Antibacterial properties of essential oils and methanol extracts of sweet basil Ocimum basilicum occurring in Bangladesh

Abstract

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

Introduction

Illness caused by the consumption of contaminated foods has a broad economic and public health impact worldwide (Mead et al., 1999). Many pathogenic microorganisms such as Listeria monocytogenes, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Salmonella sp., and Pseudomonas aeruginosa have been reported as the causal agents of food-borne diseases (McCabe-Sellers & Samuel, 2004). A variety of different chemical and synthetic compounds have been used as antimicrobial agents to inhibit bacteria in foods. Due to the identified and potential toxicity of chemical food preservatives, there have been increased demands for food preservatives from natural sources. The demands for more natural antimicrobials have driven food scientists to investigate the effectiveness of inhibitory compounds such as essential oils (Demirci et al., 2008), and extracts from plants (Nasar-Abbas & Halkman, 2004). Essential oils are a complex mixture of compounds, mainly monoterpenes, sesquiterpenes, and their corresponding oxygenated derivatives (alcohols, aldehydes, esters, ethers, ketones, phenols, and oxides) from plants, which are widely known for their scents and flavors. Plant-derived essential oils have been long used as flavoring agents or preservatives in food, beverage, and confectionery products and also have a broad spectrum of in vitro antimicrobial activities (Conner, 1993). Thus, essential oils and plant extracts are promising natural antimicrobial agents with potential applications in food industries for controlling food-borne pathogens and spoiling bacteria.

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 (Sajjadi, 2006). Basil essential oil has been utilized extensively in the food industry as a flavoring agent, and in the perfumery and medical industries (Simon et al., 1990). The O. basilicum essential oils exhibit a wide and varying array of chemical compounds, depending on variations in chemotype, leaf and flower color, aroma, and origin of the plant (Sajjadi, 2006).

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 (Adigüzel et al., 2005; Hussain et al., 2008), the result of this study differed from previous reports.

Materials and methods

Plant materials

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.

Isolation of the essential oils

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 Na₂SO₄ and preserved in a sealed vial at 4°C until further analysis.

Preparation of methanol extract and its various subfractions

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).

Gas chromatography-mass spectrometry (GC-MS) analysis

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 (Adam, 2001).

Microorganisms

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.

Antibacterial activity assay

The antibacterial test was carried out by agar disc diffusion method (Murray et al., 1999) using 100 μL of standardized inoculum suspension containing 10⁷ CFU/mL of bacteria. Whatman No. 1 sterile filter paper discs (6 mm diameter) were impregnated with 10 μL of essential oils of 1:5 (v/v) dilution with methanol and 10 μL of 30 mg/mL extracts or subfractions (300 μg/disc) and placed on the inoculated agar. Negative controls were prepared using the same solvents employed to dissolve the samples. Standard antibiotic, streptomycin (20 μg/disc), was used as positive control for the tested bacteria. The plates were incubated microaerobically at 37°C for 24 h. Antibacterial activity was evaluated by measuring the diameter of the zones of inhibition against the tested bacteria. Each assay in this experiment was replicated three times.

Minimum inhibitory concentration (MIC)

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 (NCCLS, 2008). Active cultures for MIC determination were prepared by transferring a loopful of cells from stock cultures to flasks and inoculating in LB medium and incubating at 37°C for 24 h. The cultures were diluted with fresh LB broth to achieve an optical density of 10⁷ CFU/mL for the test organisms at 600 nm using ultraviolet-visible (UV/Vis) spectrophotometers Optizen 2120UV and Optizen III (Shin et al., 2007). Dilutions to obtain the final concentrations ranging from 0 to 2000 μg/mL of essential oils and methanol extracts in LB broth medium were prepared in 96-well microplates. Finally, 20 μL inoculum of each bacterial strain (10⁷ CFU/mL) was inoculated onto the microplates and the tests were performed in a volume of 200 μL. The plates were incubated at 37°C for 24 h. The lowest concentrations of test samples which did not show any visible growth of test organisms after macroscopic evaluation were determined as MICs, expressed in μg/mL.

Results

Chemical composition of the essential oils

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	(Z)-5,7-Octadine-4-one-2,6-dimethyl	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

^aRetention time (min) of authentic compounds on VF-5 capillary column.

tr, trace (< 0.1%).

Antibacterial activity

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 oil^b		MeOH extract^c	Subfraction of MeOH extract^d			SM^e
	Leaf	Stem		Hexane	CHCl3	EtOAc
B. cereus	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
B. subtilis	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
B. megaterium	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
S. aureus	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
L. monocytogenes	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
E. coli	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
S. boydii	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
S. dysenteriae	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
V. parahaemolyticus	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
V. mimicus	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
S. typhi	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
S. paratyphi	nd	nd	nd	nd	nd	nd	18.2 ± 0.8
P. aeruginosa	nd	nd	nd	nd	nd	nd	15.3 ± 1.1

^aDiameter of inhibition zone (mm) around disc (6 mm).

^b10 μL/disc, oil was diluted with MeOH, 1:5 (v/v).

^cMeOH extract (300 μg/disc).

^dSubfraction of MeOH extract (300 μg/disc).

