Phytochemical, antibacterial activity, inhibition zone, Eclipta alba, Tylophora indica alcoholic and aqueous extracts.

Herbal medicines, also called botanical medicines or phytomedicines, refers to the use of any plant’s seeds, berries, roots, leaves, bark or flowers for medicinal purposes have long being practiced. As up to-date analysis and research shows their value in the treatment and prevention of disease, herbal drugs are more mainstream products.
Any part of the plant may contain active components like alkaloids, saponins; tannins, essential oils etc. Polyphenols and tannins in plants inhibit insect growth [3] disrupting digestive events of ruminal animals. They are also sometimes added to food meant for pregnant and nursing mothers for medicinal purposes. The increasing prevalence of multidrug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the specter of untreatable bacterial infections and adds urgency to the search for new infections- fighting strategies [5] .
Aerial parts of a well known Indian hepatoprotective herb, Eclipta alba (Asteraceae ) are used traditionally for the treatment of several diseases of liver, skin and stomach. The shoot extract of E. alba showed antimicrobial [1,10,28] antifungal activity [26] and weak cytotoxicity against the M-109 cell lines by alkaloids Verazine [2] , antiviral activity against Ranikhet disease [11] , antibacterial [12] . The leaves and roots of Tylophora indica (Asclepiadaceae) have emetic, cathartic, laxative, expectorant, diaphoretic and purgative properties. It has also been used for the treatment of allergies (asthma), cold, whooping cough, dysentery, hay fever and arthritis. It has reputation as an alterative and as a blood purifier, often used in rheumatism and syphilitic rheumatism. Root or leaf powder is used in diarrhoea, dysentery and intermittent fever. It is regarded as one of the best indigenous substitute for ipecacuanha. The roots are suggested to be a good natural preservative of food. Phytochemical literature reveals the presence of tylophorin, alkaloids, tannins, saponins, flavonoids in Tylophora indica [6,9,23] T. indica is used in the preparation of homeopathy medicines.
However, up to date, research has not been done to investigate various pharmacological activities and antimicrobial activities of only crude extracts of these traditionally used plants.
Present investigation reveals the study of antimicrobial activity of Eclipta alba and Tylophora indica which are found in the medicinal garden of Biotechnology Department of GIET, Gunupur, against Escherichia coli, Micrococcus roseus and Pseudomonas flavescens.

The leaves, stems and roots of E. alba and T. indica were collected from medicinal garden of Biotechnology Department, GIET, Gunupur. Fresh plant materials were shade dried then kept in hot air oven at 60°C for 24 hours. and homogenized into fine powder.

The fine powder was taken and subjected to different solvents to get phytochemical extracts. The solvents used were methanol, petroleum ether and water. 1gm powder of E. alba and T. indica was taken for extraction with 100 ml of different solvents in Soxhlet apparatus. The extracts were filtered through muslin cloth and concentrated by evaporation at room temperature until the solvent gets evaporated completely. Extraction was carried out with all solvents separately. Then extracted materials lyophilized by occasional shaking for 24 hrs. The extracts were preserved in refrigerator for further studies.

Phytochemical screening of plant extracts were carried out qualitatively for the presence of glycosides, alkaloids, flavonoids and tannins.

Antimicrobial assay of solvent extracts were performed by Disc diffusion method. Lawn culture of Escherichia coli, Micrococcus roseus and Pseudomonas flavescens were developed on nutrient agar plates using sterile cotton swabs. The sterile disc (5mm) was saturated with various plant extracts. Disc with solvents were used as control and the respective antibiotic disc Gentamycin used as positive control. The sterile impregnated disc with plant extract were placed on the agar surface with flamed forceps and gently pressed down to ensure complete contact of the disc with the agar surface. After the incubation at 37°C for 24h the size of the inhibition zone were measured.
Antibacterial activity was determined by measuring the diameter of the zone of inhibition For each strain, controls were included that comprised pure solvents instead of the extract. The experiments were repeated three times and the mean values were presented.

Bacterial Strains used in this study were purchased from MTCC, Chandigarh. These are Escherichia coli, Micrococcus roseus and Pseudomonas flavescens All these strains were confirmed by cultural & biochemical characteristics and maintained in slants for further use.

The minimum inhibitory concentration values were determined by broth dilution assay for the plant showing better result. Varying concentrations of the extracts (200mg/mL, 150mg/mL,100mg/mL,75mg/mL,50mg/mL and 12.5mg/mL) were prepared. 0.1mL of each concentration was added to each 9mL of nutrient broth containing 0.1mL of standardized test organism of bacterial cells. The tubes were incubated at 37°C for 24h. Positive controls were equally set up by using solvents and test organisms without extracts. The tube with least concentration of extract without growth after incubation was taken and recorded as the minimum inhibitory concentration.

