Collection, authentication, and extraction process of plant sample
The whole plant of Begonia barbata was collected from Moulvibazar Hill Tracts, Bangladesh, and was taxonomically identified (DACB-43432) by a scientific officer in Bangladesh National Herbarium. The collected plant samples were washed with water followed by air drying for several days. The dried plant parts were then turned into a fine powder. The powdered sample was macerated in methanol for 7 days. The filtrate was concentrated to dryness using a rotary evaporator. Following the technique of solvent-solvent partitioning protocol [13], the dried methanol extract of B. barbata (MEBB) was fractionated with petroleum-ether, dichloromethane, and ethyl acetate successively to separate the compounds in extracts according to their polarity. All these plant samples, PEF, DCMF, and EAF were analyzed for the assessment of the pharmacological properties of B. barbata.
Chemicals and reagents
Ascorbic acid, streptokinase, acetylsalicylic acid, morphine, diclofenac sodium, glibenclamide, fluoxetine, thiopental sodium, and loperamide were collected from Square Pharmaceuticals Ltd. and Beximco Pharmaceuticals Ltd., Bangladesh. All reagents and chemicals such as gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), tert-butyl-1-hydroxytoluene (BHT), vincristine sulfate, acetic acid, and Tween 80 were of analytical grade.
TPC (total phenolic content) analysis
The amount of phenolic compound was determined for each plant sample using Folin-Ciocalteu method [14]. The absorbance was recorded using the UV-Vis spectrophotometer at 760 nm. Gallic acid solution was prepared to construct a calibration curve. The amount of phenolic compound was expressed as milligram of gallic acid equivalent (GAE)/g of dry extract.
Antioxidant activity assay
The antioxidant activity of plant extract was estimated by DPPH scavenging assay by the protocol of Brand-Williams et al. [15]. Briefly, 3.0 ml solution of DPPH in methanol (20 μg/mL) was mixed with plant samples at different concentrations (500.0 to 0.977 μg/mL). The reaction mixture was vortex and was left in the dark for 30 min. Then, a UV-Vis spectrophotometer was used to measure the absorbance value of each mixture at 517 nm. The percentage of inhibition (I%) was measured against blank:
$$ \left(I\%\right)=\frac{A_{blank}-{A}_{sample}}{A_{blank}}\times 100\% $$
where A is the absorbance for each group. After that, the IC50 value (50% inhibition) for each plant sample was estimated from a graph of % inhibition of DPPH scavenging vs. concentration of the test materials.
Thrombolytic activity
The clot lysis ability of the extract was tested following an established protocol [16] where streptokinase (SK) was used as standard. One milliliter of human blood was transferred to clean Eppendorf tubes which were then kept at room temperature until blood clot forms. The serum was finally removed from the clot. The weight of the clot formed in Eppendorf tube was obtained by subtracting the weight of the tube alone from the weight of clot-containing tube. The plant sample was added individually to the blood clot in each tube, followed by incubation at room temperature for 90 min to examine the clot lysis. The free liquid after incubation was decanted and the tubes were once more weighted to monitor the weight variation before and after clot lysis. The weight variation which was expressed as percentage of clot lysis is calculated by following the underneath equation:
$$ \%\kern0.5em \mathrm{clot}\kern0.5em \mathrm{lysis}=\frac{Weight\kern0.34em of\kern0.17em the\kern0.34em lysis\kern0.17em clot}{Weight\kern0.17em of\kern0.17em clot\kern0.17em before\kern0.17em lysis}\times 100\% $$
Membrane stabilizing effect
The test materials were subjected to evaluate the membrane-stabilizing activity against heat- and hypotonic solution-induced hemolysis of human blood [17].
Animals
Swiss albino mice were kept under regular laboratory environment with 12 h dark and 12 h light cycle. The study protocol was permitted by the Institutional Animal Ethics Committee [18].
Animal experimental design
For in vivo experiment, albino mice were divided into the following groups with three mice each. Group I serving as negative control received the vehicle (1% Tween 80 + normal saline), group II (positive control) was treated with standard drug for the respective experiment. Groups III and IV received PEF (200 and 400-mg/kg); groups V and VI received DCMF (200 and 400 mg/kg); and group VII and VIII were loaded with EAF (200 and 400 mg/kg).
