotic diffuses through the agar, the type of medium used and many other factors. If a clear zone appears in which there is No microbial growth around the disk, it is called as the zone of inhibition, even though cleanup may take a leak occurred in this zone.(A) Antibacterial ActivityIn our authorized study, antibacterial activity was carried verboten by the agar dispersal 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 b acteria i.e. Enterococci, Staphylococcus aureus and two gram negative bacteria i.e. Escherichia coli Shigella species were taken. Petridishes, cork borer, beakers, crosspatch syringes and test tubes were sterilise by dry heat sterilization at 160C for 1hr in hot air oven.All the synthesized compounds were dissolve in DMF to make the concentrations of 40g/ml. dressing 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 sterilise 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 die petridishes. They were allowed to cool at room temp. Until the agar medium all sol idified. 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 turn tail 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) fungicidal 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 Amphotericin 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 albumin 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.Materialsbovid serum albumin (sigma)Buffer tablets (7.4 pH)DMFIbuprofen (standard)Distilled water (q.s.)METHODThe test compounds were dissolve d in minimum amount of dimethyl formamide (DMF) and diluted with phosphate yield (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 concoction at 601C in water can 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).