The antibacterial potential of essential oils and methanol extracts of sweet basil Ocimum basilicum L. (Lamiaceae) was evaluated for controlling the growth range of food-borne pathogenic bacteria. Essential oils extracted by hydrodistillation from the leaves and stems were analyzed by GC-MS. Fifty-seven compounds representing 94.9 and 96.1% of the total leaf and stem oils, respectively, were identified, of which methyl chavicol (36.7 and 29.9%), gitoxigenin (9.3 and 10.2%), trimethoquinol (10.3 and 8.4%), β-guaiene (3.7 and 4.1%), aciphyllene (3.4 and 3.0%), alizarin (3.2 and 4.4%), naphthaline (2.2 and 3.8%), (–)-caryophyllene (2.0 and 1.9%), and mequinol (1.6 and 1.8%) were the major compounds. The essential oils (10 μL/disc of 1:5, v/v dilution with methanol) and methanol extracts (300 μg/disc) of O. basilicum displayed a great potential of antibacterial activity against Bacillius cereus, B. subtilis, B. megaterium, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Shigella boydii, S. dysenteriae, Vibrio parahaemolyticus, V. mimicus, and Salmonella typhi with their respective zones of inhibition of 11.2–21.1 mm and MIC values of 62.5–500 μg/mL. The results of this study suggest that the natural products derived from O. basilicum may have potential use in the food and/or pharmaceutical industries as antimicrobial agents.
Keywords: Ocimum basilicum L.; essential oils; methyl chavicol; methanol extracts; food-borne pathogens; antibacterial activity; GC-MS
Illness caused by the consumption of contaminated foods has a broad economic and public health impact worldwide ([
Sweet basil, Ocimum basilicum L. (Lamiaceae), is an annual herb which grows in the tropical and subtropical regions of Asia, Africa, and South America. This popular herb is used as both a fresh and a dried food spice, and in traditional medicine. Among more than 150 species of the genus Ocimum, basil (O. basilicum) is the major essential oil crop around the world and is cultured commercially in many countries ([
In this study, we examined the chemical composition of the essential oils of sweet basil (O. basilicum) from Bangladesh and tested the efficacy of oils and methanol extracts against a range of food spoilage and food-borne pathogens. Although the antibacterial properties of O. basilicum essential oils or extracts from different origins have been reported ([
The leaves and stems of sweet basil (O. basilicum L.) were collected from the northern area of Dhaka in Bangladesh, in December 2007, and identified by Professor Saidul Islam, Department of Botany, University of Rajshahi, Bangladesh. A voucher specimen (BOT-477) has been deposited at the Department of Botany, University of Rajshahi, Bangladesh.
The air-dried leaves and stems (250 g of each) of O. basilicum were subjected to hydrodistillation for 3 h using a Clevenger type apparatus. The oils were dried over anhydrous Na
The air-dried leaves and stems of O. basilicum were pulverized into a powdered form. The dried powder (50 g) was extracted three times with 70% methanol in water (v/v) (200 mL × 3) at room temperature and the solvents from the combined extracts were evaporated by vacuum rotary evaporator at 50°C. The methanol extract (10.5 g) was suspended in distilled water (300 mL) and extracted successively by hexane, chloroform, and ethyl acetate, which resulted in 3.66 g slurry by hexane, 4.11 g slurry by chloroform, 2.98 g slurry by ethyl acetate, and 1.65 g slurry by residual methanol subfractions, respectively. Solvents (analytical grade) for extraction were obtained from commercial sources (Sigma-Aldrich, St. Louis, MO, USA).
GC-MS was carried out using total ion monitoring mode on a Varian 3800 gas chromatograph interfaced to a Varian Saturn ion trap 2200 GC-MS spectrometer. The temperatures of the transfer line and ion source were 280°C and 275°C, respectively. Ions were obtained in electron ionization mode. A VF-5 capillary column (30 m length, 0.25 mm I.D., 0.25 µm film thickness) was used. A 20% split injection mode was selected with a solvent delay time of 3 min with injection volume 0.2 μL. The initial column temperature was 50°C for 1 min, then programmed at 8°C/min to 200°C, and heated to 280°C at 10°C/min. The injection port was set at 250°C. Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. Molecular ions were monitored for identification (mass range: 40–500 m/z). The relative percentage of the oil constituents was expressed as percentage by peak area normalization.
