2.1 Collection of sample G. lucidium for current study was collected from a mushroom farm situated at Rangpur district Bangladesh and identified by the taxonomist of Botany department, University of Rajshahi - 6205, Bangladesh.
2.2 Extract Preparation The collected samples were washed properly with distilled water to remove dirty materials and were allowed to dry for seven to ten days. The dried materials were ground into fine powder using a grinding machine (Jaipan, India). These powder samples were kept in air tight poly bag for further use. About 200 gm of powdered materials were taken in 1L conical flasks and allowed for soaking the materials with 500 mL of de-ionized water. The conical flasks with sample materials were sealed and kept on orbital shaker (Digital rotator, Taiwan) for continuous shaking at 180 rpm for 24 hours. Sonication was also done with help of an ultrasound sonicator machine (Soniprep 150, U.K) for breaking the cell walls completely. The conical flasks were again kept on orbital shaker for 6 hours and the mixtures were filtered through vacuum pump filtration system using Whatman No.1 filter paper. The filtrated samples were kept into fridge dryer for evaporating the solvent and after 16-24 hours, the solvents were completely evaporated and the extracts became ready for experiment.
2.3 Chemicals and Reagents 1,1-diphenyl-2-picrylhydrazyl, (DPPH, Sigma chemical company, USA), Ethanol and Methanol (Sigma chemical company, USA), Ascorbic Acid (Merck, Germany), De-ionized water, Sodium Chloride (NaCl), Benedicts reagents, Biuret reagents, Wagner’s reagents, Sulfuric Acid (H2SO4), Copper Sulphate (CuSO4) solution, Sodium Hydroxide (NaOH) solution, Acetic anhydride solution, Hydrochloric Acid (HCl), 5% Ferric Chloride (FeCl3) solution, Standard Antibiotic (Amoxicillin), Nutrient medium (Luria broth, LB and Luria agar, LA Medium).
2.4 Ethical clearance This research work was certified by the Institutional Animal, Medical Ethics, Bio-safety and Bio-security Committee (IAMEBBC) for Experimentations on Animal, Human, Microbes and Living Natural Sources, memo no: 57/320/IAMEBBC/IBSc, Institute of Biological Sciences, University of Rajshahi-6205, Bangladesh.
2.5. Phytochemical Screening The bioactive constituents of medicinal plants are widely used for remedying various human diseases and have a prominent role in healing illness. The phytochemical screening of various plants is a great important work in biochemical and pharmaceutical industries for the formulation of the new medicine against various dangerous diseases. In these experiment, the aqueous extracts of G. lucidum was determined for the qualitative estimation of main active constituents like proteins, fats, carbohydrates, alkaloids, flavonoids, tannins, saponins, glycosides etc. The phytochemical analysis of the experimental extract was done followed by standard methods with slide modification described by various scientists.
2.6 Antioxidant activity Assay DPPH (1,1-diphenyl-2-picrylhydrazyl) is a free radical that produces a violet color solution in alcohol and this color is reduced due to the persistence of antioxidant molecules. DPPH was used to evaluate the free radical scavenging activity of plant extract. The DPPH free radical scavenging activity of extracts was evaluated by using the method which was described by Brand Williams et al. [48] with a small modification. In this experiment, 1.0 mL of methanolic solution of G. lucidium and Ascorbic Acid solution (standard) at different concentrations (10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL) were taken in different test tubes. Afterward 1.5 mL of methanolic solution of DPPH (1 mg/25 mL) was added into each of these test tubes and allowed them to incubation for 30 minutes in dark place at room temperature to complete the reaction. The absorbance of reaction mixture was measured at 517 nm using a spectrophotometer (GENESYS 10S UV-Vis, USA) against blank. Ascorbic acid was used as standard to see the level of antioxidant activity of plant extract. The inhibition percentage (%) of plant extract was calculated using following equation,
I % = {(Ao – A1) ÷Ao} × 100%
(Where, A0 is the absorbance of the control and A1 is the absorbance of the extract/standard). The 50% inhibition concentration of the extract, IC50 was calculated using regression line developed from plotting a graph of scavenging percentage against the different concentrations of the extract.
2.7 Brine shrimp lethality test Brine shrimp lethality assay is one of the most important bioassay which is capable of determining a wide range of bioactivity present in plant extracts. It has been used with great success as a primary study of cytotoxicity as well as an important tool for the isolation of biologically active compounds from plant extracts. This assay was carried out to detect the cytotoxicity of plant extracts using brine shrimps (Artemia salina). These shrimps were hatched in a 1L beaker filled with NaCl solution at the concentration of 38 gm/L. In different 10 test tubes, 20 nauplii were taken for each by the help of a glass capillary. Plant extract was added to these 10 test tubes at 10 different concentrations (20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 µg/mL) and incubated these test tubes at room temperature for 24 hours with suitable aeration system. After passing 24 hours, the shrimps were counted as live or dead and LC50 (50% lethal concentration) value was evaluated by using regression line developed from a graph plotting mortality percentage against different concentration.
2.8 Antibacterial activity test The antibacterial activity of aqueous extracts of G. lucidium species was determined using the disc diffusion method and this method was conducted to detect the bacterial susceptibility to aqueous extracts. There were 10 bacterial strains were used for this experiment such as Bacillus subtilis, E. coli, Acinetobacter sp., Staphylococcus aureus, Pseudomonas sp., Acetobacter cloacae, Bravibacillus bravis, Salmonella typhi, RVM (Rhizobium for vigna mungo) and RCA (Rhizobium for cicer arietinum), and all the bacterial cultures were provided by Microbiology Laboratory, Department of Genetic Engineering and Biotechnology, University of Rajshahi-6205, Bangladesh. The stock solution, Luria broth (LB), Luria agar (LA) Medium and culture plates were prepared as per standard protocol. These bacterial strains (100 μL) were inoculated on the surface of solid agar medium in different 10 petridishes. The aqueous extracts from G. lucidium at three different concentrations (50, 100 and 200 μL/disc) were impregnated from 1mg/mL stock solution on paper disc. The agar plates with the bacterial cultures were incubated at 37°C for 24 hours. The inhibition activity of bacterial strains was calculated by measuring the diameter (mm) of clear zone around each disc. The disc of Amoxicillin (10μL/disc) was used as control.
2.9 Statistical analysis All the statistical analyses were accomplished in triplicates and all data are expressed as mean ± SD. The tests of significance were performed by free software named SPSS-16 using one way ANOVA followed by Dunnett Post hoc test compare with control. The significant test were set up at 5% level, 1% level and 0.1% level where P* = 0.05, P** = 0.01 and P*** = 0.001 respectively. The statistical and graphical presentations of data were completed with free software named Microsoft Excel 2007.