Last updated: July 17, 2019
Topic: Food
Sample donated:

The ague toxic category method set out in this Guideline is a bit-by-bit process with the usage of 3 animate beings of a individual sex per measure. Depending on the mortality and/or the moribund position of the animate beings, on mean 2-4 stairss may be necessary to let judgement on the acute toxicity of the trial substance. This process is consistent, uses really few animate beings and is able to rank substances in a similar mode to the other acute toxicity proving methods ( Test Guidelines 420and 425 ) . The acute toxic category method is based on biometric ratings with fixed doses, adequately separated to enable a substance to be ranked for categorization intents and hazard appraisal. The method as adopted in 1996 was extensively validated in vivo against LD50 informations obtained from the literature, both nationally and internationally.

Guidance on the choice of the most appropriate trial method for a given intent can be found in the Guidance Document on Acute Oral Toxicity Testing. This Guidance Document besides contains extra information on the behavior and reading of Test Guideline 423.

5.1.2. Experimental Protocol ( Acute toxic category method in rats )

In the present survey the acute unwritten toxicity of the synthesized compounds were performed by acute toxic category method. In this methods the toxicity of the synthesized compounds were tested utilizing a measure wise process, each measure utilizing three rats of a individual sex. The rats were fasted prior to dosing ( nutrient but non H2O should be with held ) for three to four hour. Following the period of fasting the animate being should be weighed and the synthesized compounds ( S1-S10 ) were administered orally at a dosage of 2000 mg/Kg organic structure weight. Animals were observed separately after dosing at least one time during the first 30 min ; sporadically during the first 24 hour with particular attending given during the first 4 hour and day-to-day thereafter, for a sum of 14 yearss. As no mortality was observed with the above dosage. So, 200 mg/Kg organic structure weight dose were selected for the farther pharmacological rating.


Acute unwritten toxicity surveies were performed harmonizing to the OECD guideline 423 method.

This method has been designed to measure the substances at the fixed doses and provides information both for jeopardy appraisal and substances to be ranked for hazard categorization intents.

The synthesized compounds were administered ab initio at a get downing dosage of 2000 mg/kg b.w. in 1 % CMC ( p.o. ) and observed 14 yearss mortality due to acute toxicity.

Careful observation were made atleast twice a twenty-four hours for the consequence on CNS, ANS, Motor activity, Salivation, Skin colour and other general marks of toxicity were besides observed and recorded.

Since, no mark of toxicity observed at 2000 mg/kg b.w. to the group of animate beings, the LD50 value of the rubric compounds ( S1-S10 ) expected to transcend 2000 mg/kg b.w. and represented as category 5 ( 2000 mg/kg & A ; lt ; LD5 & A ; lt ; 2500 mg/kg ) .

From the toxicity surveies, the informations revealed that all the synthesized compounds proved to be non-toxic at tested dosage degrees and good tolerated by the experimental animate beings as their LD5 cut of values & A ; gt ; 2000 mg/kg b.w.

Evaluation OF ANTI-INFLAMMATORY ACTIVITY [ 53 ] [ 62 ]


It comprises systemic response ( affecting nervous and hormonal accommodations and proliferation of the lymphoreticulo system ; and local response ( hurting. inflammation, heat and swelling ) . Anti-inflammatory agents are substance which modifies the inflammatory reaction. The chief anti-inflammatory agents are the glucocorticoids and the non steroidal anti-inflammatory drugs ( NSAIDs ) .

Mechanism of action

NSAIDs act by suppressing arachidonic acid-metabolizing activity of COX which leads to suppression of production of PG and thromboxanes.

The cyclo oxygenase enzymes are bi functional, holding two distinguishable activities that are:

Gives PGG2

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 merely chief cyclo oxygenation reaction. Both COX-1 and COX-2 are associated with the membrane and each consists of long channel with a crook at the terminal. The channel being wider in COX-2. The gap of channel is mostly hydrophobic. Arachidonic acid enters, is twisted round the crook and has two Os inserted and a free group extracted, ensuing in the 5-carbon ring feature of the PGs.

COX-1 suppression, in general, is instantaneous and competitively reversible. COX-2 suppression is clip dependent, i.e. it is increasing with clip. Some NSAIDs produced other actions besides suppression of COX may lend to the anti-inflammatory effects such as,

Reactive O groups produced by neutrophils and macrophages are implicated in tissue harm in some conditions, and NSAIDs such as Clinoril that have peculiarly strong oxygen-radical scavenging effects every bit good as COX inhibitory activity may diminish tissue harm.

