Printer Friendly

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

INTRODUCTION

The discovery of antibiotics in the mid-nineties appears to have solved most of the common problems in microbial infections. But the risk lies in the pathological cases in which the microbes are resistant to antibiotics or develop antibiotic resistance (1,2). The massive use of antibiotics in the past 80 years both appropriate and inappropriate has led to fast antibiotic resistance. Bacteria are able to resist various antibiotics through horizontal gene-transfer or mutation (3). Furthermore, other bacteria are intrinsically resistant to antibiotic, for example not having antibiotics target sites or having low permeability to antibiotics (4,5). The resistance mechanisms for antibiotics occur by enzymatic inactivation, site target modification, active elimination, or absorption inhibition (6). Estimated report indicate that the resistance of bacteria to antibiotics and their loss of ability to treat many bacterial infections will be the first killer in year 2050, where deaths are expected to reach 10 million per year, which means they are higher than deaths due to cancer (7).

In fact antibiotic resistance has become the third threat to public health in the 21st century (8). Moreover, antimicrobial resistance causes high economic burden including high therapeutic expenses and longer hospital stay (9,10). Accordingly, it is necessary to develop anti-microbial agents as an alternative to treat infectious diseases. Several studies showed that plants substances are a promising source for the discovery of novel antimicrobial agents (11-13).

Lamiaceae is one of most famous medicinal family plants, contains 236 genera and about 6000 species (14). Ocimum sp. is one of the important geneses in this family; it contains about 150 species that greatly differ in their morphology, aromatic composition, essential oil content, and chemical composition (15). Ocimum basilicum is an annual herb grows in different regions of the world (16) and is known as Basil or Sweet Basil also named as king of the herbs (17,18). This aromatic plant traditionally used as fresh or dried leaves as food flavoring, food preservative, medicinal plant and perfumery (19-21). Several studies reported that Sweet Basil leave extract were able to cure various illnesses and symptoms (22,23). Forthermore, Sweet Basil has activity against inflammation, viruses, bacteria and fungi (24-26). It has pharmacological effects against several diseases, with great antioxidant, anticancer and anti-aging properties (27,28). Moreover, basil leaves essential oils and extracts were found to be a rich source of phytochemical compounds such as chavicol, linaloon, methyl ether, estagole, eugenol, methyl eugenol, and methyl chavicol (29-31).

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.

MATERIALS AND METHODS

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[degrees]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[degrees]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[degrees]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 1-2*[10.sup.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 [micro]L 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[degrees]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 1-2[10.sup.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[degrees]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[degrees]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-[micro]m df) was used to screen the bioactive compounds of Sweet Basil plant extracts. Hundred micro-milliliters of plant sample were diluted using 1400[micro]l of DMSO. One [micro]L 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[degrees]C and ion-source temperature 250[degrees]C. The oven temperature was programmed from 50[degrees]C to 300[degrees]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 solvent type affect the inhibitory activity (Table 1). In general, the methanol extract showed the highest inhibitory activity against Gram positive bacteria and Gram negative bacteria follow by ethanol extract then water extract. However, all solvents were equal in their impact on the yeasts. The results showed that S. aureus and E. coli were resistant to the Erythromycin (E15 mcg) antibiotic 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 25[micro]g/mL then the range became progressively narrower with ethanol extract from 6.25 to 25[micro]g/mL and water extract from 12.5 to 25[micro]g/mL (Table. 2). B. subtilis was the most sensitive microbe to both alcoholic extracts, methanol extract with MIC 3.125[micro]g/mL and ethanol extract with MIC 6.25[micro]g/mL. All three O. basilicum extracts had inhibitory effect on both yeasts. C. albicans was more sensitive at MIC 12.5[micro]g/mL than C. tropicalis with MIC 25[micro]g/mL. MBC results showed that the ethanol extract have the highest value at 100[micro]g/mL against E. coli also water extract against E. coli and S. aureus. The highest MFC value was 50[micro]g/mL against C. tropicalis in all types of solvent compared to 25[micro]g/mL against C. albicans. The result showed that the maximum ratio between MIC and MBC or MFC was three-fold (Table 2).

