World J Microbiol Biotechnol (2008) 24:27372740 DOI 10.

1007/s11274-008-9758-7

SHORT COMMUNICATION

Antimicrobial activity of saponin fractions of the leaves of Gymnema sylvestre and Eclipta prostrata
Venkatesan Gopiesh khanna Krishnan Kannabiran

Received: 9 August 2007 / Accepted: 19 April 2008 / Published online: 6 May 2008 Springer Science+Business Media B.V. 2008

Abstract The antimicrobial activity of saponin fractions from the leaves of Gymnema sylvestre and Eclipta prostrata was evaluated against pathogenic bacteria and fungi in an in vitro condition. A series of concentrations of crude and pure saponin fractions were tested for antimicrobial activity by zone of inhibition method. The pure saponin fractions were found to be more effective against tested bacterial pathogens when compared to crude saponin fractions. The minimum inhibitory concentration (MIC) exhibited by the pure saponin fraction of G. sylvestre was found to be in the range of 6001,200 mg/l against bacterial strains and 1,400 mg/l for fungal isolates. In the case of E. prostrata, the range was 1,0001,200 mg/l for bacteria and 1,400 mg/l for fungal isolates. The susceptibility of bacterial pathogens for saponin fractions was in the order of P. aeruginosa, E. coli, S. typhi, K. pneumoniae, P. mirablis, S. aureus and for fungal pathogens A. fumigatus followed by A. niger and A. avus. Whereas, A. niger was more susceptible to inhibition by E. prostrata saponin fractions, followed by A. avus and A. fumigatus. The antimicrobial potential of saponin fractions was compared with antibiotics, Chloramphenicol and Amphotericin-B with respect to bacteria and fungi. The present study suggests that the saponin fractions G. sylvestre and E. prostrata possess signicant antibacterial and antifungal activity. Our results further suggest that saponins of G. sylvestre and E. prostrata can be used as a potential fungicide against pathogenic fungi.

Keywords Antimicrobial activity Crude saponin Pure saponin Antifungal susceptibility testing Minimum inhibitory concentration

Introduction Plants have been considered as a valuable source for natural products and explored continuously for therapeuticals for human well being. The use of plant products for pharmaceutical purposes has been gradually increasing. According to World Health Organization, medicinal plants would be the best source for obtaining a variety of drugs (Santos et al. 1995). About 80% of individuals from developed countries use traditional medicines, derived from medicinal plants (Ellof 1998). The use of plant extracts and phytochemicals, with known antimicrobial properties, can be of great signicance in treatment of various microbial infections. In the last decade, numerous studies have been conducted in different countries to prove such efciency in number of medicinal plants. Most of the studies are restricted with crude extracts (Reddy et al. 2006; ErdoUrul 2002; Ate et al. 2003). In depth studies with puried plant compounds are very scanty. The objective of this study was to evaluate the antimicrobial potential of pure saponin fractions from G. sylvestre and E. prostrata on selected bacterial and fungal pathogens. The ethanolic extract of G. sylvestre leaves have been reported to possess antimicrobial activity against Bacillus pumilis, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus and inactivity against Proteus vulgaris and Escherichia coli (Satdive et al. 2003). The methanolic extract from the leaves, barks, and roots of E. prostrata have been shown to possess signicant antibacterial and antifungal activity (Wiart et al. 2004).

V. Gopiesh khanna K. Kannabiran (&) School of Biotechnology, Chemical and Biomedical Engineering, VIT University, Katpadi-Ranipet Road, Vellore, Tamil Nadu 632 014, India e-mail: kkb@vit.ac.in; kkb_67@yahoo.com