Aspirin has been shown to suppress look of the written text factor NF-kB, which has a cardinal function in the written text of the cistrons for inflammatory go-betweens.

Evaluation Methods

The three of import facets of redness that render themselves readily to measuring are erythema, hydrops and formation of granulation tissue. Compounds claimed to posses anti-inflammatory activity can be evaluated either by their ability to cut down one or more of these phenomena in by experimentation induced redness or by proving their anti-inflammatory activity in experimental arthritis produced in animate beings. The normally employed methods are:

Erythema checks

In this method, irradiation of shaven back tegument of guinea hog with UV light causes erythema which can be reduced by anti-inflammatory agents.

Edema checks

The hydrops can be produced in experimental animate beings by the local injection of substance like, methanal, carrageenin, histamine, dextron and egg white.

Granuloma checks

There are two types of granuloma checks such as cotton wool pellet and granuloma pouch method.

Experimental arthritis checks

Poly arthritis induced in rats by injection of dead tubercle B suspended in liquid paraffin is often used method. Kaolin, talc and even quicksilvers have besides been injected straight into articulations of rats and pigeons to bring on arthritis.


Localised inflammatory reaction can be produced in rats by,

Intrapleural injection of gum terpentine

Intraperitonial injection of methanal

Experimental Protocol ( Carrageenan induced paw hydrops method in rats )

Anti-inflammatory activity was performed by carrageenan- induced paw hydrops method in rats. Diclofenac Na ( 20 mg/kg, i.p ) was administered as standard drug for comparing. The synthesised compounds were administered at dose degrees of ( 200 and 400 mg/kg ) orally 30 min. prior to the disposal of 0.1ml/kg organic structure weight of carrageenin in saline ( 1 % W/V ) into the sidelong malleolus of the sub-planter part of the left hind paw. The paw volumes were measured utilizing the quicksilver supplanting technique with the aid of a plethysmograph instantly before and 30 min, 1, 2 and 3 hour. after carrageenan injection. The per centum suppression of paw odema was calculated by utilizing the undermentioned expression and the consequences are depicted in Table.

Percentage protection = [ ( Control-Test ) /Control ] X 100


The anti-inflammatory activity of the synthesized compounds was evaluated by Carrangeenan induced paw hydrops 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 informations 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 Na 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 & A ; gt ; S1 & A ; gt ; S2 & A ; gt ; S4 & A ; gt ; S10 & A ; gt ; S7.

Compound S1 holding methoxy group at para place of phenyl ring enhances the anti-inflammatory activity, which might function as new templets in the synthesis and development of potent therapeutics.



Peptic Ulcer Disease ( PUD ) embracing stomachic and duodenal ulcer is the most prevailing GI upset. The pathophysiology of PUD involves an instability between violative factors like acid, pepsin and defensive factors like hydrogen carbonate, prostaglandins.

Ulcer has been classified as,

Gastric ulcer

Duodenal ulcer

Oesophageal ulcer

Meckel ‘s Diverticulum ulcer


The chief mechanism of action of anti-ulcer drugs is found to be

Inhibition of H+ , K+ -ATPase ( proton pump )

Histamine H2- Receptor Antagonist

EXPERIMENTAL PROTOCOL [ 54 ] , [ 55 ] , [ 56 ] , [ 63 ]

( Aspirin Induced Pylorus Ligation Method )


The animate beings were divided into eight groups of 6 animate beings each and placed in coops with grating to avoid coprophagia and fasted for 24hrs and kept in environmentally controlled suites with free entree to H2O and nutrient. Synthesized compounds and standard anti-ulcer drug Omeprazole ( OMZ ) were prepared in 1 % w/v of Na carboxy methylcellulose suspension as vehicle and administered orally one time in a twenty-four hours at a dosage of 10 mg/kg organic structure weight severally. Control group of animate beings were treated with 1 % w/v of Na carboxy methylcellulose suspension and the negative control group is treated with acetylsalicylic acid at a dosage of 200 mg/kg organic structure weight with vehicle likewise to experimental animate beings.

Group I: Negative Control

Group II: Treated with Aspirin

Group III to XII: Treated with Synthesized Compounds

Group XIII: Treated with Omeprazole


To analyze the anti-ulcer activity, the animate beings were fasted over dark and anaesthetized with anaesthetic quintessence. Aspirin at a dosage of 200 mg/kg organic structure weight was used for the initiation of ulcers. Aspirin was administered orally to the rats after 30 proceedingss of pyloric ligation. The synthesized compounds and standard drug were administered orally 5 proceedingss prior to pyloric ligation.