GC-MS analysis of O. basilicum extracts showed total components in ethanol extract, methanol extract and water extract were 18, 23 and 23 respectively (Table 3). The compounds higher than 4.0% were Propane, 3-chloro-1,1,1-trifluoro (19.84%), 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- (19.26), n-Hexadecanoic acid (9.68%), 9,12,15-Octadecatrienoic acid, ethyl ester, (8.59%) and phytol (4.96%) in ethanol extract. In the methanol extract the compounds found were 4,5-Dichloro-1,3-dioxolan-2-one (24,23%), 9,12,15-Octadecatrienoic acid, (Z,Z,Z)- (10,47%), gamma--Sitosterol (11,52%), 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z) (6.30%), and stigmasterol (4.94%). And compounds found in water extract were 4,5-Dichloro-1,3-dioxolan-2-one (49.87%), phenol (6.22%), Propane, 3-chloro-1,1,1-trifluoro (4.26%) and gamma-Sitosterol (4.19%).

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 3.125[micro]g/mL and 6.25[micro]g/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-1-01, 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 (73-75). 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 broad-spectrum 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.

REFERENCES

(1.) Lockhart S.R., Iqbal N., Cleveland A.A., Farley M.M., Harrison L.H., Bolden C.B., et al. Species identification and antifungal susceptibility testing of Candida bloodstream isolates from population-based surveillance studies in two U.S. cities from 2008 to 2011. J. Clin. Microbial., 2012; 50(11): 3435-42.

(2.) Fankam A.G., Kuiate J.R., Kuete V. Antibacterial activities of Beilschmiedia obscura and six other Cameroonian medicinal plants against multi-drug resistant Gram-negative phenotypes. BMC Complement Altern. Med., 2014; 14: 241.

(3.) Toma A., Deyno S. Overview on Mechanisms of Antibacterial Resistance. Int. J. Res. Pharma. and Bio. Sci., 2015; 2(1): 27-36.

(4.) Yoneyama H., Katsumata R., Antibiotic resistance in bacteria and its future for novel antibiotic development. Biosci. Biotechnol. Biochem., 2006; 70: 1060-75.

(5.) Wise R.A review of the mechanisms of action and resistance of antimicrobial agents. Can. Respir. J., 1999; 6: S20-2.

(6.) Soares G.M.S., Figueiredo L.C., Faveri M., Cortelli S.C., Duarte P.M., Feres M. Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs. J. Appl. Oral Sci., 2012; 20(3): 295-309.

(7.) O'Neill J. Review on antimicrobial resistance. Tackling Drug-Resistant Infections Globally: Final report and recommendations. https://amr-review.org/sites/default/files/160518_Final%20paper_with%20cover. pdf, 2016.

(8.) World Health Organization; Antimicrobial resistance: global report on surveillance 2014. http://www.who.int/drugresistance/documents/surveillancereport/en/

(9.) Founou R.C., Founou L.L., Essack S.Y. Clinical and economic impact of antibiotic resistance in developing countries: a systematic review and meta-analysis. PLoS One, 2017; 12(12): 1-18.

(10.) Morgan J., Meltzer M.I., Plikaytis B.D., Sofair A.N., Huie-White S., Wilcox S., et al. Excess mortality, hospital stay, and cost due to candidemia: a case-control study using data from population-based candidemia surveillance. Infect. Control. Hosp. Epidemiol., 2005; 26(6): 540-7.

(11.) Bereksi M.S., Hassa|ne H., Bekhechi C., Abdelouahid D.E. Evaluation of Antibacterial Activity of Some Medicinal Plants Extracts Commonly Used in Algerian Traditional Medicine against Some Pathogenic Bacteria. Pharmacog J., 2018; 10(3): 507-12.

(12.) Janta1 K., Thaharn W. Antibacterial activity of medicinal plant extracts against some pathogenic bacteria causing skin diseases. RMUTT Journal, 2018; 8(1): 141-51.

(13.) Yunana B.T., Bukar B.B., Aguiyi J.C. In vitro and in vivo evaluation of antibacterial activity of Bridelia ferruginea extracts on some clinical isolates. J. Phytopharmacol., 2018; 7(4): 392-8.

(14.) CaroviT-Stanko K., Petek M., Grdi a M., Pintar J., BedekoviT D., Custic M.H., Satovic Z. Medicinal Plants of the Family Lamiaceae as Functional Foods - a Review. Czech J. Food Sci., 2016; 34(5): 377-90.

(15.) Makri O., Kintzios S. 'Ocimum sp. (Basil): Botany, Cultivation, Pharmaceutical Properties, and Biotechnology', Journal of Herbs, Spices & Medicinal Plants, 2008; 13(3): 123-50.

(16.) Cardoso N.N., Alviano C.S., Blank A.F., Romanos M.T., Fonseca B.B., Rozental S., et al. Synergism effect of the essential oil from Ocimum basilicum var. Maria Bonita and its major components with fluconazole and its influence on ergosterol biosynthesis. Evid Based Complement Alternat Med., 2016; 123(11): e65-e66.

