Phytochemical Screening, Antimicrobial Activity of Phytochemical Screening, Antimicrobial Activity of Gemmotherapeutically White Mulberry (Morus Alba) Leaves Gemmotherapeutically White Mulberry (Morus Alba) Leaves

The present study was designed to find out phytochemical analysis and antimicrobial activity of leaves of Mulberry plant. The phytochemical analysis was performed on quantitative and qualitative basis. The qualitative basis indicated the presence of alkaloids, glycosides, flavonoids, steroids, tannins, saponins and Anthraquinone. The quantitative analysis showed alkaloids (40%), glycosides (20.05%), flavonoids (14%), steroids (3.5%), tannins (11.9%) and saponins (11.5%) and anthraquinone (0.5%) presence. Antimicrobial activity against bacteria has been observed in the leaves of Mulberry ( Morus alba ) and showed zone of inhibition, Escherichia coli (7±3.3) mm, Staphylococcus aurous (8±3.1), Bacillus subtilis (9±2.5), Pasturella multocida (8±3.1) mm.


INTRODUCTION
Plants are rich source of phytochemicals and are used to kill the micro-organism by inhibiting their growth (Hammer et al., 1999).The phytochemicals possess antimicrobial properties and are used from anicent time as western medicine (Akerele et al., 1993). Traditional practitioner still use plants to cure infectious diseases or healing of wound. Medicinal plants on scientific analysis showed antibiotics properties against pathogens (Levy et al., 2006). Among medicinal plants, the Moraceae family is widely used for their antimicrobial activities against pathogenic organisms (Dev, 2010). In family Moraceae, the genus Morus is worldwide in distribution due to fast growing nature, produces cheap source of fruit and possess antioxidant properties (Newman et al., 2007).The white mulberry (Morus alba) is native plant of South Asia and found abundantly in warmer habitat. In Asian countries, this plant is widely used as herbal tea, feeding of silk worm (sericulture), fodder, making baskets and grown in orchards for fruits (Datta, 2000). The current research was designed with objective to check the phytochemical constituents and antimicrobial properties of fresh leaves of M. alba.

MATERIAL AND METHODS
Fresh leaves of M. Alba were collected from different areas of Pothwar. These fresh leaves were washed thoroughly with distilled water and then weighed. These leaves were dried by evaporation in oven. This material was blended in alcohol and glycerin mixture in the ratio of (2:1). The mixture was placed in cool and shaded environment for one month followed by shaking at different intervals to help maceration process. Stock of this material was prepared by filtering it under constant pressure and kept for further 48 hours, followed by another filter. The alcohol was removed by evaporation in rotary and further kept at 65 o C temperature in an incubator to evaporate extra alcohol.

Phytochemical Screening
The powdered leaves were subjected to phytochemical screening for the presence of the alkaloids, tannins, saponins, steroids using standard photochemical protocol used by Brain and Turner (1975)

Quantitative
Estimation of Phytochemicals i.

Extraction of Alkaloids
The Paste of plant material (25 g) was mixed with 5 % Na 2 CO 3 solution and transferred to a 500 ml flask, by adding 50 ml of chloroform. The solution was refluxed for 20 minutes, cooled, filtered and transferred to the agitator for 5 minutes. The upper layer was removed and made volume up to 5 ml.
Further 1% H 2 SO 4 (25 ml) was added and extracted using 20 ml of CHCl3. The aqueous phase was separated, and ammonium hydroxide was added to alkaline it and then extracted with 10 ml portions of CHCl 3 successively. Then chloroform layers were washed with water (5 ml) followed by reducing volume (5 ml) by distillation. The absolute alcohol (2 ml) was added to the residues and evaporated at 100° C to dryness and solid residue obtained were crude alkaloids. The percentage yield of alkaloids was determined.
% yield of alkaloids= Weight of alkaloids obtained × 100/ Total weight of Sample

ii. Extraction of Flavonoids
The paste plant material (25 g) was mixed with 100 ml ether in flask and refluxed for 1 hour at temperature 60°C, filtered and dried at 20° C. The filtrate was mixed with methanol of concentration 100 ml, refluxed (2 hour), filtered and evaporated. The crude flavonoids were determined by the following formula (Vongsak, 2013).
% yield of flavonoids = Weight of flavonoids obtained × 100 / Total weight of Sample

iii. Extraction of Tannic Acid
The paste (25 g) of plant material was mixed with acetone and water (70% and 30%) and employed for extraction. Purification of this material was carried out in order to remove the pigments and phenolics by following Hagerman (1996). % yield of Tannic acid = Weight of tannic acid obtained × 100 / Total weight of Sample

iv. Saponins Extraction
The plant material (25 g) was placed in soxhlet extractor with solvent (200 ml) i-e ether for six hours at temperature 60°C to be defatted. The residue was kept in open air for overnight to evaporate the solvent. This plant material was then placed in the thimble of soxhlet extractor again alongwith methanol solvent (200 ml) till the colorless extraction. The solvent was evaporated and calculated percent yield of saponins by following Sharma et al., (1982).
% yield of saponins = Weight of saponins obtained × 100 / Total weight of Sample