Identification of compounds of the essential oil was based on GC retention time on the VF-5 capillary column, computer matching of mass spectra with those of standards (Wiley 6.0 data for GC-MS system), and, whenever possible, by co-injection with authentic compounds ([
The following food-borne pathogens were used in the antibacterial test: Bacillius cereus, Bacillius subtilis, Bacillius megaterium, Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Shigella boydii, Shigella dysenteriae, Vibrio parahaemolyticus, Vibrio mimicus, Salmonella typhi, Salmonella paratyph, and Pseudomonas aeruginosa. The strains were obtained from the Department of Pharmacy, University of Dhaka, Bangladesh. Cultures of each bacterial strain were maintained on Luria–Bertani (LB) agar medium at 4°C.
The antibacterial test was carried out by agar disc diffusion method ([
Minimum inhibitory concentrations (MICs) of essential oils, the methanol extract, and the derived subfractions of hexane, chloroform, and ethyl acetate were tested by standard National Committee for Clinical Laboratory Standards method ([
GC-MS analyses of the oils led to the identification of 57 different compounds, representing 94.9 and 96.1% of the total oils from leaves and stems, respectively. The identified compounds are listed in Table 1 according to their elution order on a VF-5 capillary column. The oils contained a complex mixture consisting of mainly oxygenated mono- and sesquiterpenes, and mono- and sesquiterpene hydrocarbons. The major compounds detected in the leaf and stem oils, respectively, were methyl chavicol (36.7 and 29.9%), gitoxigenin (9.3 and 10.2%), trimethoquinol (10.3 and 8.4%), β-guaiene (3.7 and 4.1%), aciphyllene (3.4 and 3.0%), alizarin (3.2 and 4.4%), naphthaline (2.2 and 3.8%), (–)-caryophyllene (2.0 and 1.9%), and mequinol (1.6 and 1.8%), as shown in Table 1. Also, phenylethyl alcohol, camphor, isoledene, globulol, and leolene alcohol were found to be minor components of O. basilicum leaf and stem oils in the present study (Table 1).
Table 1. Chemical composition of the essential leaf and stem oils of Ocimum basilicum L.
No. Rt (min)a Compound Leaf (%) Stem (%) 1 7.29 3-Octanol tr tr 2 8.07 Eucalyptol 0.1 0.1 3 8.79 Tetrahydrofuran-2-ol,3,4-di(1-butyl) tr tr 4 9.09 Mequinol 1.6 1.8 5 9.26 Santolinatriene tr tr 6 9.58 Phenylethyl alcohol 0.7 0.5 7 10.33 Camphor 0.5 0.4 8 10.81 ( 7.6 10.5 9 12.11 2-Isopropylidene-5-methylhexa-4-enal tr tr 10 12.32 Octadecane tr tr 11 12.54 Phenol-4-ethyl-2-methyl 0.2 tr 12 12.83 Nonadecane 0.1 tr 13 12.83 Heptadecane 0.1 0.1 14 13.78 α-Cubebene tr tr 15 14.13 Trimethoquinol 10.3 8.4 16 14.65 (–)-Caryophyllene 2.0 1.9 17 14.65 β-Guaiene 3.7 4.1 18 14.09 Methyl chavicol 36.7 29.9 19 15.47 γ-Elemene 0.5 0.4 20 15.51 γ-Neoclovene 0.2 0.6 21 15.74 Azulene 1.0 0.9 22 15.75 Naphthaline 2.2 3.8 23 15.81 Agarospirol tr tr 24 15.94 Cycloisolongifolene 1.2 1.4 25 16.10 Isoledene 0.9 1.0 26 16.12 Hexadecane 0.2 0.1 27 16.13 1-Chloroeicosane 0.4 0.2 28 16.24 Patchoulene 1.0 1.1 29 16.34 Aciphyllene 3.4 3.0 30 16.57 Epizonarene 1.2 1.1 31 17.06 Hinesol 0.4 0.1 32 17.11 Cubenol 