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 420and 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)
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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 hrs. Following the period of fasting the animal should be weighed and the synthesized compounds (S1-S10) 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 hr with special attention given during the first 4 hr 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.
5.1.3. RESULTS AND DISCUSSION
Acute oral toxicity studies were performed according to the OECD guideline 423 method.
This method has been designed to evaluate the substances at the fixed doses and provides information both for hazard assessment and substances to be ranked for hazard classification purposes.
The synthesized compounds were administered initially at a starting dose of 2000 mg/kg b.w. in 1% CMC (p.o.) and observed 14 days mortality due to acute toxicity.
Careful observation were made atleast twice a day for the effect on CNS, ANS, Motor activity, Salivation, Skin coloration and other general signs of toxicity were also observed and recorded.
Since, no sign of toxicity observed at 2000 mg/kg b.w. to the group of animals, the LD50 value of the title compounds (S1-S10) expected to exceed 2000 mg/kg b.w. and represented as class 5 (2000 mg/kg< LD5< 2500 mg/kg).
From the toxicity studies, the data revealed that all the synthesized compounds proved to be non-toxic at tested dose levels and well tolerated by the experimental animals as their LD5 cut of values > 2000 mg/kg b.w.
EVALUATION OF ANTI-INFLAMMATORY ACTIVITY
It comprises systemic response (involving nervous and hormonal adjustments and proliferation of the lymphoreticulo system; and local response (pain. redness, warmth and swelling). Anti-inflammatory agents are substance which modifies the inflammatory reaction. The main anti-inflammatory agents are the glucocorticoids and the non steroidal anti-inflammatory drugs (NSAIDs).
Mechanism of action
NSAIDs act by inhibiting arachidonic acid-metabolizing activity of COX which leads to inhibition of production of PG and thromboxanes.
The cyclo oxygenase enzymes are bi functional, having two distinct activities that are:
A peroxidase action which converts PGG2 to PGH2
There are two types of COX (COX-1 and COX-2). Recently COX-3 has been described by Chandrasekaran. Both COX-1 and COX-2 inhibitors inhibit only main cyclo oxygenation reaction. Both COX-1 and COX-2 are associated with the membrane and each consists of long channel with a bend at the end. The channel being wider in COX-2. The opening of channel is largely hydrophobic. Arachidonic acid enters, is twisted round the bend and has two oxygens inserted and a free radical extracted, resulting in the 5-carbon ring characteristic of the PGs.
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COX-1 inhibition, in general, is instantaneous and competitively reversible. COX-2 inhibition is time dependent, i.e. it is increasing with time. Some NSAIDs produced other actions besides inhibition of COX may contribute to the anti-inflammatory effects such as,
Reactive oxygen radicals produced by neutrophils and macrophages are implicated in tissue damage in some conditions, and NSAIDs such as sulindac that have particularly strong oxygen-radical scavenging effects as well as COX inhibitory activity may decrease tissue damage.
Aspirin has been shown to inhibit expression of the transcription factor NF-kB, which has a key role in the transcription of the genes for inflammatory mediators.
The three important aspects of inflammation that render themselves readily to measurement are erythema, edema and formation of granulation tissue. Compounds claimed to posses anti-inflammatory activity can be evaluated either by their ability to reduce one or more of these phenomena in experimentally induced inflammation or by testing their anti-inflammatory activity in experimental arthritis produced in animals. The commonly employed methods are:
In this method, irradiation of shaven back skin of guinea pig with UV light causes erythema which can be reduced by anti-inflammatory agents.
The edema can be produced in experimental animals by the local injection of substance like, formaldehyde, carrageenan, histamine, dextron and ovalbumin.
There are two types of granuloma assays such as cotton wool pellet and granuloma pouch method.
Experimental arthritis assays
Poly arthritis induced in rats by injection of dead tubercle bacilli suspended in liquid paraffin is frequently used method. Kaolin, talc and even mercury have also been injected directly into joints of rats and pigeons to induce arthritis.
