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Antibacterial activity of hydroxyalkenyl salicylic acids from sarcotesta of Ginkgo biloba against vancomycin-resistant Enterococcus
Fitoterapia 80 (2009) 18–20
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Antibacterial activity of hydroxyalkenyl salicylic acids from sarcotesta of Ginkgo biloba against vancomycin-resistant Enterococcus J.G. Choi a, S.I. Jeong b, C.S. Ku c,⁎, M. Sathishkumar d, J.J. Lee e, S.P. Mun f, S.M. Kim g a
National Agricultural Products Quality Management Service, Ministry of Agriculture and Forestry, Jeonju 561-823, South Korea Jeonju Biomaterials Institute, Jeonju 561-360, South Korea c Agriculture Scientific Technique Research, Chonbuk National University, Jeonju 561-756, South Korea d Department of Environmental and Chemical Engineering, Research Institute of Industrial Technology, Chonbuk National University, Jeonju 561-756, South Korea e Faculty of Bioresources Science, College of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, South Korea f Division of Forest Science, College of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, South Korea g Department of Clinical Pathology, Wonkwang Health Science College, Iksan 570-750, South Korea b
a r t i c l e
i n f o
Article history: Received 24 August 2008 Accepted 4 September 2008 Available online 11 September 2008 Keywords: Ginkgo biloba Hydroxyalkenyl salicylic acids Antibacterial activity
a b s t r a c t A chloroform fraction prepared from the sarcotesta of Ginkgo biloba showed potent inhibitory activity against vancomycin-resistant Enterococcus (VRE). The active compounds were elucidated to be 2-hydroxy-6-(8-pentadecenyl) salicylic acid (1) and 2-hydroxy-6-(10heptadecenyl) salicylic acid (2) based on their spectral analysis. Compounds 1 and 2 showed significant antibacterial activities against VRE. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Widespread antimicrobial use continues to cause significant increases in resistant bacteria, particularly resistant Gram-positive organisms. Enterococci may acquire resistance against vancomycin during use of the drug to treat diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). The rapid emergence of vancomycin-resistant Enterococcus (VRE) is believed to be caused by the excessive use of vancomycin. Currently, it has been emerging as one of the most important hospital and community pathogens worldwide. The emergence of these resistant bacteria has caused a major concern and thus the urgent need for new antibacterial agents [1–3]. Plants are known to have defense systems against phytopathogenic bacteria [4]. Medicinal plants that are commonly used may be a good source for safe antibacterial agents [5].
⁎ Corresponding author. Tel.: +82 63 2703566; fax: +82 63 2703571. E-mail address: [email protected] (C.S. Ku). 0367-326X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2008.09.001
Recently, we found the sarcotesta of Ginkgo biloba to have antibacterial activity against VRE. G. biloba is one of the oldest living tree species, dating back over 300 million years. In China, the ginkgo trees have been used for 5000 years to treat lung ailments such as asthma and bronchitis, and also as a remedy for cardiovascular diseases [6]. Recently, clinical studies stimulated interest in a G. biloba extract for improving symptoms of Alzheimer-type dementia [7,8]. Phytochemically, G. biloba contains anacardic acid, bilobol, and cardanol. At present, however, the pharmaceutical arsenal available to control VRE is limited. In the present study, we report new antibacterial hydroxyalkenyl salicylic acids against VRE from the G. biloba extract. 2. Experimental 2.1. Plant The dried sarcotesta of G. biloba was collected in September 2002 from Chonbuk National University Garden, Republic of Korea. This plant was identified and authenticated by Prof. Young-Sung Ju at the College of Oriental Medicine,
J.G. Choi et al. / Fitoterapia 80 (2009) 18–20
