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com

1995. p. 262-3. heartwood of Garcinia indica. Phytochemstry 1977;16:148-9.

12. Badami RC, Razdan MK. Isolation and Identification of L- leucine 18. Lakshmi C, Akshayakumar K, Dennis TJ. Polyprenoylated
as DNP. L –leucine hydrochloride in the leaves of Garcinia indica. benzophenobes from Garcinia indica. J Ind Chem Soc 2002;79:968-9.
J Indian Chem Soc 1972;49:583. 19. Singhai M, Benerjee AK. Investigation on Garcinia indica seed oil.
13. Jayaprakasha GK, Sakariah KK. Determination of organic acids in Asian J Chem 1994;3:720-1.
leaves and rinds of Garcinia indica (Derr) by L.C. J Pharm Biomed 20. Babu V, Ali SM, Sultana S, Ilyas M. A biflavonoid from Garcinia
2002;28:379-84. nervosa. Phytochemistry 1988;27:3332-5.
14. Kantha AR. A chemical technique for determining the configuration of 21. Bigger E. Hand book of bacteriology. London: Biller Indall and Lox;
natural fats, operation of specific restricted random distribution rule B in 1943. p. 35.
Garcinia indica and Vateria indica seed fats. Indian J Chem 1964;2:199-203.
15. Nayak CA, Srinivas P, Rastogo NK. Charaterization of anthocyanins Accepted 18 August 2011
from Garcinia indica Choisy. Food Chem 2010;118:719-24. Revised 19 July 2011
16. Krishnamurthy N, Leuis YS, Ravindranath B. On the structures of
Received 08 October 2010
garcinol, isogarcinol and camboginol. Tetrahedron Lett 1981;72:793-6.
17. Coiterill PJ, Scheinnamn F, Puranik GS. Phenolic compounds from Indian J. Pharm. Sci., 2011, 73 (4): 470-473

Evaluation of Antimicrobial Efficacy of Flavonoids of

Withania Somnifera L.
G. SINGH* AND P. KUMAR
Laboratory of Plant Tissue Culture and Secondary Metabolites, Department of Botany, University of Rajasthan,
Bapu Nagar, Jaipur-302 055, India

Singh and Kumar: Antimicrobial Efficacy of Withania Somnifera L.

In the present study antimicrobial activity of Withania somnifera L. Dunal (Solanaceae) has been evaluated against
selected pathogens. Free and bound flavonoids of different parts (root, stem, leaf and fruit) of W. somnifera have been
studied for their antimicrobial activity using disc diffusion assay against three Gram negative bacteria (Escherichia coli
MTCC 46, Proteus mirabilis MTCC 3310 and Pseudomonas aeruginosa MTCC 1934), one Gram positive bacteria
(Staphylococcus aureus MTCC 3160) and three fungi (Candida albicans MTCC 183, Aspergillus flavus MTCC
277 and Aspergillus niger MTCC 282). Minimum inhibitory concentration (MIC) of the extracts was evaluated
through micro broth dilution method, while minimum bactericidal/fungicidal concentration was determined by sub
culturing the relevant samples. C. albicans was found to be the most susceptible organism followed by S. aureus,
P. mirabilis, E. coli and P. aeruginosa. Out of the tested organisms, A flavus and A. niger were observed to be
resistant as none of the tested extracts showed activity against them. Total activity (TA) of extracts (ml/g) against
each sensitive pathogens was also evaluated. Bound flavonoid extract of root showed best activity against C. albicans
(IZ 30, MIC 0.039, MFC 0.039, respectively). However all the microorganisms were found to be sensitive against
the extracts tested. Total activity of bound flavonoid extract of root was found to be same for E.coli, P. mirabilis,
S. aureus and C. albicans (153.84 ml/g). Results of the present study reveal that extracts of W. somnifera showing
great antimicrobial potential against test microorganisms may be exploited for future antimicrobial drugs.

