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Chapter - VI 6.1 Introduction & Literature review 6.2 Antibacterial activity 6.3 Antimycobacterial activity 6.4 Anticonvulsant activity 6.5 References 188 Chapter - VI 6.1. Introduction & Literature review:Biological evaluation is that phenomenon which explains the role of drugs in living organisms. Biological evaluation depends upon the study of various bacteria present in cell membrane of living system. In fact first life on the earth was started by bacteria called cyanobacteria,, later to it eukaryotic life started and evolved biological life like animals and humans as well. And one most important thing with bacteria is its inclusion into the eukaryotic system was nothing but the most important organelle or of cell called mitochondria. Bacteria are the simplest prokaryotic organisms. They are highly adaptable and can service extremes of temperature, temperature pH, oxygen tension and osmotic / atmospheric pressure and are therefore found in almost all natural sources. so • Size of Bacteria:Bacteria: Bacteria are very small size organisms. Which are barely visible under the light microscope. The smallest bacteria are about 0.1 million in diameter while the largest may be 60 x 6 microns. • Shape of Bacteria: Bacteria:-Bacteria occurs in three main shape Fig.6.1. Shape of Bacteria 189 Chapter - VI 1. Spherical ⇒ Spherical bacteria are called Cocci. 2. Rod-like ⇒ Rod like bacteria is called Bacilli. 3. Spiral ⇒ Spiral bacteria are called Spirilla. The following table gives some of disease causing bacteria in human being. Sr. No. Disease Bacterium 01 Meningitis Neisseria meningitides 02 Lobar pneumonia 03 Boils 04 Scarlet fever 05 Food poisoning 06 Tetanus 07 Diphtheria 08 Tuberculosis 09 Plague 10 Typhoid Salmonella typhii 11 Cholera Vibrio comma 12 Syphilis Treponemapallidum Streptococcus pneumonia Staphylococcus sp. Streptococcus scarlatinae Clostridium botalinum Clostridium tetani Cynobacterium diphtheria Mycobacterium tuberculosis Pasturellapestis Biological activity:In pharmacology, biological activity describes the beneficial or adverse effect of a drug on living matter. When a drug is a complex chemical mixture, this activity is exerted by the substances active ingradient or pharmacophore but can be modified by the other constituents. Activity is generally dosage-dependant and it is not uncommon to have effects ranging from beneficial to adverse for one 190 Chapter - VI substance when going from low to high doses. Activity depends critically on fulfillment of the ADME criteria. Where as a material is considered bioactive if it has interaction with or effect on any cell tissue in the human body. Biological activity is usually taken to describe beneficial effect i.e. the effect of drug candidates. The main kind of biological activity is substances toxicity. Antimicrobial:An anti-microbial is a substances that kills or inhibits the growth of micro-organisms (1) . Such as bacteria, fungi or protozoans. Antimicrobial drugs either kill microbes (micro-biocidal) or prevent the growth of microbes (microbiostatic). Disinfectants are antimicrobial substances used on non-living objects or outside the body. Technically, antimicrobials are only those substances that are produced by one microorganism, of course, in today’s common usage; the term of antimicrobial is used to refer to almost any drug that attempts to rid your body of a bacterial infection. Antimicrobials include not just antibiotics, but synthetically formed compounds as well. The discovery of antimicrobials like penicillin and tetra cycline paved the way for better health for millions around the world. Before penicillin became a viable medical treatment in the early 1940s, no true cure for gonorrhea, strep throad or pneumonia existed. Patients with infected wounds often had to have a wounded limb removed, or face death from infection now, most of these infections can be cured easily with a short course of antimicrobials. However with the development of anti-microbials, microorganisms have adapted and become resistant to previous 191 Chapter - VI antimicrobial agents. The old antimicrobial technology was based either on poison or heavy metals, which may not have killed the microbe completely, allowing the microbe to survive change and become resistant to the poisons and / or heavy metals. 