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Advances in Biological Research 1 (1-2): 67-71, 2007 ISSN 1992-0067 IDOSI Publications, 2007

Antimicrobial Properties and Phytochemical Constituents of an Antidiabetic Plant Gymnema montanum


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K.M. Ramkumar, 1P. Rajaguru and 2R. Ananthan

Department of Biotechnology, School of Engineering and Technology, Bharathidasan University, Tiruchirapalli-620 024, Tamil Nadu, India 2 Food Chemistry Division, National Institute of Nutrition, Hyderabad-500 007 Andhra Pradesh, India
Abstract: In the present study, five different organic solvents and aqueous extracts of Gymnema montanum leaves were screened for their phytochemical composition, antimicrobial and free radical scavenging activities. Among the different extracts tested, ethanol and hexane extracts showed significant antimicrobial and radical scavenging activities. The most susceptible microorganisms were found to be Salmonella typhi, Pseudomonas aeruginosa and Candida albicans. Phytochemical analysis of the leaf extracts revealed that the antimicrobial and the radical scavenging activities are mainly due to the presence of the phenolic compounds especially alkaloids. The results obtained suggest that G. montanum could be exploited in the infectious management of various diseases. Key words: Gymnema montanum % antioxidant % antimicrobial % free radical % phytochemical analysis INTRODUCTION Accumulated evidences indicate that free radicals involve in the pathophysiology of various diseases such as diabetes, cancer and cardiovascular diseases [1]. The antioxidant compounds can neutralize free radicals, thus playing an important role in the prevention of these diseases [2]. In recent years, many studies evidenced that plants containing high content of antioxidant phytochemicals can provide protection against various diseases [3]. The free radical scavenging activity of these phytochemicals is predominantly determined by their structures [4]. In particular, phenolic phytochemicals have antioxidative, antidiabetic, anticarcinogenic, antimicrobial, antiallergic, antimutagenic and anti-inflammatory activities [5, 6]. The antioxidant and antimicrobial properties of various plants have been reported by several studies [7-10]. In recent years the popularity of complementary medicine has increased. Dietary measures and traditional plant therapies as prescribed by ayurvedic and other indigenous systems of medicine are used commonly in India [11]. The World Health Organization has also recommended the evaluation of the plants effective in conditions where safe modern drugs are lacking [12]. Recently an intensive search for novel types of antioxidants has been carried out from numerous plant materials. Gymnema montanum Hook (Asclepiadaceae) is an endemic plant species found mainly in Western Ghats, India [13] that has been used in our study. In our earlier studies, G. montanum was found to have modulatory effect on rate-limiting enzymes of glycolysis and gluconeogenesis in liver of diabetic rats [14], antihyperlipidemic effect [15] and anti-peroxidative effect in alloxan-induced diabetic rats [16-18]. In addition, it has been demonstrated for preventing the cholinergic neural and retinal complications of hyperglycemia in diabetes [19]. In continuation of our earlier studies on medicinal properties of G. montanum, this study was undertaken to screen the phytochemical composition, antimicrobial and radical scavenging activities of different solvent extracts of G. montanum leaves. MATERIALS AND METHODS Plant material: Fresh leaves of G. montanum Hook were collected from the Shola forests of Western Ghats, Gudalur, The Nilgiri Biosphere Reserve at an altitude of 900 - 1500 MSL on November, 2004. The plant was identified with the Herbarium of Botanical Survey of India, Southern Circle, Coimbatore, India (Accession No. 32561-65) and was deposited in the Department of Biotechnology, Bharathidasan University.

Corresponding Author: K.M. Ramkumar, Department of Biotechnology, School of Engineering and Technology, Bharathidasan University, Tiruchirapalli 620 024, Tamil Nadu, India

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Advan. Biol. Res., 1 (1-2): 67-71, 2007

The leaves were dried and powdered. They were extracted using different solvents such as by ethanol, hexane, chloroform, ethyl acetate and methanol by soxhlet apparatus. The extracts were condensed to dryness by rotary evaporator. Preliminary phytochemical screening: The different solvent extracts of G. montanum leaves (GLEt) were analyzed for the phytochemical composition by qualitative methods. Dragendorfs reagent and Mayers reagent were used to indicate the presence of alkaloids by the formation of a precipitate [20] and glycosides using the KellerKilliani test [21]. Extracts were tested for saponins using the haemolysis test [22] with a Columbia Blood Agar Base [23] and for tannins using the Gelatinsalt Block test [24]. Antioxidant assay with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical: DPPH is a free radical which when dissolved in ethanol has a blue-violet color. When it reacts with the reducing agent, the solution loses color which depends upon the number of electrons taken up. Hence, the loss of color indicates radical scavenging activity of test material. DPPH was measured by the method of Cervato et al. [25]. Three ml of 60 M DPPH in ethanol was added to 1 ml GLEt and then incubated at room temperature for 15 min. Absorbance was read at 517 nm using a Genesys-5-spectrophotometer (Spectronic Instruments, Rochester, NY). The antioxidant activity was calculated as inhibition (%) of DPPH radical formation:
Inhibition (%) =
Extract A Control A 517 517 Control A 517