Analgesic activity
Tail immersion test
Central analgesic activity of the plant extracts was determined by the tail-immersion procedure in Swiss albino mice [19]. For this test, the animals were treated orally with the soluble fractions of MEBB at a dose of 200 and 400 mg/kg body weight. After treatment, the tail of each of the mice up to 5 cm was then dipped into a water bath maintained at 55 ± 0.1 °C. The time taken for the mouse to withdraw the tail in seconds was considered as the pain reaction time (PRT) and was measured at 0, 30, 60, and 90 min following the loading of the test samples. Finally, the percentage of time elongation of tail immersion was calculated in respect to standard morphine by the equation below.
$$ \%\mathrm{time}\ \mathrm{elongation}=\frac{T_{\mathrm{test}}-{T}_{\mathrm{control}}}{T_{\mathrm{control}}}\times 100\% $$
Here Ttest is the pain reaction time for the test group and Tcontrol is the pain reaction time for the control group.
Acetic acid writhing test
The peripheral analgesic activity of B. barbata was evaluated by acetic acid abdominal constriction tests [20, 21]. Like the tail-immersion test, both standard diclofenac sodium and plant samples were administered to the experimental animals by oral route. Forty minutes after the administration of all treatments, 1% acetic acid was injected intraperitoneally to each animal to induce the writhing (abdominal constrictions). Ten minutes after acetic acid injection, the number of writhing was counted for 5 min for each mouse. The plant sample possessing analgesic activity will decrease the number of writhing in animals. The percentage of writhing inhibition of the treated group was then calculated by using the following equation:
$$ \%\mathrm{inhibition}\ \mathrm{of}\ \mathrm{writhing}=\frac{N_{\mathrm{Control}}-{N}_{\mathrm{Test}}}{N_{\mathrm{Control}}}\times 100\% $$
where N is the mean number of writhing for each group.
Hypoglycemic effect
Glucose tolerance test is employed to evaluate the hypoglycemic effect of B. barbata in mice model [22]. Briefly, 30 min after oral administration of plant extract/drug, a 10% glucose solution was given orally to mice of all test groups. Then blood glucose level was recorded by glucometer for each animal prior to administration of extract/drug (at 0 min) and then at 1, 2, and 3 h after the glucose load. The percent reduction in blood glucose level of B. barbata can be estimated by the equation below:
$$ \%\mathrm{reduction}\ \mathrm{in}\ \mathrm{blood}\ \mathrm{glucose}=\frac{B{G}_{\mathrm{control}}-B{G}_{\mathrm{test}}}{B{G}_{\mathrm{control}}}\times 100\% $$
where BG is the average blood glucose level for each group.
Anti-diarrheal effect
The plant samples were also subjected to verify the anti-diarrheal effect in mice by castor oil-induced diarrhea model [23, 24]. Briefly, 30 min after the administration of the respective doses and treatments, 0.5 ml castor oil was injected to induce diarrhea in each mouse. Anti-diarrheal effect of the plant sample was observed for 4 h. The percent reduction in frequency of defecation by the test samples was determined according to the equation:
$$ \%\mathrm{inhibition}\ \mathrm{of}\ \mathrm{defecation}=\frac{D_{\mathrm{control}}-{D}_{\mathrm{test}}}{D_{\mathrm{control}}}\times 100\% $$
where D is the mean number of diarrheal episode in each group.
Anxiolytic effect
Thiopental sodium-induced sleeping time test was conducted to estimate the anxiolytic potential of B. barbata in albino mice [25]. Thirty minutes after loading of plant samples and standard fluoxetine orally, each mouse was induced to sleep by thiopental sodium. Then, the anxiolytic effect was evaluated through the estimation of percent inhibition of sleeping time using the equation:
$$ \%\mathrm{inhibition}\ \mathrm{of}\ \mathrm{sleeping}\ \mathrm{time}=\frac{T_{\mathrm{control}}-{T}_{\mathrm{test}}}{T_{\mathrm{control}}}\times 100\% $$
where T is the average sleeping time in each group.