Woosuk University. A voucher specimen (No. JSI 61) has been deposited at the Faculty of Bioresources Science, College of Agriculture and Life Science, Chonbuk National University, Jeonju, Republic of Korea. 2.2. Extraction and isolation Air-dried and pulverized sarcotesta (222 kg) of G. biloba was extracted with MeOH for 3 days at room temperature to give an extract (129 g) which was suspended in H2O and partitioned with n-hexane, CHCl3 and EtOAc, successively. The active CHCl3 fraction (23 g) was subjected to silica gel (Merck, Kieselgel 60; 0.063–0.2 mm particle size; 11 × 100 cm) column chromatography. The fraction was eluted with nhexane/EtOAc: 20:80 (10 × 250 ml), followed by MeOH. Fractions of similar composition determined by TLC analysis were pooled. A portion of the active fraction (Fr. 3–4: 500 ml) was subjected to a preparative HPLC [(Phenomenex C18, 10 × 250 mm, particle size 10 μm, CH3CN, 5.0 mL/min, fluorescence detector at excitation (284 nm) and emission wave (316 nm)] to give compounds 1 (78 mg, Rt 8.43 min, purity 96%) and 2 (56 mg, Rt 12.74 min, purity 98%). The structures of the two compounds were determined using spectroscopic techniques including UV, mass spectrometry, 1 H and 13C NMR, DEPT, HMBC and HMQC spectrometry (Fig. 1). 2.3. Preparation of bacterial cells 18 VRE strains were isolated from humans and identified by Shin-Moo Kim in the intensive care unit (ICU) of Wonkwang University Hospital, Iksan, Korea. Enterococcus faecalis CDC-286 (vanA), E. faecalis CDC-583 (vanB), E. gallinarium CDC-45 (vanC) (VRE) and E. faecalis ATCC 29212, the vancomycin susceptible strain (VSE) were used as reference. The confirmation of the identification was done by detection of the vancomycin resistance gene (vre) with PCR analysis. 2.4. Determination of anti-VRE activity The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) were determined using the agar dilution method. All strains were grown in tryptic soy broth for 24 h at 37 °C. After incubation, they were diluted with the same medium to give a concentration of approxi-
Fig. 1. Structures of compounds 1 and 2.
19
Table 1 Antibacterial activities of methanol extract and solvent soluble fractions from the sarcotesta of Ginkgo biloba against Enterococcus faecalis CDC-286 Samples
MIC
MBC
MeOH extract n-Hexane fraction CHCl3 fraction EtOAc fraction
8 16 4 16
16 32 8 32
MIC and MBC, μg/ml.
mately 108 colony forming units (CFU/ml). The isolated compounds were dissolved in dimethyl sulfoxide (DMSO) and two-fold serial dilutions were made before adding the same to tryptic soy agar plates. Bacterial cell suspensions were inoculated onto the plates using a bacteria planter (5 μl). The final inoculum concentration of CFU inoculated onto the agar plates was 5 × 105 for all strains. An agar plate containing only DMSO served as control. 3. Results and discussion To search for a traditionally used medicinal herb with potent antibacterial properties against VRE, we screened many herbs which are prescribed for gasterointestinal infections in South Korea. Among the herbal extracts tested, G. biloba extract was selected due to its higher antibacterial activity. To obtain the active component related to the antibacterial activity, G. biloba was extracted with MeOH and fractionated successively with n-hexane, CHCl3, and EtOAc. Antibacterial activities of these fractions against E. faecalis CDC-286 were tested (Table 1). The CHCl3 fraction showed significant MIC and MBC values in comparison with the other fractions. Therefore, the CHCl3 fraction was employed for
Table 2 Antibacterial activities of compounds 1 and 2 isolated from the sarcotesta of Ginkgo biloba against clinical isolates of 18 VRE, standard VSE and 3 VRE reference strains Strains
E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E.
faecalis CDC-286 (vanA) faecalis CDC-583 (vanB) gallinarium CDC-45 (vanC) faecalis ATCC29212 (VSE) faecium 1 faecium 2 faecium 3 faecium 4 faecium 5 faecium 6 faecium 7 faecium 8 faecium 9 faecium 11 faecalis 1 faecalis 2 faecalis 3 faecalis 4 faecalis 5 faecalis 6 faecalis 7 faecalis 10
MIC and MBC, µg/ml.
