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Animals: Swiss albino mice weighing 20 - 25 gms were used for the study. Animals were fed a standard pellet and water and maintained at 24-28OC temperature, 60 - 70% relative humidity and 12 hr day and night cycle. Animals described as fasted were deprived of food for 4 days, but had free access to water.
Acute oral toxicity studies were performed (Ecobichon, 1997) according to OECD (organization for economic Co-operation and department). Swiss albino male mice (n=3/each dose) were selected by random sampling technique were employed in this study. The animals were fasted for 4 hr with free access to water only. Ethanolic extract of 1,5 Benzodiazepine derivative (suspended in CMC) were administered orally at a dose of 5 mg/kg initially to separate group of mice and mortality was observed for 3 days. Here no mortality is observed at the dose of 5mg/kg ,so same procedure is repeated for 50 mg/kg then animal showing some action but no mortality is seen. Now next, same procedure is repeated for 300 mg/kg but mortality was observed in 2/3, 3/3 animals, then this dose administered was considered as toxic dose. The following general behaviors were observed for first one hour and after 24 hrs of test drug administration.
Effect of the derivative (50mg/kg) on General behaviors in mice:
Signs & Symptoms
Weight & Marking
Loss of righting reflux
Decreased motor activity
Increased motor activity
Change in skin colour
"-" Absent & "+" Present
From the study of acute toxicity it has been observed that there was no mortality after 36 hours and also there was no change in general behaviour of the animal. So the drug is safe.
Isoniazid induced convulsions test: -
Isoniazid can precipitate convulsions in patients with seizure disorders. The compound is regarded as a GABA-synthesis inhibitor (Costa et al. 1975). Clonic tonic seizures are elicited in mice which are antagonized by anxiolytic drugs.
Six mice in each group of either sex with a weight of 18 to 22 gm are taken. Group I control animals received 30% aqueous PEG 400 only, group II received standard (diazepam 10 mg/kg) i.p. Group III-VII are treated with the test compound (4a-4e) by oral administration. The synthesized compounds (4a-4e) were suspended in 0.1% solution of SCMC and administered orally in a standard volume of 0.5 ml/20 g body weight at 30 mg kg-1 doses. 30 min after i.p. or 60 min after p.o. treatment the animals are injected with a subcutaneous dose of 300 mg/kg isoniazid (isonicotinic acid hydrazide).The occurrence of clonic seizures, tonic seizures and death is recorded.
Anticonvulsant activity38 was expressed as percentage of tonus and clonus mortality.
The one way ANOVA test performed by using Graph pad Insat software. The results obtained are tabulated in Table 14.
Table 14: Anticonvulsant activity of compounds (4a-4e).
Latency of clonus (min.)
% of clonus
% of tonus and clonus mortlity.
3.20 Â± 0.1797
8.57 Â± 0.2144**
4.03 Â± 0.1806*
4.52 Â± 0.1366
7.53 Â± 0.1438**
8.15 Â± 0.0551**
6.40 Â± 17.84*
n=6; dunnets t test; * P<0.05; ** P<0.01; ***P<0.001 when compared with control.
All the compounds i.e. 4a to 4e showed a significant decrease in % of latency of clonus and % of tonus and clonus mortality. The compounds 4c, 4d,4e showed good anticonvulsant activity and compound 4a and 4b showed moderate activity.
MATERIALS REQUIRED IN MEM:
Monolayer culture bottle of Hep 2 cells
5ml, 10ml serological pipette
Minimal essential media (MEM) with 10%, 2% foetal calf serum
TPVG (Trypsin, PBS, versene, glucose)
Discarding jar, inverted microscope, dessicator
Gloves, spirit, cotton, label pad, marker pen
MATERIALS REQUIRED IN CYTOTOXICITY ASSAY:
Monolayer culture in log phase
Drug extract (different concentrations)
MEM without FCS
5ml sterile storage vial
Tissue paper, spirit, cotton, marker pen and gloves
Micropipette and tips
MINIMAL ESSENTIAL MEDIA PREPARATION:
Media is defined as a complex source of nutritional supplementation vital for the growth proliferation and maintenance of cells in vitro.
The MEM vial is dissolved in the pre sterilized Millipore distilled water and mixed well, closed and sterilized at 15lbs 121°c for 15mins. Allow ingredients in the quantity, depending on the concentration of foetal calf serum (2% or 10%) mix well by shaking. Take care avoid spills pass CO2 using sterile pipette, Shake the bottle, check Ph and adjust to 7.2 to 7.4. The MEM bottles are kept for 2 days at 37°c and checked for sterility, PH drop and floating particles they are then transferred to the refrigerator.
