Survey site selection
The region selected for this research purpose has an area of 6116.13 km2 and is located in between 22°27′ and 23°44′ north latitudes and in between 91°56′ and 92°33′ east longitudes. Approximately 5,08,182 people live here and the main indigenous communities that live here are Chakma, Monipuri, Tripura, Khumi, Marma, Tanchangya, Santal, Mro, and many more.
Ethics and consent to participate
The purpose of our study was elaborately explained to each informant and verbal consent was taken to avoid any misunderstanding. We have also taken permission from the local authority to conduct the survey. As our study only dealt with the medicinal plants with no intention to conduct any trials on human, formal institutional consent for this study is not required. In addition, Bangladesh Medical Research Council Ethical Guidelines for Conducting Research Studies Involving Human Subjects deemed ethics approval unnecessary for this kind of study on page 39, section 10.5.2.
Data analysis
All the data were listed alphabetically and ordered by the plant’s scientific name, local name, plant part used, name of the disease, and mode of preparation of the plant. Also, the data were analyzed in the “IBM SPSS Statistics 25” software and graphical presentations were made.
Antibacterial activity test
Test organisms
The pathogenic test organisms used in this assay are as follows: Salmonella typhi, Streptococcus pneumoniae, Staphylococcus aureus, Shigella flexneri, and Escherichia coli, all are known diarrhea-causing bacteria. The bacterial cultures were obtained from the Biotechnology and Molecular Biology Laboratory of the Department of Mathematics and Natural Sciences at BRAC University and the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR, B).
Sample collection and processing
With the help of some local practitioners, parts of the Heptapleurum hypoleucum plant were taken and identified. The stem of the plant was used as the sample for this research work, obtained from the rural areas of Rangamati district. The stem was washed, cleaned, and air dried under open light for several days. As soon as it dried, it was mashed into powder.
Preparation of extracts using different solvents
Ethanolic extraction
Ten grams of powdered sample was dissolved in 100 ml of absolute ethanol in a conical flask, covered with aluminum foil and then kept at 37 °C in a shaker incubator at 120 rpm for 24 h. Using an autoclaved filter paper, the filtrate was collected slowly in a conical flask and then evaporated using a rotary evaporator till the final volume was reduced to one-fourth of the original volume of the solvent used. This concentrated extract solution was poured on a sterile petri dish lid and kept in the incubator at 55 °C for 20 min. Finally, a sticky semi-solid extract appeared on the surface of the plate when all the solvent was evaporated. The extract was collected and stored in a McCartney bottle that was autoclaved and weighed. The extract inside the bottle was weighed, recorded, and the exact amount of the extract was calculated by subtracting the mass of the empty bottle. Then the bottle was labeled and stored at 4 °C in the refrigerator.
Methanolic extraction
Ten grams of powdered sample was dissolved in 100 ml of absolute methanol in a conical flask, covered with aluminum foil and then kept at 37 °C in a shaker incubator at 120 rpm for 24 h. The filtrate was collected slowly in a conical flask using an autoclaved filter paper. It was then evaporated using a rotary evaporator till the final volume was reduced to one-fourth of the original volume of the solvent used. Then the concentrated extract solution was poured on a sterile petri dish lid and kept in the incubator at 55 °C for 20 min. Finally, a sticky semi-solid extract appeared on the surface of the plate when all the solvent was evaporated. The extract was collected and stored in a McCartney bottle that was autoclaved and weighed. The extract inside the container was weighed, recorded, and the exact amount of the extract was calculated by subtracting the mass of the empty bottle. Then the bottle was labeled and stored at 4 °C in the refrigerator.
Aqueous extraction
Ten grams of powdered sample was weighed and mixed with 100 ml of distilled water in a conical flask, covered with aluminum foil. Then kept at 37 °C in a shaker incubator at 120 rpm for 24 h. Using an autoclaved Whatman No.1 filter paper, the filtrate was collected slowly in a conical flask and then stored inside autoclaved falcon tubes at 4 °C in the refrigerator.
