Collection of plant sample The leaves of six medicinal plants were collected from different location in and around Sylhet Agricultural University campus and Tilagor Eco Park, Sylhet. On the basis of morphological characteristics and monograph of leaves, flowers and roots, plant samples were identified and confirmed in the department of Plant and Environmental Biotechnology, Sylhet Agricultural University.
Preparation of leaves The leaves of the selected plants were separated using new scissor and dusts were removed by washing under running tap water, finally rinsed with distilled water. The plant samples were dried in sunbathing for 48 hours to remove water content of leaves and the dry leaves were crushed by electric grinder machine to prepare dry leaves powder. Then, the dried powder was stored in plastic container for experimental use.
Preparation of water, ethanol and methanol extract: Water, ethanol and methanol solvents were used to prepare three different extract. For the preparation of aqueous, ethanol and methanol extract of each individual plant sample, 5g of dry leaves powder were homogenized with respective solvent using mortar and pestle for 15 minutes. Respective solvent (Water/Ethanol/methanol) was further added to the blended paste in 250 ml conical flask and adjusted to 200ml with different solvent. After that, the sample containing flask was kept at room temperature for 72 hours. Then it was filtered with markin cloth followed by filtering with Whatman’s No.1 filter paper. The extract was ready for phytochemical testing.
Phytochemical screening test of the plant Terpenoids Test: 5 ml of extract was mixed with 2 ml of CHCl3 in a test tube. 3 ml of concentrated H2SO4 was carefully added to the mixture to form a layer. An interface with a reddish brown coloration was formed for the presence of terpenoids (Trease and evans, 2002). Flavonoids Test: Sodium hydroxide test and shinoda test were used for the presence of flavonoid. Precipitation of yellow colouration formed from the addition of 2ml 10% aqueous sodium hydroxide solution indicates the presence of flavonoids. Yellow colour turns into colorless on addition of dilute hydrochloric acid. In case of shinoda test, the addition of few pieces of magnesium chips along with 2 drops of conc. HCl in extract creates red or pink colour that shows the presence of flavonoids (Peach and Tracey, 1956). Alkaloids Test: A reddish-brown or orange red precipitation after the addition of few drops of Wagner’s reagents or Dragendorff’s reagent is considered as the positive test for alkaloids (Harborne, 1998; Trease and Evans, 2002).
Test for Tannins: Brownish green or a blue-black coloration obtained from the summation of 2-3 drops of 5% ferric chloride solutions to the extract shows the presence of tannin (Ciulci, 1994). Sterols (Salkowski’s test): 2ml of the extract was mixed in 2ml of chloroform. Then 2ml concentrated Sulphuric acid was carefully added to form a layer.A reddish-brown coloration of the interface was formed to show positive results for the presence of sterols.
Phenolic compound test: 3 drops of this freshly prepared mixture produced from equal amount of 1% ferric chloride solution and 1% potassium ferrocyanide was added to extract. After filtering this solution, a positive result shows the formation of a bluish-green color (William and Wenn, 1972) Cardiac Glycosides (keller-kiliani test): Concisely, 2 ml of extract was treated with 1 ml of glacial acetic acid containing one drop of ferric chloride solution pursued by addition of 1 ml of concentrated sulphuric acid. The appearance of purple ring beneath the brown ring while the formation of a greenish ring in the acetic acid layer is considered as indicative for cardiac glycoside (Harborne, 1998). Test for Saponins (Froth test): Briefly, 2.5 ml extract was added to 10 ml of sterile distilled water in a test tube. The test tube was closed with cap and shaken vigorously for about 30 second. It was then allowed to stand for half an hour. Honeycomb froth indicated the presence of saponins (Harborne, 1998). Anthraquinone Test: 1ml benzene in combination with 1ml of 10% ammonia treated with the extract produces pink, red or violet colouration that indicates the presence of anthraquinones (Trease and Evans, 1989). Test for Quinones and Coumarins: Blue green or red precipitations confirms the presence of quinones after adding 10% NaOH into the test sample whereas yellow color developed from the same test indicates the presence of coumarin (Harborne, 1998).
Determination of Total Phenolic Content The total phenolic content (TPC) was determined using gallic acid as a standard, according the method described by Keskin-Sasic et al. (2012) with slight modification. 10mg pure standard gallic acid was mixed with 80ml distilled water in a100ml volumetric flask and final volume (100ml) was adjusted by dropwise addition of distilled water to get the standard gallic acid concentration 0.1mg/ml. Serial Dilutions was performed to prepare varying concentration (12.5, 25, 50, 75, 100 μg/ml) of gallic acid. Blank solution comprises 0.5 ml FCR, 1ml 7.5% Na2CO3 and 5.5 ml distilled water to prepare blank solution. 2N commercially available FCR reagent was diluted at a ratio of 1:10 with distilled water. To prepare 7.5% sodium carbonate solution (Na2CO3), 7.5 g Na2CO3 was mixed well with distilled water and harmonized to make the volume upto 100ml. The reaction mixture was made by mixing 0.5 ml FCR reagent, 1 ml extract or different concentration of standard, 1 ml 7.5% Na2CO3 (after 3 minutes) and 4.5 ml distilled water. It was then kept at room temperature for at 20 minutes to complete whole reaction. The blue colour intensity was recorded at 680 nm against the reagent blank. Finally, the content of total phenolic compounds was determined using a reference curve of gallic acid concentration.
Determination of Total Flavoind Content A standard Aluminium chloride colorimetric protocol was followed to estimate total flavonoid content (Chang et al., 2002). Quercetin solutions of various concentrations were used to make the standard calibration curve. 10mg of quercetin was dissolved in 100ml methanol and then serial dilution was performed to make different concentration (12.5, 25, 50, 75,100 μg/ml) of standard compound quercetin using methanol. To perform the assay for the estimation of total flavonoid content, firstly 3 ml methanol was taken to 1ml extract of different plant samples or 1ml of varying concentration of standard separately in test tubes. Then, 0.2ml of 10% aluminium chloride solution and 0.2ml of 1M potassium acetate solution and finally 5.6 ml distilled water were unified into each separated test tube containing standard or solely different plant extract and mixed well. After filtering all the prepared solutions through Whatman no-1 filter paper, their absorbance was measured. Sample blank was prepared in a similar way by replacing aluminium chloride solution with distilled water. Each test tube was incubated at room temperature for at least 30 minutes to complete reaction. The intensity of yellow color was measured at 420 nm against the suitable blank. Absorbance against concentration was plotted to prepare the calibration curve. Total flavonoid content was expressed as mg of quercetin equivalent (QE) per gram of dry leaves powder (DLP).