Disc application and incubation: Discs of 6 mm diameter were prepared from Whatman No.1 filter paper. They were sterilized by autoclaving and subsequently dried at 80C for an hour. The sterile discs were impregnated with 10 mg/ml concentration of GLEt and dried for 3-5 min. After drying, the discs were placed on the MHA agar surface with flamed forceps and gently pressed down to ensure contact with the agar surface. Streptomycin and ampicilin antibiotic discs were used as positive controls and respective solvents as vehicle control. The discs were spaced far enough to avoid both reflections waves from the edges of the petri dishes and overlapping rings of inhibition. Finally, the petridishes were incubated for 18 hrs at 37C in an inverted position. After 18 hrs, the diameter (mm) of the inhibition zone around each disc was measured. Antibacterial activities were indicated by clear zone of growth inhibition. Antifungal studies: Antifungal activity of all the GLEt was screened for the in vitro growth inhibitory activity against Candida albicans, Fusarium oxysporum, Alternaria alternata and Aspergillus niger by using the disc diffusion method. The fungi were cultured in Potato Dextrose Agar medium (PDA). The sterilized medium taken in the sterilized petri plates were inoculated with a spore suspension of C. albicans, F. oxysporum, A. alternata and A. niger (106 spores cm 3 of medium). The GLEt were dissolved in respective solvents to a final concentration of 10 mg/ml and soaked in filter paper discs (Whatman no. 4.5 mm dia.). After drying, the discs were placed on the surface of the PDA medium with flamed forceps and gently pressed down to ensure contact with the agar surface. After 4 days, the inhibition zone appeared around the discs in each plate was measured. To rule out the activity of the solvent that was used in the preparation of the test solutions, a solvent only treated plate was maintained. An untreated control plate was also maintained in order to calculate the percentage inhibition. Carbendazim was used as the standard (60 g/ disc). RESULTS Phytochemical analysis: Phytochemical analysis of various solvent extracts revealed the presence of alkaloids, glycosides, saponins and Tanins (Table 1). In particular, the ethanol and methanol extracts showed highly positive for the alkaloids but it was negative for chloroform fraction. Saponin was detected only in the aqueous extract that was negative for the presence of tannins. 68

Anti microbial studies: The different solvent extracts of G. montanum were tested for antimicrobial activity by agar disc diffusion method [26, 27]. Preparation of culture medium and inoculation: Mueller Hinton Agar (MHA) medium (38 g) was mixed with 1000 ml of sterile distilled water and sterilized by autoclaving at 120C for 20 minutes. Under aseptic conditions, in the laminar flow hood 15 ml of MHA medium was dispensed into pre-sterilized petridishes to yield a uniform depth of 4 mm. After solidification of the medium, the bacterial cultures were inoculated by spread plating technique. In this study, Salmonella typhi, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa and Proteus vulgaris were used as the test strains.

Advan. Biol. Res., 1 (1-2): 67-71, 2007


Table 1: Phytochemical screening of extracts of the G. montanum leaves Sample Ethanol fraction Hexane fraction Ethyl acetate fraction Chloroform fraction Methanol fraction Aqueous fraction Alkaloids +++ ++ + +++ + Glycosides + + + + + Saponins + + +++ Tannins + + Hexane fraction Sample Ethanol fraction Table 3: Free radical scavenging activity of different extracts of Gymnema montanum leaves Concentration (g/ml) 10 50 100 10 50 100 Ethyl acetate fraction Table 2: Antibacterial and Antifungal activity of different extracts of G. montanum leaves Zone of Inhibition (mm) Microorganisma E S. typhi S. aureus E. Coli P. aeruginosa B. subtilis P. vulgaris F. oxysporium C. albicans A. niger A. macrospora 28 18 20 18 10 9 18 16 16 20 H 22 16 16 12 7 6 12 10 12 16 EA C 12 9 8 10 10 4 13 11 6 5 6 M 23 14 16 14 8 6 10 10 12 14 W 16 8 7 9 5 8 6 b