6.2. Antibacterial activity:Introduction:An antibacterial is a substance or compound that kills or slows down the growth of bacteria(2). The term “antibiotic” was coined by Selman in 1942 to describe any substance produced by a microorganism that in antagonistic to the growth of other microorganisms in high dilution (3) . This definition excluded substances that kill bacteria but are not produced by microorganisms (such as gastric juices and hydrogen peroxide). It also excludes synthetic antibacterial compounds such as Sulfonamides. With advances in medicinal chemistry, most of today’s antibacterials chemically are semi-synthetic modification of various natural compounds(4). These include for example, the β-lactam antibacterials, which include the penicillin, the cephalosporins and the carbapenems. Penicillin, the first natural antibiotic discovered by Alexander Fleming in 1929 in his small lab accidentally. When he was working with some fungus, he found that there is zone of inhibition in the plate. Then he isolated the active ingredient and studied it found that it is having potential to kill micro-organism called bacteria. The term antibiosis meaning “against life”, was introduced by the French bacteriologist Vuillemin as a descriptive name of the phenomenon exhibited by these early antibacterial drugs (4,5) . Antibiosis was first described in 1877 in bacteria when Louis Pasteus and Robert Koch 192 Chapter - VI observed that an airborne bacillus could inhibit the growth of Bacillus anthraces (6) . These drugs were later renamed antibiotics by Selman Waksman, an American microbiologist in 1942 (3, 4). Pharmacodynamics:Testing the susceptibility of staphylococcus aurous to antibiotics by the Kirby-Bauer disk diffusion method. Antibiotics diffuse out from antibiotic-containing disk and inhibit growth of S. aurous resulting in a zone of inhibition. The successful outcome of antimicrobial therapy with antibacterial compounds depends on several factors. These include host defense mechanisms, the location of infection, and the pharmacokinetic and pharmacodynamic properties of the antibacterial (7). A bactericidal activity of antibacterials may depend on the bacterial growth phase, and it often requires ongoing metabolic activity and division of bacterial cells (8). These findings are based on laboratory studies, and in clinical setting have also been shown to eliminate bacterial infections (7,9) since the activity of antibacterials depends frequently on its concentration (10) , in vitro characterization of antibacterial activity commonly includes the determination of the minimum inhibitory concentration and minimum bacteriacidal concentration of an antibacterial (7,11). To predict clinical outcome, the antimicrobial activity of an antibacterial is usually combined with its pharmacokinetic profile, and several pharmacological parameters are used as markers of drug efficiency. Classes:Antibacterials are commonly classified based on their mechanism of action, chemical structure or spectrum of activity. Most antibacterial antibiotics target bacterial functions or growth processes. 