Chemical Compositions of the Volatile Oils and Antibacterial Screening of Solvent Extract from Downy Lavender

The discovery of a new species exhibiting more effective antibacterial properties is necessary because of the demand on Lavandula species, which continues to increase in a variety of industries. Lavandula pubescens might be a good alternative, as it exhibits strong antibacterial activity. In this study, the chemical composition of the essential oils from different organs (flowers, leaves, stems, and roots) of L. pubescens was identified using gas chromatography-mass spectrometry. Furthermore, the antimicrobial activities of different solvent extracts (methanol, ethanol, diethyl ether, hexane, and ethyl acetate) and different organ (flower, leaf, stem, and root) extracts of L. pubescens were evaluated. Only the ethyl acetate extracts of L. pubescens exhibited antibacterial activity against all bacterial strains tested, including Staphylococcus haemolyticus, Escherichia coli (KF 918342), Aeromonas hydrophila (KCTC 12487), E. coli (ATCC 35150), Cronobacter sakazakii (ATCC 29544), and Aeromonas salmonicida (KACC 15136). In particular, the extracts exhibited significant activity against S. haemolyticus. Ethyl acetate extract of the leaf exhibited the best activity against all bacterial strains. This study provides valuable information on the chemical compositions in essential oils and antimicrobial properties of L. pubescens.


Introduction
Lavandula pubescens, also known as downy lavender, is an aromatic flowering plant belonging to the Lamiaceae family. Lavandula, consisting of approximately 39 known species, has been largely cultivated as ornamental plants for gardens and scenery use. This perennial plant is widely distributed in the Mediterranean, North Africa, Southwest Asia, western Iran, and eastern India [1]. The widely used and cultivated lavender species are Lavandula angustifolia, Lavandula officinalis, Lavandula latifolia, and Lavandula vera. Lavandula pubescens is a newly discovered species of lavender, which occurs in the Mediterranean. In recent years, the plants have been extensively studied as resources for medicine and aromatic products and used largely for their medicinal potentials, because these plants contain a number of bioactive compounds that act against human and plant pathogens [2], as well as compounds with various activities and properties [3].
Presently, Lavandula is widely studied because of its commercial use in the fragrance industry [4,5]. The plants have also been used as antibacterial, sedative, and antiviral agents in the pharmaceutical The antimicrobial activity of Lavandula has been widely studied [18][19][20]. Lavender plants have been used as a relaxant in aromatherapy [21,22]. Antioxidant and antiviral activities of Lavandula essential oil have been reported [23][24][25]. Therefore, this study aims to provide information on volatile constituents of in the essential oils from different organs (flowers, leaves, stems, and roots) of L. pubescens using gas chromatography-mass spectrometry (GC-MS) and the antibacterial activities of different plant parts of L. pubescens.

Plant Material and Extraction
The root, stem, leaf, and flower of L. pubescens were collected from the green house of Chungnam National University (Figure 1). The plants were washed under running tap water and dried under

GC-MS Analysis of L. pubescens
Volatile compounds were extracted and analyzed using a previously reported GC-MS method [27]. GC-MS analysis was performed on a 7820A GC/5977E MSD (Agilent, Santa Clara, CA, USA) with an HP-5 (30 m × 0.25 mm ID, film thickness 0.25 µm) fused-silica capillary column (Agilent, USA). Helium was used as the carrier gas at a flow rate of 1.0 mL/min. For GC-MS detection, an electron ionization system, with system energy of 70 eV, trap current of 250 µA, and an ion source temperature of 200 • C, was used. The oven temperature program was the same as that described for GC, and injections were used in the splitless mode. The column temperature was maintained at 35 • C for 2 min and programmed as follows: Increase from 50 to 250 • C at a rate of 10 • C/min and hold at 250 • C for 10 min. Fresh samples of each plant part (2.0 g) were placed in a 15 mL thermostatted vial that has a rubber septum. During the SPME extraction procedure, the SPME fiber was introduced for 12 h into the thermostatted vial (RT). For this analysis, a 1 cm, 50/30 µm polydimethylsiloxane/divinylbenzene/carboxen-coated fiber was used. The fiber was conditioned in a GC injection port for 1 min prior to use. The absorbed component was injected into a gas chromatograph by desorption at 250 • C for 2 min in the injector (splitless mode).

Disk Diffusion Method
A 0.1 OD 600 of the different overnight bacterial cultures was swabbed on a 25 mL LB agar plate. Whatman disk was then placed on the plates. A 30 µL sample of different solvent extracts of L. pubescens was added to the sterilized disk (Whatman No. 1 paper, 6 mm diameter) and incubated overnight at 37 • C. For screening of the different plant organs, the powdered samples were dissolved in ethyl acetate, and the resulting extract was added to the Whatman disk. Streptomycin (250 µg/mL) was used as the standard antibacterial agent. The experiment was performed in triplicate.

Minimum Inhibitory Concentration
The minimum inhibitory concentration (MIC) of the L. pubescens extracts was established according to the method of Abdullah Al-Dhabi et al. [28] by using 96-well plates. A 1 mg/mL extract was dissolved in water with 2% DMSO. The initial extract concentration was 50 µL crude extract, which was then serially diluted two-fold (1.5625 to 50 µL). Each well had 100 µL of LB broth. Then, extracts were added at the concentrations described above. A 5 µL suspension containing 10 8 CFU/mL of each of the six bacterial strains was added to the 96-well plate and incubated at 37 • C for 17 h. After the incubation time, the minimum inhibitor concentration was determined by the lowest visible growth in LB broth. The experiment was performed in triplicate.