(17.) Filip S. Basil (Ocimum basilicum L.) a Source of Valuable Phytonutrients. Int. J. Clin. Nutr. Diet., 2017; 3: 118-22.

(18.) Mohammad A., Moghaddam D. Shayegh J. Antimicrobial activity of essential oil extract of Ocimum basilicum L. leaves on a variety of pathogenic bacteria. J. Med. Plants Res., 2011; 5(1): 3453-6.

(19.) Ko K.Y., Geornaras I., Paik H., Kim K., Sofos J.N. Effects of plant-derived extracts, other anti-microbials and their combinations against Escherichia coli O157:H7 in beef systems. J. Food Prot., 2015; 78(6): 1090-7.

(20.) Gyawali R., Ibrahim S.A. Natural products as antimicrobial agents. Food Control, 2014; 46: 412-29.

(21.) Talaat I.M., Balbaa L.K.. Physiological response of sweet basil plants (Ocimum basilicum L.) to putrescine and trans cinnamic acid. Am. Eurasian J. Agric. Environ. Sci., 2010; 8(4): 438-45.

(22.) Umar A., Zhou W., Abdusalam E., Tursun A., Reyim N., Tohti I. et al. Effect of Ocimum basilicum L. on cyclo-oxygenase isoforms and prostaglandins involved in thrombosis. J. Ethnopharmacol., 2014; 152(1): 151-5.

(23.) Shafique M., Khan S.J., Khan N.H. Study of antioxidant and antimicrobial activity of sweet basil (Ocimum basilicum) essential oil. Pharmacology Online, 2011; 1(1): 105-11.

(24.) Sani Y.A., Cyril O., Yahaya Y., Hassan U., Ibrahim L.S. Phytochemical screening, in vitro anti-bacterial and partial TLC purification of different solvents extracts of Ocimum basilicum L. GSC Biological and Pharmaceutical Sciences, 2018; 5(2): 132-8.

(25.) Purushothaman B., Srinivasan R.P., Suganthi P., Ranganathan B., Gimbun J., Shanmugam K.A. Comprehensive Review on Ocimum basilicum. J. Nat. Remedies, 2018; 18(3): 71-85.

(26.) Al-Ali KH, El-Beshbishy H.A., El-Badry A.A., Alkhalaf M. Cytotoxic activity of methanolic extract of Mentha longifolia and Ocimum basilicum against human breast cancer. Pak. J. Biol Sci., 2013; 16(23): 1744-50.

(27.) Murali M., Prabakaran G. Effect of different solvents system on antioxidant activity and phytochemical screening in various habitats of Ocimum basilicum L. (Sweet basil) leavas. Int. J. Zool. Appl. Biosci., 2018; 3(5): 375-81.

(28.) Manosroi J., Dhumtanom P., Manosroi A. Antiproliferative activity of essential oil extracted from Thai medicinal plants on KB and P388 cell lines. Cancer Lett., 2006; 235(1): 114-20.

(29.) Joshi R.K. Chemical composition and antimicrobial activity of the essential oil of Ocimum basilicum L. (sweet basil) from Western Ghats of North West Karnataka, India. Anc. Sci. Life, 2014; 33(3): 151-6.

(30.) Shirazi M.T., Gholami H., Kavoosi G., Rowshan V. Tafsiry A - Chemical composition, antioxidant, antimicrobial and cytotoxic activities of Tagetes minuta and Ocimum basilicum essential oils. Food Sci. Nutr., 2014; 2(2): 146-55.

(31.) Dev N., Das A.K., Hossain M.A., Rahman S.M.M. Chemical compositions of different extracts of Ocimum basilicum leaves. J. Sci. Res., 2011; 3(1): 197-206.

(32.) The Herb Society of America. Basil: an herb society of America guide. Kirtland, Ohio 44094; 2003.

(33.) Neycee M.A., Nematzadeh G.H.A., Dehestani A., Alavi M. Assessment of antifungal effects of shoot extracts in chinaberry (Melia azedarach) against 5 phytopathogenic fungi. Intr. J. Agri. Crop. Sci., 2012; 4(8): 474-77.

(34.) National Committee for Clinical Laboratory Standards (NCCLS). Performance Standards for Antimicrobial Disk Susceptibility Tests. Approved Standard, NCCLS Document M2-A5, National Committee for Clinical Laboratory Standards, Wayne, Pennsylvania, USA, 1993; 13(24).

(35.) Omura S,. Pyl D.V.D., Inokoshi J., Takahashi Y., Takeshima Y. Pepticinnaminsnew farnesyl-protein transferase inhibitors produced by an actinomycete I. Producingstrain, fermentation, isolation and biological activity. J. Antibiot., 1993; 46: 222-8.

