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To study the acute toxicity of the synthesized compounds by Acute Toxic Class method [Organization of Economic Co-Operation and Development (OECD) guidelines-425]
To perform antiulcer evaluation of the synthesized compounds.
To perform anticonvulsant evaluation of the synthesized compounds.
Materials and methods
The various animals and materials used for the present study and their sources are as follow
1% Carboxy methyl cellulose
Water for injection
Wister rats weighing 150-200 gm were kept in a colony cages at 25+2oC, relative humidity of 45-55% under 12 hr light and dark cycle. All the animals were fed with standard animal feed and water ad libitum. The test compounds were administered orally in the form of suspension using 1%CMC as suspending agent. The experimental dose was selected between the minimum effective dose and maximal non lethal dose. All the animal experimentation was performed according to the protocols and recommendation of the institutional animal's ethics committee.
ANOVA followed by dunnet's test was performed to ascertain the significance of the exhibited antiulcer, anticonvulsant activities of the synthesized compounds.
Following activities were carried out to the synthesized compounds by below mentioned method.
acute oral toxicity ( acute toxic class method in Rats)
antiulcer activity (aspirin induced + pylorus ligation method in Rats)
anticonvulsant activity ( maximal electric shock method)
Evaluation of acute oral toxicity
Acute oral toxicity defines to those adverse effects occurring following oral administration of a single dose of substance or multiple doses given within 24 h. the various methods used to evaluate the acute oral toxicity are as follows:
Fixed dose procedure (OECD guideline-420)
Acute toxic class method (OECD guideline-423)
Ups and down procedure (OECD guideline-425)
OECD Guidelines- 42351
IAEC/XXXI/03/CLBMCP/2010-2011: Dated on 22/09/2010
OECD Guidelines for the Testing of Chemicals are periodically reviewed in the light of scientific progress or changing assessment practices. The original Guideline 423 was adopted in March 1996 as the second alternative to the conventional acute toxicity test, described in Test Guideline 401. Based on the recommendations of several expert meetings, revision was considered timely because:
International agreement has been reached on harmonized LD50 cut-off values for the classification of chemical substances, which differ from the cut-offs recommended in the 1996 version of the Guideline,
Testing in one sex (usually females) is now considered sufficient.
The acute toxic class method set out in this Guideline is a stepwise procedure with the use of 3 animals of a single sex per step. Depending on the mortality and/or the moribund status of the animals, on average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance. This procedure is reproducible, uses very few animals and is able to rank substances in a similar manner to the other acute toxicity testing methods (Test Guidelines 420 and 425). The acute toxic class method is based on biometric evaluations with fixed doses, adequately separated to enable a substance to be ranked for classification purposes and hazard assessment. The method as adopted in 1996 was extensively validated in vivo against LD50 data obtained from the literature, both nationally and internationally.
Guidance on the selection of the most appropriate test method for a given purpose can be found in the Guidance Document on Acute Oral Toxicity Testing. This Guidance Document also contains additional information on the conduct and interpretation of Test Guideline 423.
5.1.2. Experimental Protocol (Acute toxic class method in rats)-
In the present study the acute oral toxicity of the synthesized compounds were performed by acute toxic class method. In this methods the toxicity of the synthesized compounds were tested using a step wise procedure, each step using three rats of a single sex. The rats were fasted prior to dosing (food but not water should be with held) for three to four hours. Following the period of fasting the animal should be weighed and the synthesized compounds were administered orally at a dose of 2000 mg/Kg body weight.Animals were observed individually after dosing at least once during the first 30 min; periodically during the first 24 h with special attention given during the first 4 h and daily thereafter, for a total of 14 days. As no mortality was observed with the above dose.
So, 200 mg/Kg body weight dose were selected for the further pharmacological evaluation.
Flow Chart For Acute Toxic Class Method (OECD Guidelines 423) Starting Dose of 2000 mg/kg Body Weight/po
Peptic Ulcer Disease (PUD) is the most prevalent gastrointestinal disorder, encompassing gastric and duodenal ulcer. The pathophysiology of PUD involves an imbalance between offensive factors like acid, pepsin and defensive factors like bicarbonate, prostaglandins.
