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Molecules 2012, 17(11), 12469-12477; doi:10.3390/molecules171112469
Published: 24 October 2012
Abstract: The crude methanol extract of the dried aerial parts of Siegesbeckia glabrescens (Compositae) showed antibacterial activity against the foodborne pathogen Staphylococcus aureus. Bioactivity-guided separation led to the isolation of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid from nature for the first time.The structure was determined by spectroscopic data analysis (UV, MS, and NMR). The minimal inhibitory concentration (MIC) of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid against S. aureus was found to be 3.12 μg/mL. In addition, in a further antimicrobial activity assay against Gram-positive (B. subtilis, E. faecalis, P. acnes, S. epidermidis, S. schleiferi subsp. coagulans, S. agalactiae and S. pyrogens), and Gram-negative bacteria (E. coli and P. aeruginosa), and yeast strains (C. alibicans and F. neoformans), the antimicrobial activity of the compound was found to be specific for Gram-positive bacteria. The MIC values of the compound for Gram-positive bacteria ranged from 3.12 to 25 μg/mL. Furthermore, it was found that the 2-(isobutyryloxy)-4-methylpentanoic acid substituent may operate as a key factor in the antibacterial activity of the compound, together with the laurate group.
Antimicrobial resistance among Gram-positive bacteria has become increasingly prevalent and has resulted in serious infections worldwide over the past two decades [1,2]. Most nosocomial and community-acquired infections are caused by Staphylococcus aureus [3,4,5,6]. Herbs and spices with antibacterial activity have been widely used both traditionally and commercially to increase the shelf-life and safety of foods . With the recent increase in consumer mistrust of synthetic additives, there has been a concomitant increase in the search for new natural compounds from plants that can be used to replace existing synthetic antimicrobials .
Siegesbeckia glabrescens, well-known as “Hi-Chum” in Korea, is an annual herb that grows in Korea. The aerial portion and roots of S. glabrescens have been used as a traditional medicine to treat rheumatic arthritis, asthma, paralysis and allergic disorders. Modern pharmacological experiments showed that the extracts of S. glabrescens exhibit antioxidative, antiallergic, antihypertension, antitumor, and anti-inflammatory activities . However, the antibacterial activity of these extracts has not yet been evaluated. Therefore, we investigated the antibacterial effects of the S. glabrescens extract and characterized new bioactive compounds from S. glabrescens. Herein, we report the isolation, structural identification and antibacterial activity of a new compound, 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid, from S. glabrescens.
2. Results and Discussion
2.1. Isolation of Active Compound from S. glabrescens
The aerial parts of S. glabrescens was extracted with 80% MeOH, and fractionated successively with n-hexane, CHCl3, and EtOAc. The EtOAc extract exhibited high antibacterial activity against S. aureus in an agar disk diffusion assay. Separation of the active compound was performed by a series of silica gel and Sephadex LH-20 column chromatography steps, and then the compound was further purified by preparative and semi-preparative reversed-phase HPLC. The purified active compound (15 mg) was thus obtained from S. glabrescens.
2.2. Structure Determination of Isolated Compound
