Antimicrobial Activity and Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Saudi Arabian Ocimum basilicum Leaves Extracts

The present study evaluates the antimicrobial activity of Ocimum basilicum leaves extracts using well diffusion assay. Microbicidal or Microbiostatic activities were determined using (MBC or MFC) /MIC ratio. All O. basilicum extracts ethanol, methanol, and water possess antimicrobial activity. Methanol was the best solvent with greater inhibitory activity followed by ethanol then water against bacteria. However, all three solvents showed no difference in their inhibitory activity against yeast. The MIC and MBC values were in decreasing order methanol, ethanol then water against bacteria whereas Candida albicans was more sensitive at MIC 12.5μg/mL than C. tropicalis at MIC 25μg/mL and MFC values were lower against C. albicans than C. tropicalis at 25μg/mL and 50μg/mL, respectively in all type of solvents. The ratio of (MBC or MFC) /MIC were one to three-folds. The GC-MS result showed the presence of several important chemical compounds like terpene, steroids, phenols, esters, and fatty acids most of these compounds were reported to have an antimicrobial activity. The study indicates the importance of O. basilicum extracts as microbicidal agent with wide spectrum and high inhibitory properties in low concentrations. Therefore, O. basilicum leaves extracts may be important in the field of antimicrobial production as alternatives to antibiotics.

There are no previous studies involving this microbial group in a single study using alcoholic and aqueous O. basilicum extracts and the antimicrobial efficiency (Microbicidal or Microbiostatic). It is also noteworthy that most previous studies have highlighted the study of essential oil of O. basilicum and their anti-microbial activity while the O. basilicum extract received less attention. Therefore, the aim of this study was to compare the antimicrobial activity of three solvents of O. basilicum leaves extracts; ethanol, methanol and water, determine the antimicrobial efficiency and their phytochemical components.

Plant material
Ocimum basilicum L. (sweet basil) plant were purchased from a vegetable market in Dammam, Saudi Arabia. The plant was identified by Department of Biology, College of science, Imam Abdulrahman Bin Faisal University using The Herb Society of America 32 .

Preparation of plant extracts
Plant leaves were washed thoroughly with tap water and dried at room temperature. Dried leaves were ground to fine powder using a blender. Sixty grams of the powder plant was transferred one by one to three Erlenmeyer flasks containing 300 mL of distilled water, ethanol (80%), and methanol (80%) to obtain a final concentration of 20% g/mL. The mixtures were placed in a shaker at 300 rpm/min for 72 hours at 20°C to allow extraction of active compounds. The extract solutions were filtered with Whatman No. 1 filter paper then bacterial filters and filtrates were concentrated using an oven at 80°C. Dimethyl Sulfoxide (DMSO) was used to re-suspend the residues to a final concentration of 20% and the flasks were sealed and kept at 4°C for further use 33 .

Test microorganisms
Eight microorganisms were used three Gram positive bacteria (Staphylococcus aureus ATCC24213, S. aureus and Bacillus subtilis) three Gram negative bacteria (Escherichia coli ATCC25922, E. coli and Pseudomonas aeruginosa) and two yeasts (Candida albicans and Candida tropicali). Most microorganisms were obtained from King Fahd Hospital, AlKhobar, Saudi Arabia. Except B. subtilis was obtained from the Biology Department, College of Science, Imam Abdulrahman Bin Faisal University.

Agar well diffusion technique
Antimicrobial activity was carried out using agar well diffusion technique 34 . One mL of microorganisms cultures age 18-24 h (standard inoculums 1-2x10 8 CFU/mL 0.5 McFarland standard) were transfer to Petri plates, and 15 mL of nutrient agar was poured into the plates. After the cultures solidified, wells sizes 5 mm were punched using a sterile cork borer. Each well was filled with 50 µL of plant extracts; DMSO was used as a negative control, erythromycin (E15 mcg) as a positive control for the bacteria and nystatin (100mg) as a positive control for the yeasts. Treated plates were placed in a refrigerator for about one hour to allow diffusion of the plant extract and controls. Then plates were incubated for 48 hours at 37°C. Antimicrobial activity was recorded in millimeters by measuring the zones of inhibition around the wells. All experiments were performed with five replicates.

Determination of Minimum Inhibitory Concentration (MIC)
Method of Omura et al. 35 was preformed to determine the Minimum Inhibitory Concentration (MIC) of the plant extracts. Two-fold dilution of the plant extracts was made with nutrient broth media using 96 well microtitre plates. Standard bacterial inoculums at the concentration of 1-2x10 8 CFU/mL were added to the wells to a final concentration of 50%. Well number 11 left as positive control contain growth media and bacterial inoculum and well number 12 left as negative control contain growth media and plant extract. The MICs were read after overnight incubation at 37°C using a microtitre plate's reader at a wavelength of 630 nm. All experiments were performed in three replicate.

Determination of Minimum Bactericidal and Fungicidal Concentration (MBC and MFC)
Pour plate method was used to determine the MBC and MFC 36 . From the MIC experiment concentrations that showed no bacterial growth were sub-cultured using Petri plates, and then 12 mL of melted nutrient agar media was poured over it and gently mixed and left to solidify. The inoculated plates were incubated at 37°C for 48 hours. The lowest concentration that showed no visible colonies were recorded as MBC. All experiments were performed in three replicate.

