Biochemical and antimicrobial studies of the genus Jatropha excisa

Jatropha excisa is an ornamental plant, which is employed as a source of medicine with different antimicrobial activities and differentiation in organization of metabolites. Metabolite analysis of this plant was analyzed phytochemically and screened against different microorganisms responsible for various infections. Qualitative analysis of various primary and secondary metabolites was analyzed by Thin Layer Chromatography. Present report has shown more number of amino acids in wet leaves, carbohydrates in seeds and dry leaves, lipids in seeds and dry leaves, and secondary metabolites in flowers. The antimicrobial activity of the fresh methanolic leaf extract was tested against standard strains of bacteria and fungi using agar well diffusion method. Plant fresh methanolic leaf extract of 0.1mg/ml concentration produced different zone formation on Microorganisms. The zone formation was high in gram-negative bacteria such as Escherichia coli (16 mm), Micrococcus leuteus (15 mm) and Proteus mirabilis (14 mm) compared to the gram-positive bacteria such as Streptococcus faecalis (12 mm), Klebsiella pneumoniae (11 mm) and Bacillus subtilis (12 mm). The extracts were found to be effective against bacteria (11-16 mm) and yeast (15 mm) than multicellular, filamentous fungi such as Aspergillus niger and Penicillium chrysogenum (9-11mm).

rhizomatous subshrubs, and geophytes, distributed primarily in seasonally dry tropics.Related species not withstanding to Geographical changes have high gene exchange within wide limits under artificial conditions; phylogenetic relationships may be inferred by cross ability of the taxa 4,5 .Jatropha excisa Griseb.which has been reported in 1874, is a shrub 6 characterized by the frequent occurrence of milky sap.
Considerable attention has been focused on identifying naturally occurring chemo preventive substances 7 .Since ancient times, plants have been an exemplary source of medicine.Ayurveda and other Indian literature mention the use of plants in treatment of various human ailments.India has about 45,000 plant species and among them, several thousands have been claimed to possess medicinal properties 8 .
Medicinal plants are the most important sources of life saving drugs for a majority of the world's population.Biotechnological tools are important to select, multiply and to conserve the critical genotypes of medicinal plants 9 .Due to large demand of phytomedicines, plant tissue culture techniques are now being used to obtain large number of diverse medicinal plants, monitoring their respective secondary metabolites 10,11 .

Collection of plant materials
The plant material is largely found in Gajuwaka region of Visakhapatnam District.Whole plant was collected and the experiments were conducted during April -June 2008 (summer season).

Preparation of plant extracts
Freshly collected samples were thoroughly washed with distilled water separately.100 g of each plant materials (seeds, fresh flowers, fresh roots, fresh and dried leaves) were separately homogenized with 100 ml of Methanol and squeezed through three layers of cheesecloth to remove larger particles.The collected homogenate is used as sample for metabolite analysis.

2.
Silica gel in TLC was of "Lichrosolv" grade from Merck, India.

Procedure
Plates were made with silica gel by mixing about 30 gm of Silica gel (G60) with 60ml of double distilled water and the contents are vigorously mixed until the gel is uniformly dispersed.This procedure should be completed with in 4 minutes after addition of water.Then the slurry is poured into Stahl's mechanical spreader adjusted to 0.2 mm thickness.Using spreader the gel is layered on the glass plate and dried at room temperature for few minutes.Subsequently the plates are placed in an oven, kept at 100°C for 30 minutes and allowed to cool.(Stahl  1969)   Into respective developing chambers, solvent mixture was poured to a depth of about 10 mm.Cover the internal walls of the chambers with filter paper and wet it with the solvent system (or solvent mixture) used in that chamber.Close the lid and the tank is left for 10-15 minutes for saturation with solvent vapours.Softly, mark every TLC plate with a pencil line about 1.5 cm from the bottom.Subsequently make a mark every 1.5 cm from left to right.These marks will be used to apply for standards and samples.Hold the micropipette vertically and briefly touch the filled end to the pencil mark line.The samples prepared from field grown leaves (methanoic extract) were applied on the TLC plate in 10µl quantities.Hold the micropipette vertically and briefly touch the filled end to the pencil mark.Liquid should flow from the micropipette on to the plate to form a spot.Similarly standards are also applied on TLC plate in 10µl quantities.After loading the samples and standards, the plates are allowed to air dry.Once the spots have been dried, lower the plate carefully into the chamber.Close the lid and monitor the movement of solvent up the plate.When the solvent has advanced at least 15 cm, remove the plate from the chamber, mark the solvent front with a pencil, and allow all solvents to evaporate.Spray the plate with spraying reagent, let it dry, and heat it inside the oven at 110 °C for 15 minutes.The plates are removed and calculate the Rf values.

