Antibacterial Activities of different Fractions obtained from Methanolic Extracts of Allium sativum bulbs and Gacinia kola Seeds

— Antibacterial activities of different fractions obtained from crude methanolic extracts of Allium sativum and Garcinia kola and their time kill assay were investigated individually and in combined form. Standard methods were used and test organisms include: Bacillus cereus, B. subtilis, B. anthracis, B. stearothermophilus, Clostridium sporogenes, Corynebacterium pyogens, Staphylococcus aureus, Enterococcus faecalis, Micrococcus luteus, Escherichia coli, Klebsiella pneumoniae, Psedomonas aeruginosa, Salmonella sp., Shigella sp., and Proteus vulgaris. Chloroform fraction of G. kola exhibited broad spectrum effect on the test isolates while butanol fraction of A. sativum exhibited narrow spectrum effect on three Gram negative isolates. When the same fractions from each plant’s extract were combined at equal concentration and subjected to time kill assay, combined butanol fraction showed an excellent result with the ability of killing 100% of Staphilococcus aureus within 90 min at 0.66 mg/ml and 100% of Klebsiella pneumonia cells within 60 min at 1.32 mg/ml.Garcinia kola seeds and Allium sativum bulb extracts when combined had a broad spectrum antibacterial activity, with the combined butanol fractions being bactericidal as seen in the killing rate within short period and at a low concentration and this could be an important way of overcoming issues of antibiotic resistance.


INTRODUCTION
Plant materials had been the oldest method of combating infections and diseases with different types of plant and various parts been used in numerous parts of the world to treat human ailments (1). This is because plants contain diverse chemical substances like phenols, quinones, flavonoids, tannins and coumarins (2) which show antibacterial and antifungal effect and act as means by which plant defend themselves against pathogens (3). Continuous use of antibiotics for treating ailment in human had led to the development of resistant in bacteria and mechanism adopted include; alteration of target site, enzyme inactivation and efflux pump (4). Assessment of compounds that were biologically active from plants and antibacterial activities depend greatly on the solvent utilized during the process of extraction. Although, water is universally used as solvent to extract plants products with antimicrobial activity whereas plant extracts extracted with organic solvents had been said to have greater antimicrobial effect in comparison to water extracts (5).
A study reported chloroform (out of about twenty different solvent used) as the best solvent in extracting non-polar compounds which are biologically active (6). Another study reported chloroform extracted material to possess the highest zone of inhibition against Candida (7). Due to the aromatic nature of most organic compound found to possess antimicrobial activities, they are often extracted using methanol and ethanol while other solvent include dichloromethane, acetone, and hexane. Researchers also combine solvents in order to get the best extraction of compounds. A study was conducted on different solvent to assess their ability to dissolve antimicrobials from plants, extraction rate, ease of its removal from the extract, toxicity and acetone have highest rating in the overall tests (8).
Garcinia kola is a member of the Guittiferae's family and often called bitter kola while Allium sativumbelong to the Family Liliaceae. G. kola had been reported to be useful in treating bronchitis, liver disorders, hepatitis, diarrhoea, laryngitis, and gonorrhoea (9). A. sativum are been used in some parts of Africa due to their important ability in preventing heart, cardiovascular disease (10) and it has been said that use of garlic regularly may help in preventing cancer, to treat malaria, and to boost immunity.
Assessment of antibacterial activities of G. kola seeds showed that activity has been demonstrated for the aqueous, ethanolic, acetone and petroleum ether extracts (11) (12).
Another study also reported the antimicrobial potential of the methanol extract of G. kola seeds and fractions obtained from the extract (9).
A scientist (13) reported the antibacterial effect of various extracts of garlic and evaluated the effects of crude extracts on glycogen glucoamylase, in vitro. Another study reported the ability of garlic extract and its fractions to inhibit the growth of Enterobacteriaceae sp obtained from sprouted Mung bean (14). Therefore, this study assessed the antibacterial effect of various fractions obtained from methanolic seeds extract of Garcinia kola and Allium sativum bulb individually and in combined form on some selected bacterial isolates and also examined the rate at which these fractions killed bacterial isolates.

Collection of plant materials, preparation and process of extraction
Seeds of G. kola and bulbs of A. sativum were purchased from Central Market Ile -Ife in the year 2013 and identified at the department of Botany Obafemi Awolowo University Ile -Ife. The methanolic extraction was done by weighing 550 g of each of the powdered plant materials into flat bottom flask separately containing 60% methanol. These flasks was swirled to mix and left for four days and regularly agitated after which the mixture were filtered with Whatman filter paper No 1 to obtain clear solution of the extract. The methanolic filtrates of G. kola seeds and A. sativum bulbs were then evaporated using a rotary evaporator and lyophilized. The yield obtained from Garcinia kola seeds was 115g and that of Allium sativum bulbs was 125 g.

Separation of the crude extract into different fractions
About 50 g each of the crude extracts were dissolved in 300 ml of pre-sterilized deionized water and poured into a separating funnel and extracted with n-hexane (5×200). The n-hexane fractions were concentrated using rotary evaporatorand these did not yield anything. The resulting aqueous solution was re-concentrated in vacuo to remove traces of n-hexane. The residues were further extracted with chloroform (5× 200 ml). The chloroform fractions obtained were also concentrated and lyophilized and 8.32 g powder was obtained from Garcinia kola extract while 6.45 g was obtained from Allium sativum extract which were stored and maintained at -20 0 C for further use. The ethylacetate fraction (5.14 g for G. kola and 5.5 g for A. sativum) and butanol (7.5 g for G. kola and 8.75 g for A. sativum) fractions were also obtained by similar process. The aqueous phase remaining were freeze-dried and yielded 25.23 g for G. kola and 27.15 g for A. sativum which were stored as stated earlier (9).

