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Chapter-6 Chapter-6 Biological activity of newly prepared compounds [5a-e to 11a-e] 6.1 Introduction: In the animal kingdom, including human being, infection is a major category of disease. A skilled management is crucially important for antimicrobial drugs. In the past many natural substances were used as therapy. The Greeks used male fern (Dryopteris filix-mas) as anthelmintics. The Hindus treated leprosy with chaulmoogra (Hydnocarpus wightiana) and the Africans were using cinchona bark (Quina) against malaria. The history of modern chemotherapy did not begin until Paul Ehrlich observed that aniline dye selectively stained the bacteria. He invented the word ‘chemotherapy’ The term ‘chemotherapy’ means chemical therapy or chemical treatment. It was first introduced by ‘Paul Ehrlich’. So he is known as the ‘Father of Chemotherapy’. Ehrlich defined chemotherapy as the treatment of diseases due to micro-organisms invades by chemical compound which destroys the micro-organisms without affecting the tissues of the host. The ideal requirement of a drug is that it should act at the desired location or a part of the body when applied. In practice, however, no drug behaves in the said manner; but it tends to distribute itself almost everywhere in the tissues of the host. Some species like protozoa, bacteria, viruses, fungi are responsible for several diseases “Chemical compounds which are used to destroy micro-organism or to inhibit their growth without injuring any part of body are called – “Chemotherapeutic Agent” 197 Chapter-6 Chemotherapeutic Agents which destroy micro-organism is termed as a Cidal effect while the rest only inhabit growth of the organisms which is termed as Static effect. The first compound which was used by Ehrlich was organic dye. Then onwards organic compounds of diverse chemical structures have been used in chemotherapy. A huge amount of different drugs are derived either naturally or synthetically; and in most of the cases they are heterocyclic compounds. The heterocyclic compounds like nicotine, morphine and vitamins are well known for their drug activity. Numbers of nitrogen containing heterocyclic compounds are well known drugs also used as insecticides, fungicides, bactericides etc. [1-4]. Microbiology is a branch of science that deals with microbes. They are too small and can be seen only by magnifying them with the help of a microscope. Microbes are the micro-organisms like viruses, bacteria, algae, fungi, protozoa, etc. The term Bacteria is the plural form of the Bacterium, which means to be a single organism. The Dutch scientist Antonie van Leeuwenhoek was the first person to observe bacteria and other micro-organisms by a single-lens microscope. Apart from Viruses, Bacteria are the smallest living things on our planet Earth. Bacteria are found everywhere; in water, in air, in soil and even on plants and animals (including humans) externally as well as internally. They multiply quite rapidly and form colonies under favorable conditions. They reproduce by binary fission. In this process, a single cell divides into two identical daughter cells. Factors that affect bacteria growth are temperature, pH, humidity and oxygen supply. Bacteria can survive at a specific ideal temperature and pH range. Most disease-causing bacteria survive at temperatures close to that of the human body that is 37°C or 98.6°F; and at pH range of 6.7 to 7.5. 