v. Extraction of Steroids
The powdered plant material (10 g) and Gemmo-therapeutically treated M. alba was then weighed and transferred to a round bottom flask with ethyl acetate (100 ml). The solution was refluxed for 20 minute at 40°C and then filtered. The filtrate was mixed with 5% KOH (2-50 ml). Two layers were separated: ethyl acetate layer and steroids layer (The ethyl acetate layer was extracted with of 5% HCl (2-50 ml) by evaporation) while steroid layer persist. % yield of steroids = Weight of steroids obtained × 100 / Total weight of Sample

vi. Extraction of Glycosides
The grounded plant material (25 gram) was boiled with 90 ml ethyl alcohol and filtered. The filtrate was mixed with lead sub acetate solution (30 ml) to remove chlorophyll and other pigments. The filtrate was treated with distilled water (45 ml saturated with H 2 S) to remove lead sub acetate.
The pure filtrate was then dried on an electric water bath and the percentage (%) yield of crude glycosides was calculated. % Yield of Glycosides = Weight of glycosides obtained × 100 / Total weight of Sample

Antibacterial Activity
Antibacterial activities of alcohol and gemmo extracts of leaves of M. alba were tested against bacterial species (Staphylococcus aureus, Escherichia coli, Pasturella and Bacilli) in vitro as reported by Coventry and Allen (2001). The antibacterial activity was checked against bacteria were S. aureus, and E. coli.

Preparation of Disc and Media
Wicks paper disc of 10 mm were used. Agar media was prepared by following Cruickshank (1975).
The solution of peptone and agaragar were prepared by distilled water and yeast extract and NaCl were added. The solution was autoclaved.

Dispension of Medium
15 ml of medium was poured in Petri plates (9 cm) for gel formation (2-3 mm).
Agar media was distributed into each inoculated Petri dishes. Discs of Methanolic gemmo extracts (25 µL, 50 µL and 75 µL) were placed on agar medium. Medium were placed at 37 o C for 24 hours. Commercially available antibiotics Saparaxin and Rocephin were used as standard reference.

Phytochemical Analysis
Phytochemical analysis of M. Alba was carried out in order to determine the presence of different phytoconstituents quantitatively as well as qualitatively. Alba was shaken with distill water, considerable froth was produced which lasts for several hours (Sofowora, 1982). Saponins are used for hypercholesterolemia, antioxidant, antiinflammatory, anti-cancer & gentle blood cleanser .Quantitatively 11.5% saponins were present in M. Alba leaves.

Tanic Acid, Steroids and Anthraquinone
Plant material was extracted with 20 ml of distilled water for 5 minutes. Then, 5 drops of FeCl 3 were added .Blue black precipitates indicated the presence of

Chemicals Amount Chemicals Amount
Peptone 2.5 g pH 7.4 Yeast Extract

g
Agar-agar 20 g NaCl 2.5 g Distilled water tannins. Quantitative analysis showed that 11.9% tannins were present in M. Alba leave. In methanol extract of M. Alba 1-2 drop of Liebermann Bur chard reagent was added. Violet coloration appears which indicated the presence of steroids. Quantitative analysis showed 43.5% steroids were present in M. Alba leave. On extraction of plant material with absolute ethanol, then addition of n, n dimethyl aniline solution, No red coloration appeared. Anthraquinone was only 0.5% in M. Alba leaves.

Antibacterial Activity
The results of antibacterial activity against E.Coli methanolic extract of M. Alba leaves had showed inhibition zone 7 mm. Against (Staphlococus. Aurous) plant extract of M. Alba leaves showed 8 mm inhibition zone (Table 3). Methanolic extract of M. Alba leave showed inhibition zone 8mm against B. subtilis. It is a gram positive bacterium. Against P.multocida plant extract of M. Alba showed 9 mm inhibition zone.

DISCUSSION
The plants possess different kinds of metabolites and these serve as potential source of antimicrobial activities against pathogens and have medicinal importance due to minimum toxicity (Wang et al., 2008).The trend of phytochemicals uses as medical therapy is going to increase (Grayer and Harborne, 1994).
Our results of antimicrobial activity against bacteria Staphylococcus aurous show similar inhibition zone as reported by (Ayoola et al., 2011) inhibition zone (8±3.1) mm. the present findings of inhibition zone by Escherichia Coli (7±3.3) mm also confirm the findings of (Ayoola et al., 2011).
The present finding reports inhibition zone by leaves of (M. alba) for (Bacillus subtilis) (9±2.5) while similar study was reported that present bacterial species is more sensitive (Wang et al., 2011). The antimicrobial activity by leaves of (M. alba) for (Bacillus subtilis) is also reported by (Roessler et al., 2009).

CONCLUSION
The present findings revealed that natural plant extracts are potential source of antimicrobial activity which inhibit growth of microbial populations so these plants should be used in pharmaceutical industries for production of cheap medicine to conserve human health. These natural plants should be used as food supplements.