PREPARATION OF INGREDIENT:
Penicillin and Streptomycin: (Concentration 100IU of Penicillin and 100 Âµg 0f Streptomycin)
Dissolve both antibiotics in sterile Millipore distilled water, so as to give a final concentration 100 IU of penicillin and 100Âµg of streptomycin/ml. Mix well and distribute in 1ml aliquots. Store at -20° C Check sterility.
Fungizone (amphotericin B): (conc: 20Âµg/ml)
Dissolve in sterile Millipore distilled water so as to give a final concentration of 20Âµg/ml and distribute in 1ml aliquots in vials. Store at -20°c. Check sterility.
Weigh 3g of l-glutamine accurately and dissolve in 100ml sterile Millipore distilled water and mix well. Filter through Millipore membrane filter 0.22Âµ and distribute in 5ml aliquots in vials. Store at -20°c. Check sterility.
Weigh requisite quantity of sodium-bi-carbonate (to give 7.5% solution) accurately and dissolve in 100ml of sterile Millipore distilled water. Filter through what man filter paper No.4, distribute into bottles and at 121°c, 15lbs, 15mins. Cool and store at +4°c.
5. Foetal calf serum
Bring FCS at room temperature. Inactivated at 56°c in water bath forÂ½ hour and cool at room temperature. If floating particles are seen filter through Seitz filter. Distribute in 100ml,50ml, 20ml quantities in sterile bottles. Store at -20°c.
6. Trypsin, PBS,versene, glucose solution: (TPVG)
2% Trypsin: 100ml
Weigh 2g of trypsin accurately; dissolve in 100 ml sterile Millipore distilled water with magnetic stirrer for Â½ hour. Filter through membrane filter. Store at -20°c.
Weigh 200mg of EDTA accurately. Dissolve in 100 ml of sterile Millipore distilled water. Autoclave at 15lbs/15mins.
10% Glucose -100ml
Weigh 1g of glucose accurately. Dissolve in 100 ml of sterile Millipore distilled water and filter through whatman filter paper and autoclave at 15lbs/15mins.
PBS - 840ml
Penicillin & streptomycin -5ml
Mix all ingredients and adjust the pH to 7.4 with 0.1 N HCl or 0.1 N NaOH. Distribute in 100 ml aliquots. Store at -20°c.
MAINTENANCE OF CELL LINE:
Maintenance of cells involves the following operations:
Dispersion and Sub culturing (seeding) of cells.
Preservation of cells in repository.
Revival of cells from repository.
SUBCULTURING AND MAINTENANCE OF CELL LINE:
1. Bring the medium and TPVG to room temperature for thawing.
2. Observe the tissue culture bottles for growth, cell degeneration, pH and turbidity by seeing in inverted microscope.
3. If the cells become 80% confluent it goes for sub culturing process
4. Wipe the mouth of the bottle with cotton soaked in spirit to remove the adhering particles.
5. Discard the growth medium in a discarding jar keep distance between the jar and the flask.
6. Then add 4 - 5 ml of MEM without FCS and gently rinsed with tilting. The dead cells and excess FCS are washed out and then discard the medium.
7. TPVG was added over the cells. And incubate at 37° C for 5 minutes for disaggregation. The cells become individual and it's present as suspension.
8. Add 5ml of 10% MEM with FCS by using serological pipette.
9. Gently give passaging by using serological pipette. If any clumbs is present then repeat the process.
10. After passaging split the cells into 1:2, 1:3 ratio for cytotoxicity studies for plating method
"Seeding of cells":
After homogenize take one ml of suspension and pour in to 24 well plates. In each well add 1ml of the suspension and kept in a desiccators in 5% CO2 atmosphere. After 2 days incubation observe the cells in inverted microscope. If the cells became 80% confluent.
In order to study the antitumor activity of a new drug, it is important to determine the cytotoxicity concentration of the drug. Cytotoxicity tests define the upper limit of the extract concentration, which is non-toxic to the cell line. The concentration nontoxic to the cells is chosen for antiviral assay.
After the addition of the drug, cell death and cell viability was estimated. The result is confirmed by additional metabolic intervention experiment such as MTT assay.