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Materials and methods Plant materials The leaves of G. sylvestre and E. prostrata were collected from the medicinal garden of VIT University and voucher specimen was deposited in the VIT University, Vellore, Tamil Nadu, India. The leaves were washed with distilled water, shade dried, powdered and stored in an air-tight container separately for further use. Extraction of crude saponin The powdered sample was defatted by petroleum ether 3 9 1 h at 40C. After ltering the petroleum ether, the sample was extracted with methanol 3 9 1 h with mild heating. The combined methanol extract was concentrated and methanol extract of sample was obtained. In order to get the crude saponins extract the sample was dissolved in methanol and acetone was added (1:5 v/v) to precipitate the saponins (Yan et al. 1996). The precipitate was dried under vacuum, turning to a whitish amorphous powder named as crude saponin extract (CSE). Isolation and extraction of pure saponin To get the pure saponin fraction (PSF), certain amount of CSE was fractionated by applying to Merck silica gel-60 (230400 mesh) column chromatography and eluted successfully with chloroform- methanol-water (70:30:10) as described by Favel et al. (2005). Five fractions were collected and the solvents were evaporated under reduced temperature; fraction 1 was chosen based on the detection of the total saponin concentration. The total saponins concentration of each fraction was measured by spectrophotometer method with some modication (Baccou et al. 1977; Uematsu et al. 2000). Bacterial strains Bacteria such as Staphylococcus aureus (ATCC 700699), Escherichia coli (ATCC 11105), Pseudomonas aeruginosa (ATCC 27853), Klebsiella pneumoniae (ATCC 10273), Salmonella typhi (ATCC 700931), and Proteus mirablis (ATCC 19181) were used as test organisms. Exactly 0.2 ml of overnight cultures of each organism was inoculated into 5 ml of sterile nutrient broth and incubated for 35 h to standardize the culture to 106 cfu/ml. Mueller Hinton Agar (Hi-Media Laboratories Pvt. Ltd., Mumbai, India) was used for culturing of bacteria. Antibacterial activity Agar diffusion assay was carried out to evaluate the antimicrobial activity of saponin fractions. The plates were

incubated at 37C for 24 h during which activity was evidenced by the presence of a zone of inhibition surrounding the well. Each test was repeated three times and the antibacterial activity was expressed as the mean of diameter of the inhibition zones (mm) produced by saponin fractions when compared to controls. Fungal strains The clinical isolates such as Aspergillus fumigatus, Aspergillus avus and Aspergillus niger were obtained from Department of Clinical Microbiology, Christian Medical College, Vellore, Tamil Nadu, India. Antifungal susceptibility testing Antifungal activity of crude and pure saponin was tested by the standard method CLSI M38-A (formerly NCCLS). The fungal culture was maintained in 0.2% dextrose medium and the optical density 0.10 at 530 nm was adjusted using spectrophotometer. Each fungal inoculums were applied on plate and evenly spread on Sabourauds Dextrose agar (HiMedia) using a sterile swab. Agar diffusion assay was followed to evaluate the antimicrobial activity of crude and pure saponin along with standard antifungal antibiotic, Amphotericin B. The Petri plates were incubated at 30C for 2 days. At the end of the 48 h, inhibition zones formed in the medium were measured in millimeters (mm). All experiments were done in three replicates. Minimum inhibitory concentration (MIC) MIC was determined by the broth 2-fold macro dilution method. The saponin fractions were serially diluted in Mueller Hinton broth for bacteria and Sabourauds dextrose broth for fungi. Varying concentrations of the extracts 2,40075 mg/l for bacteria and 2,80087.50 mg/l for fungi were prepared from the stock solution (Andrews 2001). A 0.1 ml of culture was added to broth each containing crude and pure saponin fractions separately. The tubes were incubated aerobically at 37C for 24 h for bacteria and 30C for 48 h for fungi. Positive controls were prepared separately for both bacteria and fungi with respective organisms in the same culture media without crude and pure saponins. The tube with least concentration of saponin without growth after incubation was taken as the MIC value for the respective organism.