Determination of antimicrobial efficiency (Microbicidal or Microbiostatic)
The antimicrobial efficiency of the of O. basilicum extracts (ethanol, methanol and water) was determine by using the ratio of MBC or MFC/ MIC 37 .

Gas Chromatography-Mass Spectrometry (GC-MS)
Gas Chromatography-Mass Spectrometer Model QP2010 SE (Shimadzu-Japan) with 5 Sil MS 5% diphenyl/95% dimethyl polysiloxane capillary column (30 meter, 0.25 mmID, 0.25-µm df) was used to screen the bioactive compounds of Sweet Basil plant extracts. Hundred micromilliliters of plant sample were diluted using 1400µl of DMSO. One µL of diluted sample (100/1400, V/V in DMSO) was injected in the split mode with a split ratio 1:10. For GC-MS detection, electron impact ionization system with ionization energy of 70eV was used. Carrier gas was pure helium (99.999%) at a constant column flow 0.7ml/ min and total flow was 10.4 ml/min. Flow control mode was linear velocity of 29.6cm/sec. Injector temperature was set at 250°C and ion-source temperature 250°C. The oven temperature was programmed from 50°C to 300°C, hold time was 3 min, and total run time was 29 min. ACQ Mode Scan range from 35 m/z to 500 m/z with scan speed 2500. Chemical compounds were identified by National Institute of Standards and Technology (NIST 08) library match.

Statistical analysis
The antimicrobial activities of O. basilicum leaves extracts between the solvents and the microbes were analyzed using SPSS Version 23.0 38 at significance p <0.01.

ResUlts
The antimicrobial activity of O. basilicum extracts (ethanol, methanol and water) was tested through the presence or absence of clear growth zones around the well. The results clarify that  . and sensitive to all O. basilicum extracts.
The results of MIC test showed that the widest MIC range was methanol extract from 3.125 to 25µg/mL then the range became progressively narrower with ethanol extract from 6.25 to 25µg/ mL and water extract from 12.5 to 25µg/mL (Table. 2). B. subtilis was the most sensitive microbe to both alcoholic extracts, methanol extract with MIC 3.125µg/mL and ethanol extract with MIC 6.25µg/mL. All three O. basilicum extracts had inhibitory effect on both yeasts. C. albicans was more sensitive at MIC 12.5µg/mL than C. tropicalis with MIC 25µg/mL. MBC results showed that the ethanol extract have the highest value at 100µg/ mL against E. coli also water extract against E. coli and S. aureus. The highest MFC value was 50µg/ mL against C. tropicalis in all types of solvent compared to 25µg/mL against C. albicans. The result showed that the maximum ratio between MIC and MBC or MFC was three-fold (Table 2).