Distanced traveled by the sample Rf = ________________________________
Distance traveled by th solvent front

Antimicrobial activity Preparation of plant extracts
Freshly collected leaves were thoroughly washed with distilled water separately.100 g of plant material was homogenized with 100 ml of Methanol.The homogenate were thoroughly squeezed through three layers of cheesecloth to remove larger particles and then centrifuged at 10,000 xg at 4°C for 20 min.The supernatant was collected and the centrifugation process was repeated for 3 times at 10,000 xg at 4°C for 20 min.The final extract was filtered through whattman's filter paper no.1.The methanol present in the methanolic extract was evaporated under reduced pressure (Buchi vaporator) to yield the residue.The residue thus obtained was suspended in Dimethylsulfoxide (DMSO) to concentration of crude extract of 0.1mg/ ml.The extracts were kept at -20°C before use for maximum 24 hours.

Maintenance of microorganisms
The above bacterial cultures were maintained on Mueller-Hinton Agar (MHA) and fungal cultures were maintained on Sabouraud Dextrose Agar (SDA) at 4°C temperature until used for the study.Before use, the Bacterial and Fungal cultures were revived in Mueller Hinton Broth (MHB) for Bacteria and Sabouraud Dextrose Broth (SDB) for Fungi.

Antimicrobial assay
Zone Method is carried out for antimicrobial assay.MHA for bacterial growth and SDA for fungal growth was weighed and mixed in distilled water based on the composition.The media was autoclaved for 20 min.at 121°C (15 lbs pressure) and cooled to 45°C.The bacterial and fungal cultures with optical density of 0.6 were taken and 50 ml of inoculum was added per 500ml of MHA for bacteria and yeast, SDA for fungi.20 ml of the media was poured in each plate and was kept for solidification.By using gel puncture, 8 mm diameter wells had been made in the plate for the addition of plant extract at 0.1mg/ml concentration.Fresh leaf extract was added into the well and incubated for 24-48 hours for bacteria and yeast and 2 days for fungi.The zone of inhibition can be calculated based on the results obtained by scale reading.With the scale the zone formed on the plate was calculated and was tabulated.

RESULTS
The qualitative analysis of various primary and secondary metabolites was analyzed by TLC results.The Results of TLC were tabulated and listed in Tables 1 and 2.
Figure 1 shows more number of amino acids in wet leaves, more number of carbohydrates in seeds and dry leaves, more number of lipids in seeds and dry leaves, and more number of secondary metabolites in flowers.The ethnobotanical screening test of plant leaf extracts of Jatropha excisa in methanol as solvent against both human and plant pathogenic bacteria and fungi using zone technique was depicted in Table 3 and Figure 2. Two antibiotics (penicillin and Streptomycin) were conducted as standards shown different zones of inhibition and was listed in Table 4 The methanolic leaf extracts of wild plants had exhibited antimicrobial activity and the Zone of inhibition was calculated based on the results obtained.The zone formation was high in Gram negative bacteria such as E.coli (16 mm), M.luteus (15 mm) and P.mirabilis (14 mm) compared to the gram-positive bacteria such as S.fecalis (12 mm), K.pneumonia (11 mm) and B.subtilis (12 mm).The extracts were found to be more effective against bacteria (11-16 mm zone formation) and yeast (15 mm zone formation) rather than multicellular, filamentous fungi (9-11mm zone formation).

DISCUSSION
The quest for plants with medicinal properties continue to receive attention as scientists survey plants, par ticularly of ethnobotanical significance, for a complete range of biological activities, which range from antibiotic to antitumor.Thus far, plants have provided western medicine with an abundance of drugs and treatments for a variety of health problems 12 .
To define and describe the future tasks of phytomedicinal research in the new millennium, an analysis not only on the current state of development of phytomedicinal research but also on chemo synthetic pharmaceutical research was required.One advantage of phytotherapy is the availability of a wide group of medicinal drugs and preparation s that had been used over the centuries almost exclusively on the basis of empirical evidence.A reservoir of around 300,000 plant species exists, of which only about 30 percent had been investigated scientifically, inclusively the herbs and preparations of Chinese, Indian, South American and African traditional medicines 13 .
There are more number of amino acids in wet leaves, carbohydrates in seeds and dry leaves, lipids in seeds and dry leaves and secondary metabolites in flowers.Based on standards used, seeds contain isoleucine and xylose., flowers contain tryptophan., root contains glutamic acid and xylose., fresh leaves contain serine, threonine, phenylalanine and sucrose., dry leaves contain threonine and phenylalanin.
The methanolic leaf extracts of wild plants had exhibited antimicrobial activity and the Zone of inhibition was calculated based on the results obtained.The extracts were found to be more effective against bacteria (11-16 mm zone formation) and yeast (15 mm zone formation) rather than multicellular, filamentous fungi (9-11mm zone formation).The zone formation was high in gramnegative bacteria, such as E.coli (16 mm), M.luteus (15 mm) and P.mirabilis (14 mm) compared to the gram-positive bacteria S.fecalis (12 mm), K.pneumonia (11 mm) and B.subtilus (12 mm).

Fig. 1 :Fig. 2 :
Fig. 1: Graph showing number of metabolites in the samples examined by TLC