193 Chapter - VI Antibiotics that target the bacterial cell wall (such as penicillin and cephalosphorins) or cell membrane (for example, polymixins) or interfere with essential bacterial enzymes (such as quinolones and sulfonamides) have bactericidal activities. Those that target protein synthesis, such as the aminoglucosides, macrolides and tetracyclines, usually bacteriostatic. Further categorization is based on their target specificity. “Narrow-spectrum” antibacterial antibiotics target specific types of bacteria, such as gram-negative or gram-positive bacteria, where as broad-spectrum antibiotics affect a wide range of bacteria. gram-positive bacteria B.Subtilis gram-negative bacteria such as E. Coli There is great importance to bacteria in recent world to produce industrial enzymes as well as biotechnologically derived human therapeutic proteins. It has become possible by genetic engineering. 194 Chapter - VI Human genes are isolated and recombined into the bacterial genetic system and genes are expressed inside the bacteria and therapeutic proteins are isolated and purified and marketed. For example human insulin expression in E. coli. Human intestinal microflora play important role to the host by producing vitamins and and enzymes. They also induce some substances which keeps live the immunity of the host. For example Lactobacillus species converts sugar into lactic acid. Bacterial Cell Structure:Structure: The bacterial cell differs dramatically in structure and function compared to mammalian cells (12) . The bacterial cytoplasm is separated from the external environment by a cytoplasmic membrane as shown in Fig. 5.2 A. The bacterial cell wall is chemically ddistinct from mammalian cell walls and so is constructed by enzymes that often have no direct counterpart in mammalian cell construction. In addition, bacteria possess a crucial structure surrounding the entire cell, the Peptidoglycan (PG), which forms a su succulus cculus around the bacterial cell, is an essential cell wall polymer since interference with its synthesis or structure leads to lose of cell shape and integrity followed by bacterial death. FIG.6.2.A. FIG. CELL STRUCTURE 195 Chapter - VI The peptidoglycan (13) layer as shown in Fig. 5.2 B consists of a matrix of polysaccharide chains composed of alternating N Nacetylmuramic acid (MurNAc) and N-acetylglucosamine N acetylglucosamine (GlcNAc) sugar moieties cross--linked linked through pentapeptide side chains. Fig.6.2.B.PEPTIDOGLYYCAN .2.B.PEPTIDOGLYYCAN LAYER OF E.COLI Classification of Antibacterial Agents: -Based Based on the severnity of damage (14) to the bacteria they are divided into: 1. Bactericidal agenst act primarily by killing bacteria with an efficiency of >99.9%. 2. Bacteriostatic agents act primarily by inhibiting the growth of the bacteria. Based on the mode of action they can be further divided into following four main categories. 1. Cell wall synthesis inhibitors. 2. Cell Membrane Agents. 3. Protein synthesis inhibitors. i) Impairing 50S subunit. ii) Impairing 30S subunit. 196 Chapter - VI 4. Nucleic acid synthesis inhibitors. i) DNA replication and repair ii) Transcription A representative listing of the antibacterial compounds currently in clinical practice or in development along with a schematic overview of their targets (15) is shown in Fig. 5.2 C. More recently it has been shown that the bactericidal antibiotics, having distinct drug-target target interactions stimulate the production of highly harmful hydroxyl radical in gram-positive gram and gram-negative negative bacteria which finally contribute to the cell death. In contrast, c a Bacteriostatic drug tested doesn’t lead to production of hydroxyl radical (16). FIG.6.2.C.SCHEMATIC VIEW OF A BACTERIAL CELL WITH SITES OF ACTION OF VARIOUS ANTIBIOTICS. 