(36.) National Committee for Clinical Laboratory Standards (NCCLS). Performance Standards for Antimicrobial Disk Susceptibility Tests. Approved Standard M2-A6. National Committee for Clinical Laboratory Standards, Wayne, Pennsylvania, USA, 1997; 17(1).

(37.) Levison M.E., Levison J.H. Pharmacokinetics and pharmacodynamics of antibacterial agents. Infect. Dis. Clin. North Am., 2009; 23(4): 791-9.

(38.) IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY: IBM Corp. Released 2015.

(39.) Sermakkani M., Thangapandian V. GC-MS Analysis of Cassia italica leaf methanol extract. Asian J. Pharm. Clin. Res., 2012; 5(2): 90-4.

(40.) Tyagi T., Agarwal M. Phytochemical screening and GC-MS analysis of bioactive constituents in the ethanolic extract of Pistia stratiotes L. and Eichhornia crassipes (Mart.) solms. J. Pharmacogn. Phytochem., 2017; 6(1): 195-206.

(41.) Al-Marzoqi A.H., Hadi M.Y., Hameed I.H. Determination of metabolites products by Cassia angustifolia and evaluate antimicobial activity. J. Pharmacognosy Phytother., 2016; 8(2): 25-48.

(42.) Tan K.H., Nishida R. Methyl eugenol: Its occurrence, distribution, and role in nature, especially in relation to insect behavior and pollination. J. Insect. Sci., 2012; 12(1): 1-60.

(43.) Shibula K., Velavan S. Determination of phytocomponents in methanolic extract of Annona muricata leaf using GC-MS technique. Int. J. Pharmacog. Phytochem. Res., 2015; 7: 1251-5.

(44.) Jegadeeswari P., Nishanthini A., Muthukumarasamy S., Mohan V.R. GC-MS analysis of bioactive components of Aristolochia krysagathra (Aristolochiaceae). J. Curr. Chem. Pharm. Sci., 2012; 2: 226-32.

(45.) Nejad M., "zg nes H., Nes H, Basaran N. Pharmacological and Toxicological Properties of Eugenol. Turk. J. Pharm Sci., 2017; 14(2): 201-6.

(46.) Miguel V., Lestard M.E.D., Tuttolomondo M.E., Doaz S.B., BenAltabef A., Puiatti M. et al. Molecular view of the interaction of S-methyl methane-thiosulfonate with DPPC bilayer. Biochim. Biophys. Acta Biomembr., 2016; 1858(1): 38-46.

(47.) Shahina P., Shahzad A., Upadhyaya P., Yadav V. Gas chromatography-mass spectrometry analysis of methanolic leaf extract of Cassia angustifolia Vahl. Asian J. Pharm. Clin. Res., 2016; 9: 111-6.

(48.) Kumaravel S., Muthukumaran P., Shanmugapriya K. Chemical composition of Trigonella foenumg-raecum through gas chromatography mass spectrometry analysis. J. Med. Plants Stud., 2017; 5: 1-3.

(49.) SCCS (Scientific Committee on Consumer Safety), Opinion on resorcinol, 2010.

(50.) Kobayashi A., Konishi G. Synthesis and analysis of Resorcinol-Acetone copolymer. Molecules., 2009; 14(1): 364-77.

(51.) Eseyin O.A., Sattar M.A., Rathore H.A., Aigbe F., Afzal S., Ahmad A., et al. GC-MS and HPLC profiles of phenolic fractions of the leaf of Telfairia occidentalis. Pak. J. Pharm. Sci., 2018; 31(1): 45-50.

(52.) Madan R.K., Levitt J. A review of toxicity from topical salicylic acid preparations. J. Am. Acad. Dermatol., 2014; 70(4): 788-92

(53.) Tripathi N., Kumar S., Singh R., Singh C.J., Singh P., Varshney V.K. Isolation and Identification of [gamma]-sitosterol by GC-MS from Roots of Girardinia heterophylla. OJC, 2013; 29(2): 705-7.

(54.) Kumar R.N., Vasantha K., Mohan V.R. GC-MS Analysis of Bioactive Components of Tubers of Ruellia tuberosa L. (Acanthaceae). Am. J. Phytomed. Clin. Ther., 2014; 2(2): 209-16.

(55.) Hadi M.Y., Mohammed G.J., Hameed I.H. Analysis of bioactive chemical compounds of Nigella sativa using gas chromatography-mass spectrometry. J. Pharmacognosy Phytother., 2016; 8(2): 8-24.

