Anti-Shigellosis Activity of Cola anomala Water/Ethanol Pods Extract on Shigella flexneri-Induced Diarrhea in Rats

This study was undertaken to evaluate the activities of water/ethanol Cola anomala pods extract. In vitro antimicrobial susceptibility was determined by the disk diffusion method; the minimum inhibitory concentration and minimum bactericidal concentration were determined by agar dilution technique. In vivo, shigellosis was induced in healthy Wistar albino rats by oral administration of Shigella flexneri inoculum, 12 × 108 CFU/mL. At the onset of diarrhea, infected and normal control animals were subdivided into various groups treated with distilled water, with water/ethanol Cola anomala pods extract at 25, 50, or 100 mg/kg, or with ciprofloxacin, 2.5 mg/kg. After one-week treatment, rats were sacrificed, and blood and colon were collected. Blood was used for blood cell count. A portion of the colon served for histological studies while homogenate from the remaining part was centrifuged and the supernatant was collected for the determination of NO, PGE2, IL-1β, and TNF-α levels. In vitro, water/ethanol Cola anomala pods extract showed to be bactericidal, with a minimum inhibitory concentration of 2.0 mg/mL and a minimum bactericidal concentration of 3.0 mg/mL. In diarrheic rats, the extract significantly (P < 0.01) increased the white blood cells and significantly (P < 0.01) decreased stool Shigella density from the first to the seventh day of treatment. It partially restored the structure of eroded intestine epithelium and prevented weight loss; the dose dependently and significantly (P < 0.001) decreased NO, IL-1β, and TNF-α production in the colon and was found to have no significant effect on PGE2 production. These results support the use of this plant in traditional medicine in the treatment of gastrointestinal ailments.


Introduction
Diarrheal diseases result from an intestinal transit disorder characterized by loose or liquid stools, in abnormally high amounts or with an increased occurrence frequency, about three times a day during few days to few weeks depriving the body from its necessary mineral salts [1]. Diarrhea sometimes results from an increased motility and secretion of the digestive canal or decreased fluid absorption, leading to water and electrolytes (Na + , Cl − , and K + ) loss [2]. Usually, they can be a symptom of an intestinal infection caused by various microorganisms: parasites, bacteria, or viruses [2], and they remain the second main cause of death in infant not up to five, while accounting for nearly 1.7 billion cases with 525,000 deaths every year [3]. e probability of diarrhea occurrence is about 39.1% for Sub-Sahelian developing African countries, compared to 7.2% in developed countries [4].
In Cameroon, among the major causes of child mortality, diarrhea comes first. And among the leading causes of morbidity, they rank third with a prevalence of 13.6% nationwide. So, by their epidemic-endemic nature, diarrheal diseases constitute a major threat for the country [5]. Among the most dangerous diarrhea cases in humans is the bacillary dysentery or shigellosis, which has been responsible for major epidemics that have become historical in the world [6]. Shigellosis is an acute invasive intestinal infection caused by bacteria of the genus Shigella; it is endemic in most developing countries, being the leading cause of bloody diarrhea worldwide with at least 80 million cases of bloody diarrhea and 700,000 deaths each year [7]. Shigellosis is responsible for about 22% of deaths from diarrhea per year worldwide [8]. In Cameroon, shigellosis accounts for about 4.5% of diarrhea cases per year [4]. In 2010, the episode which occurred in the City of Buea had an antibiotic resistance rate of 90% [9].
Among Shigella species, Shigella dysenteriae type 1 (Sd1) is responsible for the most dangerous form of the disease and can cause large epidemics on a regional scale. Shigella flexneri is the main cause of endemic shigellosis in developing countries [10]. e main obstacle to shigellosis control is the high rate with which Shigella is transmitted from person to person and the rate of occurrence of resistance of the bacteria to antibiotics [10]. Treatment of shigellosis is based on the administration of an anti-infectious agent accompanied by rehydration and supplementation of the zinc [10]. Zinc reduces the secretion of chloride ions by inhibiting the potassium canals of the basolateral membrane, on the one hand, and improves the absorption capacity of water and electrolytes and strengthens the intestinal immune response on the other hand. Ciprofloxacin, mostly used as the main antishigellosis drug, showed to be the cause of arthropathy in children. e use of these antibiotics and other antidiarrheal is currently challenged by the cost as well as the availability of these synthesized molecules and by the development of multiresistant microbial strains [2]. is, thus, confirms the need of an alternative medicine promisingly from medicinal plants locally available for the treatment of these diseases [11,12].
Cola is a tropical African tree belonging to the Sterculiaceae family. e genus is made up of about 140 species, with the most commonly exploited being Cola acuminata, Cola nitida, and Cola anomala [13]. Cola nut, the mature fruits of the Cola sp., is a very important aspect of the tradition in Cameroon, as well as for medicinal purposes. Various medicinal and pharmacological values have been observed in species of Cola. Cola nuts are sometimes used against whooping cough and asthma [14], to treat malaria, nervous debility, weakness, lack of emotion, nervous diarrhea, depression, anxiety, fever, and as antimicrobial agents [15]. It is also used to increase physical capacity for endurance during fatigue and to stimulate weak heart [15]. Cola nut has a bitter taste and a great caffeine content [15]; the ingested fruit acts as stimulants creating an euphoric state [15]. Phytochemical screening revealed the presence of tannins, anthraquinones, alkaloids, saponins, and cardenolides in Cola species [14,15].
Traditionally, the fruit follicles, the bark, and leaves of Cola anomala are used by the population from the Western Region of Cameroon as a remedy against dysentery, coughs, diarrhea, and vomiting. e prior reports of antibacterial and antiparasitic activities [14] support the use of Cola fruit to treat diarrhea and other gastrointestinal infections. It is therefore necessary to establish scientific evidence for therapeutic use of Cola anomala, as it may potentially be a useful source of new lead compounds to be an input for the drug development process or give a clue about the strategies of standardized medicinal plant remedy. e present study, therefore, aimed to evaluate the activities of Cola anomala water/ethanol pods extract on Shigella flexneri diarrhea model induced in rats.

