Chemicals and reagents
All chemicals and reagents used in this study were of analytical grade. Aluminum chloride, (+)-catechin, potassium iodide, DPPH (2,2-diphenyl-1-picrylhydrazyl), and gallic acid were purchased from Sigma Aldrich, USA. Glacial acetic acid was purchased from Scharlau, Spain. Tannic acid was purchased from Loba Chemie, India. Ethanol, methanol, Fehling A and B, sodium hydroxide, and sodium nitrite were bought from Merck, Germany. Folin-Ciocalteu reagent was purchased from SRL, India. Sodium carbonate, chloroform, and iodine were bought from Alfa Aesar, UK. Hexane and ethyl acetate were bought from Daejung Chemicals and Duksun Pure Chemicals, South Korea, respectively.
Study location and plant materials
The experiments were conducted at the Cell Genetics and Plant Biotechnology Laboratory, Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Dhaka-1342, Bangladesh (23° 53′ 14″ N 90°15′ 56″ E). Initially, the seeds of Doigota (B. orellana) were collected from some cultivated plants in a garden located at Hemayetpur, Dhaka, Bangladesh, during November 2016. The seeds were germinated, and plants were cultivated in the laboratory garden with appropriate conditions and care during January 2017. Seeds, branches, and leaves were collected during March 2019 from these 2 years old cultivated plants and used as the experimental materials in this study.
Preparation of extracts
The plant materials (seed, branch cutting, and leaf) were sun dried for 7 days and then dried in a hot air oven (JSR, Korea) at 50 °C for 7 days. The dried plant materials were powdered in a mechanical grinder. In the extraction process, distilled water, 70% ethanol, and 70% methanol were used as solvents. The powdered materials (10 g) were taken in conical flasks with 70% ethanol, 70% methanol, and distilled water and kept in an orbital shaker for 3 days at room temperature. The extracts were filtered through Whatman No. 1 filter paper, and the filtrates were concentrated using a rotary evaporator at 45 °C. Finally, stock solutions of extracts (10 mg/mL) were prepared using 0.1 N NaCl. For qualitative screening and quantitative determination of phytochemicals, the stock extracts were diluted to the concentration of 1 mg/mL.
Qualitative screening of phytochemicals
The freshly prepared crude extracts were qualitatively tested for the presence of secondary metabolites such as carbohydrates, alkaloids, glycosides, coumarins, tannins, phenols, flavonoids, saponins, and proteins by following the methods described in our previous literature (Ahmed et al. 2018).
Carbohydrates
One milliliter of different extracts was taken into test tubes to which equal volume of Fehling’s A and Fehling’s B were added. The tubes were heated at 65 °C in a water bath for 10–15 min. Redbrick precipitate indicated the presence of carbohydrates (Ahmed et al. 2018).
Alkaloids
One milliliter of different extracts was taken into test tubes, and 4–6 drops of Wagner’s reagent [1.27 g of iodine and 2 g of potassium iodide in 100 mL of water] was added. The radish brown precipitate indicated the presence of alkaloids (Rizk 1982).
Glycosides
One milliliter of different extracts was mixed with 1 mL of glacial acetic acid in test tubes. Then, 5–6 drops of 1% ferric chloride solution were added. Brown color ring produced at the top indicated the presence of glycoside (Peash et al. 2017).
Coumarins
One milliliter of the extract was added with 1.5 mL of 10% NaOH. The chemical reaction produced yellow color and indicated the presence of coumarin (Ugochukwu et al. 2013).
Tannins
One milliliter of extracts was mixed with 1 mL of distilled water, and then, 4–5 drops of 1% ferric chloride were added. Blue and the greenish-black color indicated the presence of gallic tannin and cathecholic tannin respectively (Peash et al. 2017).
Phenols
One milliliter of ethanol was added to 1 mL of extracts. Then, 6–7 drops of 1% ferric chloride solution were added to each tube. Formation of the green or blue or purple color indicated the presence of phenol (Soloway and Wilen 1952).
Flavonoids
Three to four drops of 20% NaOH solution were added to 2 mL of extracts. The intense yellow color formed and become colorless when 4–5 drops of dilute HCl was added. This indicated the presence of flavonoids (Ugochukwu et al. 2013).
Saponins
Two milliliters of distilled water was added to 1 mL of extracts and shaken for 5 min. The presence of 1-cm-thick foam for 10 min indicated the presence of saponin (Kumar et al. 2009).
Proteins
One milliliter of the extract was treated with few drops of concentrated HNO3. The formation of yellow color indicated the presence of proteins (Yadav et al. 2017).
