Abstract
Ethyl acetate and methanol extracts of 18 Thai medicinal plants were investigated for their antibacterial activity against Propionibacterium acnes, Stapylococcus aureus, and S. epidermidis. Thirteen plant extracts were capable of inhibiting the growth of P. acnes and S. epidermidis, while 14 plant extracts exhibited an inhibitory effect on S. aureus. Based on the broth dilution method, the ethyl acetate extract of Alpinia galanga (L.) Wild. (Zingiberaceae) rhizome showed the strongest antibacterial effect against P. acnes, with minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 156.0 and 312.0 µg/mL, respectively. On the basis of bioassay-guided purification, the ethyl acetate extract was isolated to afford the antibacterial active compound, which was identified as 1′-acetoxychavicol acetate (1′-ACA). 1′-ACA had a strong inhibitory effect on P. acnes with MIC and MBC values of 62.0 and 250.0 µg/mL, respectively. Thus, 1′-ACA was used as an indicative marker for standardization of A. galanga extract using high performance liquid chromatography. These results suggest that A. galanga extract could be an interesting agent for further studies on an alternative treatment of acne.
Introduction
Acne vulgaris is a chronic inflammatory skin disorder involving the pilosebaceous follicles, characterized by comedones, papules, pustules, cysts, nodules, and often scars, chiefly on the face, neck, and upper trunk. It is produced by hyperkeratosis, which retains keratin and sebum, and the main microorganisms involved are Propionibacterium acnes and staphylococci. For many years, antibiotics have been used to treat acne vulgaris. However, antibiotic resistance has been increasing in prevalence within the dermatologic setting (CitationSwanson, 2003). To overcome the problem of antibiotic resistance, medicinal plants have been extensively studied as alternative treatments. In this study, 18 medicinal plants, which have been traditionally used as antibacterial and anti-inflammatory agents, were selected (CitationFarnsworth & Bunyapraphatsara, 1992) and investigated for antibacterial activity against P. acnes. The plant extract that exhibited the strongest effect was subjected to isolation of the active compound using bioassay-guided purification. In addition, the active compound was used as an indicative marker for standardization of the plant extract using the reverse-phase HPLC technique.
Materials and methods
Plant materials
The 18 plant materials used in this study are shown in . The plant materials were collected in Songkhla Province, Thailand, in June 2007. The plant materials were identified by Associate Professor Pharkphoom Panichayupakaranant and deposited at the herbarium of the Faculty of Pharmaceutical Sciences, Prince of Songkla University, Thailand.
Table 1. Plant materials used in this study.
Preparation of plant extracts
The plant materials were dried at 50°C for 12 h in a hot air oven and were ground to powder using a grinder and a sieve no. 45. The dried powder of the plant materials (20 g) was successively extracted with ethyl acetate (100 mL) under reflux conditions for 1 h ( × 2). The marc was then extracted with methanol (100 mL) under reflux conditions for 1 h (× 2). After filtering and evaporating the filtrates to dryness in vacuo, 36 plant extracts were obtained. The yields of the plant extracts are shown in .
Table 2. Yield of ethyl acetate (EtOAc) and methanol (MeOH) extracts of medicinal plants.
Microorganisms and media
Propionibacterium acnes (DMST 14916) was obtained from the Department of Medical Science Center, Thailand. Staphylococcus aureus (ATCC 25923) and Staphylococcus epidermidis (ATCC 14990) were obtained from the Department of Microbiology, Faculty of Medicine, Prince of Songkla University and Thailand Institute of Scientific and Technological Research, respectively. Mueller–Hinton agar and Mueller–Hinton broth were purchased from Merck. P. acnes was stored in glycerol broth at −20°C before it was used.
General instruments
1H and 13C nuclear magnetic resonance (NMR) spectra were recorded on a Fourier transform NMR spectrometer, 500 MHz, model Unity Inova, Varian. An infrared (IR) spectrum using the neat technique was recorded on a Fourier transform IR spectrometer, model Equinox 55, Bruker. Low-resolution electron ionization mass spectrometry (EIMS) was recorded on a MAT 95 XL mass spectrometer, Thermofinnigan. Quantitative determination of active substances was performed using a high performance liqud chromatography (HPLC) system, Agilent series 1100.
Antibacterial susceptibility testing
Disk diffusion method
The experiment was performed using the method of the National Committee for Clinical Laboratory Standards (NCCLS, 2008) with some modification. P. acnes was incubated in Mueller–Hinton agar with 5% blood for 72 h under anaerobic conditions, and adjusted to yield approximately 108 CFU/mL with 0.85% sodium chloride, compared to the turbidity of McFarland No. 0.5. An aliquot of molten Mueller–Hinton agar with 5% blood was used as an agar base. A prepared inoculum was streaked on the surface of the agar base with a cotton stick. A sterile paper disk (diameter 6 mm) was impregnated with 10 μL of plant extract (500 mg/mL) and the disk was placed on the agar. The concentration of each plant extract was 5 mg/disk. Dimethylsulfoxide (DMSO) was used as a negative control while a tetracycline hydrochloride disk (30 μg/disk) was used as a positive control. Plates were then incubated at 37°C for 72 h under anaerobic conditions.