(56.) Saleem M. Lupeol. A novel anti-inflammatory and anticancer dietary triterpene. Cancer Lett., 2009; 285(2): 109-15

(57.) El-Ziney M. GC-MS Analysis of Benzoate and Sorbate in Saudi Dairy and Food Products with Estimation of Daily Exposure. J. Food Technol., 2009; 7(4): 127-34.

(58.) Okoye N.N., Ajaghaku D.L., Okeke H.N., Ilodigwe E.E., Nworu C.S., Okoye F. B. beta-Amyrin and alpha-amyrin acetate isolated from the stem bark of Alstonia boonei display profound anti-inflammatory activity. Pharm. Biol., 2014; 52(11): 1478-86.

(59.) Madhavan M. Phytochemical constituents of leaves of Spatholobus parviflorus a rare threatened climber of South India. Int. J. Pharmacognosy and Phytochem. Res., 2015; 7(5): 991-4.

(60.) Duan X.J., Zhang W.W., Li X.M., Wang B.G. Evaluation of antioxidant property of extract and fractions obtained from a red alga, Polysiphonia urceolata. Food Chem., 2006; 95: 37-43.

(61.) Krishnamurthy V., Chandrashekar S.C., Kumar M.S., Sagar D. Efficiency of Crude Extract of Ocimum sanctum, Ocimum gratissimum and Ocimum basilicum Leaves against Bacterial Pathogens. Int. J. Curr. Microbiol. App. Sci., 2018; 7(4): 2609-15.

(62.) Khalil A. Antimicrobial activity of ethanolic extracts of Ocimum basilicum leaf from Saudi Arabia. Biotech., 2013; 12(1): 61-4.

(63.) Kaya I., Yigit N., Benli M. Antimicrobial Activity of Various Extracts of Ocimum basilicum L. and Observation of the Inhibition Effect on Bacterial Cells by Use of Scanning Electron Microscopy. Afr. J. Tradit. Complement Altern. Med., 2008; 5(4): 363-9.

(64.) Ababutain I.M. Impact of solvent type on antibacterial activities of Lawsonia inermis leaves. J. Food Agric. Environ., 2015; 13(1): 51-3.

(65.) Hansen C.M. Hansen solubility parameters a user's handbook. CRC Press Taylot and Francis Group; 2007. J. Am. Acad. Dermatol., 2014; 70(4): 788-92.

(66.) Rongai D., Pulcini P., Pesce B., Milano F. Antifungal activity of some botanical extracts on Fusarium oxysporum. Open Life Sci., 2015; 10: 409-16.

(67.) Dalla T., Sheboun A. Evaluation of some medical plants and their extracts as feed Additives in Broiler diets on health indicators and productive performance. Tishreen University Journal Bio Sciences Series, 2014; 36(4): 49-67.

(68.) Shweash M., khashan A.A., Farhan Y.M., Nasser S.J. Antibacterial activity of ethanolic extract of leaves sweet basil (Ocimum basilicum) against diarrhea caused by Escherichia coli in vitro. Int. J. Sci. Nat., 2014; 5(4): 713- 8.

(69.) G ez C.M., Souza R.O., Fischer P., Lelo M.F.M., Duarte J.A., Boligon A.A.. Evaluation of basil extract ( Ocimum basilicum L.) on oxidative, anti-genotoxic and antiinflammatory effects in human leukocytes cell cultures exposed to challenging agents. Braz. J. Pharm. Sci., 2017; 53(1): e15098.

(70.) Wu Y., Bai J., Zhong K., Huang Y., Qi H., Jiang Y., et al. Antibacterial activity and membrane-disruptive mechanism of 3-p-trans-coumaroyl-2-hydroxyquinic acid, a Novel phenolic compound from Pine Needles of Cedrus deodara, against Staphylococcus aureus. Molecules, 2016; 21: 1084.

(71.) Cowan M.M. Plant products as antimicrobial agents. Clin. Microbiol. Rev., 1999;12(4): 564-82.

(72.) McGaw L.J., Jnger A.K., van Staden J. Antibacterial effects of fatty acids and related compounds from plants (minireview). S. Afr. J. Bot., 2002; 68: 417-23.

(73.) Seidel V., Taylor P.W. In vitro activity of extracts and constituents of Pelagonium against rapidly growing mycobacteria. Int. J. Antimicrob. Agents, 2004; 23: 613-9.

(74.) Sun C.Q., Connor C.J., Roberton A.M. Antibacterial actions of fatty acids and monoglycerides against Helicobacter pylori. FEMS Immunol. Med. Microbiol., 2003; 36: 9- 17.