Plant Material.
In this study, the plant material was made up of pods from Cola anomala tree, which was identified at the Yaoundé National Herbarium, in comparison with the specimen referenced 50223HNC. e pods were collected from Batié (West Region, Cameroon, coordinates 9°25′17″N and 13°27′2″E) between March and April 2013 and were separated from the nut and, then, were cleaned, reduced into small pieces, shade dried, and crushed to get a powder. e powder was extracted with a water/ ethanol mixture (V/V). For this, 1000 g of powder was macerated for 72 h in 5 L of a mixture of equal volume of water and ethanol at room temperature with occasional stirring and then filtered (Whatman filter paper No. 1). Furthermore, the residue was remacerated for 72 h in the same solvent (5 L) and filtered. e two obtained filtrates were pooled and concentrated in a rotary evaporator (BÜCHI Rota vapor R-124) at 65°C resulting in 175 g of a light brown hydroethanolic extract which was lyophilized and kept at +4°C until used. Extract solutions were prepared with distilled water, before oral administration to rats or for in vitro and in vivo experiments.

Experimental Animals.
We used male and female healthy Wistar albino rats (90-200 g) obtained from the animal house of the Physiology and Phytopharmacology Laboratory of the University of Dschang. Animals were reared (1 rat/cage) at room temperature in clean metabolic cages with natural light/dark cycle and sufficient aeration. ey had free access to water and standard rat diet, the composition of which for 1 kg was maize flour (60%), wheat flour (10%), fish (12%), soya bean (15%), and kernel cake (3%) enriched with the vitamin complex [16]. In vivo tests were carried out with respect to the European Union Guidelines on Animal Care (Council EEC 86/609) [17] adopted in Cameroon by the Institutional Committee of the Ministry of Scientific Research and Innovation.

Microbial Strains.
e microbial strain used in this study was Shigella flexneri, a clinical strain obtained from the Centre Pasteur of Yaoundé, Cameroon. is microbial strain was isolated from local patients. Autoclaved (121°C, 15 min) Muller-Hinton agar medium (Diagnostic Liofilchem, Italy, Ref. 610033) was poured into sterile Petri dishes (4 mL/Petri dish, 3-4 mm depth) and allowed for solidification. Culture media were then inoculated by streaks with the microbial strain and then incubated in an oven (Memmert Model 700) at 37°C for 24 h. From this young culture, two colonies were isolated for the preparation of the inoculum.