S. aureus and S. epidermidis were incubated in Mueller–Hinton agar for 24 h at 37°C, and adjusted to yield approximately 108 CFU/mL. The procedures were the same as mentioned above except that the plates were incubated at 37°C for 24 h under normal conditions. All disk diffusion tests were performed in three separate experiments and the antibacterial activity is expressed as the mean of the inhibition diameters (mm).
Determination of minimum inhibitory and bactericidal concentration
The minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by microdilution assay (CitationNCCLS, 2008). The cultures were prepared in 24 h and 72 h broth cultures of P. acnes, S. aureus, and S. epidermidis, respectively. The MIC was defined as the lowest concentration of compound to inhibit the growth of microorganisms and the MBC was defined as the lowest concentration of compound to kill the microorganisms.
Bioassay-guided isolation
The ethyl acetate extract of Alpinia galanga rhizome (44.1 g) was fractionated with silica gel vacuum chromatography eluted with hexane, chloroform, ethyl acetate, and methanol, respectively. The collected fractions were pooled with the aid of their thin layer chromatographic chromatograms and subjected to investigation of antibacterial activity against P. acnes. The active fraction (fraction II) was further purified by Sephadex LH-20 gel filtration chromatography eluted with methanol. The active fraction (fraction I) obtained from the Sephadex LH-20 column was further purified by silica gel column chromatography eluted with a mixture of chloroform and hexane (1:1). A colorless oily liquid, AP1 (696 mg), was obtained from the pooled active fraction (fraction III).
Identification of AP1
Colorless oily liquid, IR (neat) cm−1: 1745 (C=O), 1644, 1605 (C=C, Ar), 1200 (C-O); EIMS: m/z 234 [M+], 192 [M – C2H2O]+, 150 [M – 2C2H2O]+; 1H-NMR (500 MHz, in CDCl3) δ ppm: 7.38 (2H, ddd, J = 8.5, 4.6, 2.7 Hz), 7.09 (2H, ddd, J = 8.5, 4.6, 2.7 Hz), 6.27 (1H, d, J = 5.9 Hz), 5.99 (1H, ddd, J = 17.2, 10.4, 5.9 Hz), 5.31 (1H, ddd, J = 17.2, 1.2, 1.2 Hz), 5.26 (1H, ddd, J = 10.4, 1.2, 1.2 Hz), 2.11 (3H, S), 2.30 (3H, S); 13C-NMR (125 MHz, in CDCl3) δ ppm: 169.4, 169.9, 150.4, 136.4, 136.0 (2), 128.4 (2), 121.6, 117.0, 75.5, 21.1, 21.2.
Quantitative determination of 1′-ACA
The authentic 1′-acetoxychavicol acetate (1′-ACA) isolated from A. galanga was used as the standard compound for quantitative determination. The calibration curve of 1′-ACA was established at the concentration range between 0.06 and 20 mg/mL. HPLC analysis was carried out using an Agilent series 1100 system equipped with a photodiode-array detector (PDA) and autosampler. Separation was achieved at 25°C on a 150 mm × 4.6 mm i.d. TSK-gel ODS-80Ts column. The mobile phase consisted of methanol–water (gradient elution from 40%, v/v, methanol to 55%, v/v, methanol in 20 min) and was pumped at a flow rate of 1 mL/min. The injection volume was 10 μL. The quantitative wavelength was set at 226 nm. All the samples were analyzed in triplicate.
Results and discussion
Among 36 plant extracts that were investigated for antibacterial activity, 13 plant extracts including the ethyl acetate extracts of Allium sativum L. (Alliaceae), Alpinia galanga (L.) Willd. (Zingiberaceae), Eupatorium odoratum L. (Asteraceae), Phyllanthus emblica L. (Euphorbiaceae), and Syzygium cumini (L.) Skeels (Myrtaceae) and the methanol extracts of A. galanga, E. odoratum, Gynura pseudochina var. hispida Thwaites (Asteraceae), P. emblica, Psidium guajava L. (Myrtaceae), Punica granatum L. (Punicaceae), Senna siamea (Lam.) Irwin & Barneby (Leguminosae), and S. cumini were capable of inhibiting the growth of P. acnes at the concentration of 5 mg/disk. Both ethyl acetate and methanol extracts of A. galanga, P. emblica, and S. cumini exhibited an inhibitory effect on all tested microorganisms, implying that their active components should be either less polar or polar compounds. Only the ethyl acetate extracts of A. sativum and E. odoratum exhibited an inhibitory effect on all tested microorganisms, implying that their active components should be less polar compounds, while only the methanol extract of P. guajava exhibited an inhibitory effect on all tested microorganisms, implying that its active components should be polar compounds. All antibacterial active extracts were subsequently subjected to determination of MIC and MBC values.