(75.) Farrington M., Brenwald N., Haines D., Walpole E. Resistance to desiccation and skin fatty acids in outbreak strains of methicillin-resistant Staphylococcus aureus. J. Med. Microbiol., 1992; 36: 56-60.

Ibtisam Mohammed Ababutain (iD)

Department of Biology, College of Science, Imam Abdulrahman Bin Faisal University, P.O. Box 1982-31441, Dammam, Saudi Arabia.

(*) Correspondence: iababutain@iau.edu.sa

Citation: Ibtisam Mohammed Ababutain, Antimicrobial Activity and Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Saudi Arabian Ocimum basilicum Leaves Extracts, J Pure Appl Microbiol., 2019; 13(2): 823-833. doi: 10.22207/JPAM.13.2.17

(Received: 01 April 2019; accepted: 20 May 2019)

https://dx.doi.org/10.22207/JPAM.13.2.17
Table 1. Antimicrobial activity of O. basilicum leaves extracts at 
concentration of 20% g/mL using agar well diffusion technique
Microorganisms                   Ethanol           Methanol
                                 extract           extract
Gram positive bacteria
S. aureus ATCC24213              10.7[+ or -]0.6   12.4[+ or -]0.6
S. aureus                        11.5[+ or -]0.5   11.8[+ or -]0.3
B. subtilis                      10.7[+ or -]0.6   13.7[+ or -]0.6
Gram negative bacteria
E. coli ATCC25922                11.5[+ or -]0.3   12.3[+ or -]0.6
E. coli                          11.8[+ or -]0.3   12.8[+ or -]0.3
P. aeruginosa                    11.8[+ or -]0.3   12.3[+ or -]0.3
C. albicans                      12.6[+ or -]0.3   12.5[+ or -]0.6
C. tropicalis                    11.8[+ or -]0.3   11.9[+ or -]0.3
(*) Significance                  0.00              0.002
(p[less than or equal to]0.01)
Microorganisms                   Water
                                 extract
Gram positive bacteria
S. aureus ATCC24213               7.5[+ or -] 0.9
S. aureus                        11.3[+ or -]0.3
B. subtilis                      10.5[+ or -]0.5
Gram negative bacteria
E. coli ATCC25922                10.8[+ or -]0.3
E. coli                          11.3[+ or -]0.3
P. aeruginosa                    11.4[+ or -]0.5
                                 Yeasts
C. albicans                      12.6[+ or -]0.5
C. tropicalis                    11.7[+ or -]0.5
(*) Significance                  0.00
(p[less than or equal to]0.01)
Microorganisms                   Significance
                                 (p[less than or equal to]0.01) (*)
Gram positive bacteria
S. aureus ATCC24213              0.001
S. aureus                        0.003
B. subtilis                      0.001
Gram negative bacteria
E. coli ATCC25922                0.001
E. coli                          0.001
P. aeruginosa                    0.002
C. albicans                      0.1
C. tropicalis                    0.006
 (*) Significance                -
(p[less than or equal to]0.01)
Microorganisms                   Positive          Negative
                                 control (**)      control (***)
Gram positive bacteria
S. aureus ATCC24213              14[+ or -]1.0     R
S. aureus                        R                 R
B. subtilis                      20[+ or -]1.0     R
Gram negative bacteria
E. coli ATCC25922                9.0[+ or -]1.0    R
E. coli                          R                 R
P. aeruginosa                    10.8[+ or -]0.3   R
C. albicans                      19.2[+ or -]0.8   R
C. tropicalis                    17.3[+ or -]1.0   R
(*) Significance                 -                 -
(p[less than or equal to]0.01)
(*) P-values are less than 0.01 which means that there is a significant 
difference between the tested microbes or between the used solvents.
(**) Erythromycin (E15 mcg) positive control for the bacteria and 
Nystatin (100mg) positive control for the yeasts. (***) DMSO, negative 
control.
Table 2. Determination of Minimal Inhibitory Concentration (MIC) 
[micro]g/ml, Minimal Bactericidal Concentration (MBC), Minimal 
Fungicidal Concentration (MFC) [micro]g/ml and their ratio of O. 
basilicum extracts
Micro-organisms          MIC, MBC and MFC ([micro]g/ml) of Sweet
                         Basil extract
                         Ethanol extract