GC-MS Analysis of L. pubescens
Chemical composition of the essential oils of different organs of L. pubescens, such as the roots, stems, leaves, and flowers, were identified by GC-MS analysis. Most of the essential oils from different organs were characterized by the dominant presence of terpenes, including monoterpenes, diterpenes, and sesquiterpenes ( Table 1). The oil from the leaves contained nine monoterpenes-neo-allo-ocimene , and acoradien (0.07%); and one diterpene-jolkinol D (0.16%), comprising 94.62% of the total leaf-derived essential oil.

Antimicrobial Screening of L. pubescens
The antimicrobial screening of L. pubescens was performed using five different organic solvents. The ethyl acetate extract showed more antimicrobial activity against the six pathogenic bacteria because the disk diffusion method revealed that the ethyl acetate extract produced higher activity than that by the other organic solvents. In particular, L. pubescens possessed significant activity against S. haemolyticus, followed by E. coli (KF 918342), A. hydrophila (KCTC 12487), E. coli (ATCC 35150), C. sakazakii (ATCC 29544), and A. salmonicida (KACC 15136) ( Table 2). Therefore, ethyl acetate was used for further studies on antimicrobial activities of L. pubescens because it had the strongest activity against all the bacteria. The antibacterial activity against bacterial pathogens using different plant organ extracts in agar plates is shown in Figure 2. The leaf extract exhibited the most powerful antibacterial activity against all bacterial strains, followed by the flower and stem extracts. On the other hand, the roots did not exhibit antimicrobial activity (Table 3). Table 2. Antibacterial activity of Lavandula pubescens using extracts with different solvents. Each value is the average of three trials ± standard deviation.

Minimum Inhibitory Concentration of L. pubescens
The MIC of crude Lavandula extract was studied by the micro broth dilution method and the results are shown in Table 2. Lavandula ethyl acetate extract at different concentrations inhibited all the growth of all bacterial strain broths. The MIC for E. coli was 6.25 µL and for S. haemolyticus, A. hydrophila, and A. salmonicida was 12.5 µL. Cronobacter sakazakii inhibition was observed at 25 µL. Streptomycin showed better MIC values in comparison with those of the Lavandula ethyl acetate extracts. MIC values are shown in Table 4.

Minimum Inhibitory Concentration of L. pubescens
The MIC of crude Lavandula extract was studied by the micro broth dilution method and the results are shown in Table 2. Lavandula ethyl acetate extract at different concentrations inhibited all the growth of all bacterial strain broths. The MIC for E. coli was 6.25 µL and for S. haemolyticus, A. hydrophila, and A. salmonicida was 12.5 µL. Cronobacter sakazakii inhibition was observed at 25 µL. Streptomycin showed better MIC values in comparison with those of the Lavandula ethyl acetate extracts. MIC values are shown in Table 4.
Ethyl acetate extract of L. pubescens tested in the present study showed strong antibacterial activity against the six bacterial strains tested. However, antibacterial activities of the extracts dissolved in the other solvents were not observed. This could be caused by the difference in the chemical composition of these extracts. Often, variations in chemical composition may result from differences in the extraction solvents, season, and presence of secondary metabolites [35]. Previous studies have shown that lavender plants contain a broad variety of terpenes, including monoterpenes, sesquiterpenes, and diterpenes, as well as phenolic compounds exhibiting antimicrobial activity [36][37][38].
Ethyl acetate extracts of L. pubescens possessed strong antibacterial activity against the six pathogenic bacteria, and L. pubescens leaves had the most powerful antimicrobial capacity. Our results are supported by those of previous studies showing that the essential oil from Lavandula species had antibacterial properties [39]. Hui et al. reported that lavender essential oil showed anti-bacterial activities against E. coli and Staphylococcus aureus [40]. Hossain reported that the essential oil of L. angustifolia was effective against all tested turtle-borne pathogenic bacteria: A. hydrophila, Aeromonas caviae, and Aeromonas dhakensis [41]. Furthermore, lavender essential oil nanoemulsions showed anti-bacterial activity against C. sakazakii [42]. The essential oil of L. angustifolia showed anti-bacterial activity against S. aureus, E. coli, Citrobacter freundii, Enterobacter aerogenes, Propionibacterium acnes, Proteus vulgaris, Pseudomonas aeruginosa, Shigella sonnei, and Streptococcus pyogenes [43]. Therefore, the essential oils from Lavandula plants are expected to possess important antibacterial properties against various bacterial species.
According to the World Health Organization (WHO), approximately 80% of the world's population depends on herbal remedies for their primary healthcare [44,45]. This study showed the different chemical composition in essential oils of different parts of L. pubescens and the different antibacterial activity of ethyl acetate extracts of these plant parts. These properties could be successfully exploited to treat several diseases caused by bacterial infections. This may indicate that lavender species used in traditional remedies may possess beneficial biological activity. Thus, this study suggests the potential use of L. pubescens roots, stems, leaves, and flowers in traditional herbal medicine applications.

Conclusions
In this study, ethyl acetate was the most effective solvent for extracting a broad variety of compounds from L. pubescens, and its extract possessed strong antibacterial activity against Escherichia coli (KF 918342), Staphylococcus haemolyticus (KCTC 3341), Aeromonas hydrophila (KCTC 12487), Escherichia coli (ATCC 35150), Cronobacter sakazakii (ATCC 29544), and Aeromonas salmonicida (KACC 15136). In particular, the leaves exhibited the strongest antimicrobial activity against these bacteria among the different plant parts tested. Therefore, this study suggests that L. pubescens could be considered a good source for human health.
Author Contributions: S.U.P. designed the experiments and analyzed the data. C.H.P., H.J.Y., Y.E.P., S.W.C., and S.S.L. performed the experiments and analyzed the data. C.H.P. and Y.E.P. wrote the manuscript. All authors read and approved the final manuscript.