0.2 0.9 33 17.19 (–)-Spathulenol tr tr 34 17.73 Gitoxigenin 9.3 10.2 35 17.77 β-Myristoylolene 0.9 0.6 36 18.08 Calarene epoxide 0.2 tr 37 18.11 Farnesyl bromide 0.4 0.8 38 18.17 Selina-6-en-4-ol 0.1 tr 39 18.45 γ-Himachalene 0.7 0.9 40 18.76 Agarospirol 1.2 1.0 41 19.40 Leolene alcohol 0.8 0.4 42 20.02 Ledene oxide 0.4 tr 43 20.16 Calarene epoxide tr 0.9 44 21.06 Globulol 0.5 0.9 45 21.12 Phytol 0.2 tr 46 22.37 Longifolenaldehyde tr 0.1 47 24.49 Dotriacontane 0.3 0.1 48 27.68 Hetadacane 0.1 0.9 49 28.08 9-Eicosyne tr 0.2 50 28.09 3-Eicosyne 0.1 0.1 51 30.40 Octadeccanoic acid tr 1.1 52 32.93 9-Octadecyne tr tr 53 43.22 Tritetracontane 0.1 0.2 54 46.74 1-Decanol-2-hexyl tr tr 55 46.93 Caryophyllene oxide tr 0.9 56 47.87 Alizarin 3.2 4.4 57 49.31 Carotene tr 0.1 Total 94.9 96.1
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The in vitro antibacterial activity of the essential oils, methanol extract, and methanol derived subfractions of O. basilicum against the employed bacteria was qualitatively assessed by the presence or absence of inhibition zones. The oils exhibited antibacterial activity against all five Gram-positive and six Gram-negative bacteria at the concentration of 10 μL of 1:5 (v/v) dilution with methanol. The oils exhibited a potent inhibitory effect against B. cereus, B. subtilis, B. megaterium, S. aureus, L. monocytogenes, V. parahaemolyticus, and S. typhi, with diameters of inhibition zones ranging from 14.0 to 20.1 mm, as shown in Table 2. The methanol extract of O. basilicum and its chloroform and ethyl acetate subfractions also revealed a great potential of antibacterial activity against all Gram-positive and six Gram-negative bacteria, at the concentration of 300 μg/disc (Table 2). The methanol extract showed the strongest antibacterial effect against B. cereus, B. subtilis, B. megaterium, S. aureus, L. monocytogenes, V. parahaemolyticus, and V. mimicus, with their respective diameter zones of inhibition of 20.1, 17.2, 18.1, 16.1, 16.2, 15.4, and 15.2 mm. On the other hand, chloroform and ethyl acetate subfractions showed moderate to high antibacterial effects against most of the bacteria tested (inhibition zones: 13.0–20.2 mm). The hexane fraction displayed a moderate inhibitory effect against some of the bacteria. In this study, in some cases, the oils, methanol extract, and its ethyl acetate subfraction exhibited higher or similar types of antibacterial activity compared with that of streptomycin against Gram-positive bacteria. However, the residual methanol subfraction did not show any activity against all the bacterial strains tested (data not shown). The blind control did not inhibit the growth of the bacteria tested. The methanol extract and its ethyl acetate subfraction showed higher activity compared with the hexane and chloroform subfractions. Also, stem oil had a higher antibacterial effect than leaf oil. No inhibitory effect was observed against S. paratyphi and P. aeruginosa by the essential oils.
Table 2. Antibacterial activity of essential oils and methanolic extracts of Ocimum basilicum L. against food-borne pathogenic bacteria.