DisCUssiON
Lamiaceae family contains natural bioactive components that may be considered a promising field in the discovery of new antimicrobial agents. The present study showed that all tested bacteria and yeasts exhibit sensitivities towards O. basilicum extracts shown by the size of inhibition zone. The results of the statistical analysis showed significant differences between the three solvents (p-values < 0.01), except for the effect of the solvents on C. albicans, which did not have significant differences (p-value > 0.01. There are also significant differences among the microbes. This result is in regard with a previous study 61 who found that water extract of O. basilicum inhibit the growth of both Gram positive and Gram negative bacteria. Moreover, Khalil 62 reported that ethanolic extract of O. basilicum inhibit the growth of E. coil and S. aureus. Also, our study partly agrees with another study found that aqueous extract of O. basilicum have stronger inhibitory activity comparing to ethyl acetate, methanol, and n-hexane extracts against Gram positive and Gram negative bacteria 24 . It is worth noting that the O. basilicum extracts showed inhibitory activity against E. coil and S. aureus that were resistance to the Erythromycin (E15 mcg), Indicating the superiority of the O. basilicum extracts on Erythromycin.
The present study showed that both yeasts were inhibited by ethanol, methanol and water extracts of O. basilicum this result are not consistent with a study conducted by Kaya et al. 63 where they found that acetone, chloroform and methanol extracts of O. basilucum did not show inhibitory activity against yeasts. These differences may be due to microbial strains, growth conditions, secondary metabolites, or extraction methods. The present result indicated that methanolic, ethanolic and water extracts of O. basilucum posses an antifungal activity.
The present results showed differences in inhibitory capability depending on the type of solvent in which the inhibitory activity for methanol extract was higher than ethanol and water against bacteria. This result is consistent with previous studies that confirm the effect of the solvent on the inhibitory capability of plant extracts and may return to solvent polarity 64 , although the solvents used in this study are polar but vary in their strength in descending order water, methanol then ethanol 65 . In this study polar solvents have been chosen due to the ability of extracting many active compounds such as polyphenolic compounds 66 .
The determination of MIC and MBC or MFC is important to measure the efficiency of the plant extract as antimicrobial agents 67 . The MIC and MBC result showed that O. basilicum methanol extract had the lowest values of 3.125µg/mL and 6.25µg/mL against B. subtilis, respectively. In general, the present result showed that the MIC and MBC values of methanol extract are lower than those of the ethanol extract and the later are lower than the values of the water extract on the tested bacteria. This result supports the conclusion that antibacterial agents with low antibacterial activity have higher MIC and MBC values compared with more effective antibacterial agents 68 . However, the result showed that yeasts respond differently to inhibitory activity of O. basilicum extracts where the C. albicans was more sensitive than C. tropicalis in all the extracts. Where, the values of MIC and MFC required to inhibit the C. albicans are higher than the C. tropicalis.
Antimicrobial agents are considered as Microbicidal agents if the MBC or MFC is less than four times the MIC and Microbiostatic agent if the MBC or MFC is more or equal than four times the MIC 37 . Calculation of the ratio between MIC and (MBC or MFC) is shown to be less than three-fold. Accordingly inhibiting activity of O. basilicum extracts may be considered as Microbicidal agent.
The current result of GC-MS analysis of O. basilicum extracts showed the presence of several important chemical compounds like terpene, steroids, phenols, esters, and fatty acid. These entire compounds were reported to have a variety of biological activities (Table 3). This result is consistent with other studies reported that O. basilicum is rich in polyphenols like Phenolic acids 17,69 . It is interesting that the current study is not consistent with Murali and Prabakaran 27 whom found that O. basilicum methanol extract contains 13 compounds that did not resemble the chemical compounds found in this study. This may be due to the difference in the environment in which the plant is grown, the age of the plant or the extraction method.
The current study showed that the alcoholic and water extracts of O. basilicum were able to inhibit the growth of bacteria and yeast alike, and this may be due to the richness of O. basilicum with Phenolic compounds like Eugenol, Methyleugenol, Benzoic acid, Benzoic acid 4-hydroxy-, Salicylic acid, and Phenol. Phenolic compounds were reported to have several mechanisms of action against microbes, including changing the permeability of microbial cell membrane through accumulation of hydrophobic groups in the phospholipids bilayer disrupting the membrane integrity and leading to leakage of intracellular components and finally cell death. Also Phenolic compounds can bind to the enzymes inhibiting their functions like proteins, DNA and RNA synthesis 70,71 . Also the results showed that the O. basilicum extract contains terpene compounds like Phytol, 3,7,11,15-Tetramethyl-2-hexadecen-1ol, Lupeol and beta-Amyrin that have an effect on microbial cell membranes by disrupting membrane efficiency.
Additionally, the antimicrobial activity may be related to the presence of fatty acids. The current result showed that O. basilicum extract were rich in saturated fatty acid and unsaturated fatty acid both are with long carbon chain 16 and more (Table 3). McGaw et al. 72 reported that Gram negative bacteria are less susceptible to fatty acids than Gram positive bacteria. Moreover, they reported that fatty acids carbon chain lengths play a very important role in their antimicrobial activity. Fatty acids containing 6 and less carbons inhibit Gram negative bacteria whereas Gram positive bacteria is inhibited by fatty acids with carbon chains longer than 12 and yeasts inhibited by fatty acids containing between 10 to 12 carbons. Several studies reported that unsaturated fatty acids with long carbon chains like linolenic acid, linoleic acid, and oleic acid have bactericidal effect on Methicillin-resistant Staphylococcus aureus, Helicobacter pylori, and Mycobacteria, while saturated fatty acids with long carbon chain like stearic acid and palmitic acid, are less active [73][74][75] . However, the primary molecular target of fatty acid is still unknown.

CONClUsiONs
The current study showed that O. basilicum extracts have a broad inhibitory spectrum against Gram negative bacteria, Gram positive bacteria and yeast. The results showed that the O. basilicum extracts were able to inhibit E. coil and S. aureus that were resistant to Erythromycin (E15 mcg). Moreover, the results of the determination of the inhibitory efficiency showed that O. basilicum extracts possesses microbicidal properties. The results also showed that the type of solvent is important in increasing the inhibitory capacity especially against the bacteria. The results of GC-MS showed that O. basilicum extracts are rich in compounds like Phenolic acids, fatty acid and terpene with inhibitory properties against wide range of microorganisms. The results of this study may provide new information on the importance of O. basilicum extracts in the production of broadspectrum anti-microbial agents with micro-bicidal properties that are environmentally friendly, have no side effects compared to antibiotics and low cost.

ACKNOWLEDGMENTS
The author would like to thank the research units at Al Rayyan campus -College of Science-Imam Abdulrahman Bin Faisal University for providing place and devices which were required for this experiment. And also like to thank Dr. Ahmed Alsayyah, Dr. Reem AlJindan, and Msr. Nouf Alromaihi for providing us with the tested microorganisms. Finally, we also like to thank Dr. Mariem Zouch for her assistant in the Statistical analysis.

FuNDING
None

DATA AvAILABILITy
All datasets generated or analyzed during this study are included in the manuscript.

ethiCs stAteMeNt
This article does not contain any studies with human participants or animals performed by any of the authors.