Localised inflammatory reaction can be produced in rats by,
Intrapleural injection of turpentine
Intraperitonial injection of formaldehyde
Experimental Protocol (Carrageenan induced paw oedema method in rats)
Anti-inflammatory activity was performed by carrageenan- induced paw oedema method in rats. Diclofenac sodium (20 mg/kg, i.p) was administered as standard drug for comparison. The synthesised compounds were administered at dose levels of (200 and 400 mg/kg) orally 30 min. prior to the administration of 0.1ml/kg body weight of carrageenan in saline (1% W/V) into the lateral malleolus of the sub-planter region of the left hind paw. The paw volumes were measured using the mercury displacement technique with the help of a plethysmograph immediately before and 30 min, 1, 2 and 3 hr. after carrageenan injection. The percentage inhibition of paw odema was calculated by using the following formula and the results are depicted in Table.
Percentage protection = [(Control-Test)/Control] X 100
RESULTS AND DISCUSSION
The anti-inflammatory activity of the synthesized compounds was evaluated by Carrangeenan induced paw oedema method in rats. The activity was studied at 200 mg/kg b.w., p.o. and their effects were measured at 30, 60, 120 and 180 min.
From the data shown in Table (I) and (II) the observations were made as follows,
Most of the synthesized compounds exhibited moderate to good anti-inflammatory activity.
All the synthesized compounds were exhibited highest activity at 120 min.
When compared to standard drug (Diclofenac sodium 20 mg/kg i.p.), compounds S1, S2 and S9 were found to exhibit good anti-inflammatory activity.
Compounds S4, S7 and S10 were found to exhibit moderate activity.
The anti-inflammatory activity shown by the synthesized compounds were S9>S1>S2>S4>S10>S7.
Compound S1 having methoxy group at para position of phenyl ring enhances the anti-inflammatory activity, which might serve as new templates in the synthesis and development of potent therapeutics.
EVALUATION OF ANTI-ULCER ACTIVITY
Peptic Ulcer Disease (PUD) encompassing gastric and duodenal ulcer is the most prevalent gastrointestinal disorder. 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
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Inhibition of H+,K+ -ATPase (proton pump)
Histamine H2- Receptor Antagonist
(Aspirin Induced Pylorus Ligation Method)
The animals were divided into eight groups of 6 animals each and placed in cages with grating to avoid coprophagy and fasted for 24hrs and kept in environmentally controlled rooms with free access to water and food. Synthesized compounds and standard anti-ulcer drug Omeprazole (OMZ) were prepared in 1% w/v of sodium 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 sodium carboxy methylcellulose suspension and the negative control group is treated with aspirin at a dose of 200 mg/kg body weight with vehicle similarly to experimental animals.
Group I : Negative Control
Group II : Treated with Aspirin
Group III to XII : Treated with Synthesized Compounds
Group XIII : Treated with Omeprazole
ASPIRIN INDUCED PYLOROUS LIGATION METHOD
To study the anti-ulcer activity, the animals were fasted over night and anaesthetized with anesthetic ether. Aspirin at a dose of 200 mg/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.
Secure the rat on the operating table. Give the incision of 1cm long in the abdomen just below the sternum. Expose the stomach, pass a thread around the pyloric ligation and tie a tight knot, while putting the knot care should be taken so that no blood vessel is tied along the knot. Close the abdomen wall by putting the sutures. Clean the skin from any blood spots and bleeding. Keep the rat in separate cage and allow it to recover.
After 4 hr of the pyloric ligation sacrifice by cervical dislocation, open the abdomen and tie the oesophageal end of the stomach. Cut and remove the entire stomach from the body of the animals.
Give a small cut to the pyloric region just above the knot and collect the contents of the stomach in a graduated centrifuge tube.
Open the stomach along the greater curvature and wash it 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 1000 rpm for 10 minutes, the supernant liquids were transferred to the measuring cylinder and the gastric volume was measured.
DETERMINATION OF pH
1 ml of the gastric juice was diluted to 10 ml of distilled water and pH was measured in the pH meter.
DETERMINATION OF ACIDITY
1 ml of gastric juice was pipetted into a 100 ml conical flask and add 2 or 3 drops of topfer's reagent as an indicator and titrate against 0.01 N sodium hydroxide. Titrate until the solution turns to orange color. This volume of sodium hydroxide is the one, which responds to the free acidity. Then add phenolphthalein 2 drops as an indicator, 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 colored stomach
0.5 = Red coloration
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.