Key words: Flavonoids, minimum inhibitory concentration, minimum bactericidal concentration, minimum
fungicidal concentration, total activity, Withania somnifera

The plants are still widely used in ethno medicine biological activity. Attention has been drawn to
around the world. Today, medicinal plants have antimicrobial activity of plants and their metabolites
recently received attention of the pharmaceutical due to the challenge of growing incidences of drug
and scientific communities and various publications resistant pathogens. Some plants have shown the
have documented the therapeutic value of natural ability to overcome resistance in some organisms
compounds in a bid to validate claims of their and this has led to researches to investigate
their mechanisms of action and isolating active
*Address for correspondence compounds [1] . There is a continuous and urgent
E-mail: geetsingh600@gmail.com need to discover new antimicrobial compounds with
July - August 2011 Indian Journal of Pharmaceutical Sciences 473
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diverse chemical structures and novel mechanisms of bound flavonoids of Withania somnifera (root, stem,
action as there has been an alarming increase in the Leaf and fruits).
incidence of new and re-emerging infectious diseases.
In the present scenario of emergence of multiple drug Different parts of W. somnifera (root, stem, leaf
resistance to human pathogenic organisms, this has and fruit) were collected in the month of April
necessitated a search for new antimicrobial substances from the western parts of India (Jaipur, Rajasthan).
from other sources including plants. Higher plants A voucher specimen has been submitted in Herbarium
produce hundreds and thousands of diverse chemicals of Department of Botany, University of Rajasthan.
compounds with different biological activities[2]. The Collected plant parts were separately shade dried,
antimicrobial compounds produced by plants are active finely powered using a blender, and subjected to
against plants and human pathogenic microorganisms[3]. extraction following the method of Subramanian and
The substances that can either inhibit the growth of Nagarjan, 1969. Hundred gram of each finely powered
pathogens or kill them and have no or least toxicity sample was Soxhlet extracted with 80% hot methanol
to host cells are considered potential compounds for (500 ml) on a water bath for 24 h and filtered. Each
developing new antimicrobial drugs. filtrate was re-extracted successively with petroleum
ether (fraction I), ethyl ether (fraction II), and ethyl
Withania somnifera, commonly known as acetate (fraction III) using separating funnel. Petroleum
Ashwagandha, is an important medicinal plant that ether fractions were discarded as being rich in fatty
has been used in Ayurvedic and indigenous medicine substances, whereas ethyl ether and ethyl acetate
for over 3,000 years. In view of its varied therapeutic fractions were analyzed for free and bound flavonoids,
potential, it has also been the subject of considerable respectively. Ethyl acetate fraction of each of the
modern scientific attention. Ashwagandha roots are a samples was hydrolyzed by refluxing with 7% H2SO4
constituent of over 200 formulations in Ayruvedha, for 2 h (for removal of bounded sugars from the
Siddha and Unani medicine, which are used in flavonoids) and filtered. The filtrate was extracted
the treatment of various physiological disorders[4,5]. in ethyl acetate and washed with distilled water to
Ashwagandha is widely claimed to have sedative, neutrality. Ethyl ether (free flavonoid) and ethyl acetate
rejuvenative and life prolonging properties. It is also fractions (bound flavonoids) thus obtained were dried
used as a general energy-enhancing tonic known as in vaccuo and weighed. The extracts were stored at 4°
Medharasayana, which promotes learning and a good and were re- suspended in their respective solvents to
memory and in geriatric problems[6,7]. get 10 mg/ml for antimicrobial assay.
Pseudomonas aeruginosa, Escherichia coli,
Staphylococcus aureus, Proteus mirabilis and Candida Free and bound flavonoids were extracted by well
albicans have been proved to be major causal established method of Subramanian and Nagarjan[10]
organisms of various human infections and have which had also been followed by several others
been selected for the present study. P. aeruginosa is workers[11-14]. The extraction protocol which has been
an opportunistic pathogen that causes urinary tract carried out in the present investigation is exclusively
infections, respiratory system infections, dermatitis, meant for free and bound flavonoids. In the extraction
soft tissue infections and a variety of systemic procedure ethyl ether and ethyl acetate fractions
infections, particularly in victims of severe burns. are supposed to contain free and bound flavonoids,