The chemical structure of the active compound was studied on the basis of MS, 1H-, and 13C-NMR spectroscopic data, including HMQC, HMBC, and 1H-1H COSY experiments. The 1H-NMR spectrum of the active compound contained a triplet methyl protons signal at δH 0.89 and proton signals of a linear carbon chain at δH 1.29 (16H, br), δH 1.62 (2H, m) and δH 2.33 (2H, t) in a pattern typical of a laurate unit. Two methyl proton signals and one proton signal appeared at δH 1.19 (6H, dd, J = 7.0) and δH 2.63 (1H, m) due to an isobutyryloxy moiety, as well as two methyl proton signals and three proton signals at δH 0.93 (3H, d, J = 6.5), δH 1.01 (3H, d, J = 6.5), δH 2.16 (1H, m), δH 5.11 (1H, dd, J = 7.5, 7.0), and δH 5.20 (1H, d, J = 4.5) due to a typical methylpentanoic acid, respectively. The 13C-NMR spectrum revealed 22 carbons, including three ester carbonyl (δC 169.8, 173.5 and 176.3), two oxygenated methines (δC 72.0 and 76.5), five methyls [δC 13.3, 17.3, 2 (18.0) and 18.4], ten methylenes [δC 22.6, 24.9, 29.0, 29.2, 29.3, 29.4, 2 (29.6), 31.9 and 33.9] and two methines (δC 29.0 and 33.9). These facts were consistent with a molecular formula of C22H40O6, which was supported by HR-EIMS data (m/z 400.2820, [M]+) and ESIMS (m/z 423.8, [M+Na]+). The 1H-1H COSY spectrum of active compound showed proton correlations of δH 5.20 (H-2) with δH 5.11 (H-3); δH 5.11 (H-3) with δH 5.20 (H-2) and δH 2.16 (H-4); δH 2.16 (H-4) with δH 5.11 (H-3), δH 0.93 (H-5) and δH 1.01 (H-6); δH 0.93 (H-5) with δH 2.16 (H-4); δH 1.01 (H-6) with δH 2.16 (H-4); δH 2.63 (H-8) with δH 1.19 (H-9 and H-10). The structure of the active compound was further established according to its HMBC spectrum, in which 1H-13C long-range correlation signals were between δH 5.20 (H-2) and δC 169.8 (C-1), δC 76.5 (C-3), δC176.3 (C-7); δH 5.11 (H-3) and δC 173.5 (C-1'), δC 169.8 (C-1), δC 72.0 (C-2), δC 29.0 (C-4), δC 17.3 (C-5), δC 18.4 (C-6); δH 2.16 (H-4) and δC 72.0 (C-2), δC 76.5 (C-3), δC 17.3 (C-5), δC 18.4 (C-6); δH 0.93 (H-5) and δC 76.5 (C-3), δC 29.0 (C-4), δC 18.4 (C-6); δH 1.01 (H-6) and δC 76.5 (C-3), δC 29.0 (C-4), δC 17.3 (C-5); δH 2.63 (H-8) and δC 176.3 (C-7), δC 18.0 (C-9 and C-10) (Table 1).
|Table 1. 1H- and 13C-NMR spectral data of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid from S. glabrescens.|
|Position||13C (δ)||DEPT||1H (δ) a (multiplicity, J)||1H-1H COSY||HMBC b (1H→13C)|
|2'||33.9||CH2||2.33 (t, 7.0, 7.5)||H-3'||C-1', C-3', C-4'|
|3'||24.9||CH2||1.62 (m)||H-2', H-4'||C-1', C-2', C-4'|
|8', 9'||29.6||2(CH2)||1.29 (br)|
|12'||13.3||CH3||0.90 (t, 7.0)||H-11'||C-11', C-10'|
|2||72.0||CH||5.20 (d, 4.5)||H-3||C-1, C-7, C-3|
|3||76.5||CH||5.11 (dd, 7.5, 7.0)||H-2, H-4||C-1', C-1, C-2, C-4, C-5, C-6|
|4||29.0||CH||2.16 (m)||H-3, H-5, H-6||C-3, C-2, C-5, C-6|
|5||17.3||CH3||0.93 (d, 6.5)||H-4||C-3, C-4, C-6|
|6||18.4||CH3||1.01 (d, 6.5)||H-4||C-3, C-4, C-5|
|8||33.9||CH||2.63 (m)||H-9, H-10||C-7, C-9, C-10|
|9, 10||18.0||2(CH3)||1.19 (dd, 7.0)||H-10, H-8||C-7, C-8|
a: 1H directly attached to 13C determined from HMQC experiment; b: 1H-13C long-range correlation (HMBC) corresponding to two- or three-bond connectivities.
From all the above information, the structure of the active compound was determined to be 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid (Figure 1), isolated from nature for the first time.
2.3. Antimicrobial Activity of Isolated Compound
Long-chain fatty acids are known as surface-active anionic detergents . In general, fatty acid sensitivity is considered to be a characteristic of Gram-positive bacteria, with few Gram-negative species being susceptible . In addition, lauric acid, which is the most effective among the saturated fatty acids, has been reported to show the antimicrobial activity against six strains of S. aureus . In regards to the chemical structure, 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid has both the structural properties described above, as it contains a saturated long laurate chain. These structural properties suggest that the compound should display antibacterial activity against Gram-positive bacteria.
