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Covenant Journal of Physical & Life Sciences (CJPL) Vol. 6 No. 2, Dec. 2018

An Open Access Journal Available Online

Antibacterial Activity of the of guineense against Selected Enteric Bacteria

*Ajiboye Adeyinka Elizabeth1, Babatunde Shola Kola2, Adedayo Majekodunmi Rachael1, Ajuwon Israel Busayo1, Odaibo Diane Adebobola3 & Ihesie Idinmachukwu Ugochinyere1

1College of Pure and Applied Sciences, Department of Biosciences and Biotechnology, Microbiology Unit, Kwara State University, Malete, P.M.B. 1530,Ilorin, Kwara State, . 2Department of Biological Sciences, Faculty of Science, Kings University, Ode-omu, Osun State, Nigeria 3University of Ilorin Teaching Hospital, Ilorin, Kwara State, Nigeria *[email protected]; [email protected]

Abstract: This study was aimed at evaluating the antibacterial activity and screening the phytochemical composition of the seed of Dialium guineense. The aqueous, methanolic and ethanolic extracts of the seed of Dialium guineense were analyzed against some clinical isolates. The phytochemical composition and antibacterial sensitivity testing were carried out using standard methods. The clinical isolates were Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Salmonella typhi. Broad spectrum antibiotics were used as positive control for the antibiotic sensitivity test. Phytochemical screening of the extracts revealed the presence of saponin, tannins, alkaloid and glycoside while antimicrobial activity test at various concentrations for ethanolic and methanolic extracts showed significant results against the selected enteric bacteria with the exception of aqueous extracts which showed no antimicrobial activity against any of the isolates. The highest zone of inhibition (13.33mm) was obtained against S. typhi using methanolic extract while E. coli had a zone of inhibition of 10.67mm using ethanolic extract. P. mirabilis showed the lowest zone of inhibition (3.67mm) using ethanolic extract. Ciprofloxacin showed the highest sensitivity to the test organisms while Gentamicin showed the lowest

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sensitivity. MIC results for the methanolic and ethanolic seed extract against the microbial isolates varied. However, a higher concentration above 225 mg/ml would be required for bactericidal activity, From these findings, the seed of Dialium guineense is a potential source of bioactive compounds and may have implications in the management of infectious diseases caused by some enteric bacteria. Keywords: Dialium guineense, phytochemicals, solvent extraction, enteric bacteria, antimicrobial activity.

Introduction The pulp is edible and sweet, with Medicinal represent a rich some quantities of tannins and source of antimicrobial agents. A wide ascorbic acid. It is quite a good source range of medicinal parts is used of protein and minerals as reported by for extract as raw drugs and they Arogba et al. [10]. D. guineense is possess varied medicinal properties. called ―Awin‖ among the Yoruba Although, hundreds of plant species speaking Nigerians. It is also known as have been tested for antimicrobial ―Icheku‖ among the Igbos in the properties, the vast majority of them Eastern part of Nigeria and as have not been adequately evaluated, ―Tsamiyarkurm‖ among the Hausa considering the vast potentiality of speaking Nigerians [9, 11]. Nwosu plants as sources of antimicrobial [11] reported that the bark and leaves drugs with reference to antibacterial of D. guineense have shown some and antifungal agents. It’s been medicinal properties and are used reported globally about the against several diseases. The antimicrobial properties of various usefulness of D. guineenseas an anti- medicinal plants and their use for ulcer agent has been reported [12]. The medicinal purposes [1, 2, 3,]. Quite a antimicrobial activities of the fruit number of plants have been used pulp extracts of D. guineense against traditionally for medicinal purposes as some clinical isolates have been they contain a variety of compounds of elucidated [13]. According to Bero et known therapeutic values[4, 5, 6].It is al. [14] the leaves and stem bark are expedient that extracts from these used for the treatment of some medicinal plants showing infections such as diarrhoea, severe antimicrobial activities at specific sites cough, bronchitis, stomach aches, other than those used by synthetic malaria fever, jaundice and drugs will be active against drug- haemorrhoids. resistant microbial pathogens. Some researchers have authenticated However, information the activity of activities of the leaves and stem bark such medicinal plants is inadequate of D. guineense which include its [7]. Plants have ability to synthesize antibacterial and analgesic activities diverse aromatic secondary [15, 16,], as well as antioxidant metabolites, and these groups of properties [17]. Globally, infectious compounds show antimicrobial action diseases have been a main cause of and serve as plant defense mechanisms death and diverse kinds of disability against pathogenic microorganisms [8, which accounts for about 23% of 9].Dialium guineense (Wild) belongs worldwide disease as opined by to the family of , commonly Murray and Lopez [18]. Lomovskaya called black velvet or velvet tamarind.