Ulcer has been classified as
Meckel's Diverticulum ulcer
Mechanism of action:
The main mechanism of action of anti-ulcer drugs is found to be
Inhibition of H+,K+ -ATPase (proton pump)
Histamine H2- Receptor Antagonist
Anti helicobacter pylori agents.
Experimental protocol (Aspirin Induced + Pylorus Ligation Method):
The animals were divided into eight groups of 6 animals each and placed in cages with grating to avoid corophagy in environmentally controlled rooms with free access to water and food. The animals were fasted for 24hrs. Synthesized compounds and standard anti-ulcer drug Lansoprazole were prepared in 1% w/v of carboxy methylcellulose suspension as vehicle and administered orally once in a day at a dose of 10 mg/kg body weight respectively. Control group of animals were treated with 1%w/v of carboxy methylcellulose suspension and the negative control group is treated with aspirin at a dose of 200mg/kg body weight with vehicle similarly to experimental animals.
Group I : Control
Group II : Negative control (aspirin treated)
Group III : Standard (Lansoprazole treated)
Group IV-VIII : Test (synthesized compounds)
Aspirin induced + pylorus ligation method:
The animals were fasted for 24hr. Aspirin at a dose of 200mg/kg body weight was used for the induction of ulcers. Aspirin was administered orally to the rats after 30 minutes of pyloric ligation. The synthesized compounds and standard drug were administered orally 5 minutes prior to pyloric ligation.
Rats were anaesthesized with anaesthetic ether. The rats were secured on the operating table. An incision of 1cm long in abdomen just below sternum was made. The stomach was exposed and thread was passed around the pyloric sphincter. While ligating due care took that no blood vessel was tied along the knot. The abdomen wall was closed by putting the sutures. The skin from any blood spots and bleeding was cleaned. The rat in separate cage was placed and allowed it to recover.
After 4 hours of the pyloric ligation sacrificed by cervical dislocation, the abdomen was opened and the oesophageal end of the stomach was tied. The entire stomach from the body of the animals was separated.
A small cut to the pyloric region just above the knot was made and the contents of the stomach in a graduated centrifuge tube were collected.
The stomach along the greater curvature was opened and washed slowly under the running tap water and photographs were taken.
Estimation of biochemical parameters:
After the collection of gastric content in the centrifuge tube it was centrifuged at 1000rpm for 10 minutes, the supernant liquids were transferred to the measuring cylinder and the gastric volume was measured.
Determination of pH:
1ml of the gastric juice was diluted to 10ml of distilled water and pH was measured by the pH meter.
Determination of acidity:
1 ml of gastric juice was pipetted into a 100ml conical flask and 2 or 3 drops of topfer's reagent was added as an indicator and titrated against 0.01N sodium hydroxide, titrated until the solution turns to orange color. This volume of sodium hydroxide responds to the free acidity. Then phenolphthalein 2 drops as an indicator was added, then titration was further carried out until the solution regains pink color. This gives the total volume of sodium hydroxide, which corresponds to the total acidity.
The acidity is calculated by the following equation:
Vol. Of NaOH X normality X 100
Acidity = --------------------------------------------- mEq/1/100g
Determination of ulcer score
The curvature of the stomach were taken and washed with running tap water. Put it on the glass slide and observed under 10X magnification for ulcers.
0 = Normal coloured stomach
0.5 = Red colouration
1 = Spot ulcers
1.5 = haemorrhagic streaks
2 = ulcers > 3 but < 5
3 = ulcers > 5
Mean ulcer score for each animal is expressed as ulcer
Evaluation of Anticonvulsant activity
Epilepsy is the collective term used for a group of chronic seizure disorders which is having in common, sudden and transient episodes (seizure of loss or disturbance of consciousness), usually but always with a characteristic body movement (convulsion) and sometimes with autonomic hyper activity. The seizure nearly always correlates with an abnormal electrical discharge.
Epileptic seizure can be classified into,
1. Primarily generalized seizure:
a. Grant -mal epilepsy or Tonic-Clonic seizure
b. Tonic Seizure
c. Clonic seizure
d. Petit-mal epilepsy or absence seizure
e. Atonic seizure or drop attack.