Microorganism Zone of inhibition (mm)a Essential oilb MeOH extractc Subfraction of MeOH extractd SMe Leaf Stem Hexane CHCl3 EtOAc 18.3 ± 1.6 20.1 ± 1.6 20.1 ± 0.5 14.2 ± 0.7 20.2 ± 1.1 21.1 ± 1.2 20.2 ± 0.5 16.3 ± 1.2 18.3 ± 0.6 17.2 ± 1.1 15.1 ± 0.6 16.1 ± 1.5 19.3 ± 1.2 16.1 ± 0.7 15.3 ± 1.2 17.2 ± 1.2 18.1 ± 1.0 13.2 ± 1.2 15.1 ± 0.5 18.2 ± 1.1 17.4 ± 1.2 16.2 ± 0.7 15.1 ± 1.2 16.1 ± 0.5 13.2 ± 1.1 15.1 ± 1.2 17.1 ± 1.2 16.2 ± 1.3 15.1 ± 1.5 17.1 ± 1.2 16.2 ± 1.1 12.3 ± 1.4 14.0 ± 1.2 15.3 ± 1.6 17.3 ± 0.6 11.2 ± 0.5 12.2 ± 1.1 14.1 ± 0.5 nd 12.1 ± 1.2 14.2 ± 0.6 16.3 ± 0.6 12.3 ± 1.2 13.3 ± 1.2 13.1 ± 1.4 nd 12.2 ± 1.1 13.0 ± 0.9 20.1 ± 0.5 11.2 ± 1.1 12.3 ± 1.2 14.2 ± 0.7 12.3 ± 0.5 14.2 ± 0.9 15.2 ± 0.6 17.0 ± 0.5 14.3 ± 1.2 15.3 ± 1.2 15.4 ± 0.5 nd 15.5 ± 0.7 16.2 ± 0.6 19.3 ± 1.1 12.2 ± 1.2 13.1 ± 0.6 15.2 ± 1.1 nd 15.2 ± 1.5 15.2 ± 1.7 21.0 ± 0.7 14.0 ± 1.2 14.2 ± 1.2 13.1 ± 0.7 11.3 ± 0.6 14.2 ± 1.5 14.2 ± 1.7 16.3 ± 0.6 nd nd nd nd nd nd 18.2 ± 0.8 nd nd nd nd nd nd 15.3 ± 1.1
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As shown in Table 3, the MIC values for the oils were found to be lower for B. cereus, B. subtilis, B. megaterium, S. aureus, and L. monocytogenes (62.5–125 μg/mL) than for E. coli, S. boydii, S. dysenteriae, V. parahaemolyticus, V. mimicus, and S. typhi (250–500 μg/mL). On the other hand, the MIC values of the methanol extract and its derived subfractions of hexane, chloroform, and ethyl acetate against the tested bacteria were found to be in the range 62.5–500 μg/mL (Table 3). The methanol extract and its ethyl acetate subfraction showed higher antibacterial activity than the hexane and chloroform subfractions. In this study, the Gram-positive bacteria were found to be more susceptible to the essential oils and various solvent extractions than the Gram-negative bacteria.
Table 3. Minimum inhibitory concentrations of essential oils and methanolic extracts of Ocimum basilicum L. against food-borne pathogenic bacteria.