PERFORMANCE OF DRUG CYTOTOXICITY ASSAY
Cytotoxicity is the toxicity or damage caused to the cells on addition of drug. After the addition of the drug, cell viability is estimated by staining techniques, where by cells are treated with Trypan blue. Trypan blue is excluded by live cells, but stains dead cells blue. The results are confirmed by additional metabolic intervention experiments such as MTT assays.
Monolayer cultures in log phase.
Drug extract (different concentrations)
MEM without FCS
0.45 ï filter
5mL sterile storage vial
Tissue paper, Marker pen, Spirit, Cotton and Gloves
1 mL, 2 mL pipettes, Micropipette and tips
Discarding jar with 1 % Hypochlorite solutio
Stock drug concentration
100 mg of drug is dissolved in 10 mL of serum free MEM giving a concentration of 10mg / 1 mL. The stock is prepared fresh and filtered through 0.45 ï filter before each assay. Working concentrations of drug ranging from 10mg/ml to 0.3125mg/mlare prepared as follow
Preparation of working stock of 1 mg /mL
To 4.5 ml MEM add 0.5 mL of stock to give a working concentration of 1mg/mL
Drug concentration can be prepared from the working stock in MEM without FCS. Prepare required volume of test sample for each concentration.
48hr monolayer culture of Hep2 cells at a concentration of one lakh /ml /well (10 cells / ml / well) seeded in 24 well titer plate.
2. The plates were microscopically examined for confluent monolayer, turbidity and toxicity if the cells become confluent.
3. The growth medium (MEM) was removed using micropipette. Care was taken so that the tip of the pipette did not touch the cell sheet.
4. The monolayer of cells was washed twice with MEM without FCS to remove the dead cells and excess FCS.
5. To the washed cell sheet, add 1ml of medium (without FCS) containing defined concentration of the drug in respective wells.
6. Each dilution of the drug ranges from 1:1 to 1:64 and they were added to the respective wells of the 24 well titer plates.
7. To the cell control wells add 1ml MEM (w/o) FCS.
8. The plates were incubated at 37°c in 5% CO2 environment and observed for cytotoxicity using inverted microscope.
MTT assay is called as (3-(4, 5-dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide.MTT assay was first proposed by Mossman in 1982.
After incubation, remove the medium from the wells carefully for MTT assay.
In each well wash with MEM (w/o) FCS for 2 - 3 times. And add 200Âµl of MTT conc of (5mg/ml).
And incubate for 6-7hrs in 5% CO2 incubator for Cytotoxicity.
After incubation add 1ml of DMSO in each well and mix by pipette and leave for 45sec
If any viable cells present formazan crystals after adding solubulizing reagent (DMSO) it shows the purple color formation.
The suspension is transferred in to the cuvette of spectrophotometer and an O.D values is read at 595 nm by taking DMSO as a blank.
Cell viability (%) = Mean OD/Control OD x 100
Con. Of chemicals (Inhibition Zone in mm)
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All chemicals dissolved in DMSO (100mg/1ml) and add different dilutions of chemicals in each well filled with different dilution of chemicals such as 2.5mg, 5mg, 7.5mg and 10mg.
Control - DMSO as a control
-- indicate as negative (None zone)
Methods for antibacterial stability test
The standard Kirby-Bauer disk diffusion method was used to determine the antimicrobial chemicals. The M.tuberculosis strain were inoculated in nutrient broth and incubated at 37° C for overnight. After overnight incubation, the bacterial culture was swapped in solidified Muller hinton agar plate and made wells of 6mm diameter were punched in the plate. Then add different concentration of chemicals was dispensed in separate wells such as 1mg, 2.5mg, 5mg, 7.5mg and 10mg and add 100 Âµl of DMSO to centre well as control. The plates were incubated at 37° C for 24 hour. After incubation, the diameter of zone of inhibition around the wells were measured and recorded.
Composition of Muller hinton agar
Ingredients Gms / Litre
Beef Extract...............................................................................2 g
Acid Hydrolysate of Casein..................................................17.5 g
Starch..................................................................................... 1.5 g
Final pH 7.3 Â± 0.1 at 25Â°C
Composition Nutrient Broth
Ingredients Gms / Litre
Peptic digest of animal tissue 5g
Sodium chloride 5g
Beef extract 1.5g
Yeast extract 1.5g
Final pH ( at 25Â°C) 7.4Â±0.2