197 Chapter - VI 6.2.1. Antibacterial Screening:Some of the synthesized derivatives from chapter three and chapter four were then assayed for their in-vitro antibacterial activity against a panel of pathogenic as well as standard bacterial strains such as Staphylococcus aurous, Klebsiella pneumonia, Salmonella typhimurium, Pseudomonas aeroginosa, Bacillus subtilis, Proteus vulgaries, Xanthomonascampestri Pv, Citri, Xanthomonascampestri Pv. Malvacerum, Bacilluthurengensis and Escherichia coli. Based on previous literature and scope of the bacterial species were selected under the different scheme. Gentamicin, Kanamycin, Streptomycin and Cefotaxime sodium were procured from commercial sources. The purities and potencies of the agents recovered from commercial sources. The purities and potencies of the agents recovered from commercial preparations were documented by showing that the MICs of antibacterials were within acceptable limits against the known strains. *Determination of MIC in terms of Zone of Inhibition:The antibacterial activity was tested by agar disc diffusion method. The killing or growth inhibition property of the agents was scored as clear zone of inhibition surrounding the disc and is measured in mm scale. *Materials & Method: • The bacterial strains were inoculated in fresh sterile MHB (Muller Hinton Broth) media tube (4.5 m) and were incubated for 18-24 hrs at 370C in a B.O.D. incubator. • Standard antibiotic Gentamicin, Kanamycin, Streptomycin and Cefotaxime sodium were prepared clear solutions with final (1mg/ml). 198 Chapter - VI • The above antibiotic solutions were poured on sterile disc at a final concentration of 40 mcg/disc for Gentamicin, Kanamycin while 40 mcg/disc for Streptomycin and Cefotaxime sodium. • All discs were dried completely by incubating into hot air oven in sterile petri dishes. • On MHA (Muller Hinton Agar) plates, the bacterial suspension was poured and spread evenly with the help of glass spreader. • After drying the plates completely, the antibiotic loaded discs were kept on the plates. • All plates were incubated at 370C in a B.O.D. (Biological Oxygen Demand) incubator for 24 hours. • Results were recorded and antibiotic activity was quantified by measuring the zone of inhibition surrounded to the disc and it were measured in ‘mm’ scale and presented in the respective tables. 199 Chapter - VI *Table 6.2 (A): The antibacterial data synthesized substituted 3Methyl-7-(1-methyl-1H-indazol-3-yl-carboxamido) 8-oxo-5-thia- azabicyclo [4.2.0] oct-2-ene-e-carboxylic acid. O H N S N N O CH3 Bacteria N R1 Streptomycine O OR 100 µg/ml standard 3.2.a 3.2.b 3.2.c 3.2.d 3.2.e Zone of Inhibition Kiebsiell pneumonia 8 - - - - - Salmonella typhy 7 - - - - - Psuedomanusauroginosa 9 9 10 - 7 11 Bacillus subtilis 7 11 - 12 - - E. coli 10 10 12 10 12 11 Proteus vulgaris 11 12 - - - 10 Xanthomonuscampe strip V. Malvacerum 9 - - - 8 - Bacillus thurengensis 11 11 13 15 13 7 Xanthaomonascampe strip V. citri 6 - - - - - Staphylococcus aureus 9 10 11 14 - 8 Summary & Conclusion:We screened 3.2 (a-e) compounds for antibacterial activity. Concentrations of the compounds used in nutrient agar plates were 100 µg / ml each. Linear growths of the test bacteria were measured every day and zone of inhibition of the isolates was recorded. It is 200 Chapter - VI clear that Table able 5.5 that compound 3.2.c had maximum zone of inhibition for Bacillus thuregensis, Staphylococus aureus, Bacillus Subtilis and showed least zone of inhibition of E. coli. It did not show any zone of inhibition to remaining bacteria. Compound 3.2.b showed showe maximum zone of inhibition of Bacillus thurengensis and E. coli. Compound 3.2.a showed maximum zone of inhibition Proteus vulgaris, Bacillus Subtilis and Bacillus thurengensis. Compound 3.2.d showed maximum zone of inhibition Xanthaomonassampstrip V. Malvacerum vacerum and Compound 3.2.e showed maximum zone of inhibition at E. coli. Streptomycin e 100 µg/ml standard 3.2.a 3.2.b 3.2.c 3.2.d 3.2.e Graphical representation of antibacterial activity of compound 3.2(a-e) e) against Streptomycin as a standard 201 Chapter - VI Table 6.2 (B):-Antibacterial data of substituted 3-(acetoxymethyl7-(2-(7-methyl-2-p-tolylimidazo[1,2-a] pyridine-3yl) acetamide-8oxo-sthia-1-azabicyclo [4.2.0] oct-2-ene-2-carbaylic acid.3.3 (a – e): Bacteria Standard (Ceftaxime sodium N N N O H O 100 µg / ml S N RI OR O 3.3.a 3.3.b 3.3.c 3.3.d 3.3.e Zone of Inhibition