In vitro antibacterial properties of essential oil and organic extracts of Premna integrifolia Linn

2.1 Plant materials

The leaves of P. integrifolia were collected from the Boro Parulia, Gopalgonj; Arappur, Jhenaidah and Ambaria, Kushtia of Bangladesh, in January 2009 and identified by Dr. Oliur Rahman, Bangladesh National Herbarium, Dhaka, where a voucher specimen (DACB 35170) has been deposited.

2.2 Isolation of the essential oil

The air-dried leaves (200 g) of P. integrifolia were subjected to hydro distillation for 3 h using a Clevenger type apparatus. The oil was dried over anhydrous Na₂SO₄ and preserved in a sealed vial at 4 °C for further analysis.

2.3 Preparation of organic extracts

The air-dried leaves P. integrifolia were first pulverized into powdered form. The dried powder (50 g) was then extracted with hexane, chloroform, ethyl acetate and methanol separately at room temperature for 7 days and the solvents were evaporated by vacuum rotary evaporator temperature at 50 °C. The extraction process yielded hexane (7.3 g), chloroform (6.2 g), ethyl acetate (7.4 g) and methanol (6.5 g) extracts respectively. Solvents (analytical grade) for extraction were obtained from commercial sources (Sigma Aldrich, St. Louis, MO, USA).

2.4 Gas chromatography–mass spectrometry (GC–MS) analysis

The 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 transfer line and ion source were 280 °C and 275°C respectively. Ions were obtained by electron ionization mode. The VF-5 capillary column (30 m length, 0.25 mm I.D. and 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 started at 50 °C for 1 min, programed at 8 °C min⁻¹ to 200 °C and heated until 280 °C at 10 °C min⁻¹. Injection port was set at 250 °C. Helium was used as the carrier gas at a constant flow rate of 1.0 mL min⁻¹. Molecular ions (mass range: 40–500 m z⁻¹) were monitored for identification. The relative percentage of the oil constituents was expressed as percentage by peak area normalization.

Identification of components of the essential oil was based on their retention indices, relative to a homologous series of n-alkane (C₈–C₂₀) on the VF-5 capillary column under the same operating conditions and computer matching with the GC–MS spectra from the Wiley 6.0 MS data and literature data (Adams, 2001).

2.5 Microorganisms

The following microorganisms were used in the antibacterial test: Sarcina lutea IFO 3232, Bacillus subtilis IFO 3026, Escherichia coli IFO 3007, Pseudomonas sp. ATCC 13867, Klebsiella pneumoniae ATCC 10031 and Xanthomonas campestries IAM 1671. The strains were kindly provided by Prof. Yong Se Lee, Department of Bioresource Technology, College of Agriculture and Environmental Science, Daegu University, Korea. Cultures of each bacterial strain were maintained on Luria-Bertani (LB) agar medium at 4 °C.

2.6 Determination of antibacterial activity of essential oil and organic extracts

The dried extracts were dissolved in the same solvent used for their extraction and sterilized by filtration using 0.22 μm sterile Millipore filter (Millipore Corp., Billerica, MA, USA). Then the antibacterial test was carried out by the agar disk diffusion method (Murray et al., 1999) using 100 μL of standardized inoculum suspension containing 10⁷ CFU mL⁻¹ of bacteria. The essential oil was diluted 1:5 (v/v) with methanol and aliquots of 15 μL were spotted onto the sterile Whatman No. 1 filter paper disks (6 mm diameter); while 10 μL of 30 mg mL⁻¹ of each organic extract (300 μg disk⁻¹) was applied on the filter paper disks and placed on the inoculated LB agar. Negative controls were prepared using the same solvents employed to dissolve the samples. Standard antibiotic, streptomycin (20 μg disk⁻¹ from Sigma–Aldrich Co., St. Louis, MO, USA) was used as positive control for the tested bacteria. The plates were incubated micro aerobically 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.