RESULTS AND DISCUSSION
The anti-ulcer activity of synthesized compounds was evaluated by Aspirin induced pylorus ligation method in rats. The activity was studied at 10 mg/kg b.w.,p.o. and their effects were measured.
From the data shown in Table (III), the observations were made as follows,
Most of the synthesized compounds exhibited moderate to good anti-ulcer activity.
When compared to the standard drug (Omeprazole 10 mg/kg), compounds S2, S3, S9 and S10 were found to exhibit good anti-ulcer activity.
Compounds S1, S4 and S5 were found to exhibit moderate anti-ulcer activity.
The anti-ulcer activity shown by synthesized compounds were S10>S3>S2>S9>S1>S4.
B) IN-VITRO ANTIMICROBIAL ACTIVITY
PRELIMINARY ANTI MICROBIAL EVALUATION
The microbial world comprises of micro-organism which are microscopic in size. But these microscopic organisms have several features that are common to higher organism. Bacteria, fungi (yeast and moulds) and microscopic algae are some of micro-organism. This organism can be distinguished into two broad groups such as prokaryotes and the eukaryotes. Eukaryotes contain nucleus and organelles (such as endoplasmic reticulum, Golgi bodies, lysosome, mitochondrion and chloroplast) where as prokaryotes lacks above features.
Bacteria are the most abundant prokaryotic organism that is vital to life of living things. Bacteria are ubiquitous, place a major positive role to the life of living things but some of them cause harmful diseases to the living things (humans. animals, plants, etc.). In nature bacteria can adopt any kind of living conditions than any other groups of organisms.
Fungi are eukaryotic organism that is subdivided in to yeasts and moulds. Yeasts are unicellular eukaryotic organisms which have size of large bacteria. The yeast mainly used in the fermentation of wine and beer, and in production of bread. Moulds are long chain cells often seen as fuzzy masses on bread and other acidic food products. Bacteria and fungi are the primary decomposers of organic matters in the world. As like bacteria some of the fungi cause harmful human diseases such as athlete's foot and thrush.
The following conditions must be accomplished for the determination of proper anti-microbial activity,
There should be intimate control between the test organism and substance to be evaluated
Micro-organism should be provided with the required conditions for growth
Measurement of activity should be done correctly
Aseptic should be maintained
Conditions should be maintained unchanged throughout the study
5.4.2. MATERIALS AND METHODS
Various methods with their own advantages and limitations have been used from time to time to evaluate the anti-microbial activity of the drugs. The anti-microbial activity can be evaluated by the following techniques,
Agar streak dilution method
Serial dilution method
Agar diffusion method
Cup plate method
Paper disc method
In the present study, the paper disc diffusion method was used to evaluate the in-vitro anti-microbial activity of the synthesized compounds. The paper disc diffusion method is one of the methods that may be used for determining the relative effectiveness of the anti-microbial activity. The results obtained by this method depend not only on the toxicity of the anti-microbial agents but also on its ability to diffuse through the medium.
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, Dr. CEEAL ANALYTICAL LAB, Chennai, India. The anti-microbial activity of the synthesized compounds was screened against the following bacteria and fungi.
a. Gram-positive organisms-
1. Staphylococcus aureus (ATCC 6538 P)
2. Micrococcus luteus (ATCC 4698)
b. Gram-negative organisms-
Escherichia coli (ATCC 25922)
Pseudomonas aeruginosa (ATCC 27853 )
Aspergillus niger (ATCC 9029)
Candida albicans (ATCC 10231)
Nutrient agar medium and sabouraud dextrose agar medium (Hi-Media Laboratories, India) was used as the media for the study of anti-bacterial and anti- fungal activity respectively. The composition of the above mentioned medium is as follows,
1. Nutrient Agar Medium
Peptic digest of animal tissue
2. Sabouraud Dextrose Agar Medium
Paper Disc Diffusion Method58
The sterilized (autoclaved at 120°C for 30min) medium (40-50°C) was inoculated (1mL/100mL of medium) with the suspension (105 cfu mL-1) of the micro-organism (matched to McFarland barium sulphate standard) and poured into a petridish to give a depth of 3-4 mm. The paper impregnated with the test compounds (25, 50 and 100 µg mL-1 in dimethyl formamide) was placed on the solidified medium. The plates were pre-incubated for 1 hr at RT and incubated at 37°C for 24 and 48 hr for anti-bacterial and anti-fungal activities, respectively. Ciprofloxacin (100 µg/disc) and Ketoconazole (100 µg/disc) were used as standard for anti-bacterial and anti-fungal activities, respectively.