198 Chapter-6 Few bacteria don’t harm unless they enter the blood. Among these, Staphylococcus is responsible for the potentially fatal toxic shock syndrome. Escherichia coli are helpful in the digestive system, but it might cause various diseases like diarrhea, cramping and possibly even death if it enters into the blood. Hans Christian Gram, a Danish bacteriologist, discovered a stain known as Gram stain. With the help of the said, bacteria are divide into two classes - Gram positive and Gram negative. For Gram stain; crystal violet is used as a primary stain and safranin is used as a counter stain. In the said process, if the bacteria turn into purple colour after getting stained, they are called 'Gram positive’, and they are ‘Gram negative’ if turned into red colour when counterstained. A fungus is a member of the group of organisms that includes unicellular or multicellular microorganisms such as yeasts and molds. They are also responsible for some diseases. Bacteria and fungus can damage food or cause diseases. Most forms of food preservation; such as freezing, heating or drying are designed to kill them or make them inactivate. One of the most common methods of destroying bacteria in food is pasteurization. It is this process that the milk can be preserved for quite a long time. The current challenge for medical science is the resistance power which has been developed by these disease-causing bacteria against the antibiotics and other concerned drugs. This evolution makes them able to turn into new forms or secret certain chemicals to make the antibiotics ineffective. 199 Chapter-6 6.2 Antibacterial activity: Following bacteria were selected for in-vitro study. Bacillus subtilis: Gram positive. Staphylococcus aureus: Gram positive Klebsiella pneumoniae: Gram-negative Escherichia coli: Gram negative Materials: All the compounds [5a-e to 11a-e] described in earlier chapters were tested for their antimicrobial study. All other chemicals used were of laboratory grade. Various methods have used from time to time by several worker to evaluate the antimicrobial activity.[5-7] the evaluation can be done by the following methods [8] 1. Agar diffusion method. 2. Agar streak dilution method. 3. Serial dilution method and 4. Turbidometric method. Agar diffusion method is again of three types. Agar cup method Paper disc method and Agar ditch method In present work, Agar cup method is used. 200 Chapter-6 Culture medium preparation: Nutrient agar medium used and Chemical composition of the medium was, Peptone 1.0 gm NaCl 0.5 gm Meat extracts 0.3 gm Distilled water 100 ml pH 7.6 Agar 2.0 gm The ingredient were weighed and dissolved in distilled water, pH was adjusted to 7.6 and then agar powder was added to it. The medium was dispensed in 25 ml quantity in different test tubes. The test tubes were plugged by cotton-wool and sterilized at 121.5°C and 15 pound per square inch (psi) pressure for 15 minutes. Antibacterial susceptibility testing: The study has been conducted according to the method adopted by Cruickshank et al [9] Nutrient agar broth was melted in a water bath and cooked to 45 0C with gentle shaking to bring about uniform cooling. It was inoculated with 0.5-0.6 ml of 24 hour old culture especially and mixed well by gentle shaking before pouring on the sterilized Petri dish (25 ml each). The poured material was allowed to set (1.5 hour) and there after the “cups” was made by punching into the agar surface with a sterile cup borer and soaping out the punched part of agar. Into this “cups” test solution was added by sterile micropipette. The plates were noted. The antibacterial activities of all compounds are calculated in percentage (%) of inhibition, shown in Tables: 6.1 to 6.8. 