2.7 Minimum inhibitory concentration (MIC)

Minimum inhibitory concentrations (MICs) of the samples were tested by the standard NCCL method (NCCLS, 2008). Active cultures for MIC determination were prepared by transforming a loopful of cells from stock cultures to flasks and inoculated in LB medium and incubated at 37 °C for 24 h. The cultures were diluted with fresh LB broth to achieve optical density of 10⁷ CFU mL⁻¹ for the test organisms at 600 nm by UV/Vis Spectrophotometer Optizen 2120UV and Optizen III (Shin et al., 2007). Dilutions, to get the final concentration ranging from 0 to 1000 μg mL⁻¹ of essential oil and various organic extracts in Luria-Bertani broth medium were prepared in 96 well microplates. Finally 20 μL inoculum of each bacterial strain (10⁷ CFU mL⁻¹) 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 the test samples, which did not show any visual growth of test organisms after macroscopic evaluation, were determined as MICs, which were expressed in μg/mL⁻¹. The MICs were measured for the oil and various extracts, as well as for standards of pure α-humulene, spathuleno and eugenol, which were tested on the same cultures under identical conditions to compare their activity with those of the investigated oil and extracts.

2.8 Statistical analysis

The essential oil and different organic extracts were assayed for antibacterial activity. Each experiment was run in triplicate, and mean values were calculated. The statistical analysis was carried out employing one way ANOVA (p < 0.05). A statistical package (SPSS version 11.0) was used for the data analysis.

3.1 Chemical composition of the essential oil

GC–MS analyses of the oil led to the identification of 29 different compounds, representing 94.81% of the total oil from leaves. The identified compounds are listed in Table 1, according to their elution order on a VF-5 capillary column. The oil contains a complex mixture consisting of mainly oxygenated mono- and sesquiterpenes, and mono- and sesquiterpene hydrocarbons. The major compounds detected in the leaves oil were phytol (27.25%), α-humulene (14.21%), spathulenol (12.12%), 1-octen-3-ol (8.21%), eugenol (6.69%), phenylethyl alcohol (5.81%) and caryophyllene oxide (2.6%), as shown in Table 1. Hexadecanoic acid (1.06%), 1,8-cineole (0.93%), α-pinene (0.86%), β-pinene (1.11%), β-carryophyllene (0.92%) and 3-octanol (0.97%) were also found to be the minor components of P. integrifolia oil in the present study.

3.2 Antibacterial activity of essential oil and organic extracts

The in vitro antibacterial activity of essential oil, various organic extracts of P. integrifolia against the employed bacteria were qualitatively assessed by the presence or absence of inhibition zones. The oil exhibited antibacterial activity against all two Gram-positive and four Gram-negative bacteria at the concentrations of 15 μL of 1:5 (v/v) dilution with MeOH. The oil exhibited a potent inhibitory effect against S. lutea IFO 3232, B. subtilis IFO 3026, E. coli IFO 3007, Pseudomonas sp. ATCC 13867, K. pneumoniae ATCC 10031 and X. campestries IAM 1671 with diameter of inhibition zones ranging from of 13.1 ± 1.5 to 22.1 ± 1.2 mm, as shown in Table 2. Methanol, chloroform and ethyl acetate extracts of P. integrifolia also revealed a great potential of antibacterial activity against all bacteria, at the concentrations of 300 μg disk⁻¹ (Table 2). Methanol and ethyl acetate extracts showed the strongest antibacterial effect against S. lutea IFO 3232, B. subtilis IFO 3026, E. coli IFO 3007 and X. campestries IAM 1671 (inhibition zones: 20.1 ± 0.5 to 14.1 ± 0.5 mm). On the other hand, chloroform extract showed moderate to high antibacterial effects against most of the bacteria that have being tested (inhibition zones: 12.0 ± 1.2 to 18.2 ± 1.1 mm). Hexane fraction displayed a moderate inhibitory effect against most of the bacteria. In this study, the oil, methanol and ethyl acetate extracts exhibited higher or similar types of antibacterial activity than that of streptomycin against Gram-positive bacteria. The blind control did not inhibit the growth of the bacteria tested. Methanol and ethyl acetate extracts showed higher activity compared with hexane and chloroform extracts.

3.3 Minimum inhibitory concentration (MIC)

As shown in Table 3, the MIC values for the oil were found lower for S. lutea IFO 3232, B. subtilis IFO 3026, E. coli IFO 3007 and X. campestries IAM 1671 (62.5–125 μg mL⁻¹) than for Pseudomonas sp. ATCC 13867 and K. pneumoniae ATCC 10031 (250 μg mL⁻¹). On the other hand, MIC values of the various organic extracts against the tested bacteria were found in the range 62.5–250 μg mL⁻¹ (Table 3). In this study, the Gram-positive bacteria were found to be more susceptible to the essential oil and various solvent extractions than Gram-negative bacteria. Standard pure compounds such α-humulene, spathulenol and eugenol in this study were also exhibited potent activity as compared to oil or extracts.