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BIOSCIENCES BIOTECHNOLOGY RESEARCH ASIA, December 2013. Vol. 10(2), 891-896
An Antibacterial, Antifungal and Anthelmintic Evaluations
of Some Synthesized Chalcone Derived Benzimidazoles
I. Sudheer Babu and S. Selvakumar*
Department of Pharmaceutical Chemistry, Sir.C.R.Reddy College of Pharmaceutical Sciences, West Godavari (Dist), Eluru - 534 007, India.
DOI: http://dx.doi.org/10.13005/bbra/1213 (Received: 15 July 2013; accepted: 20 September 2013) The Chalcone nucleus based natural plant products have various biological activities
due to their highly reactive a, b unsaturated carbonyl group. If these medicinal plants not
traceable in nature we must go through new synthetic drugs as a source of chalcone analogues.
So we synthesized a series of chalcone derived benzimidazoles by condensation of 2-acetyl
benzimidazole in alkali with various aromatic aldehydes. The synthesized chalcones were
screened for their in-vitro antimicrobial activity using disc diffusion and broth micro-dilution
assays. The synthesized chalcones shows good activity against the tested fungal species. They
have better activity against gram positive bacteria but shows poor activity against gram negative
bacteria. The In-vitro anthelmintic screening of all chalcones exhibited significant potency
when compared to standard drug albendazole.
Key words: Chalcones, Benzimidazolyl chalcones, Antibacterial, Antifungal & anthelmintic.
antimalarial, inhibit eosinophilia (asthma), nucleus of various natural plant products such antiseptical 6, analgesic/anti-inflammatory 7, as flavonoids, isoflavonoids, aurones, tetralones, cyclooxygenase inhibitors 8, anti ulcerogenic, aziridines and clavicin etc.This nucleus analogue prostaglandin binding, antiallergic, anaesthetic, is an integral part of various natural medicinal plant antiplasmodial 9, hypotensive, antifibrogenic , constitutions such as terpeneoids, dicoumarol, immuno suppression,antineoplastic, cytotoxicity, flavokawain, digitalis glycosides 1 and vitamin anticancer 10, antiproliferative 11, antileukemic, K etc. However in recent years the introduction antitumor 12 ,trypsin inhibitor, anxiolytic 13 and of new synthetic chalcone derived drugs has out placed that of natural products such as warfarin for dicoumarol. The Chalcone analogues are a related to purine nucleoside bases and it is found chemical class that has depicted antioxidant2, in some natural products, such as vitamin B , antiviral, antibacterial 3, antifungal 4, insecticidal, marine natural product namely makaluvamins antitrichomonal, antitubercular 5,antileishmanial, etc. The literature survey reports have been revealed that the 2- substituted 1H-benzimidazole compounds has reported to possess antibacterial 15, antifungal 16, antitubercular , antiviral 17, antiulcer and anthelminitic 18,19 activities . The benzimidazolyl chalcones also reported to have potent antimicrobial 20 properties. So we planned to merge the chalcone nucleus with benzimidazole at 2nd position to exhibit some better bioloical activity, * To whom all correspondence should be addressed.
E-mail: [email protected] mainly to screen for their in-vitro antimicrobial and anthelminitic activities.
BaBu & SelvaKumar, Biosci., Biotech. Res. Asia, vol. 10(2), 891-896 (2013)
ExpErimEntal
antimicrobial activity
micro-organisms
Synthesis of 1-(1H-benzoimidazol-2-yl) ethanol
Klebsiella aerogenes mTCC-39, and Escherichia coli mTCC 1302. Three fungal species were diamine (2) were refluxed for one and half an hour. used, these being Aspergillus niger mTCC 2425, The mixture was cooled, 25% potassium hydroxide Candida albicans mTCC 183 and the Penicillium solution was added until the product (3) was just citrinum mTCC 1256. all bacterial strains were cultivated in mueller Hinton agar (mHa) media Synthesis of 1-(1H-benzoimidazol-2-yl)ethanone
and fungi species were cultivated in Sabouraud Dextrose agar (SDa) media. The stock culture was maintained on agar slant at 4 °C. These microbial 50% sulphuric acid and 20% potassium dichromate strains were subculture on a fresh appropriate agar mixture, refluxed for one hour and allowed to cool plate 24 hrs prior to any antimicrobial test.