E. coli 30 20 17 14 13 15 S – aureus 20 - - - - - S – typhi 25 18 15 13 13 14 B – megaterium 24 15 13 10 14 15 Summary & Conclusion:We screened all five derivatives of 3-(acetoxymethyl-7-(2-(7methyl-2-p-tolylimidazo[1, 2-a] pyridine-3yl) acetamide-8-oxo-5thia1-azabicyclo [4.2.0] oct-2-ene-2-carbaylic acid 3.3 (a-e). The concentration of derivatives used in disc were 100 µg / ml each. The activity of these derivatives were measured against four different bacterial species by using cefotaxime sodium as standard drugs. Among the tested series 3.3.a has shown maximum activity against E. coli and S. Typhi. 3.3.b also showed good activity against E. coli and S. Typhi. 3.3.e also show comparatively good activity against E. coli 202 Chapter - VI and B. Megaterium.The overall impact of all the derivatives against cefotaxime sodium was inferior against all bacterial species. 35 30 E. coli 25 20 S – aureus 15 S – typhi 10 5 B – megaterium 0 Standard 3.3.a 3.3.b 3.3.c 3.3.d 3.3.e Graphical representation of antibacterial activity of compound 3.3(a-e) against Ceftaxime sodium as a standard 203 Chapter - VI Table 6.2 (C): The antibacterial data of synthesized substituted spiro – [2H-1, 3-benzoxazine-2, 1-cyclohexan]-4 (3H)-one. 4.2(a-g) O Zone of inhibition in (mm) H N S. aureus E. coli Standard [Nor floxacin 100 µg/ml 25 24 24 24 4.2 a 20 21 20 - 4.2 b - 22 19 21 4.2 c 21 20 - 15 4.2 d 24 17 19 - 4.2 e 23 21 20 22 4.2 f 20 13 21 19 4.2 g 15 14 17 23 O R R P. B. subtilis aeruginosa Bacteria Summary & Conclusion:We screened comp. 4.2 (a-g) for antibacterial activity. The result indicates that all compounds showed good antibacterial activity. Table 6.2 C compounds 4.2e showed very good activity against S. aureus, E. coli, B. subtilis and P. aeruginosa. From the above observation it is clear that the Benzoxazine derivatives are more active and play a prominent role in the antimicrobial activity. 204 Chapter - VI 25 S. aureus 20 E. coli 15 10 B. subtilis 5 P. aeruginosa 0 Standard 4.2 a 4.2 b 4.2 c 4.2 d 4.2 e 4.2 f 4.2 g Graphical representation of antibacterial activity of compound 4.2(a-g) g) against Norfloxacin as a standard 205 Chapter - VI Table 6.2 (D): The he antibacterial data of synthesized substituted 33 (2-Bromopropionyl) Bromopropionyl) spiro [2H - 1, 3 – benzoxazine - 2, 1 cyclohexane] – 4 (3H) – one. 4.3 (a-g) O O Zone of inhibition in (mm) Br N R R S. B. P. E. coli aureus subtilis aeruginosa O Bacteria Standard [Nor floxacin 100 µg/ml 4.3 a 4.3 b 4.3 c 4.3 d 4.3 e 4.3 f 4.3 g 25 24 24 24 24 26 24 25 23 24 21 20 24 23 19 20 20 21 19 22 24 22 19 20 22 25 22 - Summary & Conclusion:Conclusion: We screened comp. 4.3(a 4.3(a-g) for antibacterial activity. All the synthesized compounds have shown mild to good activity against pathogenic bacteria.. The synthesized compounds 4.3(e) 4.3(e) have been shown to be more potent than other synthesize compou compounds. The overall impact of all the derivatives against Norfloxacin was inferior against all bacterial species. 30 S. aureus 25 20 E. coli 15 B. subtilis 10 P. aeruginosa 5 0 Standard 4.3 a 4.3 b 4.3 c 4.3 d 4.3 e 4.3 f 4.3 g Graphical representation of antibacterial antibacterial activity of compound 4.3(a-g) g) against Norfloxacin as a standard 206 Chapter - VI Table 6.2 (E):-The The antibacterial data of synthesized substituted substitu Bromo spiro base. 5.3(a-i): 5.3 Zone of inhibition in (mm) O Br O R 2 N R R1 O O CH2 R 4 CH3 S. aureus E. coli B. P. subtilis aeruginosa 3 Bacteria Standard 24 25 23 24 [Vancomycine 25 µg/ml 5.3a 21 24 22 23 5.3b 20 21 19 19 5.3c 18 23 21 18 5.3d 22 19 16 18 5.3e 16 15 17 19 5.3f 21 18 18 18 5.3g 20 17 19 21 5.3h 19 21 20 22 5.3i 22 18 18 21 Summary and conclusion: We screened 5.3(a-i) i) for antibacterial activity. The result indicates that all compounds showed good antibacterial activity. 