E. coli and P. mirabilis are the culprits for human respectively. Here bound flavonoids imply that the
urinary tract infections[8] and most of human intestinal flavonoids that are bound with sugar moiety. These
infections are due to the bacterium Escherichia coli, bounded sugar moieties are removed (from ethyl acetate
Staphylococcus aureus causes a variety of suppurative, fraction) by acid hydrolysis during extraction. If sugar
wound infections and food poisoning in human beings. part of the flavonoids is not removed, extracts do not
Major causative agent of nosocomial infections is react with spraying reagents (i.e 5% Fehling solution and
Staphylococcus aureus[9] along with Escherichia coli 1% AlCl3 solution) during TLC analysis. On the other
and Proteus mirabilis, Candida albicans is notorious hand, after removal of sugar part, flavonoids become free
for causing candidiasis and candida vaginitis. from sugars bounded to it and now react with spraying
reagent and give colour reactions during TLC analysis.
The present study investigation evaluated the Spraying reagents 5% fehling solution and 1% AlCl3
antibacterial and anticandidal effects of free and solution are exclusively used to detect flavonoids[15].
474 Indian Journal of Pharmaceutical Sciences July - August 2011
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Ethyl ether fraction contains free flavonoids i.e produced by extract/IZ produced by standard; where
flavonoids free from sugar moiety and during TLC IZ is inhibition zone.
shows positive colour reactions with the spraying
reagents. In the ethyl ether fraction there is no need Minimum inhibitory concentration (MIC) was
of acid hydrolysis as flavonoids in this fraction are determined for plant extract showing antimicrobial
not bound with sugar. activity against test pathogens. Broth microdilution
method [17] was followed for determination of MIC
Test pathogenic microorganisms: P. aeruginosa values. Plant extracts were resuspended in acetone
(MTCC 1934), E. coli (MTCC 46), P. mirabilis (which has no activity against test pathogens) to make
(MTCC 3310), S. aureus (MTCC 3160) and 10 mg/ml final concentration and then two fold serially
C. albicans (MTCC 183), A flavus (MTCC 277) and diluted; and added to broth media of 96-wells of
A. niger (MTCC 282) were procured from Institute microtiter plates. Thereafter 100 µl inoculum (for
of Microbial Technology (IMTECH), Chandigarh, bacteria 1×108 CFU/ml and 1×107 CFU/ml for yeast
India. Bacterial strains were grown and maintained on and fungi) was added to each well. Bacterial and
Muller-Hinton Agar medium, while yeast and fungi fungal suspensions were used as negative control,
was maintained on Sabouraud Dextrose Agar medium. while broth containing standard drug was used as
positive control. The microtiter plates were incubated
Disc diffusion assay[16] was performed for screening. at 37° for 24 h for bacteria and 28° for 48 h for yeast.
Muller-Hinton agar (MHA) and Sabouraud dextrose Each extract was assayed in duplicate and each time
agar (SDA) base plates were seeded with the bacterial two sets of microplates were prepared, one was kept
and fungal inoculum, respectively with inoculum for incubation while another set was kept at 4° for
size 1×10­­8 CFU/ml for bacteria and 1×107 cell/ ml comparing the turbidity in the wells of micriplate. The
for yeast). Sterile filters paper discs (Whatman MIC values were taken as the lowest concentration
no. 1, 6 mm in diameter) were impregnated with of the extracts in the well of the microtiter plate that
100 µl of each of the extract (10 mg/ml) to give showed no turbidity after incubation. The turbidity of the
a final concentration of 1 mg/disc and left to dry wells in the microtiter plate was interpreted as visible
to remove residual solvent, which might interfere growth of microorganisms. The minimum bactericidal/
with the determination. Extract discs were then fungicidal concentration (MBC/MFC) was determined by
placed on the seeded agar plates. Each extract was subculturing 50 µl from each well showing no apparent
tested in triplicate with streptomycin (1 mg/disc) and growth. Least concentration of extract showing no
candid-V6 (1 mg/ ml) as standard for bacteria and visible growth on subculturing was taken as MBC/MFC.
fungi, respectively. The plates were kept at 4° for 1
h for diffusion of extract, thereafter were incubated Total activity is the volume at which the test extract can
at 37° for bacteria (24 h) and 27° for fungi (48 h). be diluted with the ability to kill the microorganisms.
Antibacterial and antifungal activity was expressed in It is calculated by dividing the amount of extract from
terms of activity index (AI). Activity index for each 1 g plant material by the MIC of the same extract or
extract was calculated (Table 1) as, Activity index=IZ compound isolated and is expressed in ml/g[18].

