Therapeutic effects of Byrsocarpus coccineus root bark extract on bacterially and chemically induced diarrhea in the Wistar albino rat (Rattus norvegicus domestica)

Abstract Background Diarrhea can be caused by pathogenic microorganisms and chemicals. In view of this, Byrsocarpus coccineus Schum and Thonn (Connaraceae) was used to treat diarrhea induced by castor oil or bacteria in Wistar albino rats. Methods Qualitative and quantitative analyses of an aqueous root back extract of B. coccineus were made and the acute toxicity, antidiarrhea properties, and in vitro and in vivo antimicrobial activities of the extract were investigated in rats. Results The phytochemical analysis of the root bark extract revealed the presence of flavonoids, alkaloid, saponins, tannins, and phenols. The quantitative analysis showed that saponins formed 10.6% of the extract, tannins 7.6%, flavonoids 6.2%, phenol 5.8% and alkaloids 4.4%. A dose limit of 5000 mg/kg was safe to use in the rats. At a dose of 100 mg/kg, the extract decreased distance travelled by activated charcoal in the gastrointestinal tract, frequency of defecation, and number of unformed faeces caused by castor oil‐induced diarrhea, and led to 74.96% inhibition of the diarrhea effects. Escherichia coli and Salmonella pullorum were susceptible to higher concentrations of the extract with a minimum inhibitory concentration of 0.3125 mg/mL. E. coli‐infected rats showed depression, weight loss, anorexia, diarrhea, and weakness, which was ameliorated by the extract on day 2 post treatment. Observed congestion, cellular infiltration and necrosis of the liver, intestine and kidney following infection were improved by the extract. Conclusion B. coccineus extract can be used in the treatment of anaemia, and castor oil‐ and E. coli‐induced diarrhea in rats.


| INTRODUC TI ON
Diarrhea is defined as an excessive fluid loss of 200 g (200 mL) per day. 1 Diarrhea diseases cause several million deaths in the world annually, and diarrhea is the most common cause of morbidity and mortality globally, affecting many infants in the developing countries. [2][3][4] Overcrowding, poor sanitary conditions, contaminated water, inadequate food hygiene and poor nutrition predispose mainly children to the risk of diarrhea diseases. 5,6 Ingested food components or medicines such as artificial sweeteners and lactose may also result in diarrhea. 7 Evaluating the risk of diarrhea diseases requires knowledge of the complex interaction between biological, socio-economic, behavioral, and environmental factors. 8 In Nigeria, for example, the Nigeria Demographic and Health Survey reported that diarrhea prevalence is highest in the northeast (22%) and lowest in the southwest (7%). 9 Diarrhea induces dehydration and electrolyte imbalance, especially in children, infants and frail elderly patients. 10 Acute watery diarrhea usually lasts for less than 7 days, although it can last for up to 14 days. 11 Diarrhea is also caused by a wide range of bacteria such as Campylobacter jejuni, Escherichia coli, Salmonella species and Aeromonas species. 12 Loss of large quantities of intestinal electrolytes and fluid secretion occurs in patients with cholera caused by Vibrio cholera. Cholera toxin activates adenylate cyclase by causing ADP-ribosylation of the G-proteins, resulting in constitutive stimulation of the cyclase. 13 Elevated cAMP levels in turn activate protein kinase A and protein phosphorylation opens the luminal Cl-channel in secretory cells and inhibits Na + /H + exchange in absorptive cells. The net result is gross NaCl secretion. Management of diarrhea involves the use of rehydration therapy, nutritional supplements, drugs 14 and plant extracts. 15 Medicinal plants are the primary source of medication used as complementary or alternative treatments to orthodox medicine. 16 Byrsocarpus coccineus Schum and Thonn is a climbing shrub found in west and central African countries including Nigeria, Ghana, Cameroun, Ivory Coast, Togo, Central African and Congo. It is known as Onyakpechi in Igala, Tsamiyar kasa in Hausa, Oke abolo and Mybo-apapea in Yoruba and Onyakwachi in Idoma. 17 It is used in traditional African medicine for the treatment of earache, gonorrhea, impotence, jaundice, piles, diarrhea, tumour and wounds, 18 and has been shown to have anxiolytic, sedative, 19 anti-inflammatory, 20 analgesic, 21 antidiarrhea, 22 antimicrobial 23 and uterotonic activities. 24 Radostits et al have reported that colibacillosis is the major cause of death in neonatal animals, because maternal immunoglobins are not transferred transplacentally. 25 In addition, whereas castor oil obtained from Ricinus communis is used in the treatment of constipation, sometimes the oil causes diarrhea in both human and animals. Since a number of plants can be used to treat diarrhea induced by bacteria or toxicants, [26][27][28][29][30] we investigated the effects of a root bark extract of B. coccineus on chemically and bacterially induced diarrhea.