1
2
Vancomycin
MIC
MBC
MIC
MBC
MIC
MBC
2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 4 2 4 2 2 2 2
4 4 2 2 2 4 2 2 2 2 2 2 2 4 4 4 2 4 4 2 2 2
4 4 4 2 4 2 2 2 4 2 2 2 4 2 8 4 4 8 4 4 4 8
8 8 4 4 4 4 2 2 4 4 4 4 4 2 8 8 8 8 4 4 4 8
256 32 64 32 N512 N512 N512 N512 512 256 256 128 128 N512 64 256 256 128 256 64 256 64
512 64 64 32 N512 N512 N512 N512 N512 256 256 128 256 N512 128 512 512 128 512 128 256 128
20
J.G. Choi et al. / Fitoterapia 80 (2009) 18–20
further purification. Out of the first 10 fractions obtained by silica gel column chromatography of the CHCl3 fraction, Fractions 3 and 4 showed potent inhibitory activity (MIC/ MBC: 4/8 μg/ml) against E. faecalis CDC-286 (vanA). By further purifying the fractions, compounds 1 and 2 were isolated and were confirmed to be 2-hydroxy-6-(8-pentadecenyl) salicylic acid and 2-hydroxy-6-(10-hentadecenyl) salicylic acid, respectively, by the comparison of their spectral data with those in the references [9,10]. The MIC/MBC values of the isolated compounds against VRE are summarized in Table 2. Each of the clinically isolated strains was significantly resistant to vancomycin and showed MIC/MBC values similar to or higher than 64/128 μg/ml. Compounds 1 and 2 showed very potent inhibitory activity against standard VRE, VSE as well as clinical isolates of VRE, with MIC and MBC values of 2 to 4 and 4 to 8 μg/ml, respectively. These results indicated the possibility of exploitation of hydroxyalkenyl salicylic acids originated from G. biloba as effective inhibitors of VRE. This is the first report on the anti-VRE activities of hydroxyalkenyl salicylic acids in the literature. The isolated
compounds are expected to be useful for the study of anti-VRE agents in the future. However, for medicinal purposes, the safety and toxicity of these salicylic acids need to be addressed. References [1] American Society for Microbiology (ASM). Antimicrob Agents Chemother 1995;39:2. [2] Davis J. Sci 1994;264:375. [3] Spratt BG. Sci 1994;264:388. [4] Smith E, Williamson E, Zloh M, Gibbons S. Phytother Res 2005;19:538. [5] Sato Y, Suzaki S, Nishikawa T, Kihara M, Shibata H, Higuti T. J Ethnopharmacol 2000;72. [6] Perry EK, Pickering AT, Wang WW, Houghton PJ, Perry NS. J Pharm Pharmacol 1999;51:527. [7] Le Bars P, Katz MM, Berman N, Itil TM, Freedman AM, Schatzberg AF, North American EGb Study Group. JAMA 1997;278:1327. [8] Maurer K, Dierks RI, Frölich L. J Psychiatr Res 1997;31:645. [9] He XG, Bernart MW, Nolan GS, Lin LZ, Lindenmaier MP. J Chromatogra Sci 2000;38:169. [10] Itokawa H, Totsuka N, Nakahara K, Takeya K, Lepoittevin JP, Asakawa Y. Chem Pharm Bull 1987;35:3016.
Contents lists available at ScienceDirect
Fitoterapia j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / f i t o t e
Antibacterial activity of hydroxyalkenyl salicylic acids from sarcotesta of Ginkgo biloba against vancomycin-resistant Enterococcus J.G. Choi a, S.I. Jeong b, C.S. Ku c,⁎, M. Sathishkumar d, J.J. Lee e, S.P. Mun f, S.M. Kim g a
National Agricultural Products Quality Management Service, Ministry of Agriculture and Forestry, Jeonju 561-823, South Korea Jeonju Biomaterials Institute, Jeonju 561-360, South Korea c Agriculture Scientific Technique Research, Chonbuk National University, Jeonju 561-756, South Korea d Department of Environmental and Chemical Engineering, Research Institute of Industrial Technology, Chonbuk National University, Jeonju 561-756, South Korea e Faculty of Bioresources Science, College of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, South Korea f Division of Forest Science, College of Agriculture and Life Sciences, Chonbuk National University, Jeonju 561-756, South Korea g Department of Clinical Pathology, Wonkwang Health Science College, Iksan 570-750, South Korea b
a r t i c l e
i n f o
Article history: Received 24 August 2008 Accepted 4 September 2008 Available online 11 September 2008 Keywords: Ginkgo biloba Hydroxyalkenyl salicylic acids Antibacterial activity
a b s t r a c t A chloroform fraction prepared from the sarcotesta of Ginkgo biloba showed potent inhibitory activity against vancomycin-resistant Enterococcus (VRE). The active compounds were elucidated to be 2-hydroxy-6-(8-pentadecenyl) salicylic acid (1) and 2-hydroxy-6-(10heptadecenyl) salicylic acid (2) based on their spectral analysis. Compounds 1 and 2 showed significant antibacterial activities against VRE. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Widespread antimicrobial use continues to cause significant increases in resistant bacteria, particularly resistant Gram-positive organisms. Enterococci may acquire resistance against vancomycin during use of the drug to treat diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). The rapid emergence of vancomycin-resistant Enterococcus (VRE) is believed to be caused by the excessive use of vancomycin. Currently, it has been emerging as one of the most important hospital and community pathogens worldwide. The emergence of these resistant bacteria has caused a major concern and thus the urgent need for new antibacterial agents [1–3]. Plants are known to have defense systems against phytopathogenic bacteria [4]. Medicinal plants that are commonly used may be a good source for safe antibacterial agents [5].
