Antibacterial and antifungal activities of Elephantopus scaber Linn

The present study was carried out with an objective to investigate the antimicrobial potentials of petroleum ether, diethyl ether, methanol and water extracts of the root and aerial part of Elephantopus scaber Linn. by determining the zone of inhibition and minimum inhibitory concentration (MIC) against various bacterial and fungal stains. Disc diffusion technique was used to determine in-vitro antibacterial and antifungal activities. In addition, minimum inhibitory concentration (MIC) was determined by broth dilution method. The different solvent extracts showed concentration dependent antibacterial activities against four gram-positive (Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus and Bacillus cereus) and four gram-negative (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Salmonella typhi) bacteria. The MIC values against the tested Gram-positive bacteria ranged from 50 to 500 μg/ml and against Gram-negative bacteria from 100 to 500 μg/ml. The antifungal activities were found strong against three different fungi (Candida albicans, Aspergillus niger and Aspergillus clavatus). The MIC values of the extract against tested fungal stain were found in the range of 200-1000 μg/ml.


1.Introduction
In many developing countries, traditional medicine is one of the primary healthcare systems [1,2]. Traditional African, Ayurvedic and Chinese systems of medicine are amongst the oldest known, and have undoubtedly influenced modern drug development and the isolation of novel compounds with therapeutic value [3]. Ayurvedic medicine is originated in India more than 3,000 years ago and remains one of the country's traditional health care systems. In recent decades, research has shown that plants produce a diverse range of bioactive molecules for industrial interest, making them a rich source of different types of medicines and have shown a promising effect in therapeutics [4]. Aromatic and medicinal plants are known to produce certain bioactive molecules which react with other organisms in the environment and inhibit bacterial or fungal growth [5]. Thus medicinal plants have been representing a rich source of antimicrobial agent [6].
The lectotype species of Elephantopus genus, family Asteraceae, consists of 32 species of centered in the Neotropics, Europe, Asia, Australia and Africa [7][8][9][10][11][12][13]. Elephantopus scaber Linn. is one of the medicinally important species of this genus. Hiradeve and Rangari (2014) [14] has recently reviewed the ethnomedical history of E. scaber. The whole plant, its various parts and the extracts of E. scaber have been used for the treatment of a number of diseases and also as antibiotic in many countries. Literature survey indicated the antimicrobial activity of the whole plant extracts of E. scaber in certain microorganisms. However the roots and the aerial parts individual antimicrobial and antifungal potential has not been explored till date. Hence the present study has been undertaken to explore the antimicrobial and antifungal activity of various extracts of the root and aerial part of E. scaber against gram positive, gram negative bacterial and fungal stains.

Collection and authentication of plant material
The whole plant of E. scaber was collected in the month of October-November 2012, from the IJBR (2015) 6 (05) www.ssjournals.com forest of Achanakmar, Chhattisgarh, India. The collected plant was authenticated by the Dr. G. P. Sinha, Scientist D, Ministry of environment and forests, Botanical survey of India, Allahabad, Uttar Pradesh. BSI/CRC/TECH/2014-15/ voucher specimen has been preserved in our laboratory for future reference.

Preparation of plant extract
The whole plant material was washed well with water to separate the adhering soil material. The collected entire plant containing roots and aerial parts, was dried in the shade. The total loss on drying was found to be 29.6%. After complete drying the roots and aerial parts were separated from each other. Dried roots consisted of thin, long roots attached to rhizomes while the aerial parts contained leaves, stems and flowers. 1 kg of the dried roots and rhizomes of E. scaber were comminuted to form coarse powder. The coarsely powdered crude drug material was first defatted with petroleum ether (40-60 0 ) in Soxhlet extractor. Defatted dried marc of the crude drug was further subjected to sequential extraction with diethyl ether and with methanol. 500 gm of coarse powder of root and rhizomes of E. scaber was macerated with water for 24 hrs (24x3). The same extraction procedure was followed for the extraction of aerial parts to afford petroleum ether, diethyl ether, methanol and water extracts. All the extracts obtained were concentrated and dried under reduced pressure using rotary evaporator. , were chosen based on their clinical and pharmacological importance. The bacterial and fungal cultures were incubated for 24 hours at 37°C on nutrient agar and potato dextrose agar (PDA) medium respectively, following refrigeration storage at 4°C. The bacterial strains were grown in Mueller-Hinton agar (MHA) plates at 37°C (bacteria were grown in the nutrient broth at 37°C and maintained on nutrient agar slants at 4°C), whereas the yeasts and molds were grown in Sabouraud dextrose agar and PDA media, respectively, at 28°C. The stock cultures were maintained at 4°C.

