Antibacterial Activity of Phytochemicals Isolated from Atractylodes japonica against Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) has been emerging worldwide as one of the most important problems in communities and hospitals. Therefore, new agents are needed to treat acute oral infections from MRSA. In this study, antibacterial compounds from the roots of Atractylodes japonica (A. japonica) were isolated and characterized. The compounds were isolated from the root extracts using HPLC-piloted activity-guided fractionations. Four A. japonica compounds were isolated and identified as atractylenolide III (1), atractylenolide I (2), diacetylatractylodiol [(6E,12E)-tetradeca-6,12-diene-8,10-diyne-1,3-diol diacetate, TDEYA, 3). and (6E,12E)-tetradecadiene-8,10-diyne-1,3-diol (TDEA, 4), which was obtained by hydrolysis of TDEYA. The minimum inhibitory concentrations (MICs) was determined in the setting of clinical MRSA isolates. Compound 4 showed anti-MRSA activity with a MIC value of 4-32 μg/mL. The overall results provide promising baseline information for the potential use of the extract of A. japonica as well as some of the isolated compounds in the treatment of bacterial infections.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) accounts for a large proportion of hospitalacquired infections and is considered a serious problem because of its multi-drug resistant properties. Currently, vancomycin and its analog teicoplanin are the most effective antibiotics for MRSA infections, but their clinical use often results in unexpected side effects and the development of vancomycin-resistant S. aureus infections. The search for better drugs to combat MRSA infection is urgently needed.
Atractylodes japonica (Compositae) has traditionally been used for the treatment of water retention in the body. Administration of the aqueous extract of Atractylodes japonica to humans causes diuresis and its alcohol extract also shows a diuretic effect in mice [1,2]. Atractylodes japonica is known to be effective for the control of pain and treatment of arthritis. It was reported that the Atractylodes family possesses anti-inflammatory activity [3], and modulates the intestinal immune system [4]. Atractylon, a major component, and its derivatives isolated from the rhizome were shown to have antihepatic properties [5]. The sequiterpenoid diacetyl atractylodiol and its derivatives were isolated from the nonpolar fraction [2]. Recently, it was reported that A. japonica root extract protects osteoblastic MC3T3-E1 cells against hydrogen peroxide-induced inhibition of osteoblastic differentiation [6]. However, the antibacterial effects of A. japonica on MRSA has not been evaluated. In the course of our ongoing project on the detection of bioactive compound from medicinal plants, the CHCl 3 -soluble extract of roots of A. japonica was found to exhibit distinctive antibacterial activity.

Results and Discussion
Today, with the emergence of antibiotic-resistant pathogens like MRSA, a new approach to natural products must be taken. These natural products are more and more in demand due to their benefits without side effects. Therefore, our ongoing efforts to find bioactive natural products have led us to study the antibacterial activity of A. japonica, which has been known to possess a variety of pharmacological properties against arthritis, bronchitis and respiratory infectious disease and to contain more than 50 phytochemicals, including atractylon and its derivatives [7,8], sesquiterpenoids [9] and diacetyl atractylodiol including its derivatives [10,11]. To elucidate the antibacterial effects of A. japonica, the methanol extracts of A. japonica rhizome were fractionized with n-hexane, CHCl 3 , EtOAc, and n-BuOH. The fractions were tested for MIC determination using the microdilution broth method. The results were recorded as MIC values in Table 1. The CHCl 3 fraction of A. japonica roots showed good antibacterial effects against two strains of S. aureus. The MIC of CHCl 3 fraction shows equal efficacy to ampicillin at 32 μg/mL against ATCC 33591. This is an encouraging result in regards to the ability of MRSA to be resistant to most antibiotics. By further purifying the fractions, compounds 1, 2 and 3 were isolated from the CHCl 3 fraction and were confirmed to be atractylenolide III (1), atractylenolide I (2), and (6E,12E)-tetradeca-6,12-diene-8,10-diyne-1,3-diol diacetate (diacetylatractylodiol, TDEYA, 3), respectively, by the comparison of their spectral data with those in the references [12]. Also, (6E,12E)-tetradecadiene-8,10-diyne-1,3-diol (TDEA, 4) [11] was obtained by hydrolysis of TDEYA (3) (Figure 1). The isolated compounds after were tested against different strains of S. aureus as shown in Table 2. Among the obtained isolates, TDEA (4) showed antibacterial activity with MICs ranging from 4 to 32 μg/mL independently. Additionally, among the five different fractions, the chloroform fraction of A. japonica demonstrated a higher inhibitory activity against MRSA due to its bioactive constituents, in agreement with our Smart HPLC study. In conclusion, the isolated compounds are expected to be useful in the future for the study of anti-MRSA agents. However, for medicinal purposes, the safety and toxicity of these compounds need to be addressed. Also, further study is needed using vancomycin as a positive control for MRSA.

