Phytochemical Composition and Antibacterial Activities of the Ethyl Acetate Leaf Extract of Ocimum basilicum

Phytochemicals with antibacterial effects in the herb can lead molecules in developing new antibacterial drugs. Tukey’s post hoc pairwise


Introduction
Infectious diseases account for 41%, measured in Disability-Adjusted Life Years, of the global disease burden. That is far more than injuries (16%) and close to non-infectious diseases (43%) [1]. Among the major causes of this burden is the emergence of antibiotic resistance, a problem that is evolving towards being a global pandemic. It has resulted in the appearance of multidrug resistant bacteria, or superbugs [2,3]. Antibiotic resistance is a serious public health concern, especially in the developing countries with a high burden of infectious diseases [3]. Pathogens with a signi cant health threat include Methicilin resistant Staphylococcus aureus (MRSA), Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli (E. coli). These species are responsible for a number of diseases including pulmonary infections, urinary tract infections, skin and soft tissue infections, cellulitis, folliculitis and septicaemia [4][5][6][7][8]. The problem posed by these antibiotic resistant pathogens has brought to the fore the need for the development of novel antibacterial agents [3].
Biologically active compounds that could be isolated and harnessed for their antibacterial, antiviral and antifungal activities are found, naturally, in plants. These active compounds include alkaloids, tannins, terpenoids, fatty acids and avonoids. They are secondary metabolites with complex structures [9,10]. Their antimicrobial mechanisms include cell wall damage, cytoplasmic membrane damage, formation of reactive oxygen species, Deoxyribonucleic acid fragmentation, phosphatidylserine externalization, metacaspase activation, mitochondrial membrane depolarization, nuclear condensation, modulation of transcription factors, redox signaling and redox-sensitive transcription factors [11]. Exploitation of these factors can help in the development of better antimicrobial approaches. It is, therefore, conceivable that knowledge and data regarding the therapeutic potential of medicinal herbs is of great scienti c interest as effective alternatives to the battle of antibiotic resistant microorganisms. Studies into the pharmacological activities of medicinal herbs and plants aim at providing empiric evidence on the use of traditional medicine, their pharmaceutical applications and commercialization of their active components.
Species of the genus Ocimum have been studied for their medicinal properties and some have been found to possess anti-in ammatory, analgesic, antidiabeteic, antioxidant and antimicrobial activities [12,13,14]. Ocimum basilicum is a herb that grows in the sub Saharan Africa, Asia paci c among other regions. Its leaves have been used among the Mbeere people of Kenya to treat infectious diseases [15].
Based on its use in Kenya, it was compelling to determine the antibacterial potential of its leaves on different bacterial species. The phytochemical compositions of this herb was also determined and associated with its antibacterial actions.

Methodology Plant Material
Ocimum basilicum was obtained from Siakago, Kenya, on longitude 29 o and between latitudes 0 o 35'38"S and 37 0 38'12"E in January, 2017. Collection was based on its use locally. The plant was taxonomically identi ed and a voucher specimen deposited at the National Museum herbarium. The leaf samples were cleaned under running tap water and dried at room temperature to preserve heat sensitive molecules.
Dried leaves were then ground to powder using an electric mill.

Extract Preparation
Five hundred grams of the powdered O. basilicum leaves were soaked in 1.5 liters of ethyl acetate (LOBA Chemie, Mumbai, India) in a volumetric ask for 72 hrs. The solution was sieved using a muslin cloth and ltered using a Rocker 400 vacuum pump into a conical ask. Concentration to remove the solvent was done by a rotating evaporator. Percentage yield was calculated on a dry weight basis. The extract was stored at 4 o C. This procedure adopted that used by Sui et al. with minor modi cations (16).

Quantitative Phytochemical Analysis
Quantitative phytochemical analysis was done according to the procedure by Arora and Saini [17]. One gram of the extract was weighed into a 1.5 ml Eppendorf tube and dissolved using 1 ml of ethyl acetate.
The mixture was vortexed for 30 seconds and sonicated for ve minutes using a Branson 2510E-DTE sonicator. The sample was spun in a centrifuge for 5 minutes at 1300 rpm. The supernatant was transferred into 2ml auto sampler vials and analyzed using a Gas Chromatograph Mass Spectrometry (7683 Agilent Technologies, Inc., Beijing, China).