5.3a showed very significant activity against S. aureus, E. coli, B. subtilis and P. aeruginosa. From the above observation it is clear lear that the Bromo spiro base auxillaries are more active and play a prominent role in the antimicrobial activity which helpful to improve the antibacterial activity of Carbapenems. 25 S. aureus 20 15 E. coli 10 B. subtilis 5 P. aeruginosa 0 Standard 5.3a 5.3b 5.3c 5.3d 5.3e 5.3f 5.3g 5.3h 5.3i Graphical representation of antibacterial activity of compound 5.3(a-i) i) against Vancomycineas a standard 207 Chapter - VI 6.3. Anti-mycobacterial activity:Anti-mycobacterial agents are generally used in combination with other antimicrobials since treatment is prolonged and resistance develops readily to individual agents. 1. Para-amino salicylic acid (PSA) (Bacteriostatic) PSA is specific for mycobacterium tuberculosis. 2. DAPSONE (Bacteriostatic) : DAPSONE is used in treatment of leprosy. 3. Isoniazid (INZ) (Bacteriostatic). :Isoniazid inhibitsysthesis of mycolic acids. It is used in treatment of tuberculosis. Isoniazid (INZ) is an organic compounds that is the first-line antituberculosis medication in prevention and treatment. It was first discovered in 1912 and later in 1951 it was found to be effective against tuberculosis. Isoniazid never used on its own to treat active tuberculosis because resistance quickly develops. Isoniazid is prodrug and must be activated by a bacterial catalase-peroxidase enzyme that in mycobacterium tuberculosis. Isoniazid is bactericidal to rapidly-dividing mycobacteria but is bacteriostatic if the mycobacterium is slow-growing. 6.3.1: Antimycobacterial Screening:In vitro anti-mycobacterial screening was done for the synthesized compounds 2.2.2C, 2.2.2e, 2.2.2f, 2.2.2r. Screened against standard strain H37Rv and two human strain [Human strain-I and Human strain-II]. Isolated from patients suffering from pulmonary tuberculosis in different concentration from 12.5, 25, 50, 100, 200, 400 µg/ml and the isoniazid used as standard at a cone of 50 µg/ml. 208 Chapter - VI Material Required:1. Lowenstein-Jensen (LJ) medium slopes containing various concentration of the compound. 2. Control strain H37Rv. 3. Two strains of Mycobacterium tuberculosis isolated from patients suffering from pulmonary tuberculosis. Preparation of drug containing slopes:The synthesized compounds were dissolved in DMSO and added to the egg fluid salt solution in such a way that to give final concentration of 12.5, 25, 50, 100, 200, 400 µg of the compounds per ml of the medium. The above was insisted at 900C for 50 minutes only once. Preparation of the bacterial suspension:The bacterial culture of a control strain and 2 test strain about 2/3 loop full (3mm internal diameter) is mixed with 1ml sterile distilled water in BIJOU bottle containing 3-5, glass beads, shaken in a vertexed bottle for about 1mm to get a uniform suspension. Susceptibility test procedure:1 loop full of bacterial suspension was inoculated on L. J. medium slopes containing the test compounds. A drug free slope is also included as a control. All the slopes were incubated at 370C for 15 days. The result and data are given in table V and VI. 209 Chapter - VI Table 6.3.A: - Antimycobacterial activity of compound 2.2.2 (c, e, f, i):H37Rv Conc. µg/ml Human Strain I Human Strain II 2.2.2c 2.2.2e 2.2.2f 2.2.2i 2.2.2c 2.2.2e 2.2.2f 2.2.2i 2.2.2c 2.2.2e 2.2.2f 2.2.2i 12.5 +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ 25 ++ +++ +++ +++ ++ +++ +++ +++ ++ +++ +++ +++ 50 – ++ ++ + + ++ ++ + + ++ ++ ++ 100 – – – – – – + + + + + + 200 – – – – – – – – – – – – 400 – – – – – – – – – – – – –: No growth of mycobacterium tuberculosis +: Growth of mycobacterium tuberculosis below 100 colonies ++: Growth of mycobacterium tuberculosis between 100-200 colonies +++: Growth of mycobacterium tuberculosis above 200 colonies Table 6.3.B: Minimum Inhibitory Concentration Minimum Inhibitory Concentration Compounds H37Rv Human Strain I Human Strain II 2.2.2c 50 100 200 2.2.2e 100 100 200 2.2.2f 100 200 200 2.2.2i 100 