| Extraction of B. coccineus root bark
About 1.5 kg of B. coccineus root was obtained, washed with distilled water and subsequently air dried in the laboratory at room temperature. The dry root bark was pulverized into fine powder using a grinder and kept in a cellophane bag at 4°C until use. Fifty grams (50 g) of B. coccineus root powder was placed in a conical flask containing 100 mL of distilled water. The mixture was thoroughly shaken intermittently throughout the period of extraction using a stirrer, allowed to stand overnight, filtered with Whatman No.1 filter paper into a measuring cylinder, concentrated at 60°C in an incubator and stored in a refrigerator at 4°C until required. 21

| Qualitative phytochemical analysis
The aqueous extract of Brysocarpus coccineus root bark was qualitatively evaluated for the presence of alkaloids, cardiac glycosides, saponins, tannins, steroids, flavonoids, anthraquinones, total glycosides, and reducing sugars using the methods of Trease and Evans,31 Harborne 32 and Edeoga et al. 33

| Quantitative phytochemical analysis
The root bark powder of Byrsocarpus coccineus was quantitatively analyzed for the presence of alkaloid, flavonoid, phenol, saponin and tannin according to the methods described by Ordonez et al, 34 Van Burden and Robinson 35 and Skerget et al. 36

| Experimental animals
A total of one hundred and ten (110) adult Wistar albino rats of both sexes (55 males; 55 females), weighing 218 ± 28.6 g were used for the study. The animals were obtained from the National Veterinary Research Institute, Vom, Plateau State, Nigeria and were kept under standard housing conditions and allowed to acclimatize for 2 weeks before commencement of the study. They were fed with a commercial rat feed, Finisher ® produced by Grand Cereals and Oil Mills Ltd. Clean water was provided ad libitium.
The research was carried out according to protocols and procedures approved by the Department of Veterinary Biochemistry, Physiology and Pharmacology Ethical Committee, Federal University of Agriculture Makurdi, Nigeria.

| Acute oral toxicity study
The up and down procedure revised by Saganuwan 37 was adopted for determination of median lethal dose (LD 50 ) using five female rats. All rats were dosed at 5000 mg/kg body weight and observed for 48 hours in the following sequence. The first rat was dosed and observed for 48 hours. The second and third rats were then concurrently dosed and observed. The fourth and fifth rats were then sequentially dosed and observed. All the rats were observed for an additional 12 days for signs of delayed toxicity or death.

| Effects of B. coccineus extract on castor oilinduced diarrhea in rats
Twenty-five female rats were used for this study. The method of Offiah and Chikwendu 38 was adopted. The rats were starved overnight prior to the study, but allowed access to water. They were separated into five groups of five. The rats in groups I--III were respectively orally administered 50, 100 and 200 mg/kg body weight of the extract, while the rats in group IV were administered 2 mL of normal saline. The group V rats were administered the antidiarrheal diphenoxylate hydrochloride (5 mg/kg) by intraperitoneal route. All the rats were housed singly in separate cages lined with white blotting paper. One hour after treatment, each of the rats was treated with 1 mL of castor oil, orally. The rats were then observed for 6 hours for watery faeces. All the observations were recorded as percentage (%) protection.