Preparation of Bacterial Inocula.
From the previously prepared Shigella flexneri culture, two colonies were collected and dissolved in 5 mL of sterile physiological saline. After homogenization, the opacity of the bacterial suspension in the tube was adjusted to 0.5 Mac Farland scale corresponding to 1.5 × 10 8 CFU/mL [18]. e standard inoculum was obtained by dilution of this bacterial solution to 1/1000 th and thus to 1.5 × 10 5 CFU/mL.

Antimicrobial Susceptibility. All antibacterial tests
were performed under strict aseptic conditions to ensure the quality of the results. Antimicrobial susceptibility screening of Cola anomala pods water/ethanol extract was performed by the disk diffusion method [19]. Culture media (Müller-Hinton Agar and Salmonella Shigella Agar, Titan Biotech Ltd. India 386) were inoculated with 500 μL of newly prepared Shigella flexneri suspensions at 1.5 × 10 5 CFU/mL. Having ensured that agar entire surface is covered, the excess microbial suspension was, however, eliminated with a sterile Pasteur pipette [20]. Disks previously impregnated with extract or with ciprofloxacin were then equidistantly deposited on the surface of the agar (5 per Petri dish), 15 minutes after inoculation. After 30-minute preincubation period under the hood, this preparation in Petri dishes was allowed for 24 h incubation at 37°C in an oven. After the incubation, diameters of inhibition were measured using an electronic caliper [21]. Each trial was done in triplicate [22,23].

Determination of the Minimum Inhibitory Concentration.
e minimum inhibitory concentration (MIC) is the lowest concentration of an antibiotic which presents no visible microbial growth [24]. It was determined by agar dilution technique [2,25]. All inhibition assays were conducted in triplicate. A solution set of extract with concentrations ranging from 50.00 to 0.05 mg/mL was prepared. Aliquot of each extract solution (2 mL) was added to 18 mL of presterilized molten Müller-Hinton agar or to 18 mL of Salmonella Shigella agar at 40°C leading to a final concentration ranging from 5.00 to 0.005 mg/mL. e medium in tubes was then allowed to solidify. e surfaces of the media received a 18 h Shigella flexneri inoculum, followed by 24 h incubation at 37°C. After this incubation, tubes were observed; the first in the series presenting no visible growth was considered as the MIC [2].

Determination of Minimal Bactericidal Concentration.
e minimum bactericidal concentration (MBC) of extracts on microbes was determined from the Petri dish previously considered as MIC. In fact, the agar surface of the Petri dish which showed no visible bacterial growth was scraped and streaked with an inoculation loop on the surface of sterile MH agar. Incubation was done at 37°C for 24 h. e lowest concentration with no bacterial colony (99.99% kill) was considered as the MBC of the extract on the tested strain [2].

In Vivo Antimicrobial Studies: Shigella flexneri-Induced Diarrhea in Rats.
For shigellosis induction, Shigella flexneri inoculum was adjusted to an equivalence with the turbidity of point 4.0 of the Mac Farland scale, which approximately corresponds to 12 × 10 8 CFU/mL.
Before the experiment, all animals were deparasitized by daily administration (6 : 00 am and 6 : 00 pm) of 5 mg/kg Azithromycin (Greenstone LLC Peapack NJ07977) for 3 consecutive days. After the deparasitization, the rats were individually isolated in metabolic cages and, with the exception of those in the normal control group (NC), received per os with the aid of a gavage tube, a 24 h inoculum of Shigella flexneri, adjusted to an equivalence with the turbidity of point 4.0 of the Mac Farland scale [26]. As soon as diarrheal stools appeared (24 h after Shigella flexneri administration), diarrheic rats were divided, according to their size, into five groups of five animals each: (i) A diarrheal control group (DC) which received distilled water (10 mL/kg) (ii) A positive control group (CIP) which received ciprofloxacin (Ryan PHARMA UK) (2.5 mg/kg) (iii) ree test groups which orally received each Cola anomala extract at 25 (KEO25), 50 (KEO50), and 100 (KEO100) mg/kg, respectively e animals received treatment for one week twice daily (6 : 00 am and 6 : 00 pm). Every day, stool weight and its Shigella flexneri density were recorded, as well as the body weight variation.
Shigella flexneri density was evaluated by collecting 0.5 g of fresh stool from each rat and dissolving it in 4.5 mL of sterile saline. From this stool solution, 250 μL was further diluted in 9.750 mL of sterile saline. 50 μL of this microbe suspension was finally taken and cultured on SS agar for 24 h at 37°C. Shigella density was then determined by direct Shigella flexneri colonies counting [2].
At the end of treatment, rats, including those in the normal control (NC) group, were sacrificed under anesthesia ( iopentone, ip injection 0.1 mL/100 g bw) [27], and blood collected in EDTA tubes was used for blood cell count using a hematology analyzer (Golden Harvest Industries BC 2800 Hematology Analyzer). Colon was also collected. Part of it served in histological studies while homogenate from another portion was centrifuged at 10,000 rpm for 15 min. e supernatant was collected in Eppendorf tubes and used to determine PGE 2, IL-1β, and TNF-α levels using ELISA method and for spectrophotometric evaluation of NO level [28].