201 Chapter-6 The percentage (%) data are shown in all tables as follows; (+) = Small clearing zone (<50%), slightly active, (++) = Medium clearing zone (51-55%), moderately active, (+++) = Large clearing zone (56-60%), highly active and, (++++) = Very large clearing zone (>60%), very high activity. Table 6.1: Antibacterial activity of Tetracycline (standard) % Zone of Inhibition Sample Tetracycline Gram +Ve Gram –Ve Bacillus Staphylococcus Klebsiella subtilis aureus pneumoniae ++++ +++ ++++ E-Coli ++++ Table 6.2: Antibacterial activity of compounds (5a-e) % Zone of Inhibition Sample Gram +Ve Gram –Ve Bacillus Staphylococcus Klebsiella E-Coli subtilis aureus pneumoniae 5a + + ++++ +++ 5b ++ + ++++ +++ 5c ++ + +++ ++++ 5d ++++ ++ ++++ ++++ 5e ++++ ++ ++++ ++++ 202 Chapter-6 Table 6.3: Antibacterial activity of compounds (6a-e) % Zone of Inhibition Sample Gram +Ve Gram –Ve Bacillus Staphylococcus Klebsiella E-Coli subtilis aureus pneumoniae 6a ++ + ++++ ++++ 6b ++ ++ ++++ ++++ 6c +++ ++ +++ ++++ 6d ++++ +++ ++++ ++++ 6e ++++ +++ ++++ ++++ Table 6.4: Antibacterial activity of compounds (7a-e) % Zone of Inhibition Sample Gram +Ve Gram –Ve Bacillus Staphylococcus Klebsiella E-Coli subtilis aureus pneumoniae 7a ++ + ++++ ++++ 7b ++ + ++++ +++ 7c ++ + +++ ++++ 7d ++++ +++ ++++ ++++ 7e ++++ +++ ++++ ++++ 203 Chapter-6 Table 6.5: Antibacterial activity of compounds (8a-e) % Zone of Inhibition Sample Gram +Ve Gram –Ve Bacillus Staphylococcus Klebsiella E-Coli subtilis aureus pneumoniae 8a ++ + + + 8b ++ + ++ ++ 8c ++ + + + 8d +++ +++ ++ ++ 8e ++ +++ ++ ++ Table 6.6: Antibacterial activity of compounds (9a-e) % Zone of Inhibition Sample Gram +Ve Gram –Ve Bacillus Staphylococcus Klebsiella E-Coli subtilis aureus pneumoniae 9a + ++ + + 9b + ++ ++ ++ 9c ++ ++ + ++ 9d ++ +++ ++ +++ 9e ++ +++ ++ +++ 204 Chapter-6 Table 6.7: Antibacterial activity of compounds (10a-e) % Zone of Inhibition Sample Gram +Ve Gram –Ve Bacillus Staphylococcus Klebsiella E-Coli subtilis aureus pneumoniae 10a + ++ ++ ++ 10b ++ ++ ++ ++ 10c ++ +++ ++++ +++ 10d +++ +++ ++ +++ 10e +++ ++++ +++ ++++ Table 6.8: Antibacterial activity of compounds (11a-e) % Zone of Inhibition Sample Gram +Ve Gram –Ve Bacillus Staphylococcus Klebsiella E-Coli subtilis aureus pneumoniae 11a ++ ++ ++ ++ 11b ++ ++ ++ ++ 11c ++ ++ ++ ++ 11d ++ +++ +++ +++ 11e +++ ++++ +++ ++++ 205 Chapter-6 6.3 Antifungal activity: Following fungus were selected for fungicidal activity, Rhizopus nigricans: is also known as bread mold. It found on spoiled food and in soils. Nigrospora sp: It is also carried and transferred from place to place through the soil, the decaying plants and seeds Aspergillus niger: It cause a disease called black mould on certain fruits and vegetables such as grapes, apricots, onions, and peanuts. A.niger is less likely to cause human disease which can cause pain, temporary hearing loss and in severe cases, damage to the ear canal. Method: The antifungal activity of all the compounds (mentioned in earlier chapters) was studied at 1000 ppm concentration in-vitro. Plant pathogenic organisms used were Rhizopus Nigrican, Nigrospora Sp. and A. niger. Such activity of all the compounds was measured on each of these plant pathogenic strains on a potato dextrose agar (PDA) medium. This PDA medium was prepared from potato 200 gm., dextrose 20 gm., agar 20 gm. and water 1 liter. Five days old cultures were taken for study. The compounds to be tested were suspended (1000 ppm) in a PDA medium and autoclaved at 1200C for 15 min and at 15 atm. pressure. These media were poured into sterile Petri plates and the organisms were inoculated after cooling the Petri plates. The percentage inhibition for fungi was calculated after five days using the formula given below. Percentage of inhibition = 100 (1 – Y/X) Where X = Area of colony in control plate. Y = Area of colony in test plate. 