at room temperature and poured, in to a ice cold agar diffusion assay
water with stirring to get a product(4). Synthesis of benzimidazolyl chalcones (6a-j)
out by using 100 µl of suspension of the tested The 0.01 molar (4) in 10ml 30% potassium microorganisms containing 2.0 × 106 CFu/ml for hydroxide with 0.012 molar of various aromatic bacteria and 2.0 ×105 CFu /ml spores for fungal aldehydes namely, para-anisaldehyde (5a) , strains. This microbial suspension was used to benzaldehyde (5b) , cinnamaldehyde (5c), ortho- inoculate by flooding the surface of mHa and chloro benzaldehyde (5d), para-(dimethyl amino) SDa plates for bacteria and fungi respectively. benzaldehyde (5e), para-fluorobenzaldehyde Then sterilized discs were prepared at 30µg/disc for (5f), ortho-nitro benzaldehyde (5g), para-nitro synthesized chalcones and 10µg/disc for standard benzaldehyde (5h) , ortho-hydroxybenzaldehyde antibiotics. a discs have synthesized chalcones (5i) and 4-hydroxy 3-methoxybenzaldehyde were prepared with only the corresponding volume (5j) separately, then refluxed for 2 hours of dimethylsulphoxide was used as negative with water bath, allow cooling to the room control. The petri plates were then incubated at temperature and pouring the mixture in to a 37°C for bacteria and 28°C for fungi species. The beaker containing ice cold water, with stirring to antimicrobial activity was evaluated by measuring get a product namely, 1-(1H-benzoimidazol-2- the diameter of the zones of inhibition around the yl)-3-(4-methoxyphenyl) propenone (6a),1-(1H- benzoimidazol-2-yl)-3-Phenyl Propenone (6b), Broth micro-dilution assay
1-(1H-benzoimidazol-2-yl)-5-Phenyl Pentadienone (6c), 1-(1H-benzoimidazol-2-yl)-3-(2-Chloro (mIC) were determined by twofold serial micro Phenyl) Propenone (6d), 1-(1H-benzoimidazol-2- broth dilution method in mueller Hinton or yl)-3-(4-(dimethyl amino) Phenyl) Propenone (6e), Sabouraud dextrose broth media. The synthesized 1-(1H-benzoimidazol-2-yl)-3-(4-fluoro Phenyl chalcones and standard antibiotics were dissolved ) Propenone (6f), 1-(1H-benzoimidazol-2-yl)- in 50% dimethylsulphoxide aqueous solution. all 3-(2-nitrophenyl) Propenone (6g), 1-(1H-benzo the compounds were diluted two fold concentration imidazol-2-yl)-3-(4-nitrophenyl) Propenone (6h), from 0.2 to 204 µg and the starting inoculums of 1-(1H-benzoimidazol-2-yl)-3-(2-hydroxy phenyl) 2.0 ×107 CFu/ ml were used. The test tubes were propenone (6i) and1-(1H-benzoimidazol-2-yl)-3- incubated at 37°C for bacteria and 28°C for fungi. (4-hydroxy-3-methoxy phenyl) propenone (6j). The lowest concentration of the drug displaying no all the above products were collected by filtration visible growth was considered as the mIC. with ice cold water, dried and recrystallised from anthelmintic investigation
90%ethanol. The structures of the synthesized compounds were verified by Ir,1H-Nmr, 13C- Pheretima posthuma of 7-9cm in length and Nmr and mass spectral analysis.
0.2-0.3 cm in width were selected for the invitro anthelminitic activity due to its anatomical BaBu & SelvaKumar, Biosci., Biotech. Res. Asia, vol. 10(2), 891-896 (2013)
and physiological resemblance with the gastro bacteria, Klebsiella except the 6b and 6i. The most intestinal worm parasites of human beings. The of the chalcones exhibited better activity against earth worms washed with normal saline solution to Candida than the Aspergillus and Penicillium remove all fecal and adhering soil materials before strains. Only chalcone 6f have a potent activity they were released in to petridishes. The standard drug albendazole and tested benzimidazoles were prepared at a doses level of 10, 30,50 mg by dissolving in, about 0.5ml of dimethylsulphoxide, the volume was made-up to 15 ml with normal saline, then poured into petridishes. The five earth worms were taken in each petridishes. Then, the time taken for the induction of complete paralysis and time taken for death of individual earthworms were observed. The control group was observed that the worms were still alive up to 48 hours. rESultS and diScuSSion
benzimidazolyl chalcones were prepared and evaluated for their antimicrobial activities using disc diffusion and micro broth dilution assays, the results are displayed in Table 2. The chalcones 6d, 6f, 6g and 6h were showed effective zone of inhibition against Klebsiella. all the chalcones displayed less effective zone of inhibition against E. coli except 6f. The chalcones 6d, 6f, 6g and 6h exerted potent invitro antifungal activity against Aspergillus, Penicillium and Candida species. Observations showed that chalcones 6a, 6c, 6e and 6j had better activity against Aspergillus, Penicillium and Candida species respectively. all the tested chalcones showed good zone of inhibition against fungi species and gram positive Scheme 1:
table 1. Physical analysis of synthesised benzimidazolyl Chalcones
Solvent system (TlC): chloroform: 90% ethanol (8:2) BaBu & SelvaKumar, Biosci., Biotech. Res. Asia, vol. 10(2), 891-896 (2013)
-N(CH ) or OCH groups as substituents at para or When structure activity relationship studies meta (r or r ) position on the benzene ring, both are concerned, the antimicrobial activity might enhanced the activity. It was observed that para be increased by the presence of electronegative substituted analogues are more potent than ortho substituents such as NO , fluoro and chloro or meta substituted chalcones. moreover extended groups at ortho or para (r or r ) position on conjugated analogue (6c) also slightly stimulates the chalcone benzene ring, while polar electron donating substituent OH group at ortho or para (r In vitro anthelminitic screening results in or r ) position decreases activity. Furthermore, the Table 3, data displays chalcone 6a, 6c, 6d, 6g, 6h compounds bearing nonpolar electron donating and 6i showed appreciable activity. It indicates that table 2. antimicrobial activity & minimum inhibition concentration of
the synthesized benzimidazolyl chalcones 6 a-j and reference antibiotics Inhibition zone diameters in (mm) minimum inhibition concentration (µg) values are given an average mean, (n = 3). anti-microbial activity synthesized chalcones 6a-j (30 µg D disc) and standard antibiotics (10 µg D disc). K a - Klebsiella aerogenes, e C- escherichia coli, a N- aspergillus niger, C a-Candida albicans, P C- Penicillium Citrinum, CIPN- Ciprofloxacin, ClOe- Clotrimazole.
table 3. In vitro anthelminitic activity of the synthesized benzimidazolyl chalcones (6a-j)
each average value represents the mean ± Sem (n=5). Significance levels *P<0.5, **P<0.01 and ***P<0.001 as compared with the respective standard drug (alZ- albendazole).
BaBu & SelvaKumar, Biosci., Biotech. Res. Asia, vol. 10(2), 891-896 (2013)
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