Ethnobotanical Leaflets 14: 911-19, 2010.

Antimicrobial Screening of the Plant Extracts of

Cardiospermum halicacabum L. Against Selected Microbes

B. Thirupal Reddy¹, D. Ali Moulali¹ E. Anjaneyulu², M. Ramgopal²,

K. Hemanth Kumar², O. Lokanatha, M. Guruprasad² and M. Balaji^2*

¹Department of Botany, Government College, Anantapur, A.P. 515001, India

²Department of Biochemistry, S.V. University, Tirupati, A.P.-517502, India

*Corresponding Author: E-mail: balaji.meriga@gmail.com

Issued: August 01, 2010

Abstract

Cardiospermum halicacabum L. is a wild medicinal plant distributed in Eswaramala forests of Anantapur District of Andhra Pradesh, used against ailments like skin diseases by the local people. In the present paper, plant extracts obtained from the aerial parts of Cardiospermum halicacabum when tested for their antimicrobial activity against six bacterial and two fungal species, exhibited a broad spectrum of antimicrobial activity. Methanol extract was found to be active against all the tested bacterial and fungal species while that of chloroform extract was found to be active against all the tested microorganisms except Candida tropicalis. Benzene extract was active against all the tested microorganisms except Escherichia coli and Candida tropicalis. Water extract was effective against all but Pseudomonas aeruginosa and Staphylococcus aureus. The minimum inhibitory concentrations of different extracts against the tested microorganism was provided.

Key Words: Antimicrobial activity, Cardiospermum halicacabum L, Benzene, Chloroform, Methanol.

Introduction

Microbial infections are an important health problem throughout the world and plants are possible sources of antimicrobial agents (Burapadaja & Bunchoo 1995). The widespread use of herbal remedies and health care preparation, such as those described in the ancient text like the Bible and the Vedas has been traced to the occurrence of natural product with medicinal properties In fact; plants produce a diverse range of bioactive molecules, making them rich sources of different types of medicines (Nair et al., 2005). In recent times, attention has been reverted back to plants as sources of therapeutic agents due to their higher properties. These include among others reduced cost, relative lower incidence of adverse reactions compared to modern conventional pharmaceuticals (Karachi, 2006), and ready availability.

The increasing prevalence of multi drug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the specter of untreatable bacteria infections and adds urgency to the search for new infections fighting strategies (Sierdski et al. 1999). Cardiospermum halicacabum L (Synonym: Cardiospermum microcarpum) is a plant of a family sapindaceae. Cardiospermum is a climbing plant widespread distributed in tropical and subtropical Africa and Asia often found as a weed along roads and rivers. The whole plant has different properties like diaphoretic, diuretic, emetic, emmenagogue, laxative, refrigerant, rubefacient, stomachic and sudorific (Duke and Ayensu 1985). It is used in the treatment of rheumatism, nervous diseases, stiffness of the limbs and snakebite (Chopra. et al, 1986). A tea made from them is used in the treatment of itchy skin. In view of the paramount importance of Cardiospermum halicacabum L as a medicinal plant, the present work is aimed to isolate different solvent extracts from the plant and test their antimicrobial activity against a collection of human pathogens. Our observations demonstrated that all the solvent extracts were efficient against majority of the test organisms.

Materials and Methods

Collection and extraction of plant Material

The aerial parts of Cardiospermum halicacabum L were collected from Eswaramala forest of Andhra Pradesh, India. A specimen was deposited in the form of herbarium, Ethno botanical research Center, Govt. College, Anantapur, A.P, India. The aerial parts of Cardiospermum halicacabum L were shade dried, powdered and successively extracted with benzene, chloroform and methanol using the soxhlet apparatus. The different extracts obtained were concentrated with a rota evaporator and brought to complete dryness over water bath to yield the crude extracts.

Phytochemical analysis

Phytochemical studies were carried out qualitatively for the presence of terpenoids, tannins, flavonoids, saponins, cardiac glycosides and steroids following the described procedures (Faraz et al., 2003; Harborne, 1998; Edeogo et al., 2005).

Test Organisms Test organisms include both gram positive bacteria like Bacillus cereus, Micrococcus roseus and Staphylococcus aureus and gram negative bacteria like Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa and fungal species Aspergillus niger and candida tropicali. The cultures under study were obtained from Institute of Microbial Technology (IMTECH), Chandigarh. The strains were maintained and tested on nutrient agar for bacteria and potato dextrose (PDA) for fungi for the Antimicrobial tests.