TABLE 1: ANTIMICROBIAL ACTIVITY OF WITHANIA SOMNIFERA

Plant Extract Test microorganism
part P. aeruginosa E. coli P. mirabilis S. aureus C. albicans
IZ (mm) AI IZ (mm) AI IZ (mm) AI IZ (mm) AI IZ (mm) AI
Root E1 - - - - - - 12.5 0.595±0.014 7.83 0.559±0.031
E2 10 0.500±0.050 25.5 0.980±0.011 28.5 1.140±0.012 21.5 1.023±0.014 30 2.142±0.041
Stem E1 - - - - 11.66 0.467±0.012 11 0.523±0.027 10.16 0.726±0.031
E2 - - - - - - 8.5 0.404±0.024 8.5 0.607±0.020
Leaf E1 - - 16.5 0.634±0.033 10 0.400±0.012 10.5 0.499±0.024 19.5 1.392±0.020
E2 - - - - 11.5 0.460±0.012 15.33 0.729±0.124 8 0.571±0.021
Fruit E1 - - - - 16.5 0.660±0.035 - - 10 0.714±0.021
E2 - - - - - - 13 0.618±0.090 12.5 0.892±0.020
IZ - inhibition zone in mm (mean value; indicating 6 mm diameter of disc); AI - activity index (IZ developed by extract/IZ developed by standard); ± - SEM;
(-) - No activity; E1 - free flavonoids; E2 - bound flavonoids; Extracts assayed in triplicate, IZ of standard drug streptomycin against P. aeruginosa (20 mm),
E. coli (26), P. mirabilis (25), S. aureus (21), IZ of Candid-V6 against C. albicans (14) Intraconazol against A. flavus (15) and A. Niger (10)