Procure the rat on the operating tabular array. Give the scratch of 1cm long in the venters merely below the breastbone. Expose the tummy, pass a yarn around the pyloric ligation and bind a tight knot, while seting the knot attention should be taken so that no blood vas is tied along the knot. Close the venters wall by seting the suturas. Clean the tegument from any blood musca volitanss and shed blooding. Keep the rat in separate coop and let it to retrieve.

After 4 hour of the pyloric ligation forfeit by cervical disruption, open the venters and bind the oesophageal terminal of the tummy. Cut and take the full tummy from the organic structure of the animate beings.

Give a little cut to the pyloric part merely above the knot and roll up the contents of the tummy in a calibrated extractor tubing.

Open the tummy along the greater curvature and wash it easy under the running tap H2O and exposure were taken.


After the aggregation of stomachic content in the extractor tubing it was centrifuged at 1000 revolutions per minute for 10 proceedingss, the supernant liquids were transferred to the mensurating cylinder and the stomachic volume was measured.


1 milliliter of the stomachic juice was diluted to 10 milliliter of distilled H2O and pH was measured in the pH metre.


1 milliliter of stomachic juice was pipetted into a 100 milliliter conelike flask and add 2 or 3 beads of topfer ‘s reagent as an index and titrate against 0.01 N Na hydrated oxide. Titrate until the solution turns to orange colour. This volume of Na hydrated oxide is the 1, which responds to the free sourness. Then add phenolphthalein 2 beads as an index, so titration was farther carried out until the solution regains pink colour. This gives the entire volume of Na hydrated oxide, which corresponds to the entire sourness.

The sourness is calculated by the undermentioned equation,

Vol. Of NaOH X normalcy X 100

Acidity = — — — — — — — — — — — — — — — — — — — — — — – mEq/1/100g



The curvature of the tummy were taken and washed with running tap H2O. Put it on the glass slide and observed under 10X magnification for ulcers.

0 = Normal colored tummy

0.5 = Red colour

1 = Spot ulcers

1.5 = haemorrhagic runs

2 = ulcers & A ; gt ; 3 but & A ; lt ; 5

3 = ulcers & A ; gt ; 5

Average ulcer mark for each animate being is expressed as ulcer.


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 informations 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 & A ; gt ; S3 & A ; gt ; S2 & A ; gt ; S9 & A ; gt ; S1 & A ; gt ; S4.




The microbic universe comprises of microorganism which are microscopic in size. But these microscopic beings have several characteristics that are common to higher being. Bacteria, Fungi ( barm and molds ) and microscopic algae are some of microorganism. This being can be distinguished into two wide groups such as procaryotes and the eucaryotes. Eukaryotes contain nucleus and cell organs ( such as endoplasmic Reticulum, Golgi organic structures, lysosome, chondriosome and chloroplast ) where as procaryotes deficiencies above characteristics.

Bacterias are the most abundant procaryotic being that is critical to life of life things. Bacterias are omnipresent, topographic point a major positive function to the life of living things but some of them cause harmful diseases to the life things ( worlds. animate beings, workss, etc. ) . In nature bacteriums can follow any sort of life conditions than any other groups of beings.

Fungus kingdoms are eucaryotic being that is subdivided in to barms and molds. Yeasts are unicellular eucaryotic beings which have size of big bacteriums. The barm chiefly used in the agitation of vino and beer, and in production of staff of life. Molds are long concatenation cells frequently seen as fuzzed multitudes on staff of life and other acidic nutrient merchandises. Bacteria and Fungis are the primary decomposers of organic affairs in the universe. As like bacteria some of the Fungi cause harmful human diseases such as jock ‘s pes and thrush.

The undermentioned conditions must be accomplished for the finding of proper anti-microbial activity,

There should be adumbrate control between the trial being and substance to be evaluated

Microorganism should be provided with the needed conditions for growing

Measurement of activity should be done right

Aseptic should be maintained

Conditionss should be maintained unchanged throughout the survey


Assorted methods with their ain advantages and restrictions have been used from clip to clip to measure the anti-microbial activity of the drugs. The anti-microbial activity can be evaluated by the following techniques,

Agar streak dilution method

Consecutive dilution method

Agar diffusion method

Cup home base method

Cylinder method

Paper phonograph record method

Turbidimetric method

Turdidometric methoed

In the present survey, the paper phonograph record diffusion method was used to measure the in-vitro anti-microbial activity of the synthesized compounds. The paper phonograph record diffusion method is one of the methods that may be used for finding the comparative effectivity of the anti-microbial activity. The consequences obtained by this method depend non merely on the toxicity of the anti-microbial agents but besides on its ability to spread 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 section of microbiology, Dr. CEEAL ANALYTICAL LAB, Chennai, India. The anti-microbial activity of the synthesized compounds was screened against the undermentioned bacteriums and Fungis.

