PHYTOCHEMICAL SCREENING AND ANTIBACTERIAL ACTIVITY OF CYNODON DACTYLON AGAINST HUMAN PATHOGENS

Arthur Robin Raj V. N 1 , Mohammed A. Almalki 2 , Rakesh Varghese 3 and * Ponnuswamy Vijayaraghavan 4 . 1. The Salvation Army Catherine Booth Hospital, Nagercoil, Kanyakumari District, Tamilnadu, India. 2. Biological Sciences Department, College of Science, King Faisal University, Saudi Arabia. 3. Department of Industrial Biotechnology, Bharath University, Selaiyur, Chennai 73, India 4. Bioprocess Engineering Division, Smykon Biotech Pvt Ltd, Nagercoil, Kanyakumari District, Tamil Nadu, India. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

Cynodon dactylon is a medicinally essential plant used for the treatment of various infectious diseases. The study of phytochemical and antibacterial activities of the extract of Cynodon dactylon showed potent activity against human pathogens. Four different solvents such as acetone, ethanol, methanol, chloroform and water were used to extract the compounds from the leaves of C. dactylon to evaluate the phytochemical components and antibacterial activity. The phytochemicals such as proteins, carbohydrate, flavonoids and alkaloids were detected in this study. The extracts showed 4 mm to 15 mm zone against the tested bacteria. Ethanol and methanol extract showed considerable activity than other solvents and hot water. The methanol extract of C. dactylon showed high activity against S. aerus and P. aeruginosa. However, ethanol extract was highly active against E. coli and B. subtilis. C. dactylon extracted with water, ethanol and methanol showed significant activity against Enterobacter sp. This study concluded that extracts of Cynodon dactylon have potent phytochemicals and significant activities against various human pathogens.

Introduction:-
Medicinal plants are rich in various potential drugs and it holds harmless and healthier alternate to various synthetic drugs (Rai et al., 2007). In recent years, there is a growing interest among the medical properties of plants in terms of antibacterial activity and antifungal activity. In addition, these medicinal plants are rich of bioactive compounds and play critical role in the development of novel drugs. Extracts of some of the medicinal plants are highly useful in the treatment of various health problems such as peptic ulcers, bacterial infections, arthritis and inflammation (Patwardhan et al., 2010). Medicinal plants cure various diseases which are useful for investigation of potential sources of novel antimicrobial agents (Beegum and Devi, 2003). Cynodon dactylon is known as "Garike hullu" (Kanada), Dhoorva (Marathi), "Aruvaum pullu" (Tamil), "Garike and Thella gariki" (Telulgu) and "Doob" (Hindi). Different parts of medicinal plants such as root, leaf, stem, seed, fruit and park are mainly used to obtain several phytochemical constituents of plants. The extracts of medicinal plants are useful in the treatment of inflammatory diseases (Shah et al., 2011).

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Cynodon is Bermuda grass and belongs to the family, Poaceae. It is native to East Asia, Africa, Southern Europe and Australia. Cynodon is generally considered as a weed and has been found to possess various potential medicinal properties (Singh et al., 2009). The plant is widely used in India as potential agent to control diabetes. The extract of C. dactylon has been widely reported to be anti-diabetic, hypolipidemic and antioxidant efficacy, healing of minor injuries (Oudhia, 1999), hepatic antioxidant and immunomodulatory activities. The aqueous extract of C. dactylon rhizome was used for diuretic, purifying agent and dysentery (Sadki et al., 2010). This plant extract has also potent application in secondary syphilis and dropsy (Kesari et al., 2006), cardio protective (Garjani et al., 2009) and wound healing properties (Oudhia and Pal, 2000). The extracts of C. dactylon had been reported to be effective for antimicrobial activity against fungal pathogens and bacterial pathogens (Kanimozhi and Rathbai, 2012). In this study, Cynodon dactylon was screened for its potential antibacterial activity.

Materials and methods:-
Plant material:

C. dactylon
Fresh shoots of C. dactylon was collected locally. The collected shoots were cleaned, washed with double distilled water and dried for 10 days (air drying) at room temperature (30 ± 2 °C). Then the plant material was powdered using a mixer grinder and stored in a clean plastic containers to protect from heat, light and moisture until further use.

Solvent extraction of antibacterial substances:-
Plant material was extracted with various solvents such as acetone, ethanol, methanol, chloroform and water. About 5 gm of dried powder was weighed and suspended in 50 ml of solvent separately. After 24 h, the suspension was filtered using Whatman No.1 filter paper. Then it was centrifuged for 5 min at 5000 rpm. This extraction procedure was repeated three times and pooled. The residual plant material was air dried completely to remove the respective solvents and the phytochemical components were analyzed.
Preparation of hot water extract from C. dactylon:-About 5 gm of powdered plant material was weighed and suspended in 200 ml of double distilled water. Then, this mixture was heated continuously with stirring at 35 ºC to 45 ºC for 30 min. Further, the water extract was filtered using Whatman No.1 filter paper. Then it was centrifuged for 5 min at 5000 rpm and used for the analysis of antibacterial properties.