2. Partial seizure:
a. Simple Partial seizure
b. Complex Partial seizure
3. Secondarily generalized seizures or cortical focal epilepsy or Jacksonian epilepsy.
Mechanism of action
The main mechanism of action is as follows
1. Enhancement of GABA action by
a. Activating GABAA- receptor
b.Inhibition of GABA transaminase enzyme
2. Inhibition of sodium channel function
3. Inhibition of calcium channel function
a. Ethosuxemide specially block T type calcium channel
b. GABA pentan may act on L type calcium channel
The other mechanism is inhibition of glutamate release and block of Glutamate receptors.
The various evaluation methods are as follows
MES (Maximal Electric shock) Method
Rota rod Test
Drugs which antagonised convulsion induced by MES, Leptazol and Kindling Method are usually useful in Grand-Mal.
The experimental protocol (MES Method in Rats)
In the present study anti-convulsant activity were screened by MES method. Albino rats of either sex were selected by random sampling technique was used for study. The animals were divided in three groups containing 4-5 animals in each group. One group was used as control, second as standard drug (phenytoin) and third was test group (synthesized compounds)
Different stages of the convulsions were noted as (a) tonic flexion, (b) tonic extensor phase, (c) clonic convulsions, (d) stupor, and (e) recovery or death. The animal was held properly and corneal electrodes were placed on the cornea and the prescribed current(150 mA for 0.2 seconds) was applied after half an hour administration of the test compounds. Phenytoin at the dose of 25 mg/kg i.p. was administered as standard drug for comparison. The test compounds at two dose levels were administered orally. The time spent by animal in each phase of convulsion was recorded. The reduction in time and abolition of tonic extensor phase was recorded.
Evaluation of in vitro anti-bacterial activity
The microbial world comprises of various micro-organisms which are microscopic in size. Bacteria, fungi (yeast and moulds) and microscopic algae are some of microorganisms. These are distinguished into two broad groups such as prokaryotes and eukaryotes. Eukaryotes contain nucleus and organelles (such as chloroplast, lysosome, endoplasmic reticulum, mitochondrion and golgi bodies) whereas, prokaryotes lacks the above features.
Bacteria are most abundant prokaryotic organism that is vital to life of living things. In nature bacteria can adapt to any kind of living conditions than any other group of organisms.
The following conditions must be accomplished for the determination of proper antimicrobial activity:
There should be proper control between the test organism and the substance to be evaluated.
The required conditions for the growth of the microorganism should be provided. Measurement of activity should be done correctly.
Aseptic should be maintained.
Study conditions should be maintained unchanged throughout the experiment.
Materials and methods
Various methods have been used to evaluate the antimicrobial activity of the drugs. The activity can be evaluated by the following techniques.
Agar streak dilution method.
Serial dilution method.
Agar diffusion method.
Cup plate method
Paper disc method.
The standard strains were procured from the American type culture collection (ATCC), Rockville, USA, and the pathological strains were procured from the department of microbiology, CEEAL analytical lab, Chennai, India. The anti-microbial activity of the synthesized compounds was screened against the following bacteria.
Staphylococcus aureus (ATCC 6538P)
Bacillus substillis (ATCC 6633)
Gram negative organism:
Escherichia coli (ATCC 25922)
Pseudomonas aeruginosa (ATCC 25619)
Nutrient agar medium (hi-media laboratories, India) is used as the media for the study of anti-bacterial activity. The composition of the medium is as follows.
Peptic digest of animal tissue
Ampicillin is a beta-lactam antibiotic used extensively to treat bacterial infections. The chemical name of ampicillin is 6-(2-amino-2-phenylacetamido)-3,3-dimethyl-7-oxo-thia-1-aza-bicyclo heptanes-2-carboxylic acid. It is effective against both gram positive and gram negative bacterial infection.
The anti-bacterial activity of the compounds Ia-Va and Ib-Vb were studied by the paper disc diffusion method. The test compounds were used in the concentration of 25µg/ml, 50µg/ml, 100µg/ml. Ampicillin 50µg/ml was used as standard.