Microorganism Minimum inhibitory concentration (MIC)a Essential oil MeOH extract Subfraction of MeOH extract Leaf Stem Hexane CHCl3 EtOAc 62.5 62.5 62.5 125 62.5 62.5 125 125 125 250 125 125 125 125 62.5 250 125 62.5 125 62.5 62.5 250 62.5 62.5 125 125 125 500 125 125 500 250 125 >1000 250 125 250 250 500 >1000 250 250 500 250 250 500 250 250 500 250 500 >1000 500 250 500 250 500 >1000 500 500 250 500 500 >1000 500 250 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000
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Since ancient times, aromatic plant extracts have been in use for many purposes, such as food, drugs, and perfumery. Historically, many plant oils and extracts have been reported to have antimicrobial properties. Essential oils are odorous and volatile products of plant secondary metabolism, which, because of their antibacterial, antifungal, antioxidant, and anticarcinogenic properties, can be used as natural additives in many foods, as well as being of pharmaceutical interest ([
The use of essential oils may improve food safety and overall microbial quality. If essential oils were to be more widely applied as antibacterials in foods, the organoleptic impact would be important. Foods generally associated with herbs, spices, or seasonings would be the least affected by this phenomenon, and information on the flavor impact of oregano essential oil in meat and fish supports this. The flavor, odor, and color of minced beef containing 1% (v/w) oregano oil improved during storage under modified atmosphere packaging and vacuum at 5°C and were almost undetectable after cooking ([
The pharmaceutical and therapeutic potential of essential oils and their individual constituents have been evaluated. The essential oil can be used alone, or as part of a therapeutic pharmaceutical composition, which includes at least one antimicrobial compound and a pharmaceutically acceptable carrier. The essential oils and their volatile constituents can be used via inhalation or massage therapy. In the field of complementary and alternative respiratory medicine, inhalation of peppermint essential oil vapors has been suggested as an adjunct in combined multi-drug therapy in patients with disseminated and infiltrative pulmonary tuberculosis ([
In this study, the essential oils and methanol extracts exhibited potential activity against some of the representative food-borne pathogenic bacteria such as B. cereus, B. subtilis, B. megaterium, S. aureus, L. monocytogenes, E. coli, S. boydii, S. dysenteriae, V. parahaemolyticus, V. mimicus, and S. typhi. In our opinion, major components of sweet basil essential oils, methyl chavicol, trimethoquinol, β-guaiene, aciphyllene, alizarin, naphthalene, and (–)-caryophyllene, have key roles for their antibacterial activities. Antibacterial activities of these compounds have been reported by others ([
In both the food and pharmaceutical industries there is a continuing need to find new and improved antimicrobial agents, especially in view of the increasing incidence of antibiotic resistance. One of the areas that is subject to considerable interest is plant extracts, and in particular their essential oils. Also, the increasing consumer demand for effective and safe natural products means that quantitative data on plant oils and extracts are required. Reports on the analysis of essential oils from sweet basil from South-Asian regions and their antimicrobial activity are still scare. Therefore, screening of the medicinal plant, O. basilicum growing in Bangladesh, for antimicrobial activity and phytochemicals is important in finding potential new compounds for medicinal or other uses.
The composition of essential oils extracted from O. basilicum growing in Bangladesh showed remarkable differences from the same species cultivated in Pakistan, Kenya, Iran, and Croatia, based on comparison with published results. It is often quite difficult to compare the results obtained from different studies, because the compositions of the essential oils can vary greatly depending upon the geographical region, the variety, the age of the plant, the method of drying, and the method of extraction of the oil ([
Plant essential oils are a potentially useful source of antimicrobial compounds. Results reported from different studies are difficult to compare, presumably because of different test methods, bacterial strains, and sources of antimicrobial samples used. A previous report showed that the methanol extract of O. basilicum occurring in Turkey weakly inhibited the growth of some bacterial strains in the genera Bacillus, Micrococcus, Escherichia, and Staphylococcus with inhibition zones 7–12 mm, using the concentration of 300 μg/disc, whereas only Acinetobacter was inhibited strongly (17 mm) ([
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In conclusion, the results of this study suggest that O. basilicum mediated oils and organic extracts could be a source of natural antimicrobial agents for use in the food and/or pharmaceutical industries against food-borne or pathogenic microbes. However, further research is needed in order to obtain information regarding the practical effectiveness of essential oils or extracts to prevent the growth of food-borne and spoiling microbes under specific application conditions.
The authors are grateful to M. Ali, Head, Chemistry Division, Atomic Energy Centre, Dhaka, Bangladesh for her constant encouragement and helpful suggestions. They are also grateful to Mr. Zahidul Islam and Mr. Ayub Ali for their help to preparation the sweet basil Ocimum basilicum samples.
The authors report no conflict of interest. The authors are responsible for the content and writing of the paper.
By M. Amzad Hossain; M. J. Kabir; S. M. Salehuddin; S. M. Mizanur Rahman; A. K. Das; Sandip Kumar Singha; Md. Khorshed Alam and Atiqur Rahman
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