Antibacterial Thymol Derivatives Isolated from Centipeda minima

Two new monoterpenoids, 8,10-dihydroxy-9(2)-methylbutyryloxythymol (1) and 10-hydroxy-8,9-dioxyisopropylidene-thymol (2), together with five known thymol derivatives: 8,9,10-trihydroxythymol (3), thymol-beta-glucopyranoside (4), 9-hydroxythymol (5), 8,10-dihydroxy-9-isobutyryloxythymol (6), and 8-hydroxy-9,10-diisobutyryloxythymol (7), were isolated from Centipeda minima. Their structures were identified by means of spectroscopic analyses. Interestingly, compound 2 is not an extraction artifact according to a close HPLC examination of material after extraction by analytical MeOH at ambient temperature. The antibacterial activities of compounds 1-7 were evaluated against eight microbial strains by the agar dilution method.


Introduction
The loss of quality and safety of food largely results from microorganisms.The use of antimicrobials and antiseptics is a common alternative to control bacteria in food [1].However, the widespread use of antibiotics in human medicine and agriculture has caused serious problem of bacterial resistance [2].Therefore, plant derived antimicrobial agents with high potency and low mammalian toxicity, useful for food preservation and human health, have gained special interest in recent decades [3][4][5].China is a country well known for its utilization of traditional medicine; for centuries, indigenous people have been using herbal medicines to treat various diseases, including a wide range of infectious ones.Interestingly, there is not much documented data on microbial resistance.This indicates that traditional Chinese medicine is a potential source for discovering promising antibacterial substances.Centipeda minima (L.) (Compositae) is found spread throughout China, Japan, Korea, India, Malaysia, and Oceania [6], and is a commonly used Chinese folk medicine for colds, nasal allergy, diarrhea, malaria and asthma [7].Previous studies on C. minima revealed that its flavonoids, sesquiterpene lactones and amides could block histamine release, and therefore contribute to its anti-allergy effects [8,9].Sesquiterpenoids were also found to have antagonistic activities for platelet activating factor and antibacterial activities [10,11].During our search for new antimicrobial components from traditional Chinese medicine, two new monoterpenoids, 8,10-dihydroxy-9(2)-methylbutyryloxythymol (1), 10-hydroxy-8,9-dioxyisopropylidenethymol (2), along with five known thymol derivatives: 8,9,10-trihydroxythymol (3), thymol-β-glucopyranoside (4), 9-hydroxythymol (5), 8,10-dihydroxy-9-isobutyryloxythymol (6), and 8-hydroxy-9,10-diisobutyryloxythymol (7) were isolated from the whole plants of C. minima (Figure 1).In this paper, we report the isolation and structural identification of the new compounds, and the antibacterial properties of compounds 1-7.