                         MIC     MBC    Ratic (*)
                                 or
                                 MFC
Gram positive bacteria
S. aureus                12.5    50     2
ATCC24213
S. aureus                12.5    50     2
B. subtilis               6.25   12.5   1
Gram negative bacteria
E. coli                  12.5    50     2
ATCC25922
E. coli                  12.5   100     3
P. aeruginosa            12.5    50     2
Yeasts
C. albicans              12.5    25     1
C. tropicalis            25      50     1
Micro-organisms          MIC, MBC and MFC ([micro]g/ml) of Sweet
                         Basil extract
                         Methanol extract
                         MIC      MBC     ratio
                                  or
                                  MFC
Gram positive bacteria
S. aureus                12.5     25      1
ATCC24213
S. aureus                12.5     25      1
B. subtilis               3.125    6.25    1
Gram negative bacteria
E. coli                  12.5     25      1
ATCC25922
E. coli                  12.5     50      1
P. aeruginosa            12.5     25      1
Yeasts
C. albicans              12.5     25      1
C. tropicalis            25       50      1
Micro-organisms          MIC, MBC and MFC ([micro]g/ml) of Sweet
                         Basil extract
                         Water extract
                         MIC    MBC   Ratio
                                or
                                MFC
Gram positive bacteria
S. aureus                25      50   1
ATCC24213
S. aureus                25     100   2
B. subtilis              25      50   1
Gram negative bacteria
E. coli                  25      50   1
ATCC25922
E. coli                  25     100   2
P. aeruginosa            25      50   1
Yeasts
C. albicans              12.5    25   1
C. tropicalis            25      50   1
Ratio (*) (MBC or MFC)/MIC
Table 3. GC-MS analysis of O. basilicum extracts and their biological 
activities
No   Compound name              Peak Area%
                                EL           ML       WL
 1   9,12,15-Octadecatrienoic   19.26        10.47    1.28
     acid, (Z,Z,Z)-
 2   n-Hexadecanoic acid         9.68         3.54    1.14
 3   9,12,15-Octadecatrienoic    8.59         3.81    0.3
     acid, ethyl ester, (Z
 4   Phytol                      4.96         1.17    0.2
 5   Phytol, acetate             2.69         0.5     0.27
 6   Methyleugenol               2.42         2.25    0.3
 7   3,7,11,15-Tetramethy1       2.04         0.81    0.27
     -2-hexadecen-1-ol
 8   Eugenol                     0.67         0.72    0.19
 9   Hexadecanoic acid,          0.32         2.56    0.2
     methyl ester
10   S-Methyl                    0.28         0.21    1.03
     methanethiosulphonate
11   Benzeneacetic acid          0.15         0.27    0.78
12   Vitamin E                   0.79         0.29    -
13   Resorcinol                  0.21         0.13    -
14   Propane,                   19.84        -        4.26
     3-chloro-1,1,1-trifluoro
15   Hexadecanoic acid,          4.9         -        0.22
     ethyl ester
16   9,12-Octadecadienoic        0.11        -        0.22
     acid (Z,Z)-
17   Benzoic acid, 4-hydroxy-    2.05        -        -
18   Salicylic acid              0.12        -        -
19   4,5-Dichloro               -            24.23   49.87
     -l,3-dioxolan-2-one
20   gamma-Sitosterol           -            11.52    4.19
21   9,12,15-Octadecatrienoic                 6.3     0.71
     acid, methyl ester,
     (Z,Z,Z)
22   stigmasterol                             4.94    0.78
23   9,12-Octadecadienoic       -             2.12    0.13
     acid (Z,Z)-,methyl
     ester
24   Octadecanoic acid                        0.67    0.45
25   Lupeol                     -             0.23    0.61
26   Benzoic acid               -             0.22    0.55
27   beta-Amyrin                -             1.19    -
28   1-Heptatriacotanol                       0.64
29   Phenol                     -            -        6.22
No   Molecular Formula
 1   [C.sub.18][H.sub.30][O.sub.2]
 2   [C.sub.16][H.sub.32][O.sub.2]