206 Chapter-6 The fungicidal activities displayed by various compounds are shown in Tables: 6.9 to 6.16. The Zone of inhibition data are presented in all tables as follows; (+) = Zone of inhibition (≤ 59%), slightly active, (++) = Zone of inhibition (60-64%), moderately active, (+++) = Zone of inhibition (65-69%), highly active and, (++++) = Zone of inhibition (≥ 70%), very high activity. Table 6.9: Antifungal activity of Ketoconazole (standard) Sample Ketoconazole Zone of inhibition at 1000 ppm (%) Rhizopus nigrican Nigrospora sp. A. niger ++++ ++++ ++++ Table 6.10: Antifungal activity of compounds (5a-e) Sample Zone of inhibition at 1000 ppm (%) Rhizopus nigrican Nigrospora sp. A. niger 5a + ++ ++ 5b + ++ + 5c ++ ++ + 5d +++ ++++ ++ 5e ++++ +++ +++ 207 Chapter-6 Table 6.11: Antifungal activity of compounds (6a-e) Sample Zone of inhibition at 1000 ppm (%) Rhizopus nigrican Nigrospora sp. A. niger 6a ++ +++ +++ 6b + +++ ++ 6c +++ ++ ++ 6d +++ ++++ +++ 6e ++++ ++++ +++ Table 6.12: Antifungal activity of compounds (7a-e) Sample Zone of inhibition at 1000 ppm (%) Rhizopus nigrican Nigrospora sp. A. niger 7a + ++ ++ 7b + ++ ++ 7c ++ ++ + 7d +++ +++ ++ 7e +++ +++ +++ 208 Chapter-6 Table 6.13: Antifungal activity of compounds (8a-e) Sample Zone of inhibition at 1000 ppm (%) Rhizopus nigrican Nigrospora sp. A. niger 8a ++ + + 8b ++ +++ ++ 8c ++ ++ + 8d ++++ ++++ +++ 8e ++++ ++++ ++++ Table 6.14: Antifungal activity of compounds (9a-e) Sample Zone of inhibition at 1000 ppm (%) Rhizopus nigrican Nigrospora sp. A. niger 9a + ++ ++ 9b + ++ + 9c ++ ++ + 9d ++++ +++ ++++ 9e ++++ ++++ ++++ 209 Chapter-6 Table 6.15: Antifungal activity of compounds (10a-e) Sample Zone of inhibition at 1000 ppm (%) Rhizopus nigrican Nigrospora sp. A. niger 10a +++ +++ +++ 10b +++ ++ +++ 10c +++ +++ +++ 10d ++++ ++++ ++++ 10e ++++ ++++ ++++ Table 6.16: Antifungal activity of compounds (11a-e) Sample Zone of inhibition at 1000 ppm (%) Rhizopus nigrican Nigrospora sp. A. niger 11a ++ +++ +++ 11b ++ ++ +++ 11c +++ +++ +++ 11d ++++ ++++ ++++ 11e ++++ ++++ ++++ 210 Chapter-6 6.4 Results and discussion: The activity of all the newly compounds on Bactria and Fungi are shown in Tables: 6.1 to 6.8 and 6.9 to 6.16 respectively. The compounds tested for antibacterial activity are listed in Tables-6.1 to 6.8 show percentage (%) of zone of inhibition of bacterial growth of Gram-positive bacterial strains B.subtilis and S.aureus and Gram-negative bacterial strains K. pneumoniae, S. typhi and E.Coli. Comparison of antibacterial activity of synthesized compounds with that of standard antibacterial drug against all four bacterial strains. Out of 3-((dialkylamino)methyl)-5-((2-((dimethylamino)methyl)-1H-benzimidazol-1yl) methyl)-1,3,4-oxadiazole-2(3H)-thione[5a-e] (Table 6.2) compounds 5e and 5d show good antibacterial activity. Among 4-(((1-((4-((dialkylamino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2yl)methyl)-1H-benzimidazole-2-yl)methyl)amino)-N-(pyrimidin-2-yl)benzene sulfonamide[6a-e] (Table 6.3) compounds 6d and 6e show good antibacterial activity. Compounds 7e and 7d show good antibacterial activity than the other compounds of 3-((dialkylamino)methyl)-5-((2-((ethyl(methyl)amino)methyl)-1H-benzimidazol-1yl)methyl)-1,3,4-oxadiazole-2(3H)-thione.