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Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Phenolic constituents from the aerial parts of Glycyrrhiza inflata and their antibacterial activities a
Biao Zhou & Chuan-Xing Wan
a
a
Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Group, College of Life Science, Tarim University, Alar 843300, China Published online: 15 Oct 2014.
To cite this article: Biao Zhou & Chuan-Xing Wan (2014): Phenolic constituents from the aerial parts of Glycyrrhiza inflata and their antibacterial activities, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.966095 To link to this article: http://dx.doi.org/10.1080/10286020.2014.966095
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.966095
Phenolic constituents from the aerial parts of Glycyrrhiza inflata and their antibacterial activities Biao Zhou and Chuan-Xing Wan*
Downloaded by [University of Southern Queensland] at 10:52 16 October 2014
Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Group, College of Life Science, Tarim University, Alar 843300, China (Received 18 April 2014; final version received 11 September 2014) Chemical investigation on 90% ethanol extracts of the aerial parts of Glycyrrhiza inflata afforded two new phenolic constituents, 2-(3-methyl-2-butenyl)-3,5,40 trihydroxy-bibenzyl (1) and (2S)-6-[(E)-3-hydroxymethyl-2-butenyl]-30 ,40 ,5,7-tetrahydroxy-dihydroflavanone (2) along with seven known dihydroflavanones (3 – 9). Compounds 1 –9 were tested for their minimum inhibitory concentration (MIC) values of inhibiting Staphylococcus aureus and Staphylococcus epidermidis. Compound 1 showed moderate antibacterial activities against both S. aureus (MIC of 50.00 mg/ml) and S. epidermidis (MIC of 12.50 mg/ml). The analysis of structure – activity relationships revealed that the antibacterial activity of dihydroflavanones (2 – 9) was significantly affected by the position of prenyl group. Keywords: Glycyrrhiza inflata; phenolic constituents; antibacterial activity
1.
Introduction
Licorice, a popular Chinese herbal medicine derived from Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata, is widely used in the food and tobacco industries as a sweetener, traditional Chinese medicine, and complementary medicine [1], which shows a variety of pharmacological functions such as antiulcer, antiviral, antioxidant, antibacterial, antifungal, antimalarial, antihepatotoxic, antispasmodic, and antiinflammatory activities [2]. Phytochemical investigation of licorice has shown the presence of triterpene saponins, flavonoids, and other phenolic compounds [3]. Moreover, new dihydrostilbene derivatives in the leaves of G. glabra have also been reported [4,5]. In this paper, two new phenolic constituents, 2-(3-methyl-2-butenyl)-3,5,40 -trihydroxy-bibenzyl (1) and isolicoleafol (2) along with seven known
dihydroflavanones (3– 9), were reported from the aerial parts of G. inflata, together with their antibacterial activities against Staphylococcus aureus and Staphylococcus epidermidis (Figure 1).
2.
Results and discussion
Compound 1 was obtained as a yellow – orange oil. UV absorptions at 212, 231, and 280 nm, ascribable to aromatic ring chromophores, were indicative of stilbenoid compounds [4]. The signals at dH 1.65, dH 1.73 (each 3H, s), 3.30 (2H, d, J ¼ 6.6 Hz) and one triplet partially overlapped at dH 5.11 (1H, t, J ¼ 6.6 Hz) in the 1H NMR spectrum (Table 1) of 1 showed the presence of one prenyl (3methyl-2-butenyl) substituent [6]. This prenyl moiety is located at C-2 based on the HMBC correlations (Figure 2) of H-7 at dH 3.30 (2H, d, J ¼ 6.6 Hz) to C-2 (dC
