Antibacterial Activity of Citrus Aurantifolia Leaves Extracts Against Some Enteric Bacteria of Public Health Importance

The study was conducted to determine the phytochemical composition and antibacterial activity of Citrus aurantifolia leaves extracts against clinical isolates of some enteric bacteria of public health importance. The result of phytochemical screening of the leaves extracts shows the presence of alkaloid, glycoside, saponin, tannin, flavonoid, steroids, terpenoid and phenol. The result of antibacterial efficacy of the extracts against the isolates indicated that the extracts were active against the isolates with higher activity in ethanol extract (with average zone of inhibition of 14.91mm) when compared to aqueous extract (12.67mm). The result of susceptibility of the isolates to the extracts showed Shigella was more sensitive to the extract with average zone of inhibition of 14.90mm, followed by Klebsiella (14.49mm), Escherichia coli (13.77mm) and Salmonella typhi with average zone of inhibition of 12.01mm. The minimum inhibitory concentration (MIC) of the extracts showed that dilutions of various concentrations of aqueous and ethanol of leaves extracts can inhibit the growth or kill the isolates at a concentration of between 2.125-20mg/ml. Statistical analysis of the results indicated that there is no significant different in the activity of the extracts against the isolates used at p<0.05. Findings from this study support the use of Citrus aurantifolia leaves extracts for medicinal purposes.


Introduction
Natural products such as plants have been integral part traditional medicine system in ancient such as Chinese, Ayurvedic and Egyptian [1]. Several published reports had described the antimicrobial activity of various crude extracts of plants either in single or in combination [2]. It has been estimated that about 2.5 million species of higher plants and their therapeutic values are yet to be determined. However, herbal extracts are becoming popular as natural medicine; preservatives and additives [3]. According to World Health Organization [4] medicinal plants contain substances in one of its organs such as stem, root, leaves, rhizomes, fruits, flower and seeds that can be used for therapeutic purposes or which are precursor for chemo-pharmaceutical semi-synthesis [4]. The medicinal plants are employed in the treatment or control of disease condition due to the presence of biochemical components called phytochemical [5]. Phytochemicals are considered as bioactive substances of plant origin and are of medical importance. They are otherwise known as secondary metabolites as they are of little important to the plant who manufactured them. These secondary metabolites synthesized naturally in various organs of plants such as leaves, stem, root, flower and seed [6]. Most of the modern drugs used today were originally obtained from plants [7]. Many researches confirmed the herbal extract boost immune system by stimulating the production of white blood cell which fight diseases [8]. Citrus aurantifolia belongs to Rutaceae, it is a polyembroyonic plant cultivated in several part of the world especially hot subtropical or tropical region such as India, USA, Nigeria, Mexico, West indies and Egypt [9]. The plant is shrub in nature and height of about 2 meter tall, evergreen with dense and irregular branches which possess short and stiff spines. The Citrus aurantifolia fruits are ovoid berry of about 3-6cm in diameter and sometimes possess apical papilla. When ripe, the fruits turn yellow from initial blue [10]. The plant is used in traditional medicine for treatment of several diseases such as cold and stomach ailment. It can also be used as an antiseptic, mosquito repellant, antifungal, antibacterial and antiviral agent. The health benefits of Citrus aurantifolia plant are highly associated with the high amount of bioactive constituents it contained such as phenols, flavonoids, carotenoid, vitamins and minerals [11]. Limes contain unique flavonoid compounds that have antioxidant and anti-cancer properties. The flavonoids help to inhibit cell division in many cancer cell lines in addition to its antimicrobial efficacy [12]. The plant also demonstrated bioactive activities for cold, fever, sinusitis, sore throats, asthma and bronchitis [13].