Results and discussion The antimicrobial activity and MIC exhibited by the saponin fractions of G. sylvestre against tested bacterial

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and fungal pathogens was given in Table 1. Both crude and pure saponin fractions of G. sylvestre exhibited signicant antimicrobial activity. The extent of inhibition was greater in the case of pure saponin fraction than the crude saponin fraction. The zones of inhibition observed for bacteria with the pure saponin fraction (10,000 mg/l) were equivalent with that of Chloramphenicol (25 mg/disc). Gram-negative bacteria P. aeruginosa was more susceptible (20 mm) to saponin followed by S. aureus (19 mm), E. coli (16 mm), S. typhi (16 mm), P. mirablis (14 mm) and K. pneumoniae (10 mm). A signicant observation was that both crude and pure saponin fractions from the leaves of G. sylvestre showed signicant (2- to 3-fold) antifungal activity than the fungicide Amphotericin-B. Aspergillus fumigatus was more susceptible to inhibition (20 mm) followed by A. avus (16 mm) and A. niger (16 mm). The MIC values of pure saponin fraction of G. sylvestre were ranging from 600 to 1,200 mg/l for tested bacterial pathogens. Gram-negative bacteria P. mirablis was more susceptible to inhibition by the least concentration (600 mg/l) of saponin. Crude ethanolic extract of G. sylvestre leaves has been shown to possess signicant antibacterial activity against B. pumilis, B. subtilis, P. aeruginosa and S. aureus (Satdive et al. 2003). The zones of inhibition and MIC of microbial growth by the saponin fractions from E. prostrata were given in Table 2. Similar to the results of G. sylvestre Gram-negative bacteria was more susceptible to inhibition by the saponin fractions. Antifungal activity of pure saponin fraction was more pronounced (nearly 3-fold) when compared to Amphotericin-B. Aspergillus niger was more susceptible to inhibition (22 mm) followed by A. avus

and A. fumigatus. Gram-negative bacteria P. aeruginosa was more susceptible (20 mm) to inhibition by the pure saponin fraction than the other tested bacterial pathogens. Gram-positive bacteria S. aureus were less susceptible to inhibition by the saponin fraction. Comparison of antifungal activity of saponin fractions with that of standard antibiotics revealed that the antifungal activity of saponin was more signicant than the commercial antibiotics. Aqueous, ethanol, hexane and ethyl acetate extracts of E. prostrata leaves has been shown to possess signicant antimicrobial activity against E. coli, K. pneumoniae, S. dysenteriae, S. typhi, P. aeruginosa, B. subtilis, S. aureus. Among the organic solvent extracts ethanolic extract was found to be more signicant against all tested bacterial strains (Karthikumar et al. 2007). Pseudomonas aeruginosa is the most prevalent pathogen capable of causing life-threatening illnesses and has been implicated in several infections of humans and animals. Due to multi-resistancy of P. aeruginosa and lack of active antibiotics against this bacterium increasing incidence of nosocomial infections and high mortality have been reported (Giamarellos-Bourboulis et al. 1999). Several reports are available in support of antimicrobial activity of plant extracts was due to the presence of saponins (Soetan et al. 2006). Several reports have shown that the plant saponins possess signicant antibiotic, antifungal, antiviral, hepatoprotective, anti-inammatory and antiulcer activities (Oakenfull and Fenwick 1981). Gram-negative bacteria and fungi was reported to be more resistant to antibiotics treatment (Hugo and Russell 1983). Hence, it is important to identify an effective plant drug having broad spectrum of inhibition against Gram-

Table 1 Antimicrobial activity of crude and pure saponin fractions from the leaves of G. sylvestre Microorganisms Standarda zone of inhibition (mm) (25 mg/disc) c 20 20 12 12 12 Standard (25 mg/disc) Aspergillus fumigatus Aspergillus avus Aspergillus niger
a b c b

MIC (mg/l)

Crude saponin zone of inhibition (mm) (10,000 mg/l) 12 10 14 14 14 10

MIC (mg/l)

Pure saponin zone of inhibition (mm) (10,000 mg/l) 20 16 16 10 14 19 20,000 (mg/l) 20 16 16

MIC (mg/l)

Pseudomonas aeruginosa Escherichia coli Salmonella typhi Klebsiella pneumoniae Proteus mirablis Staphylococcus aureus

c 4 0.2 0.2 0.2 0.2

1,200 1,000 1,200 1,200 1,200 1,000 20,000 (mg/l) 20 8 12 MIC (mg/l) 2,000 2,000 1,600