| Effects of B. coccineus extract on gastrointestinal motility
The method of Chime et al 39 was used to test the effect of the extract on gastrointestinal motility. Twenty-five female rats were fasted overnight, with access to water. They were then divided into five groups of five. The rats in group I were orally administered 2 mL normal saline (0.9%), those in group II were administered 3 mg/kg of the antidiarrheal atropine intraperitoneally, and those in groups III--V were orally administered 50, 100, and 200 mg/kg of the extract, respectively. One millilitre (1 mL) of castor oil was administered to each animal, before administration of the drug and the extract. Thirty minutes after the drug and extract administration, 1 mL of 5% activated charcoal suspension in a 10% aqueous solution of acacia powder was administered, and the rats were sacrificed 30 minutes later. The abdomens were opened and the distances travelled by the charcoal meal were measured and expressed as a percentage of the total length of the intestine from pylorus to caecum. 40

| Effects of B. coccineus extract on castor oilinduced enteropooling
The intraluminal fluid accumulation due to the effect of castor oil was determined by the method of Robert et al 41 Twenty-five male rats, separated into five groups of five, were used for this study. The rats were fasted overnight, before the commencement of the study.
Group I rats were orally administered 2 mL normal saline, group II rats were administered atropine (3 mg/kg) intraperitoneally, and rats in groups III--V were orally administered 50, 100 and 200 mg/kg of the extract, respectively. After 1 hour, castor oil was administered to each animal. The animals were sacrificed immediately and the small intestine removed, tied on both ends with thread and weighed. The intestinal content was collected by milking and the quantity measured.

| Determination of antibacterial activity of B. coccineus extract
To test the antibacterial activity of the B. coccineus extract, laboratory isolates of pure cultures of two gram-positive species

| Determination of in vitro antibacterial activity
The well diffusion method described by the National Committee

| Minimal inhibitory concentration
The minimal inhibitory concentration of the extract was determined using the dilution method described by Greenwood. 43 Serial dilutions of the extract were carried out to obtain concentrations of 2 MacFarland scale. Colony count was performed on the diluted culture to achieve a titre of 3.0 × 10 6 CFU/mL. 44 All the rats were starved overnight, before the experimental infection. Each rat was inoculated orally with 0.5 mL of the inoculum. The uninfected control rats were inoculated with sterile broth.

Experimental groups and E. coli inoculation
Thirty male rats were randomly divided into six groups (I-VI) of five and kept in separate cages. Groups I, II and III were inoculated with E. coli and treated with the extract at doses of 50, 100 and 200 mg/kg, respectively. Group IV was inoculated with E. coli and treated with 5 mg/kg of 5% oxyteyracycline (positive control), and group V was inoculated but untreated (negative control).
Group VI served as the normal control group (not inoculated and non-treated). Oxytetracycline (5 mg/kg, manufactured by Hebe Yuanzheng Pharmaceutical Limited) was used as the standard antibiotic. Treatment with the extract and standard antibiotic commenced 3 days post infection and lasted for a period of 7 days.

| Clinical signs and treatments
Rats from all the groups were closely observed for clinical signs of infection. The morbidity and mortality in all the groups was recorded.
The body weights of the rats were recorded at days 3, 7, 12 and 18 post-infection.

| Hematology
Blood samples obtained through the media canthus of the eye were used for the determination of hematological parameters such as red blood cell (RBC) count, packed cell volume (PCV), hemoglobin concentration and white blood cell (WBC) count. 45

| Histopathological examination
Two rats randomly selected from each group, making a total of 12, were sacrificed using phenobarbitone (

| Statistical analysis
The results of the quantitative analysis of phytochemical contents are presented as percentiles. The frequencies of droppings, gastrointestinal transit time, enteropooling, antimicrobial zones of inhibition and average organ weight gain were calculated using one-way analysis of variance. The least significance difference was set at the 5% level. The minimum inhibitory concentration of the test microorganism, average weight gains of the infected rats, RBC, PCV and WBC counts, hemoglobin, total blood volume, red cell volume, and plasma volume were calculated using two-way analysis of variance. Fisher's test was used to detect significant differences, set at the 5% level. 47

| Qualitative phytochemical analysis
Results of the qualitative phytochemical analysis of the aqueous root bark extract of B. coccineus revealed the presence of flavonoids, tannins, saponins, alkaloids and phenols. The analysis also revealed the absence of steroids, terpenoids, cardiac glycosides and anthroquinones (Table 1).