Determination of the MIC and MBC values of the herbal extracts demonstrated that the ethyl acetate extract of A. galanga showed the strongest antibacterial activity against P. acnes, with MIC and MBC values of 156 and 312 µg/mL, respectively ( and ). Although the ethyl acetate extract of A. sativum showed interesting inhibitory activities on S. aureus and S. epidermidis with MIC values of 78 and 19.5 µg/mL, the MIC and MBC values against P. acnes were higher than those of A. galaga. In the case of the methanol extract, only P. emblica exhibited interesting inhibitory activities on all tested microorganisms, with MIC and MBC values of 312 µg/mL for S. aureus and S. epidermidis and MIC and MBC values of 312 and 625 µg/mL, respectively, for P. acnes ( and ).
Table 3. Minimum inhibitory concentration of the herbal extracts.
Table 4. Minimum bactericidal concentration of the herbal extracts.
In this study, the ethyl acetate extract of A. galanga was selected and subjected to isolation of the antibacterial active compound using bioassay-guided purification. Isolation of the ethyl acetate extract using silica gel vacuum chromatography gave 10 fractions of the isolate. Investigation of the antibacterial activity against P. acnes of each fraction showed that fraction II exhibited antibacterial activity stronger than any other of the fractions ().
Table 5. Antibacterial activity of the pooled fractions isolated by silica gel vacuum column against P. acnes.
Further purification of fraction II by Sephadex LH-20 gel filtration column gave two pooled fractions. Pooled fraction I showed an inhibitory effect with inhibition zone of 34 mm. Thus, fraction I was subjected to further purification using silica gel column chromatography to produce five pooled fractions. A colorless oily liquid (AP1) was obtained from fraction III, which gave the highest inhibitory effect with an MIC value of 78 µg/mL (). On the basis of the spectroscopic data, AP1 was identified as a phenylpropanoid compound named 1′-acetoxychavicol acetate (1′-ACA) ().
Table 6. Antibacterial activity of the pooled fractions isolated by silica gel column against P. acnes.
Figure 1. Structure of 1′-acetoxychavicol acetate.
It has been reported that 1′-ACA possesses various biological activities, such as antitumor (CitationItokawa et al., 1987; CitationKondo et al., 1993; CitationMoffatt et al., 2000; CitationZheng et al., 2002; CitationIto et al, 2005; CitationAzuma et al., 2006; CitationCampbell et al., 2007), anti-inflammation (CitationNakamura et al., 1998; CitationMorikawa et al., 2005), antifungal (CitationJanssen & Scheffer, 1985), antiviral (CitationYe & Li, 2006), antioxidative (CitationKubota et al., 2001), and xanthine oxidase inhibitory activity (CitationNoro et al., 1988). Although it has been reported that the ethanol extract of A. galanga exhibits antibacterial activity against S. aureus (CitationOonmetta-aree et al., 2006), the antibacterial activity of 1′-ACA has not yet been reported.
Thus, 1′-ACA was subjected to evaluation of antibacterial activity against P. acnes, S. aureus, and S. epidermidis. It was found that 1′-ACA possessed antibacterial activity against P. acnes, S. aureus, and S. epidermidis with MIC values of 62, 250, and 250 µg/mL,respectively, and MBC values of 250, 1000, and 1000 µg/mL, respectively. The results indicated that P. acnes was more sensitive to 1′-ACA than were S. aureus and S. epidermidis. This is the first report on the antibacterial activity of 1′-ACA against anaerobic bacteria. These results indicated that A. galanga extract possessed antibacterial activity against an acne involved microorganism through the active constituent, 1′-ACA. Thus, 1′-ACA could be recommended as an indicative marker for the standardization of A. galanga extract.
The optimal conditions for quantitative analysis of 1′-ACA in A. galanga extract were examined using reverse-phase HPLC. Baseline separation of 1′-ACA was achieved within 30 min. The retention time of 1′-ACA was 26 min (data not shown). The identity of the 1′-ACA peak was confirmed by comparison of its absorption spectrum produced by photodiode-array detector with the authentic compound. The linearity of the HPLC method was evaluated using standard samples at six concentrations between 0.6 and 20.0 µg/mL. It exhibited good linearity over the evaluated range with correlation coefficient 0.9994. On the basis of the HPLC analysis, the ethyl acetate extract of A. galanga was composed of 1′-ACA 76.1 ± 0.62%, w/w.
The results from this study indicate that A. galanga extract might be an antibacterial agent for an alternative treatment of acne, and 1′-ACA is recommended as the standard marker for standardization of the extract.
Declaration of interest
The authors wish to thank the Graduate School, Prince of Songkla University, for support in the form of a research grant. The authors alone are responsible for the content and writing of the paper.
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