1. Bacterias

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 )

2. Fungi

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 survey of anti-bacterial and anti- fungous activity severally. The composing of the above mentioned medium is as follows,

1. Food Agar Medium



Peptic digest of carnal tissue


Beef infusion


Sodium chloride




2. Sabouraud Dextrose Agar Medium



Mycological peptone






Paper Disc Diffusion Method58

The sterilized ( autoclaved at 120 & A ; deg ; C for 30min ) medium ( 40-50 & A ; deg ; C ) was inoculated ( 1mL/100mL of medium ) with the suspension ( 105 cfu mL-1 ) of the microorganism ( matched to McFarland Ba sulphate criterion ) and poured into a petridish to give a deepness of 3-4 millimeter. The paper impregnated with the trial compounds ( 25, 50 and 100 µg mL-1 in dimethyl formamide ) was placed on the coagulated medium. The home bases were pre-incubated for 1 hour at RT and incubated at 37 & A ; deg ; C for 24 and 48 hour for anti-bacterial and anti-fungal activities, severally. Ciprofloxacin ( 100 µg/disc ) and Ketoconazole ( 100 µg/disc ) were used as standard for anti-bacterial and anti-fungal activities, severally.


Standard DRUG

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 bacteriums. It acts by suppressing the reproduction of bacterial DNA gyrase ( topoisomerase II ) during growing and reproduction.



Standard DRUG

Ketaconazole60 is orally active broad-spectrum anti-fungal agent. It acts by suppressing the steroid alcohol 14a-demethylase, a microsomal cytochrome P-450 dependant enzyme system. Therefore they impair biogenesis of ergosterol for the cytol membrane and lead to accretion of 14a-methyl steroid alcohols.


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 fix stock solution of trial compounds. These were incorporated to specified sum of molten unfertile agar ( alimentary agar for anti-bacterial and sabarause dextroglucose for anti-fungal activity ) . A medium 40 to 500C which incorporating trial compound was poured in to petridish up to 3-4 millimeter deepness. The deepness of medium insures by kept on apparent surface.The contain of petridish allowed to solidify. The suspension of micro-organism were prepared to incorporate about 105 cfu mL-1 and applied to the home bases with serially diluted compounds in dimethyl sulfoxide, and are tested and incubated at 370C for 24 hour and 48 hour for bacteriums and Fungis, severally. The MIC was preferred to be lowest concentration of the trial substance demoing no seeable growing of matching bacteriums or Fungis on the home bases. The ascertained MIC is presented in the Table ( VII ) .


The synthesized compounds were ( 50, 100 and 150 ?g mL-1 ) screened for anti-microbial activity by paper phonograph record diffusion method.

From the informations shown in ( Table VIII ) , the observation were made as follows,

Most of the synthesized compounds exhibited moderate to good anti-microbial activity against tested microorganism.

When compaired to standard drug ( Ciprofloxacin and Ketoconazole for anti-bacterial and anti-fungal severally ) , compounds S1, S2 and S9 ( 100 ?g mL-1 ) were exhibit good anti-microbial activity.

The MIC of the synthesized compounds were screened by agar run dilution method.

From the informations ( 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 scope of 13-54 ?g mL-1.

The compound ( S1 ) as found to exhibit the highest anti-microbial activity against S. auereus ( MIC: 13 ?g mL-1 ) , M. luteus ( MIC: 16 ?g mL-1 ) , E.Coli ( MIC: 20 ?g mL-1 ) , P.aeruginosa ( MIC: 24 ?g mL-1 ) , A. Niger ( MIC: 26 ?g mL-1 ) , C.Albicans ( MIC: 30 ?g mL-1 ) .

The compounds were active against all the tested microorganism with a scope of MIC values of S. auereus ( MIC: 24 – 47 ?g mL-1 ) , M. luteus ( MIC: 16 – 40 ?g mL-1 ) , E.Coli ( MIC: 20 – 42 ?g mL-1 ) , P.aeroginosa ( MIC: 24 – 51 ?g mL-1 ) , A. Niger ( MIC: 26 – 54 ?g mL-1 ) , C.Albicans ( MIC: 30 – 46 ?g mL-1 ) .

The powerful anti-microbial activity exhibited due to the negatron retreating substituent on phenyl ring at 2nd place of the benzimidazole ring, so all the compounds exhibited more anti-bacterial and anti-fungal activity.