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Ajiboye A. Elizabeth, et al CJPL (2018) 6(2) 1-10 and Bostian [19] have suggested that Extraction of Plant Material improvement of the efficacy of Forty grams of D. guineense powdered available antibiotics might be a sample was soaked in 1000ml of reasonable and sustainable option due sterile distilled water, 160 ml of 95 % to the challenge between the slow ethanol and methanol respectively for development of new drugs and the fast 24 hours at room temperature on emergence of resistant strains. This orbital shaker at 160 rpm. The content may raise some hope rather than was filtered using muslin cloth and making the future management of evaporated to dryness using water bath infectious diseases look bleak. at 60 oC. The extracts were collected Although, various works have been and stored at refrigerator temperature done to investigate the antimicrobial until when needed. and phytochemical screening of D. Antimicrobial Sensitivity Method guineense plant using its leaves, bark, Antimicrobial activity study of the and roots but fewer or no researches crude extracts of ethanol, methanol have been recorded in investigating the and aqueous extract of Dialium potential antimicrobial effects of the guineense seed was carried out on plant . Escherichia coli, Proteus mirabilis, This work is therefore aimed at Klebsiella pneumoniae and Salmonella screening the phytochemicals and typhi. The agar well diffusion method conducting antimicrobial activity tests of Collins et al. [20] with slight on the seed of Dialium guineense. modification was adopted for this

Materials and Methods assay. The antibiotic discs of Plant Collection Ofloxacin, Gentamicin and The of Dialium guineense were Ciprofloxacin were placed aseptically purchased at Ketu in Lagos State, on seeded plate of the isolates with the Nigeria. The seeds were separated aid of a sterile pair of forceps. from the fruit and were identified at Determination of Minimum the herbarium section of the Inhibitory Concentration (MIC) Department of Plant Biology, The MIC of the plant extracts were University of Ilorin, Nigeria. determined by using the broth dilution

Collection of Microbial Isolates method [20]. One ml of 24 hours The bacteriall isolates were obtained culture of test organisms (107cfu/ml) from the Department of Microbiology adjusted to McFarland turbidity and Parasitology, University of Ilorin standard was incubated in test tubes Teaching Hospital, Ilorin, Kwara with varying concentrations of 125, State, Nigeria. The pure isolates 150, 175, 200 and 225 mg/ml of plant collected were as follows; Salmonella extracts in normal saline at 37 oC for typhi, Escherichia coli, Klebsiella 24 hours. The concentration with the pneumoniae and Proteus mirabilis. lowest dilution and no detectable The isolates were sub-cultured and the bacterial growth was considered as the culture was maintained at 4 oC. minimum inhibitory concentration However, further sub-culturing was (MIC) [21]. done to keep the organisms viable.

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Determination of Minimum the highest zones of inhibition on the Bactericidal Concentration (MBC) enteric bacteria. Also, the ethanolic The minimum bactericidal crude extract showed a different range concentration was determined by first of inhibition on the isolates with E. selecting plates that showed no growth coli being the most sensitive with during MIC determination and 10.67mm diameter zone of inhibition incubated for another 24 hours at 37 followed by S. typhi with 10.00 mm oC. The minimum bactericidal diameter zone while K. pneumoniae concentration was considered as the showed the lowest zone of inhibition lowest concentration that could not with 2.67mm (Figure 1). produce a single bacterial colony [20, Minimum Inhibitory Concentration 21]. About five-fold dilutions was done

Qualitative Phytochemical from the crude methanolic and Screening ethanolic extracts with the The method described by Adebayo and concentration of 225 mg/ml, 200 Sofowora [22] was used to test the mg/ml, 175 mg/ml, 150 mg/ml and presence of saponins, tannins, 125 mg/ml for each extract phenolics, alkaloids, steroids and respectively. The MIC (minimum glycoside. inhibitory concentration) of the