Assessment of antimicrobial activity of plant's material
The agar-wells diffusion assay as described previously (9) was used to confirm the antibacterial effects of different fractions of the plant extracts on the test isolates. Eighteen hours old broth culture of the test isolates were standardized (0.5 McFarland) before use and sub-cultured on Mueller-Hinton agar by uniformly seeding the agar plates with the inoculums suspension. Plant extract solutions (35 mg/ml) were dispensed into wells bored with the use of sterilized cork borer (6 mm diameter) in the agar medium separately and in-combination (35 mg/ml) and streptomycin (1 mg/ml) as positive control.
The plates were left on the bench for 1 h to allow solution diffuse properly into the medium before incubating the plates for 24 h at 37 o C. Zones of inhibition were examined on the plates and measured in millimeter (using ruler). The experiments were performed in duplicates.

Dermination of minimum inhibitory concentrations (MICs) exhibited by the extract on test bacteria
The MICs of the extracts were assessed as previously described (15

Determination of the rate at which the active fractions killed the test bacteria
The rate and extent to which the active fractions of the plant extract killed the test bacteria were determined using the method described earlier by (16). Experiments were carried out by using each of the active fractions on S. aureus and K. pneumoniae as representative of each of the groups of organisms in relation to Gram reaction. Viable counts on the test organisms were pre-determined. About 0.5 ml of a known cell density (by viable counts 10 6 cFu/ml) from each test bacteria suspensions were dispensed into 4.5 ml of the active fractions at various concentrations. The solutions were mixed very well and held at 28-30 0 C and the rate of killing were determined within 2 h of exposure. An aliquot of 0.5 ml of the solution were withdrawn at set time interval and dispensed into 4.5 ml nutrient broth that contain 3% "Tween 80" as recovery media so as to neutralize the antimicrobial effect from the test solutions. The suspensions were mixed very well and then diluted up to 10 -5 in sterilized normal saline with 0.5 ml of the final dilution of the test organisms transferred into sterile nutrient agar at 45 0 C and plated out. The plates were left to solidified and incubated for 48 h at 37 0 C. Control experiment without the addition of extracts was set up. Viable counts were carried out in replicates for the sample. Reduction in the cell counts means killing of the cells by the antibacterial compounds.

III. RESULTS AND DISCUSSION
The chloroform fraction of G. kola exhibited antibacterial effect on all the used bacteria with the exception of C. pyogenes. The zones of inhibition obtained with the chloroform fraction ranges from 10 ± 1.41 mm and 27 ± 1.41 mm. Butanol and Ethylacetate fractions were active against seven bacterial isolates used and with inhibitory zones that ranges from 12 ± 0.00 mm to 21 ± 0.71 mm and 15 ± 1.41 mm to 18 ± 0.00 mm respectively ( Since chloroform fraction of the methanolic G. kola extract and butanol fraction of the crude extract of the A. sativum bulbs were the most active fractions against the used isolates, antibacterial potential of the combined chloroform fraction and combined butanol fraction of the two extracts were determined. Combined butanol fractions of G. kola and A. sativum have antibacterial effect on all the tested bacteria with the exception of C. sporogenes. Zones of inhibition ranged between 12±0.00 mm and 21±1.06 mm. All the tested bacterial isolates were sensitive to the antimicrobial activity of the combined chloroform fraction of G. kola and A. sativum except C. pyogenes with zones of inhibition ranging between 12±0.00 mm and 21±1.41 mm. These implied broad spectrum activities by the combined fractions.  Key: LIO= Locally Isolated Organisms, NCIB= National Collection of Industrial Bacteria, mm ** = mean of two replicates, 0= Not sensitive, AC= Chloroform fraction, AE= Ethylacetate fraction, AB= Buthanol fraction, C-CHLORO = Combined Chloroform and C-BUT = Combined Buthanol fraction. Minimum inhibitory concentrations shown by combination of chloroform and butanol fractions of the crude extracts against the isolates were as in Table 3 (Table 4). Considering the MICs stated above, the result of this study is still in agreement with the result of (19) who stated that, extract showing MIC below 100 mg/ml has a good activity.  (Fig. 2).
The effectiveness of an antibacterial agent is said to be measured by its bactericidal and bacteriostatic ability (20). In vitro rate of killing analysis are shown as the rate at which a particular concentration of an antibacterial agents inhibit visible growth of cells and its one of the most vital ways to determine tolerance (20). The effect of the different fractions obtained from crude methanol G. kola and A. sativum extracts against the organisms individually and in combination in this study appears to depend on time and concentration. Increase in contact time of the bacterial cells with the bioactive fractions as well as increase in concentration led to increase in the number of cells killed and this agrees with the findings of (9) from his study on antibacterial potential of red grape juice and red wine on Listeria monocytogenes.
From the result of the rate of kill analysis, it was discovered that the combined butanolic fractions of G. kola and A. sativum exhibited the highest percentage of killing against both S. aureus (Gram positive) and K. pneumonia (Gram negative). This justifies the utilization of alcohol in  extracting bioactive components from plants in traditional medicine. Higher activity of the combined butanol fraction also confirm the findings of another researcher (21) who reported that alcoholfraction of plant extract showed better activities and that this might be attributed to the higher polarity of butanol and thus more affinity for the active components of G.kola.

IV. CONCLUSION
All the fractions obtained from the methanolic extracts of G. kola and A. sativum individually and even in combined form possess the potential of providing relief to antibiotic resistance experienced in the recent times with pathogenic organisms as seen in this study. With the antimicrobial potential exhibited by these plant extracts especially when combined, there is need to assess the main mechanisms of action of these plants extracts in combination and antimicrobial activities of a wider strains of each organisms that shows susceptibility to these plants so as to adequately explore their potential.