[7a-e] ( Table 6.4) In 3-((dialkylamino)methyl)-5-((2-((diethylamino)methyl)-1H-benzimidazol-1yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[8a-e] (Table 6.5) compound 8d exhibit good antibacterial activity. In 3-((dialkylamino) methyl)-5-((2-((dipropylamino)methyl)-1H-benzimidazol-1yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[9a-e] (Table 6.6), compounds 9d and 9e show moderate antibacterial activity. 211 Chapter-6 Among 3-((dialkylamino) methyl)-5-((2-((diphenylamino)methyl)-1H-benzimidazol1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione [10a-e] (Table 6.7) compound 10e show good antibacterial activity. In synthesized compounds, 3-((dialkylamino) methyl)-5-((2-((ethyl(phenyl)amino) methyl)-1H-benzimidazol-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[11a-e] (Table- 6.8) compound 11e show good antibacterial activity. The compounds tested for fungicidal activity are listed in Tables-6.9 to 6.16 show Zone of inhibition at 1000 ppm (%) for Rhizopus Nigrican, Nigrospora Sp. and A. Niger. Comparison of antifungal activity of synthesized compounds done with Ketoconazole as standard compound against all three fungal strains. Among newly synthesized compounds 3-((dialkylamino)methyl)-5-((2-((dimethyl amino)methyl)-1H-benzimidazol-1-yl) methyl)-1,3,4-oxadiazole-2(3H)-thione[5a-e] (Table 6.10) compounds 5d and 5e show high antifungal activity. Out of 4-(((1-((4-((dialkylamino)methyl)-5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl) methyl)-1H-benzimidazole-2-yl)methyl)amino)-N-(pyrimidin-2-yl)benzene sulfonamide[6a-e] (Table 6.11) compound 6d and 6e show good antifungal activity. In 3-((dialkylamino)methyl)-5-((2-((ethyl(methyl)amino)methyl)-1H-benzimidazol-1yl) methyl)-1,3,4-oxadiazole-2(3H)-thione[7a-e] (Table 6.12) compounds 7d and 7e show high antifungal activity. In 3-((dialkylamino)methyl)-5-((2-((diethylamino)methyl)-1H-benzimidazol-1-yl) methyl)-1,3,4-oxadiazole-2(3H)-thione[8a-e] (Table 6.13) compound 8d and 8e exhibit good antifungal activity. 212 Chapter-6 Out of 3-((dialkylamino) methyl)-5-((2-((dipropylamino)methyl)-1H-benzimidazol-1yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[9a-e] (Table 6.14) compounds 9d and 9e show good antifungal activity. Among 3-((dialkylamino) methyl)-5-((2-((diphenylamino)methyl)-1H-benzimidazol1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione [10a-e] (Table 6.15) compounds 10d and 10e exhibit good antifungal activity. Among 3-((dialkylamino) methyl)-5-((2-((ethyl(phenyl)amino)methyl)-1Hbenzimidazol-1-yl)methyl)-1,3,4-oxadiazole-2(3H)-thione[11a-e] (Table 6.16) compounds 11d and 11e show good antifungal activity. In conclusion all the compounds are toxic, more or less to selected bacteria as well as fungi. More particularly the compounds d and e in all series are more toxic, this might be responsible due to presence of phenyl ring. 213 Chapter-6 References: [1] B.P.Patel,P.J.Shah and H.S.Patel, J.Saudi chemical Soc.,17,307 (2013). [2] A.R.Shah, H.R.Dabhi and A.K.Rana.,Der Pharma Chemica, 7(3),158(2015) [3] J.D.Bhatt, K.S.Nimavat and K.B.Vyas., J.Chem.Pharm.Res., 5(10),327(2013). [4] M.Sharda and G.D.Acharya., Der Pharma Chemica, 7(8), 25(2015) [5] C. Robert, “Medical Microbiology”, ELBS, Livingston, 11th edition,.815, 901 (1970). [6] G. D. Sujatha, Ind. J. Expt. Biol., 13, 286 (1975). [7] R.J.Patel and K.S.Trivedi, Experimental Microbiology,Aditya Publication,7th Edition (2014) [8] S.A.Walksman, “Microbial Antagonism and Antibiotic Substances”, Commonwealth Fund, N.Y., 2nd edition, 72 (1947) [9] R.Cruickshank, J.P.Dugid, D.P.Marmion and R.H.A.Swain,"Medical Microbiology”, Churchil-Livingstone, Edinburgh, London, Vol. 2, 12th edition (1975). 214