| Acute oral toxicity study
Oral administration of the aqueous root bark extract of B. coccineus to rats produced no visible signs of toxicity at 5000 mg/kg body weight. Hence, the median lethal dose (LD 50 ) value for the extract was estimated to be above 5000 mg/kg.

| Effects of B. coccineus extract on gastrointestinal transit of charcoal
The effect of the extract on gastrointestinal transit of charcoal is shown in Table 4. There was a significant (P < .05) decrease in the intestinal transit of charcoal in the extract-treated groups compared to the control group. The charcoal travelled very rapidly along the intestine in the control group, while the rate of movement was significantly (P < .05) reduced in the rats treated with the extract. The rats treated with 3 mg/kg of atropine had a slower rate of charcoal movement along the small intestine compared to the extract-treated rats ( Table 4). The transit of charcoal in the groups treated with the extract appeared to be statistically similar to the group treated with atropine.

| Effects of B. coccineus extract on castor oilinduced enteropooling
The effect of the extract on castor oil-induced enteropooling is shown in Table 5. The results showed that there was a significant

| Clinical signs
Rats from infected groups showed signs of depression and anorexia at

TA B L E 6
In vitro antimicrobial activities of higher doses of the aqueous root bark extract of Byrsocarpus coccineus without control There was a significant (P < .05) decrease in the body weights of the extract-treated groups compared to those of oxytetracyclinetreated and uninfected control groups. The body weights of the rats in the infected untreated group were the lowest among all the groups.

| Effects of E. coli infection and the extract treatment on hematological parameters of rats
The

TA B L E 1 2 Effects of the aqueous root bark extract of Byrsocarpus coccineus on white blood cells (WBC) of rats infected with E. coli
and the values appear to be similar to that of the uninfected control group (Table 13) (Table 13). hepatocytes (H) ( Figure 1C). In the intestine, there was severe villous collapse, with matting and fusion of the villi (Figure 2A).

TA B L E 1 3 Effects of the aqueous root bark extract of Byrsocarpus coccineus on differential white blood cell counts of rats infected with E. coli
In the kidney, there was marked congestion, shrunken glomeruli with a wide bowman capsule, tubular necrosis and cellular infiltration in rats infected with E. coli but not treated ( Figure 3B). The kidney of the infected rats had severe lesions. The rats treated with 200 mg/kg of the extract showed evidence of recovery ( Figure 3C).
The kidney from the control was normal ( Figure 3A). Generally, the extract-treated groups had less severe lesions compared with the infected untreated groups.  49 Flavonoids present in the extract have been reported to possess antioxidant properties. 50 Quercetin, kaempferol and quercitrin are flavonoids commonly present in plants. Tannins have astringent properties, which hasten wound healing and reduce mucus production in inflamed tissue. 51 This activity is due to their ability to precipitate proteins, thereby protecting underlying tissues.