Antibacterial Assays
Four concentrations of the extract (1 g/ml, 0.75 g/ml, 0.50 g/ml and 0.25 g/ml) were prepared by dissolving in 4% Dimethyl sulphoxide (DMSO). These concentrations were put in sterile bijou bottles and refrigerated at 4 0 C during use. Microbial cultures from the stock suspensions were inoculated onto the surface of Mueller Hinton Agar and incubated for 18 hrs to obtain fresh growing colonies. A fresh growing bacterial colony was picked from a petri dish using a sterilized wire loop, inoculated into 4 ml of sterile peptone water and incubated at 37°C for 4 hrs. The bacterial suspensions were then adjusted in order to obtain turbidity comparable to 0.5 McFarland's standard, which corresponds to about 1-2×10 8 colony forming units/ml. These suspensions were used within fteen minutes of preparation.
Determination of antibacterial susceptibility patterns was done by the agar disc diffusion method as per Njeru et al. with slight modi cations [19]. Gentamycin (40 mcg/ml) and Neomycin (200 mcg/ml) were used as positive controls. Paper discs were prepared from a Whatman lter paper, placed in bijou bottles and sterilized. Aliquots of 0.1 mL of the bacterial suspensions were aseptically pipetted and inoculated by spread plate method on the Mueller hinton agar. The paper discs were impregnated with 20 µl of each concentration of the plant extract and control antibiotics and placed on the surface of the inoculated petri dishes using sterile forceps. Dimethyl sulphoxide at 4% was used as the negative control. Each disk was pressed against the agar medium to ensure level and complete contact. The agar plates were inverted and incubated at 37 o C for 24 hrs. After the speci ed time, diameters of zones of inhibition formed around discs were measured in millimeters (mm) to determine the activity of test samples against different strains. The scale used for determining the strength of antibacterial activities was 8-13 mm: low inhibition; 14-19 mm: moderate inhibition; ≥ 20 mm: high inhibition [20].

Determination of Minimum Inhibitory Concentrations and Minimum Bactericidal Concentration
The broth microdilution method [21] was used to determine the minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC). A bacterial colony of each test bacterium was picked using a sterilized wire loop and placed into 4 ml of Mueller Hinton broth medium in test tubes and incubated at 37 o C for 4 hours. The bacterial suspensions were then adjusted to 0.5 McFarland's standard. One hundred µl aliquots of the inoculated broth at McFarland's standard were placed into each well of the 96 well microtiter plates. One hundred microliters of the 1 g/ml concentration of the extract was pipetted and added into each of the rst well of the inoculated microtiter plate and serial diluted eight times. The extract dilutions obtained ranged from 1 g/ml, to 3.90624 mg/ml. One hundred microliters of gentamycin were pipetted into the control wells and serial diluted to concentrations that ranged from 20 mcg/ml to 0.15625 mcg/ml. Serial dilutions of neomycin were also made. Dimethyl sulphoxide was used as the negative control. The microtiter plates were incubated at 37 o C for 24 hours.
The concentrations that inhibited growth were taken as the MIC.
For MBC determination, fty µl aliquots from each inhibited well were pipetted and sero-diluted to the ninth dilution. Fifty µl aliquots of the ninth dilution were pipetted and inoculated onto the surface of the agar plates. Fifty µl aliquots of the bacterial suspensions in the wells immediately above and below the MIC well were also serial-diluted and inoculated onto the surface of the agar plates. The inoculated plates were incubated at 37 o C for 24 hrs. The lowest concentrations that showed complete inhibition of bacterial growth were taken as the minimum bactericidal concentrations.

Data Analysis
The antibacterial experiments were done in triplicates. Statistical analyses were done by One way analysis of variance (ANOVA) and students T-test. Data is presented as Means ± Standard deviation from the mean. Signi cance was set at p < 0.05. Statistical analyses were carried out using Minitab version 17.

Percentage Yield
The  Table 1 and Fig. 1 below. The antibacterial activities of the extract are presented in Tables 2-6. A broad spectrum antibacterial activity was exhibited by the extract of O. basilicum. The zones of inhibition ranged from 17.33 ± 0.58 mm to 27.00 ± 2.00 mm in diameter. Gram negative bacteria showed more susceptibility to the organic extract compared to the gram positive bacteria. Among the gram negatives, P. aeruginosa had the highest zone of inhibition (27.00 ± 2.00 mm), while among the gram positives, MRSA had the highest zone of inhibition (25.00 ± 1.73 mm) at 1 g/ml concentration of the organic extracts. There was a gradual decrease in the antibacterial activity of the extract that coincided with a decrease in the concentration of the extract. Two Sample T tests for the determination of Signi cance differences between strains The clinical isolates exhibited a reduced susceptibility to the organic extract compared to the quality control strains. However, this difference was not signi cant between the ATCC strains and the clinical isolates. A notable reduction in the zones of inhibition was observed in tandem with decreasing concentrations of the extract. These results are presented in Tables 3, 4 and 5.

Minimum Inhibitory Concentration and Minimum Bactericidal Concentration
At a concentration of 62.5 mg/ml, the extract exhibited its MIC on E. coli, S. aureus and MRSA while on P. aeruginosa, MIC was manifested at a concentration of 125 mg/ml. The leaf extract exhibited an MBC of 62.5 mg/ml on the S. aureus strain and an MBC of 125 mg/ml on MRSA, E. coli and P. aeruginosa. These results are presented in Table 6 below.