⁎ Corresponding author. Tel.: +82 63 2703566; fax: +82 63 2703571. E-mail address: [email protected] (C.S. Ku). 0367-326X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2008.09.001
Recently, we found the sarcotesta of Ginkgo biloba to have antibacterial activity against VRE. G. biloba is one of the oldest living tree species, dating back over 300 million years. In China, the ginkgo trees have been used for 5000 years to treat lung ailments such as asthma and bronchitis, and also as a remedy for cardiovascular diseases [6]. Recently, clinical studies stimulated interest in a G. biloba extract for improving symptoms of Alzheimer-type dementia [7,8]. Phytochemically, G. biloba contains anacardic acid, bilobol, and cardanol. At present, however, the pharmaceutical arsenal available to control VRE is limited. In the present study, we report new antibacterial hydroxyalkenyl salicylic acids against VRE from the G. biloba extract. 2. Experimental 2.1. Plant The dried sarcotesta of G. biloba was collected in September 2002 from Chonbuk National University Garden, Republic of Korea. This plant was identified and authenticated by Prof. Young-Sung Ju at the College of Oriental Medicine,
J.G. Choi et al. / Fitoterapia 80 (2009) 18–20
Woosuk University. A voucher specimen (No. JSI 61) has been deposited at the Faculty of Bioresources Science, College of Agriculture and Life Science, Chonbuk National University, Jeonju, Republic of Korea. 2.2. Extraction and isolation Air-dried and pulverized sarcotesta (222 kg) of G. biloba was extracted with MeOH for 3 days at room temperature to give an extract (129 g) which was suspended in H2O and partitioned with n-hexane, CHCl3 and EtOAc, successively. The active CHCl3 fraction (23 g) was subjected to silica gel (Merck, Kieselgel 60; 0.063–0.2 mm particle size; 11 × 100 cm) column chromatography. The fraction was eluted with nhexane/EtOAc: 20:80 (10 × 250 ml), followed by MeOH. Fractions of similar composition determined by TLC analysis were pooled. A portion of the active fraction (Fr. 3–4: 500 ml) was subjected to a preparative HPLC [(Phenomenex C18, 10 × 250 mm, particle size 10 μm, CH3CN, 5.0 mL/min, fluorescence detector at excitation (284 nm) and emission wave (316 nm)] to give compounds 1 (78 mg, Rt 8.43 min, purity 96%) and 2 (56 mg, Rt 12.74 min, purity 98%). The structures of the two compounds were determined using spectroscopic techniques including UV, mass spectrometry, 1 H and 13C NMR, DEPT, HMBC and HMQC spectrometry (Fig. 1). 2.3. Preparation of bacterial cells 18 VRE strains were isolated from humans and identified by Shin-Moo Kim in the intensive care unit (ICU) of Wonkwang University Hospital, Iksan, Korea. Enterococcus faecalis CDC-286 (vanA), E. faecalis CDC-583 (vanB), E. gallinarium CDC-45 (vanC) (VRE) and E. faecalis ATCC 29212, the vancomycin susceptible strain (VSE) were used as reference. The confirmation of the identification was done by detection of the vancomycin resistance gene (vre) with PCR analysis. 2.4. Determination of anti-VRE activity The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) were determined using the agar dilution method. All strains were grown in tryptic soy broth for 24 h at 37 °C. After incubation, they were diluted with the same medium to give a concentration of approxi-
Fig. 1. Structures of compounds 1 and 2.
19
Table 1 Antibacterial activities of methanol extract and solvent soluble fractions from the sarcotesta of Ginkgo biloba against Enterococcus faecalis CDC-286 Samples
MIC
MBC
MeOH extract n-Hexane fraction CHCl3 fraction EtOAc fraction
8 16 4 16
16 32 8 32
MIC and MBC, μg/ml.
