Study of In-vivo Analgesic Activity | Experiment

A) ANIMALS

Swiss albino mice (20-25 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. All of the animals were left for 2 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.

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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 standard laboratory conditions (24 ± 2°C; 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 STUDIES

The acute oral toxicity studies were performed to study the acute toxic effects and to determine 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 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 the first three hours for any toxic manifestation. Thereafter, 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 ACTIVITY

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A) Method 1: Hot plate method

Heat is used as a source of pain. Animals were individually placed on the hot plate maintain at constant temperature (55°C) 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 dissolved in the Carboxy Methyl Cellulose (0.5% suspension). Control, standard 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 reference drug for comparison. The result 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 Tukey’s Multiple Comparison Test where p < 0.05 was accepted to be a significant difference.

B) Method 2: Acetic Acid Induced Writhing Method

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Analgesic activity was determined by calculating total number of writhings, following intraperitoneal (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 sterile 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 volume 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 control – No. of writhings for test compound

—————————————————————– *100

No. of writhings for control

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II) ANTI-PYRETIC ACTIVITY STUDIES:

Albino rats of Wistar strain of either sex weighing between 170-190g were used. For induction of fever in rats, 20% w/v of brewer’s yeast in distilled water was administered by subcutaneous injection. All animals were induced pyrexia by injection of 10 ml/kg of brewer’s 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 thermometer to a depth of 2 cm into the rectum. The rise 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 minutes post treatment. Decrease in rectal temperature post treatment indicated antipyretic effect. The difference in body temperature was recorded.

III) ANTI-INFLAMMATORY ACTIVITY:

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The anti-inflammatory activity of compounds on carrageenin-induced rat paw oedema was determined according to the method described by Winter et al. (1962). The experimental animals were divided into ten groups, each containing five animals. First 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 3^rd to 10^th 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 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 (Kouadio et al., 2000):

% Inhibition = 1-× 100

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.

IV) ANTIMICROBIAL ACTIVITY

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Antimicrobial 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 series of antimicrobials.

The principle use of antibiotics is to help the body fight bacterial and/or fungal infections. The course of an infection is often linked to a race between the pathogen’s ability to grow in the host tissue and the tissue’s ability to capture and destroy the invading pathogen. Antibiotics are given to weaken or kill some of the invading Pathogens; hopefully, the body’s 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 clear 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 Activity:

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In 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 species were taken. Petridishes, cork borer, beakers, glass syringes and test tubes were sterilized by dry heat sterilization at 160ºC for 1hr in hot air oven.All the synthesized compounds were dissolved in DMF to make the concentrations of 40µg/ml.

Preparation of nutrient agar media:

Preparation 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 121ºC for 15mins.

Preparation of agar plates:

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The sterilized nutrient media was cooled to 45º-46ºC 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 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. All the plates were incubated for 24hrs at 37ºC. At the end of incubation period, diameters of the zone of inhibition were measured and recorded.

(B) Antifungal Activity:

The 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 160ºC for 1hr in hot air oven. Each sample compound was dissolved in DMF to make the concentrations of 40µg/ml. Clotrimazole and Amphotericin B were used as standard dugs.

Media for fungi:

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Sabouraud Dextrose Agar : 65g procured from Himedia, Mumbai

Distilled water : 1000ml

Preparation of agar media:

The preparation of the media involves the following steps:-

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• 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 121ºC for 15mins.

The sterilized nutrient media was cooled to 45º-46ºC 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 25ºC for 48hrs. The zone of inhibition was measured and recorded.

V) IN-VITRO ANTI-INFLAMMATORY ACTIVITY

Method followed: In vitro inhibition of albumin denaturation:

Denaturation 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.

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Materials:

• Bovine serum albumin (sigma)
• Buffer tablets (7.4 pH)
• DMF
• Ibuprofen (standard)
• Distilled water (q.s.)

METHOD:

The test compounds were dissolved in minimum amount of dimethyl formamide (DMF) and diluted with phosphate buffer (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 27º±1ºC for 15 min. Denaturation was induced by keeping the reaction mixture at 60º±1ºC 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).

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