Collection and extraction of plant material
Leaves with barks of the plant Campsis radicans L. were collected from Curzon Hall, University of Dhaka, Bangladesh during the month of December 2016. The plant was taxonomically identified by the experts of Bangladesh National Herbarium, Mirpur, Dhaka (DACB; Accession No- 43433). The properly cleaned leaves with barks were air-dried and ground to a coarse powder. The powdered materials were macerated in methanol for few days and finally, filtered through Whatman filter paper number 1. The filtrate was concentrated using a rotary evaporator (Heidolph, UK) at low temperature (40 °C) and pressure. About 5 g of the concentrated extract designated as the crude methanol extract of Campsis radicans was subjected to solvent-solvent partitioning following the modified Kupchan method [13] to petroleum-ether (PESF), dichloromethane (DMSF) and ethyl acetate (EASF) soluble fractions. Then the crude extract and its Kupchan fractions were studied separately for the evaluation of their biological activities.
Drugs and reagents
All regaents and chemicals such as 1,1-diphenyl-2-picrylhydrazyl (DPPH), tert-butyl-1-hydroxytoluene (BHT), gallic acid, acetic acid and Tween 80, castor oil were of analytical grade. Ascorbic acid, streptokinase, acetyl salicylic acid, morphine, diclofenac sodium, glibenclamide, fluoxetine, thiopental sodium, castor oil and loperamide were obtained as gifts from Square Pharmaceuticals Limited, Kaliakair, Dhaka and Beximco Phamraceuticals Limited, Tongi, Dhaka.
In vitro studies
Determination of total phenolic content
The total phenolic content was determined by the Folin-Ciocalteau’s spectrophotometric method [14, 15]. Here, gallic acid was used as the standard and different concentrations of gallic acid solution were prepared in the range of 0.391 μg/ml and 100 μg/ml. 1.0 ml plant extract (2 mg/ml) was mixed with Folin-Ciocalteau’s phenol reagent, 2.0 ml of 7.5% Na2CO3 solution was then added to the reaction mixture followed by the addition of 13 ml of deionized distilled water. The mixture was allowed to stand in the dark for 20 min. The absorbance was recorded at 760 nm. A calibration curve was prepared from the data of gallic acid solution and it was used to determine total phenolic content of the plant extracts. The results were expressed as mg of gallic acid equivalent (GAE) per g dry weight extract.
Free radical scavenging by DPPH method
The DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of PESF, DMSF and EASF on the stable DPPH radical was estimated by the method established by Brand-Williams et al [16]. Briefly, plant extract was dissolved in methanol to produce stock solution of 1.0 mg/ml. Serial dilution was performed on the stock solution to produce solutions of 0.977 μg/ml to 500 μg/ml. To 2 ml of each diluted solution of plant extract, 3 ml of DPPH working solution (prepared in methanol, 20 μg/ml conc.) was added and then incubated in the dark for 30 min. The absorbance was then measured in a UV-Vis spectrophotometer (Shimadzu, Japan) at 517 nm. % Inhibition of DPPH was calculated and IC50 value (50% inhibition concentration) was calculated for each extract by plotting % inhibition of DPPH against concentration. Tert-Butyl-1-hydroxytoluene (BHT) and ascorbic acid (AA) were used as reference standards.
In vitro thrombolytic activity
The thrombolytic activity of PESF, DMSF and EASF was evaluated following the method developed by Prasad et al [17] using streptokinase (SK) as standard.
In vitro membrane stabilizing activity
The membrane stabilizing activity of the plant samples was evaluated by the method described in literature [18] which involved heat- and hypotonic solution-induced hemolysis.
Test animals
For in vivo biological tests, 4–5 weeks old Swiss albino mice of either sex were collected from International Center for Diarrheal Disease and Research, Bangladesh (ICDDR, B). They were kept under controlled room temperature (24 ± 2 °C; relative humidity 60–70%) in a 12 h light-dark cycle and fed ICDDR, B formulated rodent food and water ad libitum. Food was withdrawn 12 h before and during the experiment. The experiments were accomplished according to the guide for the care and use of laboratory animals. The protocols for conducting the experiments on the animals were approved by the institutional ethical committee [19].
Study design for in vivo studies
Swiss albino mice were randomly assigned to eight groups of three animals each for the different experimental models. The first group served as negative control receiving 1% Tween 80 in normal saline (10 ml/kg body weight). The second group served as positive control and was given standard drugs for the respective experiment. The third and fourth groups received PESF (200- and 400 mg/kg body weight), the fifth and sixth groups received DCM (200- and 400 mg/kg body weight), and finally seventh and eighth groups received EASF (200- and 400 mg/kg body weight) of crude methanol extract of C. radicans, respectively.