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
2
B. Zhou and C.-X. Wan OH 5 β
6' 5' HO 4'
6 α 1 8
1' 2' 3'
5'
4 3 2 OH
OH 4''
7
9 11
HO 7 2'' 6 3'' 1''
5''
10
1 9 O 2
8
5 10 4 OH O
6' 1'
2'
4' OH HO 3' OH
OH HO
O
OH
1
OH
3
2
O 3
OH OH
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HO
O
HO
O
HO
OH OH
OH
OH OH O
O
O
OH
4
5
O 6
OH HO
HO
O HO
OH
O
O OH
O OH 7
O 8
O
9
Figure 1. Structures of compounds 1 –9. Table 1. 1H and 13C NMR spectroscopic data for compound 1 (acetone-d6). 1 Position 1 2 3 4 5 6 7 8 9 10 11 A B 10 20 30 40 50 60 3-OH 5-OH 40 -OH
dC 143.0 118.1 156.7 101.3 156.7 108.5 25.0 125.7 130.1 18.1 25.9 36.5 37.6 133.8 130.0 115.9 156.4 115.9 130.0
dH (J in Hz)
6.28 d (2.0) 6.24 d (2.0) 3.30 d (6.6) 5.11 t (6.6) 1.73 br 1.65 br 2.73 br 2.73 br
s s s s
7.06 d (8.2) 6.76 d (8.2) 6.76 d (8.2) 7.06 d (8.2) 7.97 br s 7.83 br s 8.08 br s
118.1), C-1 (dC 143.0), and C-3 (dC 156.7), as well as H-8 at dH 5.11 (1H, t, J ¼ 6.6 Hz) to C-2 (dC 118.1). In particular, the analysis of the aromatic region of the spectrum led us to establish the substitution pattern of the aromatic rings. Two meta-coupled signals at dH 6.24 (1H, d, J ¼ 2.0 Hz) and dH 6.28 (1H, d, J ¼ 2.0 Hz), characteristics of a 1,2,3,5-tetrasubstituted benzene ring, were attributed to the A ring, whereas a 10 ,40 para-substitution pattern was assigned to the aromatic B ring owing to the presence of the signals of four aromatic protons at dH 6.76 (2H, d, J ¼ 8.2 Hz) and 7.06 (2H, d, J ¼ 8.2 Hz). Finally, the 1H NMR spectrum was very similar to that of 2-(3-methyl-2butenyl)-3,5-dihydroxy-bibenzyl [7], except for the appearance of an additional hydroxy signal in B ring. The HMBC correlations of OH-3 at dH 7.97 (1H, br s) to C-2 (dC 118.1), C-3 (dC 156.7), and C-4 (dC 101.3); OH-5 at dH 7.83 (1H, s) to C-4 (dC 101.3), C-5 (dC 156.7), and C-6 (dC 108.5); and OH-40 at dH
Journal of Asian Natural Products Research
3
OH OH HO
OH
HO
O OH
HO
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OH 1
HMBC
ROESY
O
2
Figure 2. Selected HMBC and ROESY correlations of compounds 1 and 2.
8.08(1H, s) to C-30 (dC 115.9), C-40 (dC 156.4), and C-50 (dC 115.9) suggested that the signals at dH 7.97, 7.83, and 8.08 were assigned to C-3 (dC 156.7), C-5 (dC 56.7), and C-40 (dC 156.4), respectively. That was also confirmed by the NOESY experiment (Figure 2) showing the cross-peak from OH-3 (dH 7.97) to H-4 (dH 6.28), from OH-5 (dH 7.83) to H-6 (dH 6.24), and from OH-40 (dH 8.08) to H-30 /50 (dH 6.76). On the basis of the above analysis, the structure of 1 was characterized as 2(3-methyl-2-butenyl)-3,5,40 -trihydroxybibenzyl. Compound 2 was achieved as a yellow – orange amorphous solid. Three double-doublets at dH 5.37 (2H, dd, J ¼ 13.0, 3.0 Hz), 3.13 (1H, dd, J ¼ 17.0, 13.0 Hz), and 2.75 (1H, dd, J ¼ 17.0, 3.0 Hz) in its 1H NMR spectrum (Table 2) suggested the characteristic signals of a dihydroflavanone [8]. In addition, the 1H NMR spectrum showed a signal due to one isolated aromatic proton at dH 6.04 (s) and three aromatic protons at dH 6.86 (2H, s), and 7.03 (1H, s). These signals were similar to those of licoleafol [8], except that the prenyl (3-hydroxymethyl-2-butenyl) was substituted at C-6 in 2, not C-8. The position of prenyl was further confirmed by the HMBC correlations (Figure 2) of H-100 at dH 3.29 (2H, d, J ¼ 7.0 Hz) to C-5 (dC 162.3), C-6 (dC