July - August 2011 Indian Journal of Pharmaceutical Sciences 475

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Antimicrobial activity (assessed in terms of inhibition MIC and MBC/MFC values (Table 2) were evaluated
zone and activity index) of the plant extracts, tested for plant extracts which had shown activity in
against selected microorganisms were recorded diffusion assay. The range of MIC and MBC/MFC of
(Table 1). In the present study total eight extracts extracts recorded was 0.039-0.625 mg/ml and 0.039-
of different parts of plant were tested for their 1.25 mg/ml, respectively. In the present investigation
bioactivity. All 8 extracts showed significant lowest MIC value 0.039 mg/ml was recorded against
antimicrobial activity against test microbes. Most E. coli, P. mirabilis, S. aureus and C. albicans
susceptible organism in the investigation was C. whereas, against P. aeruginosa lowest MIC observed
albicans against which one or other all the plant was 0.156 mg/ml, indicating significant antimicrobial
extracts showed inhibition zone in some cases even potential of test extract. MIC and MBC/MFC values
more than the standard and the best activity was were found to be same for extracts of plant.
observed for bound flavonoids of root with IZ
30 mm, AI 2.142±0.041 and MIC 0.039 mg/ml. Quantity of extract obtained per gram from plant parts
P. aeruginosa was found to be the most resistant and total activity (TA) was calculated and recorded
microbe, against which only one extract showed (Table 3). Total activity indicates the volume at which
activity. Best antibacterial activity against E. coli (IZ extract can be diluted without loosing ability to kill
25.5, AI 0.980±0.011, MIC 0.039), P. mirabilis (IZ microorganism. Most of the extracts showed high
28.5, AI 1.140±0.012, MIC 0.039) and S. aureus (IZ values of TA against E. coli, P. mirabilis, S. aereus
21.5, AI 1.023±0.014, MIC 0.039) was observed for and C. albicans. Maximum TA values calculated were
bound flavonoids of root whereas, free flavonoids 38.46, 153.84, 198.71, 153.84 and 198.71 ml against
of leaf showed best activity against E. coli (IZ P. aeruginosa, E. coli, P. mirabilis, S. aureus and C.
16.5, AI 0.634±0.033, MIC 0.078). Free flavonoids albicans, respectively.
of fruit showed bioactivity against P. mirabilis (IZ
16.5, AI 0.660±0.035, MIC 0.078) whereas, bound There is a continuous and urgent need to discover
flavonoids showed activity against S. aureus (IZ 13, new antimicrobial compounds as there is an alarming
AI 0.618±0.090, MIC 0.078). increase in the incidence of new and re-emerging

TABLE 2: MIC AND MBC/MFC VALUES OF W. SOMNIFERA AGAINST TEST PATHOGENS

Plant Extract Test microorganism
part P. aeruginosa E. coli P. mirabilis S. aureus C. albicans
MIC MBC MIC MBC MIC MBC MIC MBC MIC MFC
Root E1 - - - - - - 0.156 0.312 0.312 1.25
E2 0.156 0.312 0.039 0.039 0.039 0.039 0.039 0.078 0.039 0.039
Stem E1 - - - - 0.078 0.156 0.156 0.312 0.078 0.156
E2 - - - - - - 0.625 0.125 0.156 0.312
Leaf E1 - - 0.078 0.078 0.312 0.312 0.156 0.312 0.078 0.078
E2 - - - - 0.156 0.312 0.078 0.156 0.156 0.312
Fruit E1 - - - - 0.078 0.078 - - 0.078 0.156
E2 - - - - - - 0.078 0.156 0.039 0.039
E1 - Free flavonoids; E2 - Bound flavonoids; MIC - Minimum inhibitory concentration (mg/ml); MBC/MFC - Minimum bactericidal/fungicidal concentration (mg/ml)

TABLE 3: QUANTITY AND TOTAL ACTIVITY OF FREE AND BOUND FLAVONOIDS OF W. SOMNIFERA
Plant Extract Amount of Total activity (ml/g)
part extract mg/g
dried plant part P. aeruginosa E. coli P. mirabilis S. aureus C. albicans
Root E1 7 - - - 44.87 22.43
E2 6 38.46 153.84 153.84 153.84 153.84
Stem E1 5.5 - - 70.51 35.25 70.51
E2 3 - - - 4.80 19.23
Leaf E1 10.5 - 134.61 33.65 67.30 134.61
E2 3.5 - - 22.43 44.87 22.43
Fruit E1 15.5 - - 198.71 - 198.71
E2 6.5 - - - 83.33 166.66
E1 - Free flavonoids; E2 - Bound flavonoids; total activity = Extract per gram dried plant part/MIC

