2.7. Experimental Design

The antidiarrheal activity was performed on BALB/c mice in castor oil-induced diarrhea with some modifications in the protocols [28]. Animals were randomly assigned into four groups of five animals each. Group I was the control and received normal saline, while group II was the standard that received (loperamide) at a dose rate of 50 mg/kg (b.w). Groups III and IV were the tested groups that received the methanol extract at a dose rate of 200 mg/kg and 400 mg/kg (b.w), respectively. After the treatment of respective drugs, each animal was put in separate cages laid with papers for collecting the fecal mass. Diarrhea was induced by oral administration of castor oil (1 mL/mice). The methanol extract and loperamide were given 1h before the oral administration of the castor oil; the time is taken for the first feces excretion, and the total number of fecal outputs within 6 h of administration was recorded. The inhibition of defecation was calculated using the following equation:

where Mo = mean defecation of control and M = mean defecation of the experimental group.

Loperamide, the standard drug used, slows intestinal motility and alters the bowel water and electrolyte movement. It is a potent opiate receptor agonist in the gut wall where it inhibits the release of acetylcholine and prostaglandins, thereby reducing propulsive peristalsis and increasing the intestinal transit time. The drug has also been reported to increase the activity of the anal sphincter [29]. It thus antagonizes the diarrheal activity induced with castor oil [30].

The effect of C. oxycantha on magnesium sulfate-induced diarrhea was also determined on adult healthy BALB/c mice [31]. After overnight fasting, the animals were distributed into 4 groups of 5 animals each. Group I, the control, received only normal saline. Group II, the standard, received loperamide at a dose rate of 20 mg/kg (b.w), while groups III and IV, which were the test groups, received a methanolic extract of C. oxycantha at a dose rate of 200 and 400 mg/kg (b.w). Sixty minutes after treatment of the respective drug, all the animal groups were treated orally with magnesium sulfate at a dose rate of 2 g/kg (b.w). The frequency of defecation and the fecal material was again noted for up to 4 h. The mice were in transparent (clear) cages with pre-weighed plastic dishes for feces collection at the bottom of all cages. The weights of the plastic dishes were recorded and compared to that of the control before and after defecation.

Four standard bacterial strains: S. aureus, S. typhi, E. coli, and P. aeruginosa were used in this study. Agar nutrient medium was used for the growth of bacterial strains and was allowed to stand for a period of 24 h at 37 °C. Nutrient agar was added to a conical flask that already contained distilled water. The nutrient agar powder and distilled water were mixed in (proper ratio). The aqueous solution was made by incorporating an amount of 20 gm of agar nutrient in 1000 mL of distilled water with constant shaking for 6 min. The solution was then sterilized and transferred to Petri dishes for the inoculation of bacterial strains.

The antibacterial activity of C. oxycantha was determined individually by the agar well diffusion method [32]. Twenty milliliters of molten nutrient agar was poured into each of the Petri dishes and allowed to solidify. Overnight, a bacterial broth, standardized to 0.5 McFarland, was spread on the dry nutrient agar and spread using a spreader pre-sterilized in ethanol and flamed. With the aid of a sterile cork-borer, five 6-mm holes, about 5 cm apart, were made in the nutrient agar. Three of the wells were filled with 200 µL of the C. oxycantha plant extract dissolved in sterile distilled water, one well with the water only (the negative control), and the last with 1% standard antibiotic, doxycycline (the positive control) was dispensed into the wells in triplicates. The antibacterial activities were determined after incubation for 24 h period at 37 °C as the diameter of the inhibition zone. The zones of inhibition observed with the extract were compared with that of the standard antibiotic, doxycycline. The experiment was done in triplicates. The measured doxycycline inhibition zones’ diameters were subsequently matched with the respective standard zones’ diameters [33] for S. aureus, S. typhi, E. coli, and P. aeruginosa [34]. The C. oxycantha zone of inhibition from 9-14 mm in diameter was taken as a positive antibacterial activity based on those previously reported for Carthamus caeruleus L [35] and Carthamus tinctorius [36]. The fold change in the doxycycline and C. oxycantha zone of inhibition was measured by the following equation: (B−A)/A, where the C. oxycantha and doxycycline zone of inhibition is A and B, respectively.

Solutions of C. Oxycantha extract at varied concentrations of 1.0 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, and 5.0 mg/mL were introduced to molten agar plates and incubated for 24 h at 37 °C. The plates were then inoculated with the specific bacterial strains, incubated at 37 °C for 24 h, and the minimum inhibitory concentration (MIC) of the extract was determined against the selected bacterial strains. The MIC of the extract against the bacterial strains was found to be 0.5 mg/mL, which shows that, while increasing the concentration, their activity also increased [37].