 3   [C.sub.20][H.sub.44][O.sub.2]
 4   [C.sub.20][H.sub.44][O.sub.2]
 5   [C.sub.22][H.sub.42][O.sub.2]
 6   [C.sub.11][H.sub.40][O.sub.2]
 7   [C.sub.20][H.sub.40][O.sub.2]
 8   [C.sub.10][H.sub.12][O.sub.2]
 9   [C.sub.17][H.sub.34][O.sub.2]
10   [CH.sub.3][SO.sub.2][SCH.sub.2]
11   [C.sub.9][H.sub.10]BrN[O.sub.2]
12   [C.sub.29][H.sub.50][O.sub.2]
13   [C.sub.6][H.sub.6][O.sub.2]
14   [C.sub.3][H.sub.4][CIF.sub.3]
15   [C.sub.18][H.sub.36][O.sub.2]
16   [C.sub.18][H.sub.32][O.sub.2]
17   [C.sub.7][H.sub.6][O.sub.3]
18   [C.sub.7][H.sub.6][O.sub.3]
19   [C.sub.3][H.sub.2][Cl.sub.2]][O.sub.2]
20   [C.sub.29][H.sub.50]O
21   [C.sub.19][H.sub.32][O.sub.2]
22   [C.sub.29][H.sub.48]O
23   [C.sub.19][H.sub.34][O.sub.2]
24   [C.sub.19][H.sub.35]C[O.sub.2]H
25   [C.sub.30][H.sub.50]O
26   C([double dagger]) H ([dagger]) O,
27   [C.sub.30][H.sub.50]O
28   [C.sub.37][H.sub.76]O
29   [C.sub.6][H.sub.5]OH
No   Compound Nature and Biological activities
 1   Linolenic Omega-3 polyunsaturated fatty acid,
     Anti-inflammatory, Cancer preventive,
     Antieczemic and Nematicide (39).
 2   Palmitic saturated Fatty acid ester, Antioxidant,
     Nematicide, Hypochloesterolemi and Antiandrogenic,
     pesticide (39,40).
 3   Linolenic acid, ethyl este No activity was
     reported.
 4   Diterpene, Antimicrobial, Cancer preventive,
     Anticancer and Anti-inflammatory (39,40).
 5   Anti-inflammatory, antileishmanial and
     antitrypanosomal (41).
 6   Phenolic compounds, Anti-inflammatory,
     Antioxidant, Antimicrobial, Nematicide and
     insecticide (42).
 7   Terpene Alcohol, Antimicrobial, Antioxidant,
     Anti-inflammatory and flavoring agent (39,43,44).
 8   Phenolic compounds, Antimicrobial, Nematicide,
     insecticide and food additive (42,45).
 9   Palmitic saturated fatty acids ester, Antioxidant,
     Flavor, Hypocholesterolemic Pesticide and
     5-Alpha reductase inhibitor (39).
10   Ester and Antimutagenic agent (46).
11   No activity was reported.
12   Lapid, Anti-Alzheimer, Antiaging, Antioxidant
     and Anti-inflammatory (41,47,48).
13   1,3-isomer of dihydroxyphenol, not a
     bacterial mutagen (49,50).
14   No activity was reported.
15   Palmitic saturated fatty acids, Antioxidant,
     Flavor, Anti-androgenic, Nematicide,
     Hemolytic and Hypocholesterolemic (39).
16   Article 1. Linoleic acid polyunsaturated fatty acid,
     Anti-inflammatory, Anticancer, Antiacne and
     Nematicide (39).
17   Phenolic compounds and No activity
     reported (51).
18   Phenolic compounds, Bacteriostatic,
     Fungicidal, Anti-inflammatory and
     Antibacterial (52).
19   No activity reported
20   Steroid and Antidiabetic drug (53).
21   Linolenic polyunsaturated fatty acid methyl
     ester, Antimicrobial, Antiarthritic, Anticancer
     and Antiasthama (39).
22   Steroid, Antioxidant, Antimicrobial, Anticancer,
     Antiarthritic, Antiasthma, Anti inflammatory
     and diuretic (40,54).
23   Methyl linoleate, Analgesic, Anti-inflammatory
     and Ulcerogenic (55).
24   Stearic saturated fatty acid No activity reported
25   Article II. Triterpenoid, Antimicrobial,
     Anti-inflammatory, Anticancer, and
     Antioxidan (54,56).
26   Phenolic compounds and food additives (57).
27   Triterpenes, Anti-inflammatory (58).
28   Alcoholic compound, Antioxidant, Anticancer,
     Antimicrobial, Anti inflammatory (55,59).
29   Phenolic compound and Antioxidant (60).
COPYRIGHT 2019 Oriental Scientific Publishing Company
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2021 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:RESEARCH ARTICLE
Author:Ababutain, Ibtisam Mohammed
Publication:Journal of Pure and Applied Microbiology
Geographic Code:7SAUD
Date:Jun 1, 2019
Words:6435
Previous Article:Effect of Induced Compost by Cellulolitic (Aspergillus fumigatus) and Ligninolitic (Geotrichum sp.) Fungi Inoculum Application on Vegetative Growth...
Next Article:Effect of Glutathione, Ascorbic Acid and Multivitamins on Sensitivity of Norfloxacin against Pseudomonas aeruginosa and Klebsiella pneumoniae.
Topics:

Terms of use | Privacy policy | Copyright © 2022 Farlex, Inc. | Feedback | For webmasters |