The disk diffusion method was used to investigate the correlation between structure and antibacterial activity of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid and lauric acid. As is shown in Table 2, while 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid produced an inhibition zone diameter of 11 mm against S. aureus at 6.25 μg/mL, no antibacterial activity was observed at the same concentration of lauric acid. An amount of 50 μg/mL of lauric acid showed a similar antibacterial activity as 6.25 μg/mL of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid (Table 2), indicating that the efficacy of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid against S. aureus was approximately 8-fold stronger than that of lauric acid. These results suggest that laurate and 2-(isobutyryloxy)-4-methylpentanoic acid may operate as key factors in the antibacterial activities against S. aureus and synergistically enhance the antibacterial activity. We also observed the influence of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid on the growth of S. aureus at 1.562 μg/mL (1/2 MIC), 3.125 μg/mL (MIC), and 6.25 (2 MIC) μg/mL. As shown in Figure 2, 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid inhibited the growth of S. aureus at 3.25 μg/mL.
|Table 2. Antibacterial activity of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid and lauric acid against S. aureus measured using the disk diffusion method.|
|Compounds||Diameter of clear zone (mm)|
|Lauric acid||- b||-||-||11 c|
a: Amount of compounds (μg/mL); b: No antibacterial activity; c: Diameter of clear zone (mm), and diameter of filter disk is 10 mm.
Also, we further examined the antibacterial activities of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid against Gram-positive bacteria (S. aureus, B. subtilis, E. faecalis, P. acnes, S. epidermidis, S. schleiferi subsp . coagulans, S. agalactiae and S. pyrogens), Gram-negative bacteria (E. coli and P. aeruginosa) and yeast strains (C. alibicans and F. neoformans) by measuring the MIC, which is the lowest concentration yielding no growth. As shown in Table 3, the MIC values of the compound for the Gram-positive bacteria were between 3.12 and 25.00 μg/mL. Specifically, while the compound showed the strongest activity against S. aureus, the antibacterial activity against E. faecalis, P. acnes and S. epidermidis was relatively low. In addition, 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid did not show any antibacterial activity against Gram-negative bacteria and yeast (Table 3). These results suggest that the antibacterial activity of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid is specific against Gram-positive bacteria.
|Table 3. MIC of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid.|
|Organism||MIC (μg/mL)||Organism||MIC (μg/mL)|
|Gram-positive bacteria||B. subtilis||6.25||S. epidermidis||25.00|
|E. faecalis||25.00||S. schleiferi||12.50|
|P. acnes||25.00||S. agalactiae||6.25|
|S. aureus||3.12||S. pyrogens||6.25|
|Gram-negative bacteria||E. coli||- a||P. aeruginosa||-|
|yeast||C. alibicans||-||F. neoformans||-|
a: No activity.
The HPLC system used was comprised of Waters prep LC 2000 a multi-solvent delivery, and Waters 2487 Dual λ Absorbance detector. UV spectra were obtained on a Dual λ Absorbance detector (Waters 2487) instrument (all Waters, Milford, MA, USA). The HPLC-grade organic solvents and bulk organic solvents were purchased from the Duksan (Ansan, Korea) and J.T. Baker (Phillipsburg, NJ, USA) companies. 1H- and 13C-NMR spectra were obtained on a Bruker Avance-500 spectrometer (Bruker Spectrospin, Rheinstetten, Germany; 500 MHz for 1H- and 125 MHz for 13C-) using methanol-d4 as solvent and tetramethylsilane (TMS) as an internal standard, and the chemical shifts were reported in δ (ppm) units relative to the TMS signal and coupling constants (J) in Hz. A complete attribution was performed on the basis of the 2D-experiment (heteronuclear multiple bond correlation, HMBC). Mass spectrometry (ESI) data were measured on Agilent 1100LC/MSD trap. HRMS data were recorded on a JMS-600W spectrometer (JEOL, Tokyo, Japan).
3.2. Plant Material
The aerial parts of S. glabrescens Makino (Compositae) were purchased from Sam-Hong Pharmaceutical Co., Ltd. in Seoul, Korea.