Quantitative determination of phytochemicals
Determination of total phenolic content
The total phenolic content of the extracts was estimated according to the method described previously (Shohael et al. 2006a). 0.1 mL aliquots of extracts and standards of different concentrations were mixed with 2.5 mL deionized water followed by the addition of 0.1 mL (2 N) Folin–Ciocalteu reagent. They were mixed well and allowed to stand for 6 min before 0.5 mL of a 20% sodium carbonate solution was added. The color developed after 30 min at room temperature. The absorbance was measured at 760 nm in a UV-visible spectrophotometer (T60 UV-Visible Spectrophotometer, PG Instruments Ltd., UK). The concentration of total phenol was determined using gallic acid as standard. Total phenolic content was expressed as milligrams of gallic acid equivalent (mg GAE)/g of extract.
Determination of total flavonoid content
Total flavonoid content was determined following the method described previously (Shohael et al. 2006b). Briefly, 0.25 mL of extracts or (+)-catechin standard solution was mixed with 1.25 mL of distilled water, followed by the addition of 0.75 mL of a 5% sodium nitrite solution. After 6 min, 0.15 mL of a 10% aluminum chloride solution was added, and the mixture was allowed to stand for a further 5 min and then 0.5 mL of 1 M sodium hydroxide was added. The mixture was brought to 2.5 mL with distilled water and mixed well. The absorbance was measured immediately at 510 nm in a T60 UV-visible spectrophotometer. The concentration of total flavonoids was determined using (+)-catechin as standard. The concentration of flavonoids was expressed as milligrams of catechin equivalent (mg CE)/g of extract.
Determination of total tannin content
Total tannin content was determined using the Folin-Ciocalteu reagent as described previously (Ahmed et al. 2018). Briefly, 0.1 mL of the sample extract is added with 7.5 mL of distilled water. Then, 0.5 mL of Folin-Ciocalteu reagent and 1 mL of 35% sodium carbonate solution were added. The total volume was adjusted to 10 mL by adding distilled water. The mixture was incubated at room temperature for 30 min, and the absorbance was measured at 725 nm in a T60 UV-visible spectrophotometer. Blank was prepared with water instead of the sample. A set of standard solutions of tannic acid was read against a blank. The concentration of total tannin was determined using tannic acid as standard. The concentration of tannin was expressed as milligrams of tannic acid equivalent (mg TAE)/g of extract.
DPPH free radical scavenging activity
DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activity was carried out using the method described previously (Kabir et al. 2016). Different concentrations (800, 400, 200, 100, 50 μg/mL) of extract were dissolved in methanol. Three milliliters of a 0.004% DPPH solution was added to each test tube. The mixture was incubated for 30 min in dark condition. The absorbance was measured at 517 nm in a T60 UV-visible spectrophotometer. The percent inhibition activity was calculated from [(AC − AS)/AC] × 100, where AC is the absorbance of the control and AS is the absorbance of the sample.
Antibacterial assay
In vitro antibacterial assay was performed by using the disc diffusion method. Pure cultures of bacteria isolated from clinical specimen were obtained from the Department of Microbiology, Jahangirnagar University. The organisms were Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. The pure culture contains the test organisms on solid media. They were transferred to the sterile nutrient broth and incubated for 18 h at 37 °C in an incubator (HYSC, Korea). The optical density of the culture was determined using a T60 UV-visible spectrophotometer at 600 nm. An inoculating loop was used to aseptically transfer the bacterial strains into 5 mL of media, which was used as a starter culture. The Whatman No. 1 filter paper was used for the preparation of discs which were 5 mm in diameter. The disks were impregnated with different concentrations (10 mg/mL, 7.5 mg/mL, 5 mg/mL, and 2.5 mg/mL) of extracts. Each piece of the disc was impregnated with 50 μL of extract. So the final concentrations of extracts on the discs were 500, 375, 250, and 125 μg/disc. The prepared working discs were completely air-dried inside of the class II biological safety cabinet (ESCO, Singapore) and placed on the nutrient agar medium plate. The nutrient agar medium plate was spread with 100 μL of starter bacterial culture before the placement of the disks. The culture plates were incubated at 37 °C in an incubator (HYSC, Korea). After incubation, they were examined for the inhibition of growth. The growth inhibition was measured in terms of zones of inhibition. Zones of inhibition were measured in millimeter-scale using a digital slide caliper. OxoidTM Streptomycin antimicrobial susceptibility discs (25 μg) (ThermoFisher Scientific, USA) were used as positive control, and sterile distilled water was used as the negative control.
Statistical analysis
Significant differences among mean values were compared by Tukey’s honestly significant difference (HSD) test at a level of significance of P < 0.05. All data were displayed as the mean ± standard error of the mean (SEM) at least three independent biological replications. The statistical analysis was performed using Statistical Package for Social Science software (SPSS, version 16.0, IBM Corporation, NY).