Sample preparation
Antimicrobial activity of the extracts was tested at various concentrations ranging from 5.00-250.00 µg/ml. The roots and aerial part extracts of E. scaber from four selected solvents were weighed and dissolved in DMSO to prepare stock solution of 250.00 µg/ml concentrations. The same stock solution has been utilized to get desired concentrations of 5.00 µg/ml, 25.00 µg/ml, 50.00 µg/ml, 100.00 µg/ml and 250.00 µg/ml by the serial dilutions method.

Determination of zone of inhibition (ZOI)
The petroleum ether, diethyl ether, methanol and aqueous extracts of E. scaber roots and aerial parts were screened for antimicrobial activity by using the disc diffusion method [15,16]. In the assay each inoculums suspension (10 8 CFU/mL) was spread evenly over the entire nutrient agar surface by sterile collection swab. Then, discs having of diameter 6 mm were sterilized at 121 0 C for 15 min and loaded with prepared positive control (ampicillin, 20 µg/ml) and extract solutions of E. scaber at various concentrations. The impregnated discs were dried for 3-5 min and dispensed onto the surface of the inoculated plates with flamed forceps. Each disc was pressed down firmly to ensure complete contact with nutrient agar surface. The discs were placed suitably apart and not relocated once contacted with the agar surface. The plates were then labeled and incubated at 37 0 C for 24 hours for both bacteria and fungus. The results were measured and expressed in terms of zone of inhibition (ZI) of bacterial and fungal growth around each disc in millimeters.

Determination of Minimum inhibitory concentration (MIC)
Minimum inhibitory concentration is determined by broth dilution method with some modification [17,18]. Serial dilutions were prepared in primary and secondary screening. In primary screening 1000 µg/ml, 500 µg/ml, and 250 µg/ml concentrations of the extracts were taken. The active extracts found in this primary screening were further tested in a second set of dilution against all microorganisms. The extract found active in primary screening were similarly diluted to obtain 200 µg/ml, 100 µg/ml, 50 µg/ml, 25 µg/ml, 12.5 µg/ml, 6.250 µg/ml concentrations. The control tube containing no antibiotic is immediately sub cultured (before inoculation) by spreading a loopful evenly over a quarter of plate of medium suitable for the growth of the test organism and put for incubation at 37 0 C overnight. The tubes are then incubated overnight. The MIC of the control organism is read to check the accuracy of the drug concentrations. The lowest concentration inhibiting growth of the organism is recorded as the MIC. The amount of growth from the control tube before incubation (which represents the original inoculums) is compared.

Extractive values and preliminary phytochemical screening
The result of extractive values of petroleum ether, diethyl ether, methanol and aqueous extract of root and aerial part of E. scaber has been depicted in table 1. The various extracts obtained were subjected to preliminary phytochemical screening. The root extract of E. scaber shown the presence of various phytochemical constituent such as glycoside, steroid and terpenoid found in petroleum ether extract; alkaloid, steroid, flavanoid, terpenoid and saponin found in diethyl ether extract; carbohydrate, glycoside, alkaloid, steroid terpenoid and saponin found in methanolic extract; carbohydrate, alkaloid, flavonoid, terpenoid and saponin was found in aqueous extract. However the aerial part extracts of E. scaber shown the presence of glycoside and steroid in petroleum ether extract; steroid, flavonoid, terpenoid and saponin in diethyl ether extract; carbohydrate, flavonoid, tannin, terpenoid and saponin in methanolic extract and carbohydrate, alkaloid, tannin, terpenoid and saponin in aqueous extract.
Thin layer chromatographic study of the root extracts of E. scaber has shown three distinct spot in solvent system pet. ether: dichloromethane (6:4) by petroleum ether extract; three spot in benzene: pet. ether (9:1) by diethyl ether extract and six spot in hexane: ethyl acetate (8:2) by methanol extract. The aerial part extract of E. scaber has shown three spot in pet ether: chloroform (7:3) by pet ether extract; four spot in pet ether: ethyl acetate (6:4) by diethyl ether extract and six spot in hexane: ethyl acetate (8:2) by methanolic extract. Vanillin-H2SO4, phosphomolybdic acid, FeCl3-ethanol and iodine were used as the chromogenic reagents.

Figure 2: Graphical representation of zone of inhibition of E. scaber root and aerial part extract against
Gram positive organism at 250 μg/ml concentration

Conclusion
Various extracts of the roots and aerial parts of E. scaber has shown significant antibacterial and antifungal activity against the gram positive, gram negative and fungal strains in concentration dependent manner. In cases of gram negative and fungal strains the activity has been found to more significant than that of standards. The results of the antimicrobial and antifungal studies validate the ethnomedical utility of E. scaber in various countries for the treatment of infectious diseases.