Statistical Analysis extracts was also determined. All the Data obtained were expressed as mean organisms showed different MIC and standard deviation of triplicates concentrations of each extract. P. and were statistically analysed using mirabilis showed low MIC range at SPSS statistical package of version 150 mg/ml using methanolic extract 16.0. The results obtained were and 200 mg/ml using ethanolic extract. statistically analysed by ANOVA. However, the MIC of both the Values were considered significant at methanolic and ethanolic extract p˂0.05 against E. coli and S. typhi was 225 mg/ml. The MIC value for K. Results Antibacterial Activity of the pneumoniae was 200 and 225 mg/ml Extracts. for ethanolic and methanolic seed Ethanolic and methanolic extracts extract respectively. Using ANOVA to showed significant result against the analyse the data obtained, there was no significant difference at P ˂ 0.05. selected enteric bacteria with the exception of aqueous extracts which Minimum Bactericidal showed no zones of inhibition on any Concentration (MBC) of the isolates. All the plates that showed MIC after S. tyhpi showed the highest inhibition 18-24hours of incubation were plated to the crude methanolic extract of D. out for MBC. Minimum bactericidal guineense seed with 13.33mm zone of concentration was evaluated in order inhibition followed by E.coli with to ascertain the bactericidal effect of 11.33mm zone inhibition. P. mirabilis the methanolic and ethanolic extracts showed the lowest zone of inhibition on the enteric bacteria. Both to the crude methanolic extract with methanolic and ethanolic extracts at 3.67mm zone of inhibition. The the various concentrations used did not methanolic extract however showed show any bactericidal activity. This

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Ajiboye A. Elizabeth, et al CJPL (2018) 6(2) 1-10 implies that a higher concentration mirabilis but about 4.0mm and well above the MIC would be required 0.67mm on K. pneumoniae and S. for bactericidal activity. For all typhi respectively. K. pneumoniae concentrations plated out there was showed high sensitivity to Ofloxacin growth of organisms after 18-24hours having a zone of 14.00mm. Zones of of incubation. inhibition of 11.67mm and 11.00 mm Antibiotics Sensitivity Testing was obtained for E. coli and S. typhi Using some broad antibiotics as respectively. P. mirabilis expressed reference drugs, the sensitivity of the very low sensitivity with a zone of antibiotics, Ofloxacin (5 µg), 4.33 mm (Figure 2).

Ciprofloxacin (30 µg) and Gentamicin Qualitative Phytochemical (10 µg), were investigated on Muller- Screening of D. guineense extracts. Hinton agar plates (MHA). Reading The ethanolic, methanolic and aqueous was taken after incubation for 18- extracts of the seed of D. guineense o 24hours at 37 C. Among all, showed positive test for all the Ciprofloxacin showed the highest screened phytochemicals except for zones of inhibition to isolate K. steroid which showed a negative result pneumoniae. Gentamicin showed the for all extracts. Table 3 showed the lowest range of inhibition zones to the phytochemical screening result for organisms with zero effect i.e. no each extract. zones of inhibition on E .coli and P.

Figure 1: Zones of inhibition of the crude extracts of D. guineense on the bacterial isolates

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Figure 2: The effect of antibiotics on the bacterial Isolates.

Table 1: MIC and MBC of the ethanolic and methanolic seed extract of D. guineense Enteric Bacteria Ethanolic Conc. mg/ml Methanolic Conc. mg/ml extract extract MIC MBC MIC MBC Escherichia coli 225 - 225 - Proteus 200 - 150 - mirabilis Klebsiella 200 - 225 - pneumoniae Salmonella 225 - 225 - typhi

Table 2: Qualitative phytochemical screening of the ethanolic, methanolic and aqueous seed extracts of D. guineense Phytochemicals Ethanolic Methanol Aqueous extract extract extract Saponin +++ + ++