Antimicrobial activity

The agar disc diffusion method was used to determine the antimicrobial activity of the different plant extracts (Cruikshank, 1968). The discs (6 mm diameter) impregnated with known concentrations of the standards and extracts were placed on the surface of the Petri plates containing 20 ml of nutrient agar media for bacterial strains and potato dextrose agar media for fungal strains respectively, seeded with 100µl of microbial cultures (5 x 10 ⁵ CFU/ml). Standard antibiotics viz, ampicilin (10 µg/ml) tetracycline (30 µg/ml), vancomycin (30 µg/ml) were used as positive controls. The plates were incubated for 24 hrs at 35 ±2⁰C for bacteria and for 48 hrs for fungi at 30 ⁰C. The inhibition zones formed around the discs were measured and expressed in millimeter. Three independent trials were conducted for each concentration and the average values calculated and given in Table 2. The microbial activity was confirmed by transferring a subculture from the clear zone of inhibition to a fresh broth media and observed for the growth of microbes.

Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration was determined, using a common broth micro dilution method in 96 well micro titer plates (Camporese et al., 2003; NCCLS, 1999.) Among the previously prepared different microbial suspensions (10⁵ CFU/mL) were added to each well. Plates were incubated for 18 hr at 37⁰ C and then were examined with Elisa reader (TECAN, Sunrise, China) at 620nm and the lowest concentration of each extract showing no growth was taken as its minimum inhibitory concentrations (MIC). All the samples were tested in triplicates to confirm the activity and the values were noted (Table 2).

Results and discussion

Preliminary Phytochemical screening of different extracts of Cardiospermum halicacabum L showed the presence of bioactive components like Terpenoids, Tannins, Sugars, Saponins, Flavonoids, Glycosides, Alkaloids and Steroids (Table 1). The presence or absence of a particular component plays a major role in deciding the medicinal property of the plant extract. Antimicrobial assays performed with extracts of Benzene, chloroform, methanolic and aqueous extracts of Cardiospermum halicacabum revealed that all of these extracts exhibited antimicrobial activity against most of the tested microorganisms as shown in Table 2. Similar reports on antimicrobial activity of plant products was reported with extracts of Oscimum, Withania, Tylophora, Curcumine etc, Chopra et al, 1956, Chatterjee and Pakrasshi, 1995, Nascimento et al., 2000).

Table 1. Phytochemical analysis of the extracts of Cardiospermum halicacabum L.

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Component BE CE ME WE

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Terpenoids − + + +

Tannins WP − + −

Flavonoids + + + WP

Sugars + WP + +

Saponin + + + +

Cardiac glycoside + + + +

Alkaloids _ + + +

Steroids + + + WP

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BE = Benzene extract, CE= Chloroform extract, ME = Methanol extract, WE = Water extract,

WP = weakly positive.

Table 2. Antimicrobial activity of plant extracts.

S.NO.	Name of the Test organisms	Benzene extract		Chloroform extract		Methanol extract		Water extract		Standard Antibiotics
		ZI	MIC	ZI	MI C	ZI	MIC	ZI	MIC
1.	Bacillus cereus	12	100	12	175	12	75	30	150	22^a
2.	Micrococcus roseus	8	150	14	150	8	150	12	150	24^a
3.	Staphylococcus auereus	11	150	10	125	14	100	-	-	24^a
4.	Escherichia coli	-	-	14	100	8	175	8	75	22^b
5.	Klbsiella pneumoniae	11	100	12	100	12	150	8	100	22^b
6.	Pseudomonas aerugionsa	11	150	8	75	8	125	-	-	28^b
7.	Aspergillus niger	12	100	8	150	14	100	11	150	20^c
8.	Candida tropicalis	-	-	-	-	12	100	10	100	22^c

ZI: Zone of inhibition in mm; MIC: Minimum Inhibitory Concentration (µg/ml);

A: Ampicillin 10 µg/ml; b: Tetracycline 30 µg/ml; C: Vancomycin 30 µg/ml; - Resistant

The data presented in Table 2 shows various degrees of zones of inhibition of tested microorganisms and minimum inhibitory concentrations (MIC) of plant extracts against test microorganisms. Among all the extracts, the over all efficiency of methanolic extracts was found to be more effective against all the tested mircroorganisms. Chloroform extract was found to be active against all the tested microorganisms except candida tropicalis. Benzene extract was active against all the tested microorganisms except Escherichia coli and candida tropicalis. Aqueous extract was effective against all but Pseudomonas aeruginosa and staphylococcus aureus. Similar reports were observed with plant extracts of Morinda citrifolia L, Cassia auriculata etc, (Usha et al., 2010 Maneemegalai et al., 2010).

Among the tested gram positive bacteria, Bacillus cereus showed the maximum zone of inhibition with water extract, followed by Micrococcus roseus and Staphylococcus auereus with chloroform and methanolic extracts respectively. Among the tested gram negative bacteria E.coli and Klbsiella pneumoniae showed maximum zones of inhibition with chloroform extract followed by Pseudomonas aerugionsa with benzene extract. Our observations suggest that among the tested bacteria, gram positive bacterial strains were relatively more sensitive than the gram negative bacterial strains.