200 200 Summary & Conclusion:In vitro anti-mycobacterial screening was done for the synthesized compounds 2.2.2c, 2.2.2e, 2.2.2f, 2.2.2i. Synthesized compounds screened against standard strain H37Rv and two human 210 Chapter - VI strain isolated from patients suffering from pulmonary tuberculosis in different concentration from 12.5, 50, 100, 200, 400 µg/ml and the Isoniazid used as standard at a cone of 50 µg/ml. Synthesized compounds 2.2.2c, 2.2.2e, 2.2.2f & 2.2.2i showed significant antitubercular activity. Minimum inhibitory concentration 600 500 Axis Title 400 Human Strain II 300 Human Strain I 200 H37Rv 100 0 2.2.2c 2.2.2e 2.2.2f 2.2.2i Graphical representation of MIC of compound 2.2.2(c,e,f,i)) 211 Chapter - VI 6.4. Anticonvulsant Activity:Anticonvulsant evaluation by Maximal Electro Shock (MES) Method: The anticonvulsant activities of the compounds were evaluated by maximal electro shock method using rats,where the electro shock is applied through the corneal electrode producing optic stimulation cortical excitations. The MES convulsions are divided into five phases such as (a) Toxic flexion (b) Tonic extension (c) Clonic Convulsion (d) Stupor and (e) Recovery of death. A drug is known to possess anticonvulsant properties. If it reduces or abolishes the extensor phase or MES convulsions, for the evaluation anticonvulsant activity the total 16 groups of animals were kept fasting for 10 – 14 hrs. The animals were divided into 16 groups each containing 6 animals. In the 14 groups were served for testing the synthesized compounds, one as control and one as standard (phenytoin 25 mg/kg of body weight) was used as a standard drug. The activities of each group were measured after the intervals of half-an-hour are compounds administering including control and standard. Results and data are given in table 6.4.A. 212 Chapter - VI Table 6.4.A:- Anticonvulsant activity of some synthesized compounds by MES method: Compound Mean time in various phases of convulsions (seconds) ± standard error mean Recovery MES Test Percentage protection (% abolition of tonic extensor phase) Flexion Extensor Control 6.833 ± 0.40 10.00 ± 0.73 √ 0/6 -- 2.2.2a 3.67 ± 0.21 1.83 ± 0.83 √ 3/6 50% 2.2.2b 3.33 ± 0.21 2.16 ± 0.74 √ 2/6 33.33% 2.2.2c 4.66 ± 0.33 2.00 ± 0.69 √ 2/6 33.33% 2.2.2d 4.33 ± 0.49 4.66 ± 0.49 √ 0/6 -- 2.2.2e 5.33 ± 0.61 5.00 ± 0.57 √ 0/6 -- 2.2.2f 4.33 ± 0.42 1.00 ± 0.63 √ 4/6 66.66% 2.2.2g 6.66 ± 0.66 7.30 ± 0.61 √ 0/6 -- 2.2.2h 4.66 ± 0.49 1.00 ± 0.63 √ 5/6 83.33% 2.2.2i 5.66 ± 0.33 9.16 ± 0.74 √ 0/6 -- 2.2.2j 6.66 ± 0.33 9.83 ± 0.60 √ 0/6 -- 2.2.2k 6.16 ± 0.47 8.66 ± 0.49 √ 0/6 -- 2.2.2l 6.50 ± 0.42 9.66 ± 0.61 √ 0/6 -- 2.2.2m 4.66 ± 0.22 3.66 ± 0.33 √ 0/6 -- 2.2.2n 4.83 ± 0.47 5.33 ± 0.88 √ 0/6 -- Standard (Phenytoin) 4.00 ± 0.36 0.00 ± 0.00 √ 6/6 100 % Summary & Conclusion:All the synthesized compounds exhibit significant to moderate anticonvulsant activity. Compounds 2.2.2a, 2.2.2b, 2.2.2c, 2.2.2d, 2.2.2e, 2.2.2h, 2.2.2m, 2.2.2n showed significant anticonvulsant activity in the flexion phase, other compounds showed moderate activity. In the extensor phase 2g showed most significant activity when compared with 2.2.2a, 2.2.2b, 2.2.2c, 2.2.2d, 2.2.2e, 2.2.2h, 2.2.2m, 2.2.2n. Compounds 2.2.2i, 2.2.2j, 2.2.2k, 2.2.2l showed reduction in the duration of the flexion and extensor phase. 2.2.2e and 2.2.2g least active in the flexion and showed significant activity in extensor phase. 213 Chapter - VI Activity zone shown by synthesized indazole comp.(Table-6.2A) Activity zone shown by synthesized Imidazole comp. (Table-6.2B) Activity zone shown by synthesized Benzoxazine comp. (Table-6.2C) 214 Chapter - VI Activity zone shown by Bromoxazine comp. (Table-6.2D) Activity zone shown by Bromo spiro base comp. (Table-6.2E) 215 Chapter - VI 6.5.References: 1. M.R. Shiradkar, K.K. Murahari, H.R. Gangadasu, T. Suresh, C.C. Kalyan, D. Panchal, R. 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