Antibacterial Thymol Derivatives Isolated from Centipeda minima

Abstract

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-β-glucopyranoside (4),  9-hydroxy- thymol (5), 8,10-dihydroxy-9-isobutyryloxythymol (6), and 8-hydroxy-9,10-diisobutyryl- oxythymol (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-hydroxy- thymol (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.

Figure 1. Structures of compounds 1-7.

Figure 1. Structures of compounds 1-7.

Results and Discussion

Compound 1 was obtained as a colorless oil. Its molecular formula C₁₅H₂₂O₅ was deduced on the basis of HRESIMS showing a peak at m/z 305.1363 [M+Na]⁺ (calcd. C₁₅H₂₂O₅Na, 305.1364). The ¹H-NMR spectrum exhibited a set of signals characteristic of a 1,3,4-trisubstituted phenyl moiety. The ¹³C-NMR data (Table 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 ¹H-¹H COSY interactions of aliphatic protons, suggesting the existence of a CH₃-CH₂-CH-CH₃ 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)-methyl- butyryloxythymol.

The molecular composition of 2 was assigned as C₁₃H₁₈O₄ by HRESIMS (peak at m/z 261.1108 [M+Na]⁺ (calcd. C₁₃H₁₈O₄Na 261.1102), featuring five degrees of unsaturation. The resemblance of ¹³C-NMR spectra between 2 and 3 suggest they are analogues. However, two additional methyl signals (δ_C 25.9, 27.2) and one sp³ quaternary carbon (δ_C 110.6) were observed in the ¹³C-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-dioxy- isopropylidenethymol.

The five known monoterpenoids were identified as 8,9,10-trihydroxythymol (3) [12], thymol-β-glucopyranoside (4) [13], 9-hydroxythymol (5) [14], 8,10-dihydroxy-9-isobutyryloxy- thymol (6) [15] and 8-hydroxy-9,10-diisobutyryloxythymol (7) [15], respectively, by comparison of their spectroscopic data (¹H-, ¹³C-NMR and MS) with those reported in the literature. Thymol derivatives have been isolated from other Compositae species [16,17,18,19,20], with compounds 3-7 being thymol derivatives were isolated from C. minima for the first time.

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.

Figure 2. HPLC check of compound 2 in the plant ^a

Figure 2. HPLC check of compound 2 in the plant ^a

^a HPLC conditions: column: LiChrospher 100 RP-18e (125 x 4 mm, 5 μm), mobile phase: 22% acetonitrile aqueous, flow rate: l mL/min, detection: 220 nm; A peak at t_R14.8 min of the chromatogram of the crude extracts bears the same UV/DAD absorption and retention time as the reference compound; The proportion of peeks at 13.0 and 14.8 min is reversal when mixing reference with crude extracts.

Experimental

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. ¹H-NMR (400 MHz) and ¹³C-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 GF₂₅₄ 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% H₂SO4 in ethanol or anisaldehyde reagent followed by heating.

Plant Material

The 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.

Extraction and Isolation

Dried and powdered plant materials of C. minima (10 kg) were extracted three times with 95% EtOH under reflux and the combined solvent was evaporated in vacuo. The extracts was suspended in water and successively partitioned with petroleum ether, EtOAc and n-BuOH, respectively. The EtOAc extracts (170 g) were subjected to column chromatography (CC) over silica gel (200-300 mesh) and eluted with CHCl₂-MeOH (8:1) to give fractions 1-4. Fraction 3 (70 g) was submitted to CC on silica gel, eluting with CHCl₃-MeOH (11:1) to give fractions 3.1-3.4. Fraction 3.3 (13 g) was chromatographed on MCI gel CHP 20P eluted with MeOH-H₂O (9:1) followed by gel filtration on Sephadex LH-20 (MeOH) and repeated vacuo liquid chromatography (VLC) to yield 3 (22 mg) and 4 (52 mg). Fraction 2 (30 g) was chromatographed on Diaion HP20 (95% EtOH) to decolor, the eluents were then subjected to CC on silica gel, eluting with CHCl₃-MeOH (1:0-5:1) to give fractions 2.1-2.8. Fraction 2.2 (10 g) was passed through MCI gel CHP 20P eluted with MeOH-H₂O (9:1) to decolor, and then subjected to Sephadex LH-20 (MeOH), RP-18 (MeOH-H₂O, 50:50-100:0) and repeated VLC to yield 1 (44 mg), 2 (13 mg), 5 (28 mg), 6 (66 mg), and 7 ( 90 mg).

8,10-Dihydroxy-9(2)-methylbutyryloxythymol (1). Colorless oil; [α]_D^20.8 +13.46^o (c 0.52, CHCl₃); UV (CHCl₃) λ_max (log ε) nm : 278 (3.34), 238(3.08); IR (KBr) ν_max: 3417, 1717, 1578, 1462 cm^-1. ESIMS m/z: 305 [M+Na]⁺; HRESIMS m/z: 305.1363 [M+Na]⁺ (calcd. C₁₅H₂₂O₅Na 305.1364, error = -0.635 ppm); ¹H- and ¹³C-NMR data, see Table 1.