Discussion
Medicinal plants contribute to the development of new chemo-preventive agents. It is therefore important to determine their bioactive compounds as well as antibacterial activities [22]. Plants produce a wide collection of phytochemicals that are related to stress, defence mechanisms and antimicrobial activities [23]. The ethyl acetate leaf extract of O. basilicum had a variety of phytochemical classes and exhibited antibacterial activities.
Flavonoids are free radical scavengers and water soluble antioxidants with the ability to prevent oxidative cell damage. These metabolites protect the body against cancer, in ammation, allergens, microbes, platelet aggregation, tumors and hepatotoxins [24]. The methylated avonoid, 6,3'-Dimethoxy avone exhibits antimicrobial activities. Its derivative has broad spectrum antibacterial activities on E. coli, P. aeruginosa and S. aureus [25,26].  [27]. Synthetic derivatives of alkaloids are medicinally important for their antispasmodic, analgesic and bactericidal effects. Its physiological activities are apparent when this metabolite is administered in animal models [28].
Terpenoids represent the most diverse and largest class of chemicals among the many metabolites produced by plants. They are a group of compounds possessing an isoprene unit as their basic structure.
They are classi ed based on the number of carbon atoms. Terpenoids provide protection against pathogenic microorganisms [29]. Nootkatone is a sesquiterpene that is synthesized by the oxidation of valencene [30]. Nootkatone has been shown to exhibit antibacterial activities on Gram-positive bacteria including S. aureus, Enterococcus faecalis, Corynebacterium diphtheriae, Listeria monocytogenes and Bacillus cereus [30]. This compound exhibits antibacterial effects by targeting metabolites or structures that are speci c to Gram-positive bacteria such as the peptidoglycan component of cell walls. Synthetic retinoids contain an isoprene unit that is capable of killing MRSA by penetrating and disrupting the lipid bilayers [31]. There is a possibility that nootkatone inhibits bacterial proliferation by acting on the synthetic pathway of peptidoglycan.
Fatty acids identi ed include Oxirane, 2-methyl-2-(1-methylethyl)-; 11-Eicosenoic acid, methyl ester and 1,3-Dimethyl-5-isobutylcyclohexane. 11-Eicosenoic acid, methyl ester has been associated with antibacterial activity and anti-in ammatory effects [38]. The exact mechanisms by which fatty acids impose their antibacterial effects remain unknown. It has, however, been hypothesized that these molecules induce peroxidative processes that inhibit bacterial fatty acid synthesis. Fatty acids may also interact with cellular membranes thereby causing leakage of molecules from the cells, reduction of nutrient uptake or inhibiting cellular respiration [42]. Amines eluted from the extract include 9-Thiabicyclo  [39,40]. Aldehydes with long chain fatty alcohols such as E-11(13-Methyl) tetradecen-1-ol acetate, Tridecenol < 2E-> and tetradecanal have been shown to have antibacterial activities on S. aureus [41]. Tetradecanaal and tridecanol are long chain alcohols that exhibit their antibacterial activity by damaging cell membranes thereby leading to the leakage of K + ions together with subsequent reactions that lead to further leakage [41]. Compounds that possess an alkyl chain promote antibacterial activity and resensitize methicillin susceptible and resistant S. aureus to antibiotics [41].
Phenolic compounds with imidazole moieties such as 1H-Imidazole, 2-ethyl-4,5-dihydro-4-methyl-; 3,6,6-Trimethyl-cyclohex-2-enol and l-Alanine, N-(2-thienylacetyl)-, butyl ester possess antibacterial properties. The presence of the imidazole ring to the quinolone moiety increases its antibacterial activity [43]. The antibacterial effects exhibited by the extract were broad spectrum and could be attributed to the effects of these phytochemicals. The inhibitory effects were exhibited in a dose dependent manner with a better e cacy being exhibited on the gram positive bacteria compared to the gram negative. The difference in susceptibility indices could be attributed to the fact that gram negative bacteria have a stable peptidoglycan layer that allows bioactive compounds into the cytoplasm at a lower rate compared to the gram positive bacteria. The dose dependent active nature of the extract on the bacterial strains was due to the decreasing concentrations of active compounds. The ethyl acetate leaf extract of O. basilicum was found to have strong antibacterial properties [20].

Conclusions
The results of this study provide an important basis for the use O. basilicum in the treatment of bacterial diseases. The extract also contained various pharmacologically active compounds that could be used as Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. substantively revised the manuscript; MM conceived and designed the study, interpreted the data, substantively revised the manuscript; JOO conceived and designed the study, interpreted the data, substantively revised the manuscript .