2.1 Plant Collection and Identification
For the analysis of these two extracts, plants were collected from two different locations. For the preparation of aqueous extracts, the leaves of C. bifida were collected from Comilla City, which is surrounded by a variety of shrub species. For the preparation of methanolic extracts, the C. bifida plant was collected from the Botanical Garden in Mirpur, Dhaka, Bangladesh. The plant was identified and distinguished by the scientific officers of the Bangladesh National Herbarium, Mirpur-01, Dhaka-1216 (Accession Number DACB 88209).
The plant was harvested, rinsed with tap water, and subsequently air-dried in a shaded area rather than exposed to direct sunlight to prevent the active ingredients from evaporating. After a week of drying, the plant components were gathered and processed into a coarse powder using an appropriate grinder. Following this, the powder was sifted, and the granular portion was further ground. The powder was then carefully weighed using an analytical balance after the finely ground portion was sifted a second time. Finally, the powder was sealed in an airtight container and stored in a cool, dark, and dry location until the analysis was initiated.
2.2 Extract Preparation
The maceration method was used to prepare the crude extracts. Both extracts were collected using the cold extraction method, with methanol and water as solvents. In this process, powdered material from C. bifida leaves was macerated by soaking it in solvents in a round-bottom flask for ten days. The container and its contents were sealed and maintained with occasional agitation. The mixtures were then filtered separately using a piece of clean, white cotton material and Whatman filter paper. An electrical balance was used to weigh the filtrates after pouring them into beakers. The aqueous extract was concentrated for 2 days by evaporating water through drying using a water bath, and the methanolic extract was obtained through vacuum drying using a rotatory evaporator. The concentrated extracts were then maintained in a refrigerator at a temperature between 2 and 8°C.
2.3 Chemicals and Reagents Used
The chemicals used in the study were methanol (Merck, Germany), acetic acid (RCI Labscan), and castor oil (WELL’s Health Care, Spain). The standard drug, Loperamide, was obtained from Square Pharmaceuticals Ltd., and distilled water was obtained from the laboratory of Primeasia University, Banani, Dhaka.
2.4 Experimental Animals
In this pharmacological activity study, Swiss albino mice of either sex, aged 6-8 weeks and weighing an average of 25-30 g, were used. The mice were obtained from the animal house of Pharmacy Department in Comilla University, Cumilla, Bangladesh, housed in the animal house of Primeasia University, Banani, Dhaka and acclimated for one week with food and water ad libitum in the room under controlled conditions of temperature 25 ± 2° C, humidity 55 ± 5%, and 12:12 h light/dark cycle.
2.5 Qualitative Phytochemical Screening Tests
The extracts of C. bifida leaves were used for phytochemical screening to identify the chemical compounds present in the leaves, following standard qualitative testing procedures [9].
2.6 Evaluation of Castor Oil-Induced Anti-Diarrheal Test
The methodology outlined by Shoba and Thomas was adopted for this experiment [10]. The animals were randomly allocated into groups to investigate how the crude extract influenced castor oil-induced diarrhea. The animals were pre-screened for diarrhea before the test by administering 0.5 mL of castor oil orally. Animals with diarrhea were selected for the study. Subsequently, thirty mice were selected and divided into six groups, with each group consisting of five mice. Prior to the experiment, the mice were fasted for 18 hours with unrestricted access to water. The selected mice were grouped by the following-
- Group 1: Normal control (NC) group for both aqueous and methanolic extract received distilled water.
- Group 2: Positive control (PC) group for both aqueous and methanolic extract received standard drug, Loperamide, at a dosage of 3 mg/kg body weight (BW).
- Group 3: Test group (TG) treated with aqueous extract at a low dose of 200 mg/kg body weight (BW).
- Group 4: Test group (TG) treated with aqueous extract at a high dose of 400 mg/kg body weight (BW).
- Group 5: Test group (TG) treated with methanolic extract at a low dose of 200 mg/kg body weight (BW).
- Group 6: Test group (TG) treated with methanolic extract at a high dose of 400 mg/kg body weight (BW).
Thirty minutes after administration, all mice were given 0.5 mL of castor oil orally to induce diarrhoea and were individually placed in cages lined with non-absorbent blotting paper. The paper was changed every hour throughout the 4-hour observation period. During this time, the number of instances of diarrhoea was recorded, and the percentage of inhibition of defecation was calculated for each group of animals. The percentage inhibition of defecation from the NC group was determined using the following formula:
% inhibition of defecation = [(mean number of faeces in NC − mean number of faeces of TG) / mean number of faeces of NC] × 100
2.7 Statistical Analysis
All data were expressed as the mean ± standard error of the mean (SEM). To analyze the information obtained from this experiment, we used GraphPad Prism 6 (GraphPad Software, San Diego, California, USA, http://www.graphpad.com). Statistical analysis of normally distributed and homogenous variance data was done using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Differences were considered significant at p < 0.05.