The results of the phytochemical analysis of the different alcohol and aqueous plant extract of Eclipta alba are shown in [Table-1] The extracts mostly contained higher quantities of glycosides, followed by flavonoids and alkaloids. Alkaloids and tannins were entirely absent in most of the tested aqueous extracts.
The Phytochemical screening of the alcohol and aqueous extracts of Tylophora indica revealed the presence of glycosides, alkaloids, flavanoids and tannins in all the plant extracts studied as shown in [Table-2] . The metabolites are of various pharmacological importance. The presence of tannin in most of plant extract could be responsible for possible antitumor and anti oxidant activities [7] [Table-3] indicates that methanol, petroleum ether and aqueous leaf extracts of E. alba is more sensitive to E. coli than to other experimental bacterial strains.
In case of E. coli methanol leaf extracts shows maximum zone of inhibition (15mm) than that of petroleum ether and aqueous extracts. Where as M. roseus and P. flaescens shows more or less zone of inhibition in organic solvents and no zone of inhibition in aqueous extracts. [Table-4] indicates that the methanol, petroleum ether and aqueous stem extracts of E.alba is more sensitive to E.coli than other two experimental bacterial strains. Here the findings indicate that the aqueous stem extracts is more sensitive i.e. 12mm zone of inhibition. Methanol stem extracts of E. alba shows maximum zone of inhibition (20 mm) against P.flavescens and no zone of inhibition is seen in aqueous stem extract of E. alba against P. flavescens and M. roseus. [Table-5] indicates that the methanol root extracts of E. alba shows zone of inhibition against the three experimental bacterial strains where as petroleum ether root extracts of E. alba does not show any zone of inhibition. Here the methanol root extracts of E. alba shows maximum (20mm) zone of inhibition against P. flavescens.
The observations of [Table-6] shows that the methanol and aqueous leaf extracts of T. indica is more sensitive to E. coli than the other two bacterial strains studied. Methanol leaf extract of T.indica shows maximum zone of inhibition (8mm) against E. coli. Where as neither of the leaf extracts of T. indica shows any zone of inhibition against M. roseus. Also the aqueous leaf extracts of T. indica shows zone of inhibition (2mm) against P. flavescens.
The findings of [Table-7] indicates that the methanol, petroleum ether & aqueous stem extracts of T. indica is more sensitive to E.coli than the other two experimental bacterial strains. Among them the methanol stem extracts of T.indica shows maximum zone of inhibition (12mm) against E. coli, but no zone of inhibition is found against M. roseus. Only the petroleum ether stem extracts shows zone of inhibition against P. flvescens where as it is resistant to the other stem extracts.
The observations of [Table-8] suggests that methanol root extracts of T. indica shows zone of inhibition against all the three experimental bacterial strain showing maximum zone of inhibition (15mm) against P. flavescens and minimum zone of inhibition (4mm) against M. roseus.
The minimum inhibitory concentration ranged between 12.5mg/mL and 75mg/mL depending on microorganism and various extracts are shown in [Table-9] . The result of minimum inhibitory concentration suggests that methanol, petroleum ether and aqueous extract of E.alba leaf, stem and root could possibly act as a bactericidal agent to these microorganisms. Except P. flavescens all other microorganisms were inhibited by the lowest concentration (12.5mg/mL) of methanol and petroleum ether extract of leaf, stem and root.

The present study exhibited the antibacterial effect of various extracts of Eclipta alba and Tylophora indica against Escherichia coli, Micrococcus roseus and Pseudomonas flavescens. As a result it is`sure that these extracts can surely inhibit the growth of these microorganisms there by preventing various diseases such as asthma, whooping cough, dysentery etc. Eclipta alba and Tylophora indica extract thus provides safe, easy, effective and practical solutions to every day ailments leaving behind no toxins and creating a clean, pleasant atmosphere. The inhibitory effect of the extracts justified the medicinal use of Eclipta alba and Tylophora indica and further study is required to find out the active component of medicinal value.
Conservation of genetic resources of crop species is of prime importance for suitable production and maintenance of diversity for future generation. Approaches are made for the production of low cost medicines for rural people. This in vitro study demonstrated that folk medicine can be used as effective as modern medicine to combat pathogenic microorganisms. The millenarian use of these plants in folk medicine suggests that they represent an economic and safe alternative to treat infectious diseases. However, none of the plants are recommended in the treatment of infections produced by E.coli, M. roseus and P.flavescens.

The authors are grateful to the management of GIET, Gunupur for providing us all the facilities and requisite support for completion of this work and for their constant motivation.