800 1,000 1,000 1,200 600 1,200 MIC (mg/l) 1,400 1,400 1,400

MIC (mg/l) 0.2 0.8 0.4

6 6 8

Chloramphenicol disc (25 mg) Amphotericin-B disc (25 mg) Indicates no activity

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Table 2 Antimicrobial activity of crude and pure saponin fractions from the leaves of E. prostrata Microorganisms Standarda zone of inhibition (mm) (25 mg/disc) c 22 c 14 16 12 Standard (25 mg/disc) Aspergillus fumigatus Aspergillus avus Aspergillus niger
a b c b

MIC (mg/l)

Crude saponin zone of inhibition (mm) (10,000 mg/l) 8 16 c 8 16 10

MIC (mg/l)

Pure saponin zone of inhibition (mm) (10,000 mg/l) 20 16 14 14 08 10 20,000 (mg/l) 16 18 22

MIC (mg/l)

Pseudomonas aeruginosa Escherichia coli Salmonella typhi Klebsiella pneumoniae Proteus mirablis Staphylococcus aureus

c 4 c 0.25 0.25 0.25

1,200 1,200 c 1,400 1,200 1,000 20,000 (mg/l) 16 16 14 MIC (mg/l) 2,000 2,000 2,000

1,200 1,200 1,200 1,000 1,000 1,000 MIC (mg/l) 1,400 1,400 1,400

MIC (mg/l) c 0.4

c

c 6

Chloramphenicol disc (25 mg) Amphotericin-B disc (25 mg) Indicates no activity acid and arachidonic acid with ceftazoline on multiresistant Pseudomonas aeruginosa. Lipids 34:151152. doi:10.1007/ BF02562270 Hugo WB, Russell AD (1983) Pharmaceutical microbiology 3rd edn. Blackwell Scientic Publications Karthikumar S, Vigneswari K, Jegatheesan K (2007) Screening of antibacterial and antioxidant activities of leaves of Eclipta prostrata (L). Sci Res Essay 2:101104 Oakenfull D, Fenwick DE (1981) Saponin content of soybeans and some commercial soybean products. J Sci Food Agric 32: 273278. doi:10.1002/jsfa.2740320311 Reddy PS, Jamil K, Madhusudhan P (2006) Antibacterial activity of isolates from Piper longum and Taxus baccata. Pharm Biol 39:236238 Santos PRV, Oliveira ACX, Tomassini TCB (1995) Controle microbiogico de produtos toterapicos. Rev Farm Bioqum 31:3538 Satdive RK, Abhilash P, Fulzele DP (2003) Antimicrobial activity of Gymnema sylvestre leaf extract. Fitoterapia 74:699701 doi: 10.1016/S0367-326X(03)00154-0 Soetan KO, Oyekunle MA, Aiyelaagbe OO, Fafunso MA (2006) Evaluation of the antimicrobial activity of saponins extract of Sorghum Bicolor L. Moench Afr J Biotechnol 5:24052410 Umeatsu Y, Hirata K, Saito K, Kudo I (2000) Spectrophotometric determination of saponins in Yucca extract used as food additive. J AOAC Int 836:14511454 Wayne PA (2002) National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of lamentous fungi. Approved standard M38-A Wiart C, Mogana S, Khalifah S, Mahan M, Ismail S, Buckle M, Narayana AK, Sulaiman M. (2004) Antimicrobial screening of plants used for traditional medicine in the state of Perak, Peninsular Malaysia. Fitoterapia 75:6873. doi:10.1016/j.tote. 2003.07.013 Yan W, Ohtani K, Kasai R, Yamasaki K (1996) Steroidal saponin from fruits of Tribulus terrestris. Phytochemistry 42:14171422. doi:10.1016/0031-9422(96)00131-8

negative bacteria and fungi to overcome the ailments caused by the microorganisms. Our ndings have shown that the saponin fractions from the study plants were active against Gram-negative bacteria especially P. aeruginosa, and as well as tested Aspergillus species. Saponin may be a future drug candidate to prove its efcacy as a preventive and therapeutic agent against Gram-negative pathogenic bacteria and fungi. However, further studies are needed to identify and characterize chemical nature of saponins.
Acknowlegements The authors wish to thank the management of VIT University for providing necessary facilities for completion of this study.

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