10-Hydroxy-8,9-dioxyisopropylidene-thymol (2). Colorless crystal; mp 156-157^oC; [α]_D^20.5 +9.36^o (c 0.29, MeOH); UV (MeOH) λ_max (log ε) nm: 282 (3.32), 275(3.33), 219 (3.87), 203 (4.36); IR (KBr) ν_max: 3405, 1617, 1588, 1458 cm^-1; ESIMS m/z: 261 [M+Na]⁺; HRESIMS m/z: 261.1108 [M+Na]⁺ (calcd for C₁₃H₁₈O₄Na 261.1102, error = 1.9953 ppm); ¹H- and ¹³C-NMR data, see Table 1.

Table 1. ¹H- and ¹³C-NMR spectra data for compounds 1 and 2^a.

position	1		2
	δH (J=Hz)	δC	δH (J=Hz)	δC
1		139.9		139.5
2	6.72 br. s	118.5	6.57 br. s	117.1
3		156.4		154.6
4		119.6		127.3
5	6.94 d (7.9)	126.3	7.32 d (7.8)	128.5
6	6.67 br. d (7.9)	120.5	6.62 br. d (7.8)	120.9
7	2.29 s	20.9	2.23 s	21.1
8		78.9		86.5
9a9b	4.54 d (12.0)4.46 d (12.0)	67.3	4.40 d (9.0)4.16 d (9.0)	72.3
10a10b	3.90 d (12.0)3.81 d (12.0)	65.9	3.73 br. d (11.5)3.60 br. d (11.5)	67.3
1’		177.9		110.6
2’	2.40 m	41.0	1.27 s	25.9
3’a3’b	1.62 m1.51 m	26.6	1.52 s	27.2
4’	0.84 m	11.4
5’	1.11 d (7.7)	16.4

^a The spectra were recorded in CDCl₃ (400 MHz for ¹H, 100 MHz for ¹³C).

Table 1. ¹H- and ¹³C-NMR spectra data for compounds 1 and 2^a.

position	1		2
	δH (J=Hz)	δC	δH (J=Hz)	δC
1		139.9		139.5
2	6.72 br. s	118.5	6.57 br. s	117.1
3		156.4		154.6
4		119.6		127.3
5	6.94 d (7.9)	126.3	7.32 d (7.8)	128.5
6	6.67 br. d (7.9)	120.5	6.62 br. d (7.8)	120.9
7	2.29 s	20.9	2.23 s	21.1
8		78.9		86.5
9a9b	4.54 d (12.0)4.46 d (12.0)	67.3	4.40 d (9.0)4.16 d (9.0)	72.3
10a10b	3.90 d (12.0)3.81 d (12.0)	65.9	3.73 br. d (11.5)3.60 br. d (11.5)	67.3
1’		177.9		110.6
2’	2.40 m	41.0	1.27 s	25.9
3’a3’b	1.62 m1.51 m	26.6	1.52 s	27.2
4’	0.84 m	11.4
5’	1.11 d (7.7)	16.4

^a The spectra were recorded in CDCl₃ (400 MHz for ¹H, 100 MHz for ¹³C).

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.

Table 2. Antibacterial activity of compounds 1-7 (MIC ^a values, μg/mL).

Pathogen	1	2	3	4	5	6	7	standards b
Reference strains
Staphylococcus aureus CMCC26001	>100	12.5	>100	>100	>100	100	6.25	15	7.5
Escherichia coli CMCC44103	>100	>100	>100	>100	>100	>100	>100	7.5	7.5
Salmonella typhimurium CMCC80087	>100	>100	6.25	25	50	25	>100	7.5	7.5
Shigella flexneri CMCC51335	>100	12.5	>100	>100	>100	>100	6.25	3.25	3.25
Clinically isolated strains
Staphylococcus epidermidis	>100	25	>100	50	>100	100	50	3.25	7.5
Bacillus subtilis	>100	6.25	>100	>100	>100	50	>100	3.25	3.25
Salmonella paratyphi-A	>100	>100	>100	>100	>100	>100	>100	3.25	3.25
Salmonella paratyphi-B	>100	>100	>100	>100	>100	>100	6.25	3.25	3.25

^a Minimum inhibition concentration; ^b Left column is for cefradine and the right is for gentamycin.

Table 2. Antibacterial activity of compounds 1-7 (MIC ^a values, μg/mL).

Pathogen	1	2	3	4	5	6	7	standards b
Reference strains
Staphylococcus aureus CMCC26001	>100	12.5	>100	>100	>100	100	6.25	15	7.5
Escherichia coli CMCC44103	>100	>100	>100	>100	>100	>100	>100	7.5	7.5
Salmonella typhimurium CMCC80087	>100	>100	6.25	25	50	25	>100	7.5	7.5
Shigella flexneri CMCC51335	>100	12.5	>100	>100	>100	>100	6.25	3.25	3.25
Clinically isolated strains
Staphylococcus epidermidis	>100	25	>100	50	>100	100	50	3.25	7.5
Bacillus subtilis	>100	6.25	>100	>100	>100	50	>100	3.25	3.25
Salmonella paratyphi-A	>100	>100	>100	>100	>100	>100	>100	3.25	3.25
Salmonella paratyphi-B	>100	>100	>100	>100	>100	>100	6.25	3.25	3.25

^a Minimum inhibition concentration; ^b Left column is for cefradine and the right is for gentamycin.