^eStandard antibiotic: streptomycin (20 μg/disc).

nd, not detectable inhibition zone. Values are given as mean ± SD (n = 3).

Minimum inhibitory concentration (MIC)

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
B. cereus	62.5	62.5	62.5	125	62.5	62.5
B. subtilis	125	125	125	250	125	125
B. megaterium	125	125	62.5	250	125	62.5
S. aureus	125	62.5	62.5	250	62.5	62.5
L. monocytogenes	125	125	125	500	125	125
E. coli	500	250	125	>1000	250	125
S. boydii	250	250	500	>1000	250	250
S. dysenteriae	500	250	250	500	250	250
V. parahaemolyticus	500	250	500	>1000	500	250
V. mimicus	500	250	500	>1000	500	500
S. typhi	250	500	500	>1000	500	250
S. paratyphi	>1000	>1000	>1000	>1000	>1000	>1000
P. aeruginosa	>1000	>1000	>1000	>1000	>1000	>1000

^aMinimum inhibitory concentration (values in μg/mL).

Discussion

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 (Tsigarida et al. 2000; Busatta et al. 2008).

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 (Skandamis & Nychas, 2001). The addition of thyme oil at up to 0.9% (v/w) in a coating for cooked shrimps had no ill effects on the flavor or appearance (Ouattara et al., 2001). Individual essential oil components, many of them being approved food flavorings, also impart a certain flavor to foods. In addition, it is recommended to apply essential oils or their compounds as part of a hurdle system and to use them as antimicrobial components along with other preservation techniques, for example in combination with reduced temperature and reduced pH, or use a synergistic combination of essential oils and their compounds (Ultee et al., 2000).

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 (Shkurupii et al., 2002). Massage therapy with essential oil can be useful in the treatment of people suffering from dementia (Ballard et al., 2002). Inflammatory diseases such as allergy, rheumatism, and arthritis are often alleviated using essential oil massage therapy (Maruyama et al., 2005).

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 (Saxena & Sharma, 1999; Sartoratto et al., 2004; Inouye et al., 2006; Deba et al., 2008). Also, the antibacterial activity of individual components of essential oils such as methyl chavicol (estragol) has been reported previously (Yasuo, 2005). On the other hand, the components in lower amounts such as phenylethyl alcohol, camphor, isoledene, globulol, and leolene alcohol also contributed to antibacterial activity of the oils (El-Sakhawy et al., 1998; Lago et al., 2004; Sartoratto et al., 2004; Deba et al., 2008). It is also possible that the minor components might be involved in some type of synergism with the other active compounds (Marino et al., 2001). Further, the antibacterial activity of the methanol extract could be attributed to the presence of some bioactive phenolics (rosmarinic acid, chicoric acid, caftaric acid, etc.) in the O. basilicum plant, and these findings are in agreement with a previous report (Lee & Scagel, 2008).

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 (Daferera et al., 2000). In this study, the plant materials used were from the same season, as plant materials from the following year might be different in terms of reproducibility. In previous studies, leaf oils from Pakistan, Iran, and Croatia contained camphor at 3.1%, 0.6%, and 0.5%, respectively (Sajjadi, 2006; Politeo et al., 2007; Hussain et al., 2008). Moreover, caryophyllene was also found in oils from O. basilicum cultivated in Pakistan (1.9%), Croatia (0.3%), and Turkey (0.08%) (Politeo et al., 2007; Hussain et al., 2008; Jean-Claude & Özcan, 2008). The content of methyl chavicol in our oils (29.9–36.7%) was close to that found in the species cultivated in Iran (40.5%), while phenylethyl alcohol (0.5–0.7%) was also found as a minor constituent in the leaf oil from US O. basilicum (Seung-Joo et al., 2005; Sajjadi, 2006).

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) (Adigüzel et al., 2005). Our methanol extract of O. basilicum occurring in Bangladesh demonstrated significantly higher antibacterial activity against some food-borne pathogens in the genera Bacillius, Staphylococcus, Listeria, Escherichia, Shigella, Vibrio, and Salmonella (inhibition zones 13–20 mm) at the same concentration. Also, our basil oil (MIC: 62.5–500 μg/mL) showed stronger antibacterial activity according to minimum inhibitory concentration than that of Pakistani basil (MIC: 0.8–2.6 mg/mL) (Hussain et al., 2008).

Deans et al. (1995) observed that the susceptibility of Gram-positive and Gram-negative bacteria to plant volatile oils had little influence on growth inhibition. However, some oils appeared to be more specific, exerting a greater inhibitory activity against Gram-positive bacteria. It is often reported that Gram-negative bacteria are more resistant to plant-based essential oils (Smith-Palmer et al., 1998). The hydrophilic cell wall structure of Gram-negative bacteria is constituted essentially of a lipopolysaccharide (LPS) that screens out the hydrophobic oil and avoids the accumulation of essential oils in the target cell membrane (Bezic et al., 2003). This is the reason that Gram-positive bacteria were found to be more sensitive to the essential oil, methanol extract, and various methanol derived subfractions of O. basilicum than those of Gram-negative bacteria.

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.

Acknowledgements

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.

Declaration of interest

The authors report no conflict of interest. The authors are responsible for the content and writing of the paper.