In vivo studies
Central analgesic activity
Tail immersion procedure [20] was used to determine the central analgesic activity of the plant extracts. Morphine (2 mg/kg body weight) solution prepared in normal saline was administered subcutaneously as the standard. The organic soluble fractions of the methanol of Campsis radicans (200 - and 400 mg/kg body weight) and the negative control (1% Tween 80 in normal saline) were administered orally. For the test, 1–2 cm of tail of the mouse was dipped into a water bath containing warm water maintained a constant temperature of 55 °C. The time taken by the mouse to deflect its tail was recorded before (0 min) and at 0, 30, 60, and 90 min after administration of the treatments. The % time elongation was determined using the following equation and this was compared with the standard to evaluate central analgesic activity. The higher the elongation percentage of the group the greater is the group’s central analgesic activity.
$$ \% time\ elongation=\frac{T_{test}-{T}_{control}}{T_{control}}\times 100\% $$
Here Ttest is the average time of tail deflection in the test group and Tcontrol is the average time of tail deflection in control group.
Peripheral analgesic activity
Acetic acid-induced writhing test was performed as reported previously [21] to determine the peripheral analgesic activity of C. radicans in albino mice. Plant extracts (200 - and 400 mg/kg body weight) as well as the negative control (1% Tween 80 in normal saline) and the standard diclofenac sodium (50 mg/kg body weight) were administered to the albino mice orally. About 40 min after administration of the sample, 1% acetic acid (0.1 ml/10 g body weight) was administered to the experimental animal by intra-peritoneal route to induce writhing. Number of abdominal constrictions i.e. writhes were counted for each group of mice starting from 5 min after the injection of acetic acid up to 10 min and % inhibition of writhing was determined according to the following formula to evaluate peripheral analgesic activity:
$$ \% inhibition\ of\ writhing=\frac{W_{test}-{W}_{control}}{W_{control}}\times 100\% $$
Here, Wtest refers to the average number of writhing in the test group and Wcontrol refers to that in the control group.
Hypoglycemic activity
Hypoglycemic activity of C. radicans was determined in albino mice according to the method described in literature [22]. The soluble fractions of crude methanol extract of C. radicans (200- and 400 mg/kg body weight) and standard glibenclamide (10 mg/kg body weight) were administered to the mice orally. 1 h after the administration, all groups was treated with 10% glucose solution (2 g/kg body weight). At 1, 2 and 3 h of glucose administration, the blood was collected from tail vein of mice and blood glucose level was measured using a standard glucometer. The hypoglycemic activity of C. radicans was reflected by % reduction of blood glucose level calculated using the formula shown below:
$$ \% reduction\ in\ blood\ glucose=\frac{B{G}_{control}-B{G}_{test}}{B{G}_{control}}\times 100\% $$
Here, BGtest refers to the average blood glucose level in the test group and BGcontrol refers to average blood glucose level in the control group.
Anti-diarrheal activity
Anti-diarrheal activity of C. radicans was evaluated by the method of castor oil-induced diarrhea in mice [23]. The test groups received the soluble fractions of crude methanol extract of C. radicans at the doses of 200- and 400 mg/kg body weight while the positive control group received the standard loperamide (50 mg/kg body weight) and the negative control group received vehicle (1% Tween 80 in normal saline) at the dose of 10 ml/kg body weight orally. The number of diarrheal feces excreted by the mice was observed for up to 4 h and % reduction in diarrhea by the plant extract was calculated using the following formula to evaluate antidiarrheal activity:
$$ \% reduction\ in\ diarrhea=\frac{D_{control}-{D}_{test}}{D_{control}}\times 100\% $$
Here, Dtest refers to the average number of diarrheal defecation in the test group and Dcontrol refers to that in control group in the same duration.
CNS antidepressant activity
The CNS antidepressant activity of C. albicans was evaluated by thiopental-induced sleeping time test on Swiss albino mice [24]. The soluble fractions of crude methanol extract of C. radicans (200- and 400 mg/kg body weight) and standard fluoxetine (30 mg/kg body weight) were administered orally. After passing 30 min, thiopental sodium (25 mg/kg body weight) was administered to each mouse subcutaneously to induce sleep. Then, the sleeping time for each mouse was recorded and the CNS antidepressant activity was evaluated by determining % inhibition of sleeping time according to following formula:
$$ \% inhibiti\mathrm{o}n\ of\ sleeping\ time=\frac{T_{control}-{T}_{test}}{T_{control}}\times 100\% $$
Here Ttest is the average sleeping time in the test group and Tcontrol is that in control group.
Statistical analysis
All values were determined as mean ± standard error of mean (SEM) whenever possible. When applicable, data was statistically evaluated by means of Dunnett’s t-test using GraphPad Prims (version 7) and the p values < 0.05 was considered statistically significant.