Preparation of extract solution for antibacterial activity test
The following formula was used to determine the amount of solvent to be added for making the extract solution for antibacterial activity test.
$$ \mathrm{Amount}\ \mathrm{of}\ \mathrm{solvent}\ \mathrm{to}\ \mathrm{be}\ \mathrm{added}=\frac{100\times \mathrm{amount}\ \mathrm{of}\ \mathrm{extract}\ \mathrm{obtained}}{\mathrm{amount}\ \mathrm{of}\ \mathrm{plant}\ \mathrm{powder}\ \mathrm{used}} $$
Agar well diffusion
Antibacterial activity of aqueous and solvent extracts was determined based on agar well diffusion method developed by Clinical and Laboratory Standards Institute, USA with some modifications depending on our laboratory conditions [19].
Plates of bacterial strains were taken in the laminar hood. A loop was sterilized in the Bunsen flame and was used to scrape off the bacteria and dipped into the test tubes containing saline solution (each containing 9 ml of NaCl) to make a suspension. The test tubes were vortexed and the turbidity of the suspension was visually compared with the 0.5% MacFarland standard solution in order to keep the number of bacteria in the saline suspension within a given range for standardizing the lawn culture of antimicrobial tests. Then, an autoclaved cotton swab was dipped into the suspension and pressed against the inner walls of test tubes to remove excess liquid before taking them out. The cotton swab was rubbed horizontally across the surface of the labeled Mueller Hinton Agar (MHA) plates to conduct the lawn culture of the bacterial strains. A cork borer was heated to sterilize, cooled, and then pressed onto the MHA plates to create the required number of wells on the quadrants of the agar. After that, each well was labeled and filled with 60 μL of diluted methanolic, ethanolic, and aqueous extracts, respectively. Different antibiotic disks were used as a positive control and placed onto one quadrant. Then the MHA plates were kept in the incubator for 24 h at 37 °C and the results were observed and recorded the next day.
All the tests were conducted 3 times to obtain the average value of zones of inhibition. The zones were measured using a millimeter scale and then the activity index for each extract was calculated.
The following formula was used for calculating the activity index:
$$ \mathrm{Activity}\ \mathrm{index}=\frac{\mathrm{zone}\ \mathrm{of}\ \mathrm{inhibition}\ \mathrm{of}\ \mathrm{plant}\ \mathrm{extract}}{\mathrm{zone}\ \mathrm{of}\ \mathrm{inhibition}\ \mathrm{of}\ \mathrm{antibiotic}\ \mathrm{disc}} $$
Biochemical assays and phytochemical analysis
The plant extracts in methanolic, ethanolic, and aqueous solutions were assessed for the existence of the phytochemical compounds based on the following standard methods [20,21,22,23].
Tests for alkaloids
The methanolic extract was diluted in an acidic solution of HCl. This test solution was used for the detection of alkaloids using various reagents.
Hager’s test: 1 ml of extract was carefully mixed with 3 drops of freshly prepared Hager’s reagent in a test tube. The formation of yellow precipitates represents a positive result and the presence of alkaloids in the extract.
Wagner’s test: 1 ml of extract was mixed in a test tube with 3 drops of Wagner’s reagent prepared beforehand. The formation of brown precipitate will indicate the presence of alkaloids.
Dragendraff’s test: 2 ml of extract was taken in a test tube with 0.2 ml diluted HCl and 1 ml of Dragendraff’s reagent and left for a few minutes. A positive result is indicated by the presence of an orange-brown precipitate.
Test for steroidal compounds
Salkowaski’s test: 0.5 g of the extract was dissolved in 2 ml chloroform in a test tube. Concentrated sulfuric acid was carefully added on the wall of the test tube to form a lower layer. A reddish-brown color at the interface will indicate the presence of a steroid ring.
Test for phenolic compounds
Equal amounts of 1% ferric chloride solution and 1% potassium ferrocyanide were mixed. Then, 3 drops of this freshly prepared mixture were added to 2 ml extract. The formation of a bluish-green color will represent a positive result.
Test for flavonoids
Reaction with sodium hydroxide: 2 ml diluted NaOH solution was added to 3 ml of extract. The mixture was inspected for the production of yellow color, which is considered positive.
Tests for saponins
Froth test: 0.5 g of the extract was dissolved in 10 ml distilled water. The test tube was stoppered and then shaken vigorously for 30 s. It was then allowed to stand for 30 min. Formation and retention of honey-comb froth on the surface for 30 min is considered positive for saponins.
Tests for tannins
Lead acetate test: 5 ml of extract and a few drops of freshly prepared 1% lead acetate were dissolved together. The formation of a yellow precipitate is considered a positive result.