2.1 Plant materials
The whole plant of Drymaria diandra was collected from the wetlands of Sundarharaicha morang, during March 2018. Healthy and mature plants were selected for the study. The taxonomic identification of plant was authenticated by Dr. T. P. Gautam of Department of Botany, Mahendra Morang Adarsh Multiple Campus, Biratnagar and deposited to the department as a sample herbarium. The plants were thoroughly washed with tap water followed by second distilled water to remove the dirt and specks of dust.
2.2 Drying
The cleaned plants were cut into small pieces and were left in shade to dry for 10 days. Afterwards, they were dried in hot air oven at 40 °C for 2 h to remove the equilibrium moisture before the extraction process.
2.3 Extraction
Extraction was performed by Soxhelt extraction technique [11]. Whole plants were pulverized by using a mechanical grinder, which was later sieved through mesh size 80 to get the powder of uniform size. Around 8 g of the powder was packed in a thimble of filter paper. The apparatus was then assembled and the extractions were carried out using 250 ml each of methanol, hexane, and methanol-water (1:1) as solvent (Fig. 1). The temperature was maintained at 35 °C for methanol and hexane, and 60 °C for methanol-water (1:1). The extraction was continued for 10 h for methanol, 5 h for hexane, and 16 h for methanol-water (1:1) following the color of the solvent collected in the thimble chamber. Ten milliliters each of concentrated extract was kept for phytochemical screening and remaining extracts were dried. The dried extract was used for antioxidant and antibacterial analysis.
2.4 Phytochemical screening
Phytochemical screenings were carried out for hexane, methanol-water, and methanol soluble fractions as per the standard methods [12,13,14,15], and following tests were performed:
Test for alkaloids
- a.
Mayer’s test
To 2 ml of each extract, few ml of 2N HCl along with few drops of Mayer’s reagent was added. Gelatinous white precipitation confirms the presence of alkaloids.
- b.
Wagner test
To 2 ml of extract few drops of Wagner reagent was added, a reddish brown precipitation observed confirms the presence of alkaloids.
Test for coumarins
Few drops of FeCl3 were added in 2 ml of sample; yellow coloration confirms the presence of coumarins.
Test for saponin
Froth flotation test: 2 ml of sample was added in a test tube with few ml of water, a froth observed and persisted on constant shaking confirms the presence of saponin.
Test for tannins and phenolic compound
To a 2 ml of extract few drop of 5% FeCl3 solution was added, blue black precipitation confirms the presence of tannins and phenolic compound.
Test of cardiac glycoside
To 2 ml of plant extract, 2 ml of glacial acetic acid containing 1 drop of ferric chloride solution and 1 ml of concentrated H2SO4 was added. Appearance of a brown ring indicates the presence of cardiac glycoside.
Fehling test: 1 ml each of Fehling’s A and Fehling’s B solutions was mixed and boiled for 1 min and equal volume of test solution was added. The whole solution is heated in a boiling water bath for 5–10 min. Formation of brick red ppt. confirmed the presence of carbohydrate.
Test for anthraquinone
In 2ml plant extract, 3 ml of benzene and 5 ml of 10% NH3 were added. Appearance of pink, violet, or red coloration in ammonical layer indicates the presence of anthraquinones.
Test for glycoside (killer killiani test)
To 2 ml of extract, 1 ml of glacial acetic acid, few drop of FeCl3, and few drops of concentrated H2SO4 were added. Green/blue precipitation indicates the presence of glycoside.
Test for emodins
To 2 ml of extract, 2 ml of NH4OH and 3 ml of benzene were added. Red coloration indicates the presence of emodins.
Test for phlobatannins
To 2 ml of extract, 2 ml of 1% HCl was added and heated. Red precipitate indicates the presence of phlobatannins.
Test for terpenoids
To 2 ml of extract, 2 ml of chloroform and 2 ml of concentrated H2SO4 was added. A reddish brown coloration indicates the presence of terpenoids.