3.3. Extraction and Isolation
The air dried S. glabrescens (100 g) were cut into small pieces and extracted three times with 80% MeOH (2 L) at room temperature for 7 days, and filtered. The original 80% MeOH in H2O (500 mL × 3, 17.67 g) extract was evaporated to dryness in vacuo, and then suspended in 500 mL of water. The water suspension was partitioned three times with CHCl3 (100 mL). The CHCl3 (5.93 g) extract was evaporated to dryness in vacuo, and was then suspended in 500 mL of 80% MeOH in H2O. The 80% MeOH suspension was partitioned three times with n-hexane (500 mL). The 80% MeOH (4.46 g) extract was evaporated to dryness in vacuo, and was then suspended in 500 mL of water. The water suspension was partitioned three times with EtOAc (500 mL). The EtOAc extract (3.4 g) was evaporated to dryness in vacuo. Since high antibacterial activity was observed for the EtOAc extract, this extract was further investigated in detail. The EtOAc extract was chromatographed on a silica gel column (1:100 ratio of sample:silica gel), under medium pressure and eluted using a CHCl3-MeOH step gradient system with increasing polarity from 0%, to 2%, 4%, 6%, 8%, 10%, and 100% MeOH to give seven fractions (Fractions 1–7). Fraction 2 (1.38 g) was then subjected to Sephadex LH-20 column chromatography eluted with 80% MeOH in CHCl3 (5 mL/15 min) to give 80 fractions (Fractions No.1–80 at once). The Sephadex fractions 66–77 (30 mg) was further purified by preparative reversed-phase HPLC using a gradient from 75%–100% ACN in H2O (Luna, 250 × 21.20 mm; 5 μm particle size; 15 mL/min; UV detection at 210 nm), to afford 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid (15 mg, Rt 60.16 min). Oil; UV λmax (MeOH) 210 nm; 1H-NMR and 13C-NMR (CD3OD); see Table 1; HR-EIMS (EI+) m/z 400.2820, [M]+ (calcd. for C22H40O6, 400.2829); ESIMS (m/z): 423.8, [M+Na]+ (calcd. for C22H40O6Na, 423.3).
3.4. Antibacterial Activity Assay
Gram-positive bacteria [Staphylococcus aureus (ATCC 6538P), Bacillus subtilis (ATCC 15245), Enterococcus faecalis (ATCC 11700), Propionibacterium acnes (ATCC 6919), Staphylococcus epidermidis (ATCC 12228), Staphylococcus schleiferi subsp. coagulans (ATCC 49545), Streptococcus agalactiae (ATCC 14364) and Streptococcus pyrogens (ATCC 19615)], Gram-negative bacteria [Escherichia coli (ATCC 8739) and Pseudomonas aeruginosa (ATCC 27853)] and yeast [Candida alibicans (ATCC 10231) and Filobasidiella neoformans (ATCC 34144)] were used in these experiments. The antibacterial activity was determined using both the agar diffusion and broth dilution techniques as described previously by Cheesbrough  and Gatsing et al. . Agar diffusion susceptibility testing was performed using the disc method. A disc of blotting paper was impregnated with 50 μL of a 60 mg/mL (for crude extract) or 4 mg/mL (for pure compounds) solution of each sample dissolved in DMSO. Thus, the disc potencies were 1 mg and 200 μg for the crude extract and pure compounds, respectively. Erythromycin (Sigma, St. Louis, MO, USA) was used as the standard drug. After drying, the disc was placed on a plate of sensitivity testing agar inoculated with the test organism. Petri dishes were left at room temperature for approximately 45 min to allow the extract or the compounds to diffuse from the disc into the medium, and were then incubated at 37 °C for 24–48 h. The zones showing no growth were then noted and their diameters were recorded as the zones of inhibition.
We pre-cultured the bacterial cells for 24 h at 37 °C in 10 mL broth. Approximately 5 × 105 cfu/mL bacterial cells of the pre-cultured bacteria were inoculated into 3 mL of broth. The samples were then added into approximately 3 mL of broth containing the bacteria and cultured for 24 h at 37 °C. To determine the activity of the samples, we employed a two-fold serial dilution method. The total volume of the mixture was approximately 3 mL, with the test-compound concentrations in the tube ranging from 200 to 0.78 μg/mL and the concentration of standard compound (erythromycin) ranged from 100 to 0.78 μg/mL. After 24 h of incubation at 37 °C, the MIC value was defined as the lowest concentration that inhibited the visible growth of tested microorganism.
It was demonstrated that a new compound, 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid isolated from S. glabrescens has antibacterial activities specifically against Gram-positive bacteria. We found that the antibacterial effect of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid was due to both the 2-(isobutyryloxy)-4-methylpentanoic acid group and laurate group. In addition, the antibacterial activity of 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid was more potent than lauric acid itself. To the best of our knowledge, this is the first study demonstrating the antibacterial activity of S. glabrescens. In addition, we demonstrated that 3-(dodecanoyloxy)-2-(isobutyryloxy)-4-methylpentanoic acid is an antibacterial compound of S. glabrescens. The compound also has a specific antibacterial activity against Gram-positive bacteria and the strongest activity was observed against S.aureus, which are major foodborne pathogenic microorganisms.
Conflict of Interest
The authors declare no conflict of interest.
This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Korea (Grant No.: A103017).
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- Sample Availability: Samples of the title compound are available from the authors.
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