| D ISCUSS I ON
They also inhibit microbial growth and proliferation. 52 Saponins have been shown to possess hypolipidemic, anticancer, expectorant activities. They are used in the treatment of upper respiratory tract inflammation. 31 Volatile oils (essential oils) contribute greatly as an essence in enhancing the aroma of some plant species. 53 They are also used for the management of digestive, respiratory and urinary tract infections. 54 Alkaloids are the largest group of secondary chemical constituents in plants, containing nitrogen-based compounds synthesized from amino acids. They combine with acids to form salts. 55 Therapeutically, alkaloids can serve as central nervous system (CNS) stimulants, anaesthetics, sedatives, analgesics, anti-inflammatory agents, anticancer and muscle relaxants. 56 Acute toxicity studies of extracts of B. coccineus showed that an oral LD 50 > 5000 mg/kg is relatively safe, 57,58 indicating that the extract could be administered with some degree of safety at lower  Table 3).
The ricinoleic acid liberated from castor oil caused irritation of the gastrointestinal tract mucosa, resulting in inflammation, increased gastrointestinal secretion and enhanced motility of the gastrointestinal tract, 62 leading to castor oil-induced diarrhea. 1 The diarrhea also may result from decreased absorption of substances within the intestine. 63 Since the extract was able to inhibit castor oil-induced diarrhea, its antidiarrhea effect may partly be due to its inhibitory effect on gastrointestinal secretion and/or gastrointestinal motility. The reduction in castor oil-induced enteropooling may be due to the ability of the extract to reduce or prevent fluid and electrolyte secretion into the intestine, consequently decreasing gastrointestinal motility similarly to diphenoxylate, a synthetic piperidine opioid structurally related to pethidine, used clinically alone or in combination with atropine for control of non-specific diarrhea in small animals. 5,9 Therefore, it could be inferred from the results of this study that the decreases in frequency of defecation and distance travelled by the charcoal meal may be due to the inhibition of gastrointestinal motility by the extract.
It is possible that the effect of the extract may be mediated via α2 adrenergic or muscarinic receptor stimulation.  66 The susceptibility of E. coli and S.
pullorum to the extract could be attributed to the presence of tannins and saponins observed to be present in the extract. Generally, tannin is known for its antimicrobial activities in the bark of plants where it protects the plant against bacteria and fungi infections. It also has soothing, anti-inflammatory, antidiuretic and astringent activities. 67 Significant beneficial effects attributed to the use of tannins are seen in wound healing, bleeding, tissue injury and skin regeneration. However, high tannin consumption has been implicated in osteoporosis and anemia in animals and humans due to its effects on calcium and iron absorption. 68 Prohp and Onoagbe reported a tannin content of 12% in Triplochitin scleroxylon stem bark extract, 69 hence the quantity of tannin (7.6%) obtained in this study may be considered safe.
The improved weight gain ratio and hematological parameters observed in the present study agree with the report of Saganuwan Capsicum ftutescens, among others. 65 The pathogenicity of the E.
coli strain used in this study was confirmed based on the clinical symptoms, mortality and lesions noticed in the infected rats during the experimental period. The present findings agree with the report indicating that E. coli is a commensal bacterium of the intestine that is pathogenic to mammals. 73 The main symptoms observed in E. coli-infected rats, are not exclusive to this species; 74,75 E. coli strains associated with avian infection usually induce respiratory and septicaemic diseases. 76 The efficacy of the extract in controlling the E. coli infection induced in this study may be due to the presence of some of the phytochemical compounds in the extract such as tannins, phenols, alkaloids and flavonoids, 77 which are known to possess appreciable antibacterial activities. Tannins are known to decrease bacteria cell proliferation by blocking the enzymes of microbial metabolism, producing hydrogen bonds with the carbonyl group in the enzyme (redox reaction). An antimicrobial activity of flavonoids has also been demonstrated. 78 The presence of flavonoids in the extract may thus be responsible for the significant neutrophil counts observed in the present study, since flavonoids are reported to have profound effects on the function of immune and inflammatory cells. 67 Neutrophils play an important role in the defence mechanism of the body by providing the first line of defense against the invading microorganism, being free wandering cells in the body. 79 The eosinophilia observed may be connected with inflammation of the kidney and liver. 80 Eosinophils kill parasites, regulate the hypersensitivity reaction mediated by TgE antibodies and may promote inflammation and tissue damage. 81 The general leucocytosis observed in the E. coli-infected rats may be due to neutrophilia, eosinophilia, basophilia and monocytosis, as proven by differential leucocyte counts. Parasitic infections may be responsible for increased leucocyte counts. 70 Hence the extract may be ef-

| CON CLUS ION
The B. coccineus extract tested in this study contains flavonoids, tannins, saponins, alkaloids, and phenols, which have potent antimicrobial activities against E. coli and S. pollorum, and as such may be used in the treatment of bacterially and chemically induced diarrhea in rats. The extract of this plant is apparently safe when administered orally. Hence the results of this study confirm folkloric claims that the aqueous root bark extract of B. coccineus can be used to prevent chemical and bacterial diarrhea. The root bark extract has hematinic and plasma expander effects in rats and may therefore be used in treatment of anaemia. It also has cellular immunostimulatory, hepatoprotective and anti-inflammatory effects.

CO N FLI C T O F I NTE R E S T
None.

AUTH O R CO NTR I B UTI O N S
EAS carried out the study, whereas PAO and SAS designed the study, analyzed the data, wrote and proofread the manuscript. SAS edited the manuscript finally. All authors approved the final version of the manuscript for submission.