Qualitative analysis of phytochemicals from C. dactylon:-
Phytochemical test for the presence of bioactive chemical constituents in C. dactylon was carried out using various solvent using the standard procedures. All extracts were subject to phytochemical analysis.

Analysis of total protein:-
The presence of protein content in the shoot of C. dactylon was carried out by using standard method.

Analysis of carbohydrate:-
The presence of carbohydrates in the extract of C. dactylon was evaluated as suggested by Fehling's test. In this method, 1.0 ml of Fehling's reagent A was mixed with 1.0 ml of Fehling's reagent B and crude plant extract was added. Followed by gentle heating, the reagent mixture showed brick (Fehling, 1984).

Analysis of Flavonoids:-
To quantify flavonoids, 2 ml of 2% NaOH were added in 0.10 ml plant extract, which showed intense yellow colour and the colour disappeared for few min. Then addition of four drops of 1% aluminum solution added in each filtrate. Reappearance of yellow colour indicated the presence of flavonoids in the sample (Edeoga et al., 2005).

Analysis of Steroids:-
The plant extracts mixed with 2 ml of chloroform and Conc. H 2 SO 4 . Development of red color in the chloroform layer indicated the presence of steroids. Then few drops of acetic acid was added. Development of greenish colour indicated steroids positive.

Antibacterial test:-
Microorganisms used in the present study mainly represent pathogenic species commonly associated with human infections.

Test microorganisms:-
The following pathogenic strains were used for the antibacterial studies. The bacteria such as, Staphylococcus aureus, E. coli, Bacillus subtilis, Pseudomonas aeruginosa, and Enterobacter sp. were used.

Preparation of inoculum:-
Active cultures for experiments were prepared by transferring a loop full of cells from the stock cultures to the nutrient broth (g/l) (peptic digest of animal tissue, 5.0; beef extract, 1.5; yeast extract and 1.5; sodium chloride, 5.0). The Erlenmeyer flasks were incubated at 37 °C for 18 h and were used as the inoculum.

Antimicrobial susceptibility tests:-
Antimicrobial susceptibility testing of the crude plant extracts using well diffusion method. In this method, the antibacterial substances diffuse from the well through a solidified agar layer in a Petri plate to an extent so that the growth of added micro organisms is inhibited entirely in a circular area or zone. The antibacterial activity is expressed as the zone of inhibition in millimeters, which is measured with a zone reader. The presence of definite zone of inhibition of any size around the well indicated antibacterial activity and the zone of inhibition was measured.

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The phytochemicals such as quinines, tannin and phenols are mainly responsible for antimicrobial properties. The alkaloids of C. dactylon exhibit antimicrobial activity against human pathogens. It was previously reported that the solvent extract of C. dactylon exhibited antimicrobial activity because of the presence of gramine, tyramine, tyrptamine and alkaloids (Raman et al., 2002). The aerial parts of C. dactylon contain cynodin, triticin, hydrocyanic acid and beta carotene (Kirtikar and Basu, 1980). In the present study, well diffusion method was followed to elucidate antibacterial activity. The extracts showed 4 mm to 15 mm zone against the tested bacteria. Ethanol and methanol extract showed considerable activity than other solvents and hot water. The methanol extract of C. dactylon showed high activity against S. aureus and P. aeruginosa. However, ethanol extract was highly active against E. coli and B. subtilis. C. dactylon extracted with water, ethanol and methanol showed significant activity against Enterobacter sp. (Table 2). This result was in accordance the observations made previously with other medicinal plants. Likewise, the methanolic extract of Solanum palinacanthum was observed the zone of inhibition on B. subtilis, A. hydrophila and S. aureus (Aline et al., 2008). The petroleum ether extract of the medicinal plant, Capparis zeylanica showed more activity against K. pneumonia, P. vulgaris, S. aureus and B. subtilis. The inhibitory zone ranged from 10 to 16 mm at a concentration of 10 μg/ml (Chopade et al., 2008). Chloroform extracts of the medicinal plant exhibited least antibacterial activity against the selected bacterial pathogens. In the present study also, chloroform extract showed very less activity than other solvents and hot water extract. It was also observed that selected gram negative bacteria were more sensitive to most of the extracts tested compared to grampositive bacteria. The gram-negative bacteria were highly resistant to antibiotics than that of gram positive bacteria. And, this resistance is mainly due to the variations in their cell wall composition (Paz et al., 1995). -----9 ----11 In an another study six various organic solvents were employed to extract the antibacterial compounds from the leaves of C. dactylon against the pathogenic stain such as Streptococcus pyogens, Bacillus subtilis, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Proteus mirabillis and Pseudomonas aeruginosa. Among the solvents tested butanolic extract of Cynodon dactylon was found to be good than other tested solvents (Chaudhari et al., 2011). The present work revealed that the solvent and water extract of C. dactylon was found to be effective against various human pathogens. From this study we draw a conclusion that the traditional use of plant C. dactylon for the infectious disease is promising against bacteria.