Test for protein
To 2 ml of extract, few drops of concentrated H2SO4 was added on it. White precipitate indicates the presence of protein.
Test for steroid (Salkowski test)
To 2 ml of extract, 2 ml of CHCl3 and 2 ml of conc. H2SO4 were added on it. A reddish brown ring at the junction indicates the presence of steroid.
2.5 Heavy metal concentration test
The quantification of metals in the plant extract was done by flame AAS technique equipped in ICE 3000 series atomic absorption spectrometer. For this, 1.0 g of dried sample was taken in a 250-ml conical flask, and 5 ml of conc. HNO3 (GFS Chemicals Inc., Columbus, 69%) was added slowly. The mixture was heated on the hot plate till the brown fumes disappeared yielding the white fumes. Water was added to make the solution, and it was then filtered in a 50-ml volumetric flask. Finally, the volume was adjusted to 50 ml by adding triple distilled water up to the mark [16, 17]. This filtrate was then introduced in flame AAS for the detection of metals.
2.6 Antioxidant activity study
The antioxidant activity of methanol extract was measured by DPPH free radical scavenging assay and it was carried out by Asadujjaman et al. method [18]. The sample solution was prepared in methanol at five different concentrations (25, 50, 75, 100, and 150 μg/ml). One-milliliter sample solution of each concentration was thoroughly mixed with 2-ml DPPH solution and shaken well for complete interfusion. The solution was incubated in dark for 30 min at room temperature for complete reaction with DPPH and the absorbance was measured at 517 nm by a UV/Vis. spectrophotometer against blank. The free radical scavenging activity of sample was calibrated in terms of % age inhibition of DPPH radical and compared with ascorbic acid as standard. The % age inhibition of DPPH radical is
$$ I\%=\frac{A_o-A}{A_o}\times 100 $$
where I represents DPPH inhibition (%), Ao is the absorbance of control sample, and A is the absorbance of a tested sample at the end of the reaction. The antioxidant activity of the sample was also expressed in terms of IC50 value, which represents the concentration of sample required to inhibit 50% formation of DPPH radical [19,20,21].
2.7 Antibacterial activity study
Antibacterial activity study was used to examine the antibacterial potency of the extracted plant sample. The antibacterial study was performed in the laboratory of the Department of Microbiology at Mahendra Morang Adarsh Multiple Campus, Biratnagar. The pathogenic bacteria used in the study were collected from the biochemistry laboratory of Suraksha Hospital, Biratnagar, Nepal.
The agar diffusion test (Kirby–Bauer (KB) antibiotic testing, also called disc diffusion antibiotic sensitivity testing) is a well-known standard method for testing the antibacterial sensitivity of bacteria. The samples (methanol, hexane, and methanol-water (1:1) extract) were tested in vitro against some gram positive and gram negative human pathogenic bacteria; namely Escherichia coli, Proteus vulgaris, and Staphylococcus aureus. Well-sterilized Whatman paper discs of 5-mm size were used as antibiotic assay discs. The discs were made by punching machine and well dried. Firstly, all the glassware, paper disc, and the Mueller-Hinton agar (MHA) media were sterilized. Twenty-five milliliters of MHA was carefully poured into each petri discs and allowed to rest for an hour in the sterilized zone for solidification. Freshly revived bacterial culture was wiped all over the media using sterile swab and the discs were stuck over it. Consequently, these discs were supplied with test compounds at three different concentrations (150, 75, and 37.5 μg/μl in DMSO). One of the disc was soaked in DMSO and used as the solvent control, while amikacin (30 μg/disc) was used as positive control. The whole process was carried out under UV laminar flow to eliminate the bacterial contamination, and the loaded discs were dried within the laminar flow chamber. The inoculated plates were incubated at 37 °C for 24 h. Also, the diameter of the zone of inhibition was measured by using anti-biogram zone measuring scale. The results were interpreted using the chart of NCCLS.