Data Analysis.
e results were expressed as means (X) affected by the standard errors of the mean (SEM): X ± SEM, and they were presented in the form of tables or figures. Control and test group means were compared by one-way analysis of variance (ANOVA) followed by Dunnett's test using GraphPath Prism 7, Version 7.04 software.
e most important inhibition was 20.09 ± 0.27 mm at 6.25 mg/mL. Ciprofloxacin, the reference drug, inhibited Shigella flexneri growth with a diameter of about 28.00 ± 0.30 mm at 0.03 mg/mL.

Minimal Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC).
e water/ethanol extract of Cola anomala and ciprofloxacin in vitro inhibited Shigella flexneri growth (Table 2). MBC/MIC ratios of Cola anomala and ciprofloxacin were 1.5 and 2.0, respectively ( Table 2).

In Vivo Activities of Extract of Cola Anomala Pot and
Ciprofloxacin on Shigella flexneri-Induced Diarrhea in Rats

Effect on Animal Behavior and Stool Appearance.
Four hours after Shigella flexneri administration, rats became calm and folded into a ball with erected furs, signs of abdominal cramps. e first diarrheal stools appeared 20 h after induction. ese unmolded stools presented traces of blood and mucus (Figure 1). Treated diarrheal animals (with different extract doses or ciprofloxacin) gradually recovered mobility, with normal stools.

Effect on Shigella flexneri Stool Density.
In diarrheic control (DC) rats, Shigella flexneri load increased and was significantly (P < 0.001) high from the onset of diarrhea in the first to the third day ( Figure 2). In treated animals, the number of bacteria significantly (P < 0.001) decreased from the first day of treatment (Figure 2). e stool Shigella density of rats treated with the extract of C. anomala pods at 25, 50, or 100 mg/kg (KEO25, KEO50, or KEO100) significantly decreased (p < 0.01) during the first to the seventh day of treatment. At day seven, bacterial loads reduced of about 28.01, 39.12, 29.41, and 35.10%, respectively, by KEO25, KEO50, KEO100, and ciprofloxacin.

Weight Evolution of Rats.
From the first to the seventh day of treatment, the body weight of normal (NC) or diarrheal (DC) rats treated with extract or ciprofloxacin showed no important change (Figure 3).

Effects on Intestine Morphology of Shigella flexneri-Induced Diarrhea
Rats. Diarrheal rats' intestine epithelium (A) presented significant alteration with more mucus and total erosion of microvilli with straight and angular surface compared to normal rats (B). Treatment with ciprofloxacin (C) as well as hydroethanolic C. anomala extract at 25 (D), 50 (E), and 100 mg/kg (F) almost completely restored the epithelium structure (Figure 4).
(2) Effects on Shigella flexneri-Induced Diarrhea Rats PGE 2 Production in the Colon. Shigella flexneri infection was not found to be of great importance on PGE 2 production in rat colon. PGE 2 concentrations in various treated groups were not significantly different compared to those in normal or untreated groups ( Figure 6).