Hydrolysis of TDEYA(3)
A solution of 3 (20 mg) in 3% K 2 CO 3 (cat.)/MeOH (7 mL) was heated at 60 ºC for 10 min. After cooling, the solution was neutralized with 1 N HCl, and MeOH was extracted with Et 2 O and then washed with deionized water. The organic layer was dried over anhydrous Na 2 SO 4 and evaporated in vacuo to obtain compound 4 as a pale yellow amorphous powder (8 mg).

Chromatographic fingerprint conditions
A LC800 series (Smart LC, GL sciences, Tokyo, Japan) system equipped with binary solvent delivery pump, auto sampler, degasser, system oven and UV-visible detector was used to achieve Smart LC fingerprints. The chromatographic separation was carried out on a 2.1 mm × 50 mm, 2 μm particle, Innertsil ODS-4 C18 column (GL sciences, Japan) maintained at 40 ºC. The mobile phase consisting a mixture 0.05% aqueous phosphoric acid and acetonitrile in the ratio of 34:66 (v/v) with flow rate of 0.5 mL/min was employed. The detector wavelength was monitored at 254 nm. All injection volumes of sample and standards were 1.5 μL.

Antibiotics
All antibiotics, including ampicillin was purchased from Sigma Chemical Co. (St Louis, MO, USA).

Detection of mecA gene
Detection of the mecA gene in the MRSA strains was performed by polymerase chain reaction (PCR) amplication. For rapid extraction, one to five bacterial colonies were suspended in 300 L of cell lysis buffer and heated at 100 ºC for 20 min. After centrifugation at 12,000 rpm for 10 min, 2 L of the supernatant was used for the DNA extraction. PCR reactions were performed using a MRSA Primer Mix Kit (Genotex Co., Korea). The PCR amplification consisted of 30 cycles (94 ºC, 60 seconds; 55 ºC, 60 seconds; 72 ºC, 60 seconds). The primers used in this study were as follows: mecA forward primer, 5′-ATGAGATTAGGCATCGTTTC-3′; reverse primer, 5′-TGGATGACAGTACCTGAGCC-3′ [14]. The final PCR products were separated 0.2% agarose gel.

Minimum Inhibitory Concentration (MIC)
The standard agar dilution method was used to determine the MIC of the antibiotics alone in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines [16]. To determine the MIC of A. japonica extract and isolated compounds, the specimen was initially dissolved in minimal amount of dimethyl sulphoxide followed by water to give a range of concentrations from 0.325% to 5% with a volume of 2 mL. Each samples were diluted to give a final concentrations from 0.0625% to 1% in each agar plate. Cell suspensions (1 × 10 4 colony-forming units/mL) of 12 MRSA isolates and standard strain (ATCC 25923 and ATCC 33591) were inoculated onto agar plates and then incubated at 35 ºC for 18 h. The MICs were defined as the lowest concentration of a test compound that completely inhibited cell growth in visible.