Collection, identification and authentication of plant materials
The leaves of C. aurantifolia were used in this study. The leaves were collected from the botanical garden of School of Technology, Kano State Polytechnic. After collection, the leaves were identified and authenticated at the herbarium in the Department of Plants Science, Bayero University Kano with the following voucher number BUKHAN 0341, voucher specimen of the plant's leaves were deposited in the herbarium for future references. The leaves were then rinsed with distilled water and air dried at room temperature for 2 weeks. The dried leaves were made into fine powder under laboratory condition using sterile pestle and mortar. The powder was stored in dark and air tight container.

Indole test
Indole test was conducted according to Cheesbrough [17] using Tryptophan broth. The broth was inoculated with the isolate of the test organism, and then incubated at 37 °C for 24. About 0.5ml of Kovack's reagents was added to the broth culture.

Methyl red test
Indole test was conducted using MR-VP broth as described by Holt et al. [16] and Cheesbrough [17]. The MR-VP broth was inoculated with an isolate of the test organism with the aid of sterile inoculating wire loop. The inoculated broth was incubated at 37 °C for 24 hours. About 5 drops of Methyl-red reagent was added to the broth culture.

Voges proskauer
MR-VP broth was inoculated with an isolate of the test organism using sterile inoculating loop and incubated at 37 °C for 24 hours. Six milliliter (6ml) of 5% alpha naphthol was added followed by 0.2ml of KOH. The tube was shaken gently and remained undisturbed for 5 minutes.

Citrate utilization test
Simmon's citrate agar was streaked back and forth with inoculums of the test organism and incubated aerobically at 37 °C for 24 hours.

Preparation of the leaves extracts
Water and ethanol were used as solvent for extraction. The aqueous and ethanol extracts of the plant leaves were prepared separately. Fifty grams (50g) of leaves powder was prepared by mixing with 500ml each of distilled water and ethanol respectively in 1liter flask. The flask were kept were kept for 3 day with intermittent shaking at room temperature. Filtration of the mixture was conducted using Whatman filter paper. The ethanol filtrate was evaporated using rotary evaporator at 50 °C while the aqueous filtrate was evaporated at 40 °C in water bath until dried extract samples were obtained. All the dried extract samples were dissolved in 10% DMSO separately to the final concentration of 200mg/ml as a stock concentration. The prepared extracts were refrigerated for further use [18].

Qualitative phytochemical screening
The preliminary phytochemical screening of C. aurantifolia leaves extracts was conducted using standard laboratory methods as described by Sofowora [19] and Trease and Evans [20]. The presence of bioactive constituents such as alkaloids, reducing sugars, steroid, glycoside, phenol, Anthraquinones, terpenoids, flavonoids, saponin and tannin were determined.

Antibacterial assay of the extracts
Agar well diffusion method was used to determine the activity of the extracts against the test isolates as adopted by Nas et al.

Determination of minimum inhibitory concentration (MIC)
Broth dilution method was used for MIC determination.
During the process, double fold serial dilution of the extracts was

Determination of minimum bactericidal concentration
Minimum bactericidal concentration of the extracts was determined using method adopted by Nas et al. [21].

Statistical analysis
One-way Analysis of variance (ANOVA) was used to analyze the data obtained from the zone of inhibition produced by the isolates against the extracts. The statistical program SPSS 21.0 version (Statistical Package for the Social Science) was employed. The result was as a mean ± standard deviation. The significance level for the differences was set at p<0.05.

Preliminary phytochemical screening
The result of phytochemical screening of the extracts is presented in Table 1. The results indicate the presence of alkaloid, flavonoid, glycoside, saponin, steroid, phenols, terpenoid and tannin in both aqueous and ethanol extracts.

Antibacterial activity of ethanol extract
The result of antibacterial activity of ethanol leaf extract of C.
aurantifolia is presented in Table 3. According to the result, the zone of inhibition recorded by the isolates depends on the concentration of the extract and the types of bacteria isolate. Klebsiella pneumoniae has the highest zone of inhibition (19.27±0.31mm) at 100mg /ml. The zone of inhibition of the control (Amoxicillin 50mg/ml) ranges from to 19.34-20.34mm.