Scavenging activity (%)a 42.84 68.46 91.16 20.22 48.82 71.18 3.04 5.68 7.64 12.36 24.16 46.24 27.12 44.36 66.74 11.25 20.20 33.56

Phytochemical test: - negative and + positive, +++- quantitative presence

10 50 100

Chloroform fraction

10 50 100

----------------------------------------------------------------------STR 20 15 18 15 9 8 NT NT NT NT AMP CAR 22 14 15 16 8 6 NT NT NT NT NT NT NT NT NT NT 15 16 15 16 Standard BHT Aqueous fraction Methanol fraction

10 50 100 10 50 100

10 50 100

38.16 61.44 91.74

E- ethanol, H- hexane, EA-ethylacetate, C-chloroform, M-methanol, W-aqueous extract STR- Streptomycin (500 g/ml), AMPAmpicillin (500 g/ml), CAR- Carboxamide (500 g/ml) - No significant result a 100 l was applied to each well. Concentration of the extracts was 10 mg/ml b values are the mean of three replicates NT- not tested

a values are the mean of three replicates

Antibacterial activity: The antibacterial activity of different solvent fractions of GLEt was evaluated (Table 2). The GLEt was screened for activity against S. typhi, S. aureus, E. coli, B. subtilis, P. aeruginosa and P. vulgaris by agar disc diffusion method. Results were compared with the drugs such as streptomycin and ampicillin. GLEt at the concentration of 10 mg/ml showed appreciable zone of activity against all the bacterial pathogens tested. Among the various solvent extract of G. montanum, ethanol and hexane fractions showed higher zone of inhibition against bacteria which are comparable to that of standard drugs. No appreciable zone of inhibition was observed with other solvent fractions.

Antifungal activity: The antifungal activity of GLEt was evaluated using agar disc well diffusion method (Table 2). The GLEt was tested against A. macrospora, C. albicans, A. niger and F. oxysporium at 10 mg/ml concentration. GLEt exhibited significant activity against all the tested fungi compared with the standard drug carbendazim. Among the various solvent extract of G. montanum, ethanol and hexane fractions showed higher zone of inhibition against fungi whereas chloroform fraction showed inhibition only against C. albicans. Antioxidant activity: The antioxidant activity reflected by the DPPH radical scavenging assay was clearly observed in various solvent extracts of plant (Table 3). Among the various solvent extracts of G. montanum, ethanol and hexane fractions exhibited higher free radical scavenging activity. Lower EC50 value indicates greater antioxidant activity. Only 40 g/ml of ethanol extract was required to reduce the DPPH radicals by 50% whereas the hexane fraction needs 50 g/ml. This was significantly similar to the concentration needed for commercial antioxidant BHT. On the other hand, more than 100 g/ml was necessary for other extracts to achieve the same results.

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DISCUSSION Plant substances continue to serve as viable source of drugs for the world population and several plant-based drugs are in extensive clinical use [28]. For the past few decades, number of plants have been widely used for the treatment of various diseases due to their antioxidant properties. Antioxidants can be defined as compounds that can delay or prevent the oxidation of lipids or other molecules by inhibiting the initiation or propagation of an oxidizing chain reaction and by many other mechanisms and thus prevent disease [29, 30]. Results from our phytochemical analysis revealed that ethanol and hexane fractions showed the presence of alkaloids which could be well correlated with the antimicrobial activities. Ethyl acetate and chloroform fractions did not show remarkable antimicrobial activity. In the present study, initial antimicrobial screening of the different extracts from the G. montanum showed varying degrees of antimicrobial activity against human pathogenic bacteria such as S. typhi, S. aureus, E. coli, B. subtilis, P. aeruginosa and P. vulgaris and plant pathogenic fungi such as A. macrospora, C. albicans, A. niger and F. oxysporium. The variation in the effectiveness of the extract against different microorganisms depends upon the chemical composition of the extracts and membrane permeability of the microbes for the chemicals and their metabolism. It has been suggested that the antimicrobial activity is mainly due to the presence of essential oils, flavanoids and triterpenoids and other natural polyphenoilc compounds or free hydroxyl groups [31]. The antioxidant activity reflected by the DPPH radical scavenging assay was clearly observed in GLEt in dose dependent manner. This suggests that the physico-chemical nature of the individual phenolics in the extract may be important in contributing the antioxidant activity. The antioxidant property of the plant is well correlated with the concentration of the extract, which showed the presence of active principals in the extract. Based on the results described, we may conclude that the ethanol and hexane extracts of G. montanum posses significant free radical scavenging and antimicrobial activities. We will conduct further research to isolate the antioxidant constituents of the plant with high activity, which will pave the way for the production of bioactive prophylactic agents. 1.

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