The plant part was collected from Madhupur of Tangail forest region of Bangladesh, in between April-May 2013. Taxonomist of National Herbarium Bangladesh, Dhaka, identified the plant and an accession number was submitted (35483).

Extract was prepared from leaf part of the collected plant by usingorganic solvent Ghani, 2005. The fresh leaves of Glycosmis pentaphylla were pieced; washed and air-dried at room temperature (24 ± 2°C) for about 10 days. Dried leaves was milled into coarsepowder. Coarse powder, weighing about 200 grams, takenin a bottle and dissolved in ethanol. Then the mixture kept for 2 days with uninterrupted shaking. The extract was collected using Buckner funnel, where theethanolic mix of the powder was poured under vacuumsuction. The filtrate contained the crude drug extract ofethanol. The ethanol was evaporated and a concentrated crude drug extract of Glycosmis pentaphylla leaves was obtained, which wasweighed to be 29 grams and was preserved into alpine tubefor further use at 4°C. The percent yield was 14.5%.

DPPH scavenging assay: The DPPH scavenging activity of G. pentaphylla was measured according to the method of Liu and Zhao (2006) Liu and Zhao, 2006. The reaction mixture contained 2 ml of 95% ethanol, 0.1 M DPPH and 2 ml of the ethanolic leaf extract of G. pentaphylla (50, 75, 100, 200, 300 μg/ml). The solution was incubated at 25°C for 15 min, and the absorbance of G. pentaphylla was determined at 517 nm. The antioxidant activity of G. pentaphylla extract was evaluated according to the following formula:

Scavenging rate (%) = [1-A]/ A₀ X 100

Where A is absorbance of G. pentaphylla extract and A₀ is the absorbance of negative control (DPPH solution). Ascorbic acid used in this method as positive control, to compare the effectiveness.

In vitro Brine shrimp lethality bioassay Rahman and Rashid, 2008 technique applied, using nauplii of Artemia salina, for the determination of general toxic property of G. pentaphylla. In this method Vincristinsulphate was used as a positive control, for the comparison. Eggs kept in a small tank containing 3.8% NaCl solution for hatching, a light source was attached to that tank, we hatched eggs for 2 days and then it is ready for experiment. Four milligrams of the extract was dissolved in DMSO to geta concentration of varying concentrations 100, 50, 25, 12.50 and 6.25 μg/ml. 10 brine shrimp nauplii were then placed in each vial and allowed to stand for 24 hour. The vials were observed using a magnifying glass and the number of survivors in each vial were counted and noted. From these data, the percentage of mortality of the nauplii was calculated for each concentration and the 50% lethal concentration (LC₅₀) values were determined.

Antimicrobial Activity: Stock solution was prepared by dissolving 10 mg of the ethanolic crude drug extract in ethanol. The disk for drug dissolving was prepared using sterilized filter paper. Papers were punched uniformly to exactly 6mm in diameter. Sample solutions of desired concentrations (100, 200, 400 and 500 μg/disk) were applied with the help of the micropipette in an aseptic condition. These disks were left for a few minutes in aseptic condition for complete evaporation of the solvent. In this study, commercially prepared Kanamycin disk, K-30 disks containing 30 μg/disk, was used as a standard for comparison purpose. The in vitro disk diffusion assay (Perez C, et al., 1990), of antibacterial screening was used to determine the susceptibility of the pathogenic microorganisms to the test compound applied.

Preparation of fresh culture of the pathogenic organisms: The nutrient agar medium was prepared and dispersed in a number of test tubes to prepare slants (5 ml in each test tube). This was done to prepare (Axenic) cultures from the supplied cultures (Madigan M and Martinko J, 2005). The test tubes were sterilized at 121°C temperature and a pressure of 15lbs/sq inch for 15 minutes. After sterilization, they were kept in an inclined position, for solidification, and then was incubated at 37.5°C. The test organisms were transferred to the agar slants from the supplied cultures with the help of an inoculating loop in aseptic condition. The culture was kept at 4°C or less for bacterial growth for 12 hours. Then incubated at 37°C for 24 hours to assure the growth of test organisms. These fresh (Axenic) cultures were then used for the sensitivity test.