476 Indian Journal of Pharmaceutical Sciences July - August 2011

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infectious diseases. Medicinal plants could be a good the extracts MIC values recorded were very low,
alternative source for costly antibiotics (against which indicating strong bio efficacy of the selected plant.
microbes are developing resistance rapidly), as most
of the medicinal plants are safe with little or no side Extracts under study not only inhibit the bacterial/
effects, cost effective and have ability to affect a wide fungal growth but the IZ developed, was more or
range of antibiotic resistant microorganisms. less permanent when compared with the IZ developed
by the standard drug used, as after sometime
Present study is an effort towards this direction. In bacterial/fungal colonies could be easily seen in
the present study IZ, AI, MIC, MBC/MFC and TA IZ developed by standard drugs. In the light of
have been evaluated for each extract. For most of the fact that microorganism are becoming resistant
the extracts MIC values recorded were very low, against the drugs in use, present investigation is
indicating strong bio efficacy of the plant. Most of the of great significance, as far as the future drugs are
extracts of plant were found to be potent inhibitor of concerned and advocates uses of selected plant by the
tested microorganisms except P. aeruginosa, against pharmaceutical industries for preparing plant based
which only one extract of the plant showed activity. antimicrobials drugs.
Excellent activity was shown by bound flavonoids of
roots having low MIC and MBC/MFC values. ACKNOWLEDGEMENTS

Extracts with higher MBC/MFC values than MIC Authors are thankful to the Head of Botany Department,
values against microorganisms tested, indicate the University of Rajasthan for providing all necessary facilities
bacteriostatic/fungistatic effects of the extracts. Bound for present work. Financial assistance provided by UGC is
flavonoids of roots were found to be bactericidal gratefully acknowledged.
against E. coli and P. mirabilis and fungicidal against
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Synthesis and Antimicrobial Activities of Some New

Azetidin-2-ones and Thiazolidin-4-ones
SHOBHA R. DESAI*, U. V. LADDI1, RAJANI S. BENNUR AND S. C. BENNUR
Department of Chemistry, S. S. Arts College and T. P. Science Institute, Old P. B. Road, Sankeshwar-591 313,
1
Channabasaveshwara Institute of Technology, N.H. 206, B.H. Road, Gubbi, Tumkur-572 216, India

Desai, et al.: New Azetidin-2-ones and Thiazolidin-4-ones: Synthesis and Evaluation

Various (4-substituted) phenyl-3-β-[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino]propionylamido-1,3-thiazolidine-

4-ones (3a–x) and 1-β-[(N-benzenesulphonyl/tosyl)-4-(un)substituted anilino]-propionylamido-3-chloro-4-(4-
substituted)phenyl-azetidin-2-ones (4a–x) have been synthesised by the cyclocondensation of Schiff bases (2a-x)
with thioglycolic acid and chloroacetyl chloride, respectively. The structures of the newly synthesised compounds
have been established on the basis of their spectral data and elemental analysis. All compounds were evaluated for
antimicrobial activities against Escherichia coli, Bacillus cirroflagellosus, Aspergillus niger and Colletotrichum capsici.
Most compounds investigated exhibited significant antifungal activity against Colletotrichum capsici, comparable
to that of fluconazole, the standard used.

Key words: Azetidinones, antimicrobial activity, sulfonamide moiety, thiazolidinones

Survey of the recent literature shows that intensive anticonvulsant [6] activities. β-Lactam compounds
and indiscriminate use of antibiotics has lead to drug e.g., penicillin and cephalosporin are well known
resistant microbial pathogens necessitating the need antibiotics. Azetidinones are also reported to possess
for the search for new antimicrobial agents with antimicrobial [7], antiinflammatory [8,9], analgesic [9]
reduced resistance to pathogens and better activity. and CNS depressant [9] activities. Encouraged by
these observations, it was contemplated to
Sulfonamides possess diverse biological and incorporate the biologically active ‘sulfonamide’
pharmacological activities from antimicrobial[1] to moiety. To enable further evaluation of the potential
antitumor[2] activities. 4-Thiazolidinones are associated usefulness of thiazolidinones, azetidinones, and in
with antibacterial[3] antifungal[4], antitubercular[5] and continuation of our search for nitrogen heterocycles
of pharmacological importance[10-13], we report herein
*Address for correspondence the synthesis of some new thiazolidinones (3a-x)
E-mail: dr.shobha.desai@gmail.com and azetidinones (4a-x) with a view to achieve
478 Indian Journal of Pharmaceutical Sciences July - August 2011