mately 108 colony forming units (CFU/ml). The isolated compounds were dissolved in dimethyl sulfoxide (DMSO) and two-fold serial dilutions were made before adding the same to tryptic soy agar plates. Bacterial cell suspensions were inoculated onto the plates using a bacteria planter (5 μl). The final inoculum concentration of CFU inoculated onto the agar plates was 5 × 105 for all strains. An agar plate containing only DMSO served as control. 3. Results and discussion To search for a traditionally used medicinal herb with potent antibacterial properties against VRE, we screened many herbs which are prescribed for gasterointestinal infections in South Korea. Among the herbal extracts tested, G. biloba extract was selected due to its higher antibacterial activity. To obtain the active component related to the antibacterial activity, G. biloba was extracted with MeOH and fractionated successively with n-hexane, CHCl3, and EtOAc. Antibacterial activities of these fractions against E. faecalis CDC-286 were tested (Table 1). The CHCl3 fraction showed significant MIC and MBC values in comparison with the other fractions. Therefore, the CHCl3 fraction was employed for
Table 2 Antibacterial activities of compounds 1 and 2 isolated from the sarcotesta of Ginkgo biloba against clinical isolates of 18 VRE, standard VSE and 3 VRE reference strains Strains
E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E. E.
faecalis CDC-286 (vanA) faecalis CDC-583 (vanB) gallinarium CDC-45 (vanC) faecalis ATCC29212 (VSE) faecium 1 faecium 2 faecium 3 faecium 4 faecium 5 faecium 6 faecium 7 faecium 8 faecium 9 faecium 11 faecalis 1 faecalis 2 faecalis 3 faecalis 4 faecalis 5 faecalis 6 faecalis 7 faecalis 10
MIC and MBC, µg/ml.
1
2
Vancomycin
MIC
MBC
MIC
MBC
MIC
MBC
2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 4 2 4 2 2 2 2
4 4 2 2 2 4 2 2 2 2 2 2 2 4 4 4 2 4 4 2 2 2
4 4 4 2 4 2 2 2 4 2 2 2 4 2 8 4 4 8 4 4 4 8
8 8 4 4 4 4 2 2 4 4 4 4 4 2 8 8 8 8 4 4 4 8
256 32 64 32 N512 N512 N512 N512 512 256 256 128 128 N512 64 256 256 128 256 64 256 64
512 64 64 32 N512 N512 N512 N512 N512 256 256 128 256 N512 128 512 512 128 512 128 256 128
20
J.G. Choi et al. / Fitoterapia 80 (2009) 18–20
further purification. Out of the first 10 fractions obtained by silica gel column chromatography of the CHCl3 fraction, Fractions 3 and 4 showed potent inhibitory activity (MIC/ MBC: 4/8 μg/ml) against E. faecalis CDC-286 (vanA). By further purifying the fractions, compounds 1 and 2 were isolated and were confirmed to be 2-hydroxy-6-(8-pentadecenyl) salicylic acid and 2-hydroxy-6-(10-hentadecenyl) salicylic acid, respectively, by the comparison of their spectral data with those in the references [9,10]. The MIC/MBC values of the isolated compounds against VRE are summarized in Table 2. Each of the clinically isolated strains was significantly resistant to vancomycin and showed MIC/MBC values similar to or higher than 64/128 μg/ml. Compounds 1 and 2 showed very potent inhibitory activity against standard VRE, VSE as well as clinical isolates of VRE, with MIC and MBC values of 2 to 4 and 4 to 8 μg/ml, respectively. These results indicated the possibility of exploitation of hydroxyalkenyl salicylic acids originated from G. biloba as effective inhibitors of VRE. This is the first report on the anti-VRE activities of hydroxyalkenyl salicylic acids in the literature. The isolated
compounds are expected to be useful for the study of anti-VRE agents in the future. However, for medicinal purposes, the safety and toxicity of these salicylic acids need to be addressed. References [1] American Society for Microbiology (ASM). Antimicrob Agents Chemother 1995;39:2. [2] Davis J. Sci 1994;264:375. [3] Spratt BG. Sci 1994;264:388. [4] Smith E, Williamson E, Zloh M, Gibbons S. Phytother Res 2005;19:538. [5] Sato Y, Suzaki S, Nishikawa T, Kihara M, Shibata H, Higuti T. J Ethnopharmacol 2000;72. [6] Perry EK, Pickering AT, Wang WW, Houghton PJ, Perry NS. J Pharm Pharmacol 1999;51:527. [7] Le Bars P, Katz MM, Berman N, Itil TM, Freedman AM, Schatzberg AF, North American EGb Study Group. JAMA 1997;278:1327. [8] Maurer K, Dierks RI, Frölich L. J Psychiatr Res 1997;31:645. [9] He XG, Bernart MW, Nolan GS, Lin LZ, Lindenmaier MP. J Chromatogra Sci 2000;38:169. [10] Itokawa H, Totsuka N, Nakahara K, Takeya K, Lepoittevin JP, Asakawa Y. Chem Pharm Bull 1987;35:3016.