The minimum inhibitory concentrations of different extracts varied against the tested microorganisms. The MIC of Benzene extract was 100µg against Bacillus cereus, Klbsiella pneumoniae, Aspergillus niger and 150µg against Micrococcus roseus, Staphylococcus auereus, Pseudomonas aerugionsa. The MIC of chloroform extract was 175µg for Bacillus cereus, 150µg against Aspergillus niger, Micrococcus roseus, 125µg against Staphylococcus auereus, 100µg against Klbsiella pneumoniae, Escherichia coli and 75µg against Pseudomonas aerugionsa. The MIC of methanolic extract was 175µg against Escherichia coli, 150µg against Micrococcus roseus, Klbsiella pneumoniae, 100µg against Staphylococcus auereus, Aspergillus niger, Candida tropicalis, 125µg against Pseudomonas aerugionsa and 75µg against Bacillus cereus. The MIC of water extract was 150µg against Bacillus cereus, Micrococcus roseus, Aspergillus niger, 100 µg against Klbsiella pneumoniae, Candida tropicalis and 75µg against Escherichia coli.

Conclusion

Among the extracts of Cardiospermum halicacabum, except the aqueous extract, the extracts of Benzene, chloroform and methanol were active against the three tested gram positive bacteria (Bacillus cereus, Micrococcus roseus and Staphelococcus auereus). While the extracts of Methanol and chloroform were effective against all the tested gram negative bacteria, the extracts of Benzene and water were not active against the gram E.coli and Pseudomonas respectively. Among the tested fungal strains, all the plant extracts showed inhibitory effects against Aspergillus niger while Candida tropicalis was inhibited by methanol and water extracts only. Our results authenticate the usage of Cardiospermum halicacabum by local people for healing of wounds, skin diseases and against some bacterial and fungal infections. Our results provide the basis for further isolation and evaluation of major active principles of the plant material and test their efficiency against various infections.

Acknowledgements

Authors thank Professor. K.C. Naidu, Department of Botany, Andhra University, Visakapatnam for his cooperation, valuable suggestions and providing laboratory.

References

Burapadaja, S., and Bunchoo A., 1995. Antimicrobial activity of tannins from Terminalia citrina. Planta Medical 61 (4): 365-366.

Chatterjee A, Pakrashi Sc. The Treatise on Indian Medicinal Plants. 1995;4:208-212.

Chopra, R. N., Nayer, S. L. and Chopra, I. C. (1956). Glossary of Indian Medicinal Plants, CSIR, New Delhi.

Chopra. R. N., Nayar. S. L. and Chopra1986. I. C. Glossary of Indian Medicinal Plants (Including the Supplement). Council of Scientific and Industrial Research, New Delhi.

Duke. J. A. and Ayensu. E.S1985. Medicinal Plants of China Reference Publications, Inc. ISBN 0-917256-20-4.

Edeoga, H.O., Okwu, D.E. and Mbaebie, B.O. 2005. Phytochemical constituents of some Nigerian medicinal plants, African J of Biotechnology 4: 685-688

Faraz, M., Mohammad, K., Naysaneh, G. and Hamid, R.V. 2003. Phytochemical screening of some species of Iranian plants, Iranian J of pharmaceutical Research : 77-82.

Gislene. g. f, nascimento juliana locatelli, paulo. c, freitas1giuliana. , silva 2000 antibacterial activity of plant extracts and phytochemicals on antibioticresistant bacteria, Brazilian journal of microbiology 31:247-256

Harborne, J.B. 1998. Phytochemical methods. In A guide to modern techniques of plant analysis 3^rd ed., pp. 40-137. London: Chapman and Hall;

Karachi 2006. Importance of Medicinal Plants highlighted retrieved June 18th, from http:/www.dawn.Com/2006/01/07/Local14.htm.

Maneemegalai.S, Naveen. T 2010 Evaluation of Antibacterial Activity of Flower Extracts of Cassia auriculata, Ethnobotanical Leaflets 14: 8-20

Murray P.R. Baron EJ Pfaller MA, Tenover FC, Yolken HR 1995. Manual of clinical; microbiology, 6^th Edition, ASM Press, Washington DC, 15-18.

Nair R, Kalariya T, Chanda S 2005. Antibacterial Activity of Some Selected Indian Medicinal Flora, Turk. J. Biol. 29: 41-47.

Olurinola P F 1996. A laboratory manual of Pharmaceutical microbiology. Printed by National Institute for Pharmaceutical Research and Development, Idu, Abuja, Nigeria 69-105.

Rotimi VO, Laughon BE, Bartlet JG, Mesodomi HA 1988. Activities of Nigeria chewing stick against Bacteroides gingivalis and Bacteroides melaninogenicus. Nig. J. Microbiol. 9: 13-16.

Sieradski.K. Roberts R.B., Haber S.W., Tomasz. A 1999: The development of vancomycin resistance in a patient with methicillin – resistant Staphylococcus aureus infection, N.Eng.J.Med. 340:517-523.

Usha. R, Sangeetha Sashidharan, Palaniswamy.M 2010. Antimicrobial Activity of a Rarely Known Species, Morinda citrifolia L. Ethnobotanical Leaflets 14: 306-11.