Tannin +++ +++ +

Alkaloid ++ ++ +++

Steroid - - -

Glycoside +++ ++ ++

Key Notes: + → Positive, ++ → More positive, +++ → Most positive, - → Negative

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Discussion extracts has been revealed from the The seed of Dialium guineense had a result of the phytochemical screening significant effect on all test bacteria. of ethanolic and methanolic extracts. However, the crude aqueous extract Ethanol and methanol extracts showed did not have any antimicrobial effect a positive result for the components on enteric bacteria such as Escherichia tested except for steroid which showed coli, Proteus mirabilis, Klebsiella a negative result for both extracts and pneumoniae and Salmonella typhi. Orji also for the aqueous extracts. Fat and et al. [16] explains that the active lipids are not present in the seed of ingredient in the plant bark and leaf Dialium guineense. This could be the are more soluble in ethanol than water. reason why steriod was negative for all This may be the reason why aqueous extracts. This is in line with Arogba et extract had no effect on the enteric al. [24], who said that the edible part isolates. However, the evaluated (pulp) of ripe D. guineense fruit is phytochemicals of the aqueous extract sweet but acidic and relatively poor in of the seed of Dialium guineense protein and oil with fairly low levels of revealed the presence of saponins, ascorbic acid and tannin. However, the tannins, alkaloids and glycosides, seed had also been reported to be although, there is a relatively low mildly acidic, poor in oil but fairly presence of tannin and absence of good source of protein and minerals. steroids. This supports the trends of This could also be evident from this Gideon et al. [17] who said flavonoids, study as ethanolic, methanolic and alkaloids, tannins and saponins are aqueous extract showed a negative present in the leaf and bark of D. composition of steroid. The findings of guineense of ethanolic and aqueous this study do not agree with Orji et al. extract. Akinpelu et al. [9] also [16] who reported the antimicrobial supported the fact that there are active properties of the crude aqueous leaf components present in D. guineense. extracts of D. guineense against S. He reported that the phenolic aureus and K. pneumoniae but showed compounds from medicinal herbs and the presence of flavonoids, alkaloids, dietary plants play important role in tannin and saponin in the aqueous health; in addition to enhancing extracts. This is in conformity with the antimicrobial activities in these plants. findings from this present study as the Udoh et al. [23] reported that the aqueous extract of D. guineense seed sensitivity of aqueous extract of had these phytochemicals. Akinpelu et Lasianthera africana possess al. [9] also reported the bioactivity of antimicrobial effect against Salmonella the methanolic crude leaf extract of D. typhi, Escherichia coli and Proteus guineense on fourteen environmental vulgaris. strains of Vibro species. Their findings

However, the ethanolic and methanolic are in conformity with this study in crude extracts showed a greater range line with the antimicrobial activities of of inhibition to the microbial isolates the methanolic and ethanolic extracts with E. coli and S. typhi showing the of the seed of D. guineense against highest sensitivity and K. pneumoniae Escherichia coli, Salmonella typhi, and P. mirabilis being the least Proteus mirabilis and Klebsiella sensitive. The sensitivity of these pneumoniae. Orji et al. [16] also reported the presence of alkaloids,

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Ajiboye A. Elizabeth, et al CJPL (2018) 6(2) 1-10 flavonoids, tannins and saponins in the action on the bacterial isolates, it was ethanol extract of the stem bark of D. able to inhibit their growth. guineense. The fruit pulp of D. In conclusion, the result of this study guineense has been reported by has revealed that the methanolic and Arogba et al. [10] as antiulcer and as a ethanolic seed extract of D. guineense vitamin supplement. has antimicrobial activity against some The range of MICs in this study is microbial isolates which are enteric between 150-225mg/ml for all the Also the seed extracts were shown to isolates. Also, Orji et al. [16] reported possess significant quantities of MIC values for the aqueous and phytochemicals such as saponin, ethanolic extract of leaf and stem bark tannin, alkaloid, steroid and of Dialium guineense for S. aureus and glycosides. This study also agrees with K. pneumoniae to be 200mg/ml. This the findings of Ajiboye et al. [13] who variation could be as a result of the also reported antimicrobial activity of variety of strains of microbial isolates the fruit pulp of D. guineense on some used, varying phytochemical microbial isolates. Therefore, the seed components of plant parts and the of D. guineense can be employed as a extraction methods used as well. potential antimicrobial agent.

The present study has revealed However, further research should be evidence of the sensitivity of the tailored towards investigating the microbial isolates to some of the different parts of D. guineense such as broad-spectrum antibiotics while some leaf, root, bark, stem and fruit pulp in were resistant. E. coli and P. mirabilis relation to their phytochemical showed resistance to gentamicin while components and their effects on K. pneumoniae and S. typhi were clinical isolates. susceptible. However, the isolates Acknowledgments were susceptible to ofloxacin and We acknowledge the Laboratory ciprofloxacin, but P. mirabilis and S. Technologists of Microbiology Unit, typhi showed low zones of inhibition Kwara State University, Malete, to ciprofloxacin. Ofloxacin is a broad- Nigeria for their technical supports. spectrum antibiotic of the class quinolones and due to its mode of

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