Results and Discussion
Compound  1) were very similar to those of 6, except for two methyls at δ C 11.4 and 16.4, one methylene at δ C 26.6, and one methine at δ C 41.0 present in 1, instead of two symmetrical methyl groups at δ C 19.1, and one methine at δ C 34.4 seen in 6, which in conjunction with the 1 H-1 H COSY interactions of aliphatic protons, suggesting the existence of a CH 3 -CH 2 -CH-CH 3 segment in 1.The HMBC correlations of H-2', H-3', H-5' and H-9 all correlated with a carbonyl carbon at δ C 177.9 established the linkage of C-1'and C-9 (δ C 67.3).The absolute stereochemistry at C-8 and C-2' still remained obscure.Thus, the structure of 1 was determined to be 8,10-dihydroxy-9(2)-methylbutyryloxythymol.
The molecular composition of 2 was assigned as C 13 H 18 O 4 by HRESIMS (peak at m/z 261.1108 [M+Na] + (calcd.C 13 H 18 O 4 Na 261.1102), featuring five degrees of unsaturation.The resemblance of 13 C-NMR spectra between 2 and 3 suggest they are analogues.However, two additional methyl signals (δ C 25.9, 27.2) and one sp 3 quaternary carbon (δ C 110.6) were observed in the 13 C-NMR spectrum of 2, in conjunction with one more unsaturation and the 40 Da larger mw of 2 than 3, disclosing the presence of an isopropyl moiety.This was further confirmed by the observed HMBC interactions of H-9, H-2', and H-3' with C-1'.The cross peaks of H a-9 with H a -10 and H b -10, and H b -9 with H-2' observed in the ROESY spectrum indicated that hydroxymethyl group, H a -9, and H-3' were at the same face of the five-membered ring.The structure of 2 suggests an extraction artifact, however, the unambiguous detection of 2 by a close HPLC analysis in the raw material after extracting with analytical methanol and concentrating at ambient temperature suggested that 2 is indeed a new natural product (Figure 2).Therefore, the structure of 2 was assigned as 10-hydroxy-8,9-dioxyisopropylidenethymol.
Antibacterial tests revealed that all the agents tested exhibited antibacterial effects against all the bacteria investigated (Table 2).At the MIC of 6.25 μg/mL, compound 2 was found to be most effective against B. subtilis, compound 3 against S. typhimurium, and compound 7 against S. aureus, S. flexneri, and S. paratyphi-B.The MIC value of 2 against S. aureus is 12.5 μg/mL comparable with that of cefradine.All the tested bacteria were less sensitive to compound 1 with the MIC larger than 100 μg/mL.Thymol, as a component of volatile oil in many plants, has been proved to possess antimicrobial activities [21], the antibacterial activities of compounds 2-7 were probably resulted from their structural similarities with thymol.The results implied that C. minima could be a potential source for searching natural antibacterial substances applied for food preservation.

General
Melting points were obtained on an XRC-1 micromelting apparatus.Optical rotations were determined on a JASCO-20C digital polarimeter.UV spectra were recorded on a Shimadzu UV-2401PC spectrophotometer.IR spectra were obtained with a Bruker Tensor 27 FT-IR spectrophotometer with KBr pellets. 1 H-NMR (400 MHz) and 13 C-NMR (100 MHz) spectra were recorded on a Bruker AM-400 spectrometer with TMS as an internal reference.2D NMR spectra were measured with a DRX-500 spectrometer.EIMS (70 eV) were recorded on a VG Auto Spec-3000 spectrometer.ESIMS and HRESIMS were carried our with an API QSTAR Pulsar 1 spectrometer.Silica gel (200-300 mesh and 10-40 μm) for column chromatography and GF 254 for TLC were obtained from Qingdao Marine Chemical Factory, Qingdao, People's Republic of China.Sephadex LH-20 was obtained from Amersham Pharmacia Biotech, Sweden.RP-18 silica gel (40-63 μm) used for open column chromatography was purchased from Daiso Co., Japan.Diaion HP20 and MCI gel CHP 20P (75-150 μm) were obtained from Mitsubishikasei, Tokyo, Japan.Fractions were monitored by TLC and spots were visualized after spraying with 10% H 2 SO4 in ethanol or anisaldehyde reagent followed by heating.

Plant Material
The

Antibacterial Assay
Antibacterial properties were tested by agar dilution method [22].The bacterial strains employed were Staphylococcus aureus CMCC26001 (CMCC, National Center for Medical Culture Collections, Beijing, China), Escherichia coli CMCC44103, Salmonella typhimurium CMCC80087, and Shigella flexneri CMCC51335, and the following clinically isolated strains: Staphylococcus epidermidis, Bacillus subtilis, Salmonella paratyphi-A, Salmonella paratyphi-B.Cefradine and gentamycin were used as reference standards, plates containing only MHA medium and 1% DMSO in MHA medium served as negative and solvent controls.The tests were performed in triplicate and repeated once.

Figure 2 .
Figure 2. HPLC check of compound 2 in the plant a 1 was obtained as a colorless oil.Its molecular formula C 15 H 22 O 5 was deduced on the basis of HRESIMS showing a peak at m/z 305.1363 [M+Na] + (calcd.C 15 H 22 O 5 Na, 305.1364).
whole plants of C. minima were purchased from Yunnan Corporation of Materia Medica, Yunnan Province, P. R. China, and identified by Mr. H.Y. Sun at Yunnan Corporation of Materia Medica.A voucher specimen (No.CHYX0159) was deposited at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, P. R. China.