Antibacterial Activities of Hyphaene Thebaica (Doum Palm) Fruit Extracts Against Intestinal Microflora and Potential Constipatio

Tanzania Journal of Science 47(1): 104-111, 2021 ISSN 0856-1761, e-ISSN 2507-7961 © College of Natural and Applied Sciences, University of Dar es Salaam, 2021 Antibacterial Activities of Hyphaene thebaica (Doum Palm) Fruit Extracts against Intestinal Microflora and Potential Constipation Associated Pathogens in Yola Metropolis, Nigeria Joel Uyi Ewansiha 1*, Chidimma Elizabeth Ugo 1, Damaris Ibiwumi Kolawole 1, 2 and Lilian Sopuruchi Orji 3 1Department of Microbiology, Federal University of Technology, Yola, Nigeria. 2Department of Food Science and Technology, Universidade de Sao Paulo, Brazil. 3Bio-resources Development Centre, National Biotechnology Development Agency, Lugbe, Abuja Nigeria. Emails: [email protected]*; [email protected]; [email protected]; [email protected] *Corresponding author Received 1 Nov 2020, Revised 25 Dec 2020, Accepted 28 Dec 2020, Published Feb 2021 Abstract This study aimed at determining the antibacterial activities of Hyphaene thebaica fruit extracts against some intestinal constipation associated bacteria. Qualitative analysis of some phytochemical constituents, agar well diffusion and broth dilution methods were used to determine the zones of inhibition and minimum inhibitory concentration (MIC) of the plant extracts. Phytochemical components viz flavonoids, saponins, terpenoids, tannins, phenols, alkaloids, glycosides and steroids were detected in the plant extracts, and the test organisms were susceptible to the plant extracts. The diameter zone of inhibition (DZI) obtained with n-hexane extract ranged from 15.10 ± 0.51 mm to 2.0 ± 0.55 mm against K. pneumoniae, 10.20 ± 0.57 mm to 2.00 ± 0.35 mm against P. aeruginosa and 8.00 ± 0.35 mm to 1.00 ± 0.55 mm against S. typhi, while the DZI for aqueous extract was 7.10 ± 0.23 mm to 2.0 ± 0.35 mm against K. pneumoniae, 6.20 ± 0.31 mm to 2.00 ± 0.35 mm against S. typhi and 5.42 ± 0.55 mm to 2.05 ± 0.75 mm against P. aeruginosa. The MIC and minimum bactericidal concentration of the extracts were 100 mg/mL and 200 mg/mL, respectively. Hyphaene thebaica fruit possessed antimicrobial activities against the test organisms. Therefore, toxicity and clinical studies are recommended for possible drug development. Keywords: Doum; Constipation; Intestinal; Microflora; Fibre; Hyphaene thebaica. Introduction et al. 2005). Antimicrobial activity involves Medicinal plants play vital roles in the complex mechanisms such as prevention of cell control and prevention of various diseases such membrane, cell wall formation and inhibition as cancers, diabetes, and cardiovascular of nucleic acid and protein metabolism. diseases (Mohanta et al. 2003). Some Hyphaene thebaica is an oval fruit that medicinal plants have been used for production belongs to the mint family (Arecaceae). It is a of various drugs and as principal raw materials desert palm fruit native to Nigeria, Senegal, for production of other conventional medicines Egypt, Tanzania, Kenya, Arabian Peninsula due to their antimicrobial activities (Srivastava and West India (Orwa et al. 2009). This fruit 104 http://journals.udsm.ac.tz/index.php/tjs www.ajol.info/index.php/tjs/ Tanz. J. Sci. Vol. 47(1) 2021 serves as source of minerals, (calcium, Materials and Methods potassium, phosphorus, sodium, and Preparations of plant materials magnesium), carbohydrate, B-complex The fresh fruits of Hyphaene thebaica were vitamins, and fibres that are essential for good harvested during the rainy season, precisely in nutrition (Hsu et al. 2006). The root is used in May, 2019 at Jambutu farmland Yola, the treatment of bilharzia, while the fruit pulp Adamawa State, Nigeria. The samples were is used in controlling hyperlipidaemia and kept in polyethylene bags, transported to the hypertension (Coopoosamy and Magwa 2007). laboratory, and were identified by Dr Changya Aboshora et al. (2014) reported on the of the Department of Plant Science in Modibbo antioxidant, anticancer, antimicrobial, and anti- Adamawa University of Technology, Yola, inflammatory activities of Hyphaene thebaica Nigeria. The epicarp of doum palm fruit was fruit due to the high amounts of phenol, and cleaned, scrapped with sterile (70% ethanol flavonoids. cleaned) scapel, and dried at room temperature Constipation is a condition of digestive (26 °C) until constant weight was obtained. system that makes bowel movements become The fruits were pulverized by pounding with less frequent and result in hard stools that are mortar and pestle to increase the surface area difficult to excrete. Studies by Zhao and Yu with the aim of enhancing the penetration and (2016) have shown that constipation is caused extraction by the extracting solvents. by disturbances in composition and stability of gut microbiota. Zhao and Yu (2016) stated that Sample collection changes or alterations of intestinal microbiota A total of 50 stool samples from patients with in patients with chronic constipation can be cases of constipation were collected from characterised by a relative decrease in obligate Peace Hospital, Jimeta, Adamawa State, bacteria (e.g., Bifidobacterium, Lactobacillus Nigeria and transported to the laboratory using and Bacteroides species) and parallel increase transport medium (Cary-Blair) for analysis. of potentially pathogenic microorganisms such as Campylobacter jejuni and Pseudomonas Extraction aeruginosa. Thus, this alteration might have Cold maceration method as described by influenced on intestinal mobility and secretory Obeidat (2011) was used for aqueous extract functions by changing the available amount of with distilled water as extracting solvent and physiological active substances and metabolic Soxhlet extraction procedure for the n-hexane environment (Khalif et al. 2005). Constipation extract as described by Ewansiha et al. (2017). can result from inadequate intake of fibres, and For aqueous extract, 250 g of plant material changes in diet (Khalif et al. 2005). were weighed and transferred into 600 mL of The increase in resistance of distilled water, the mixture was homogenised microorganisms to many commercial synthetic and left at room temperature (26 °C) for three products has led to search of plant materials days. After three days, the aqueous solution that can be used in developing new was filtered with muslin cloth, and the filtrates antimicrobial agents that can solve this were evaporated to dryness in a water bath set problem (Zhao and Yu 2016). Hence, this study at 60 °C. The dried extracts were stored in determined the antibacterial activities of doum screw capped bottles and kept in the palm (Hyphaene thebaica) fruit extracts against refrigerator at 4 °C until further analysis. intestinal microflora and potential constipation associated pathogens. Identification of phytochemical constituents The presence of flavonoids, tannins, phenols, alkaloids, and saponins were identified according to the methods reported by Akoma and Olawepo (2002). The flavonoids were 105 Ewansiha et al. - Antibacterial activities of Hyphaene thebaica (doum palm) fruit extracts … identified by dissolving 0.5 mg of the extract overnight culture of bacteria was gram stained, into 1 mL of ethanol and filtered. After observed through the microscope and was filtration, 2 mL of 1% HCl and magnesium grouped based on Gram’s reaction and ribbon were added. Colour changes of red or morphology. pink colour indicated the presence of flavonoids. Tannins were identified by Biochemical characterization dissolving 0.5 mg of dried plant extract into 1 The biochemical identification was done on the mL of distilled water, filtered and 2 mL of isolates using the method described by FeCl3 was added to the filtrate. Appearance of Chessbrough (2002). The tests included blue or black colour indicated the presence of catalase, lactose, oxidase, indole, citrate, tannins. mannitol tests and were done after microscopic Determination of alkaloids: The Alkaloids identification of the isolates to confirm their were determined by adding 0.5 mg of plant genus and species. extract to 1 mL of methanol, filtered and 1% of HCl was added to the filtrate and the solution Antibacterial susceptibility test was heated. Colour changes from red to pink Standardization of inoculum after the addition of Mayor’s reagent confirmed McFarland standard (0.5) was made by mixing the presence of alkaloids. 0.05 mL of 1.175% barium chloride dihydrate Determination of phenols: Phenols were also (BaCl2.H2O) with 9.95 mL of 1% sulfuric acid determined by dissolving 50 mg of dried (H2SO4) and its turbidity was used as a extract into 5 mL of distilled water, 2-3 drops standard for the test organisms. This was done of neutral ferric chloride solution was added, according to Chessbrough (2002). and a dark green colour indicated the presence of phenols. Preparation of extract concentrations Determination of saponins: Saponins were Different concentrations of the plant extracts identified by dissolving 0.5 mg of dried plant were made by using distilled water as diluent extract into 1 mL of methanol and filtered. and the n-hexane extract in 10% dimethyl Distilled water was added, the filtrate was sulfoxide. Each concentration of extracts (100 shaken for few minutes and persistent frothing mg/mL, 200 mg/mL, 300 mg/mL ,400 mg/mL, indicated the presence of saponins. and 500 mg/mL) were prepared by dissolving 1000 mg, 2000 mg, 3000 mg, 4000 mg and Isolation and identification of test organisms 5000 mg of extracts in 10 mL of water, Samples were prepared based on a method respectively. described by Pradhan and Tamang (2019) with brief modifications. Briefly, 1 g of stool sample Antibacterial assay was homogenised with 9 mL of distilled water The antibacterial test was done using agar well and 1 mL of the diluent was inoculated into diffusion method as described by Akoma and MacConkey agar, Salmonella-Shigella agar, Olawepo (2002). Mueller Hinton agar was used cetrimide agar and incubated at 37 °C for 24 and 0.1 mL of standardized inoculum was hours. After incubation, the plates were inoculated on the agar with even distribution. A observed for growth. Colonies from each plate well was made on the medium using a 6 mm were sub-cultured unto freshly prepared diameter sterile cork bearer.

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