Ciprofloxacin59 is chemically 1-cyclopropyl 6-fluoro 1,4-dihydro 4-oxo 7(1- piperazinyl) 3-quinoline carboxylic acid. It is active against both Gram-positive and Gram-negative bacteria. It acts by inhibiting the replication of bacterial DNA gyrase (topoisomerase II) during growth and reproduction.
Ketaconazole60 is orally active broad-spectrum anti-fungal agent. It acts by inhibiting the sterol 14a-demethylase, a microsomal cytochrome P-450 dependent enzyme system. Thus they impair biosynthesis of ergosterol for the cytoplasm membrane and lead to accumulation of 14a-methyl sterols.
5.4.3. Determination of MIC
Agar Streak Dilution Method61
MIC of the synthesized compounds was determined by streak dilution method. Dimethyl sulfoxide was used to prepare stock solution of test compounds. These were incorporated to specified amount of molten sterile agar (nutrient agar for anti-bacterial and sabarause dextrose for anti-fungal activity). A medium 40 to 500C which containing test compound was poured in to petridish up to 3-4 mm depth. The depth of medium insures by kept on plain surface .The contain of petridish allowed to solidify. The suspension of microorganism were prepared to contain approximately 105 cfu mL-1 and applied to the plates with serially diluted compounds in dimethyl sulfoxide, and are tested and incubated at 370C for 24 hr and 48 hr for bacteria and fungi, respectively. The MIC was preferred to be lowest concentration of the test substance showing no visible growth of corresponding bacteria or fungi on the plates. The observed MIC is presented in the Table (VII).
5.4.4. RESULTS AND DISCUSSION
The synthesized compounds were (50, 100 and 150 μg mL-1) screened for anti-microbial activity by paper disc diffusion method.
From the data shown in (Table VIII), the observation were made as follows,
Most of the synthesized compounds exhibited moderate to good anti-microbial activity against tested micro-organisms.
When compaired to standard drug (Ciprofloxacin and Ketoconazole for anti-bacterial and anti-fungal respectively), compounds S1, S2 and S9 (100 μg mL-1) were exhibit good anti-microbial activity.
The MIC of the synthesized compounds were screened by agar streak dilution method.
From the data (MIC Table VII), the observation were made as follows,
All synthesized compounds exhibited moderate to good anti-bacterial and anti-fungal avtivity with an MIC range of 13-54 μg mL-1.
The compound (S1) as found to exhibit the highest anti-microbial activity against S. auereus (MIC: 13 μg mL-1 ), M. luteus (MIC: 16 μg mL-1 ), E.Coli (MIC: 20 μg mL-1 ), P.aeruginosa (MIC: 24 μg mL-1 ), A. Niger (MIC: 26 μg mL-1 ), C.Albicans (MIC: 30 μg mL-1 ).
The compounds were active against all the tested micro-organisms with a range of MIC values of S. auereus (MIC: 24 - 47 μg mL-1 ), M. luteus (MIC: 16 - 40 μg mL-1 ), E.Coli (MIC: 20 - 42 μg mL-1 ), P.aeroginosa (MIC: 24 - 51 μg mL-1 ), A. Niger (MIC: 26 - 54 μg mL-1 ), C.Albicans (MIC: 30 - 46 μg mL-1 ).
The potent anti-microbial activity exhibited due to the electron withdrawing substituent on phenyl ring at second position of the benzimidazole ring, so all the compounds exhibited more anti-bacterial and anti-fungal activity.