Disc diffusion method
A suspension of Staphylococcus aureus was added to the sterile nutrient agar at 45°C in aseptic environment, the mixture was transferred to sterile petridishes and allowed to solidify. Sterile discs of Whatmann filter paper 6mm in diameter was dipped in solutions of compounds Ia-Va and Ib-Vb, standard were placed on the surface of the agar plates.
All the plates were allowed to stand at room temperature for 1 hour, (This was as a period of preincubation diffusion to minimize the effects of variation in time between the application of the different solutions) then plates were placed for incubation for 18 hours at 37 ± 1°C and observed for the antibacterial activity. In which plate zone of the inhibition observed diameter of that was measured.
Similar procedures was carried out for studying the antibacterial activity of the compounds Ia-Va and Ib-Vb against. The average area of the zone of inhibition was calculated and compared with the standard.
In vitro Evaluation of Anti-fungal activity of the synthesized compounds
A fungus is a colorless plant which is lack of chlorophyll. Fungi that cause infections may be like yeast or hyphi (mould) and these are called fungal infections.
These are of two types
Superfacial mycotic infection
Systemic mycotic infection
Because of the large widespread prevalence and airborne and soilborne transmission of the fungal pathogens, sanitary methods are not sufficient to eradicate the disease caused. So the search for the new and improved agents continues.
Material and Method
By Disc Diffusion Method the antifungal activity of the synthesized compounds Ia-Va and Ib-Vb were studied. For this following organisms were used.
Aspergillus fumigates (ATCC 46645)
Candida albicans (ATCC 10231)
Compounds Ia-Va and Ib-Vb were used in the concentration of 25µg/ml, 50µg/ml, 100µg/ml using DMF as solvent. Fluconazole (50µg/ml) was used as standard.
The Disc Diffusion Method was employed for the screening of antifungal activity by using Sabouraud Dextrose Agar medium (30gms of sabouraud dextrose agar and 1000ml of water heating).
Disc Diffusion Method
A suspension of micro-organism (Aspergillus nigar) was added to sterile Sabouraud dextrose agar medium at 45°C and the mixture was transferred to sterile petridishes and allowed to solidify. Sterile disc of Whatmann filter paper of 6mm in diameter dipped in solutions of synthesized compound Ia-Va, Ib-Vb and standard were placed on the surface of agar plates.
All the plates were allowed to stand at room temperature for 1hour. Then the plates were incubated at 37 ± 1°C for 18 hours and observed for antifungal activity. The diameter of zone of inhibition was measured. Similar procedure was carried out for studying antifungal activity of compounds against Aspergillus fumigates. The average area of zone of inhibition was calculated and compared with that standard.
Determination of Minimum Inhibitory Concentration
The MIC (minimum inhibitory concentration) of the synthesized compounds Ia-Va, Ib-Vb were determined by the agar streak dilution method. The MIC was determined against the bacteria and fungi.
Staphylococcus aureus (ATCC 6538P)
Bacillus Substilis (ATCC 6633)
Escherichia coli (ATCC 25922)
Pseudomonas aurogenousa (ATCC 25619)
Aspergillus fumigates (ATCC 46645)
Candida albicans (ATCC 10231 )
Media used for bacteria was nutrient agar and for fungi saboraud dextrose agar. All the culture media were sterilized by autoclaving at 15lbs for 20 min.
Agar Streak Dilution:
The stock solutions (1mg/ml) of the synthesized compounds were made by using DMF as solvent. From this stock solution, required quantities of drug solution were mixed with the known quantities of the molten sterile agar media aseptically to provide the following concentration 1,2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50 and 100µg/ml. About 20ml of the media containing the drug was dispensed into sterile petridishes. Then the media were getting allowed to solidify.
Over the surface of the agar plates, 1µl of standardized micro-organism (1-105 CFU/ml) were poured aseptically. After inoculation, all the plates were incubated at 37 ± 10C for 24 hours. Then all the plates were observed for the growth of the organism. The lowest concentration of the synthesized compounds inhibiting the growth of the bacteria/ fungi were taken as the minimum inhibitory concentration of the test compounds against that bacteria/ fungi.