The test plates were prepared for the disc diffusion test of the test samples. Bacterial suspensions were transferred to the sterile petri dishes in an aseptic area. The petri dishes were rotated several times, first clockwise and then anticlockwise to assure homogenous distribution of the test organisms. The media was poured into petri dishes in such a way, in order to give a uniform depth of approximately 4mm.

Finally, the medium was cooled to room temperature in laminar airflow unit and it was kept in refrigerator at (4°C) and the sample impregnated discs and standard disc were seeded, in the sub-solidified medium. The medium was congealed to room temperature in laminar airflow unit, then refrigerate at (4°C) for 24 hours in order to provide sufficient time to diffuse the antibiotics into the medium. Hence, the zones of inhibition of different samples were compared Brown and Kothari, 1975.

Extracts were checked by thin layer chromatography (TLC) on analytical plates over silical gel. The solvent systems used was H-EA = 2:1 where, H = hexane, EA = ethyl acetate. In this case, the spots were visualized by exposure of the plates to UV lamp. Different bands were observed and corresponding Rf values are determined. Rf value of each spot was calculated, Rf = (Distance traveled by solute/Distance traveled by solvent)

The statistical analysis was performed using Graph-Pad Prism-6 software. Values are represented in tabular sheet as mean ± SD and ANOVA was performed for anti-microbial assay. The significant limit for that particular case was set p<0.05.

DPPH is a relatively stable free radical and the assay determines the ability of ethanolic extract of G. pentaphyllato reduce DPPH free radicals to the corresponding hydrazine by converting the unpaired electrons to paired ones. Antioxidant can act by converting the unpaired electron to paired one. The dose dependent inhibition of DPPH radicals ( Figure 1 ) indicates that selected extract causes reduction of DPPH radical in a stoichiometric manner Murray, 1999Sanchez-Moreno, 2002Vani et al., 1997; with the inhibitory concentration (IC₅₀) 204.91 ± 2.223 μg/ml; where the comparable standard have 56.182 ± 2.016 μg/ml of IC₅₀value ( Table 1 ). From this point of view, it is clear that the extract have moderate antioxidative capacity, through which it can yet reduce the exacerbation free radicals.

The antimicrobial activity of the ethanolic extract of-leaves of G. pentaphylla was measured by disc diffusion method. Different concentrations of 100 μg/disk, 200 μg/disk, 400 μg/disk, and 500 μg/disk were measured and compared with the zone of inhibitions, which was produced by the standard. The zones of inhibition were seen against selective bacteria at a particular concentration ( Table 2 ). The studied ethanolic extract of leaves of plant G. pentaphylla showed higher activity against E. coli. At higher concentrations of 400 μg/disc and 500 μg/disc, the extract also showed goodinhibitions against other studied microorganism. However, the extract showed negligible or no activity against S. dysenteriae, which is a gram-negative bacteria.

In cytotoxic test activity, percent of mortality increased gradually with the increase in concentration of the test samples. LC₅₀ values obtained from the best-fitline slope ( Figure 3 ) were 30.49 ± 1.976 μg/ml and 24.879 ± 2.413 μg/ml for G. pentaphylla and vincristine sulphate, respectively.

The brine shrimp lethality bioassay is very useful toassess the bioactivity of the plant extracts, which inmost cases correlates reasonably well with cytotoxicand anti-tumor properties McLaughlin et al., 1993. LC₅₀ values of G. pentaphylla revealed its considerable cytotoxic potency. Sufficient amount of phenolics and flavonoidsmay be present and it might be responsible for its promising cytotoxic activity Moreira et al.,2007Okwori, 2007 and the possible mechanism of cytotoxicityagainst brine shrimp nauplii due to poisonous effecton cell mitosis.

Observation of the TLC plates under UV lamp results following ( Table 3 ). Four non-polar compounds were present with Rf values of 0.12, 0.15 and 0.23 and 0.33. Two compounds are in between polar and non-polar with Rf value of 0.44 and 0.53. Two polar compounds were present with Rf values of 0.63 and 0.70. A fluorescent compound with an Rf value of 0.81 could also be detected. Three non-chromatophoric compounds with Rf values of 0.04 (nonpolar) and 0.23 (nonpolar) and 0.64 (partially polar or polar). Thus, many compounds were present and isolation of pure compound is necessary.