Discussion
is work was carried out to verify the scientific claim of the use of Cola anomala in traditional medicine against diarrheal diseases. C. anomala water/ethanol extract antidiarrheal activities were investigated, in vitro on Shigella flexneri growth inhibition and in vivo on Shigella flexneri diarrhea model in rats. e extract in vitro inhibited S. flexneri growth with an MBC/MIC ratio ≤4, thus indicating bactericidal effect of the extract [26]. In vivo, the extract protected infected rats against the deleterious effects of S. flexneri infection. S. flexneri is responsible for acute bloody diarrhea through invasion and destruction of the colonic epithelium. is sometimes leads to fever and stomach cramps or to the formation of microulcers and inflammatory exudates and causes inflammatory cells and blood or mucous to appear in stools [7]. Stomach cramps in shigellosis may results from Shiga-toxin action [29]. In this study, symptoms of shigellosis in infected treated rats significantly reduced with respect to the decrease of S. flexneri stools load. is confirms the in vitro and vivo antimicrobial activity of the extract which showed to have bactericidal activity. e reduction of stools bacterial load in treated groups was further associated with a decrease of NO, IL-1β, and TNF-α colon production. is treatment had no significant effect on PGE 2 level. Bacterial antigen, lipopolysaccharides (LPS), is an endotoxin in cell walls of Gram-negative bacteria. It is responsible for the inflammation of colon mucosa and can initiate a great number of hemodynamic and metabolic changes [30]. LPS is responsible for the activation of macrophages resulting in the secretion of biologically active substances, including prostaglandins, an arachidonic acid metabolite; nitric oxide; and cytokines [30]. Furthermore, LPS is thought to increase inducible NO synthase (iNOS) expression with NO production in muscularis resident macrophages. Excess NO level stimulates gastrointestinal tract secretion by the increase of intracellular cAMP and cGMP levels [30]. In this study, C. anomala water/ethanol extract reduced NO concentration in colon homogenate.
is activity probably may be the result of the inhibition of iNOS activity or of the inhibition of its production. LPS also can alter the normal influx/efflux ratio and particularly reduce the lumen-to-blood fluid influx. However, this extract showed no significant effect on PGE 2 concentration indicating that the extract may not have any activity on cyclooxygenase 2 (COX-2). e sequence of events in S. flexneri infection starts with the invasion of the bacteria by translocation through M cells from the lumen of the colon into the submucosa; phagocytosis of Shigella by macrophages; escape of bacterial vacuolar from the phagosome into the cytoplasm of the macrophage; production of invasion plasmid antigen B (IpaB); the distribution of IpaB through the macrophage's cytoplasm; binding and activation of interleukin-1β converting enzyme (ICE); and induction of apoptosis and cleavage of IL-1β [31]. C. anomala and ciprofloxacin in treated animals slightly reduced IL-1β production in the colon. is extract might contain bioactive substances which have direct effect on S. flexneri destruction or which may inhibit one of the infections steps previously described. Similar results were obtained by Kamgang et al. with Euphorbia scordifolia [19] or by Noubissi et al. with Crinum jagus [2]. In fact, phytochemical studies have revealed the presence of alkaloids and phenolic compounds in Cola sp. [32], where phenolic compounds are shown to be responsible for bacteria plasma membrane rupture, thus increasing its permeability [33].
Early cytokine production (TNF-α and IL-1β) correlated with weight loss and histopathological affection of colon mucosa is implicated in the pathogenesis and S. flexneri infection outcomes [34]. C. anomala and ciprofloxacin treatment protected diarrheic rats from excessive weight loss, colon mucosa damage, and, furthermore, slightly decreased TNF-α production. is may result from a direct bactericidal effect on luminal S. flexneri confirmed by decreased S. flexneri count in stools. Ciprofloxacin is a synthetic bactericidal antibiotic which belongs to fluoroquinolone family and is recognized for its great anti-S. flexneri activity [29]. It inhibits DNA gyrase activity and, thus, prevents the supercoiling of the bacterial chromosome [29]. C. anomala may contain potential bioactive molecules acting via the same mechanism.

Conclusion
In vivo, Cola anomala extract inhibited bacterial growth and showed to be bactericidal. In infected treated rats, it decreased bacterial load and protected them against weight loss and colon mucosal damage. Furthermore, the extract decreased PGE 2 , IL1β, TNF-α, and NO production. ese results support the use of the plant in traditional medicine in the treatment of gastrointestinal ailments and could therefore be a potential candidate for the production of improved forms of traditional medicines, cheap and available for local population for effective treatment of shigellosis. In future studies, extract activities will be evaluated on other models of experimentally induced diarrhea. Also, detailed phytochemical profiles of the extract will be explored so as to elucidate the major compounds underlying the observed activities.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.