108.7), and C-7 (dC 164.8), as well as H-200 at dH 5.49 (1H, t, J ¼ 7.0 Hz) to C-6 (dC 108.7). Furthermore, the geometry configuration of this substituent was concluded to be E by comparison of the 13C NMR spectrum of 2 with that of the prenyl Table 2. 1H and 13C NMR spectroscopic data for compound 2 (acetone-d6). 2 Position
dC
dH (J in Hz)
2 3
79.9 43.7
5.37 dd (13.0, 3.0) 2.75 dd (17.0, 3.0) 3.13 dd (17.0, 13.0)
4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 5-OH 7-OH 30 -OH 40 -OH
197.3 162.3 108.7 164.8 95.3 162.0 103.2 131.7 114.7 146.4 146.0 116.0 119.2 21.2 123.1 135.9 68.4 13.8
6.04 s
7.03 s 6.86 s 6.86 s 3.29 d (7.0) 5.49 t (7.0) 3.89 s 1.77 s 12.47 br s 9.74 br s 8.09 s 8.14 s
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4
B. Zhou and C.-X. Wan
group in licoleafol [(S)-8-[(E)-3-hydroxymethyl-2-butenyl]-30 ,40 ,5,7-tetrahydroxyflavanone] [8]. This was also confirmed by the ROESY correlation from H-100 at dH 3.29 (1H, d, J ¼ 7.0 Hz) to H-500 at dH 1.77 (1H, s). The absolute configuration at C-2 of 2 was assigned to be S by the negative Cotton effect of CD spectrum at 297 nm (D1 ¼ – 12.5) and the positive Cotton effect at 336 nm (D1 ¼ þ 4.8) [8]. Accordingly, the structure of 2 (isolicoleafol) was established to be (2S)-6-[(E)-3-hydroxymethyl-2-butenyl]-30 ,40 ,5,7-tetrahydroxydihydroflavanone. In addition, the known compounds, licoleafol (3) [8], 5,7,30 ,40 -tetrahydroxy-8(30 ,30 -dimethylallyl)-flavanone (4), 5,7,30 , 40 -tetrahydroxy-6-(30 ,30 -dimethylallyl)-flavanone (5) [9], 8-prenylnaringenin (6), 6-prenylnaringenin (7) [10], 5,7-dihydroxy-8-(3 0 ,3 0 -dimethylallyl)-flavanone (8), and 5,7-dihydroxy-6-(30 ,30 -dimethylallyl)-flavanone (9) [11], were identified by comparing their spectral data with those previously reported. Compounds 1– 9 were investigated on their antibacterial activities against S. aureus and S. epidermidis (Table 3). Compound 1 showed moderate antibacterial activities against both S. aureus (MIC of 50.00 mg/ml) and S. epidermidis (MIC of 12.50 mg/ml). MIC of other compounds ranged from 12.50 to 100.00 mg/ml. The Table 3. Minimum inhibitory concentration (MIC) values of compounds 1 – 9 against S. aureus and S. epidermidis. MIC (mg/ml) Compounds
S. aureus
S. epidermidis
1 2 3 4 5 6 7 8 9
50.00 25.00 100.00 12.50 12.50 12.50 . 100.00 25.00 . 100.00
12.50 25.00 .100.00 25.00 50.00 12.50 .100.00 25.00 .100.00
structure –activity analysis revealed that the bioactivity was strongly affected by the position of prenyl (3-methyl-2-butenyl) group. Comparing the MICs of 4 and 5, 6 and 7, and 8 and 9 revealed that the dihydroflavanones with a prenyl group substituted at C-8 position showed stronger activity than those of C-6 substitutions (Table 3). However, if the prenyl moiety possessed a hydroxyl group, the results were reversed, indicated by the MICs of 2 and 3. 3.
Experimental
3.1 General experimental procedures UV spectra were recorded using a UV2450 UV – visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan). IR spectra were performed on a Nicolot 380 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). CD spectra were determined with a JASCO P-1020 digital polarimeter (Shimadzu). NMR spectra were measured in acetone-d6 on a Varian 400M NMR spectrometer (Varian, Palo Alto, CA, USA) with TMS as the internal standard. HR-ESI-MS data were obtained on a G1969A TOF-MS instrument (Agilent Technologies, Inc., Palo Alto, CA, USA). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Qingdao, China) were employed for TLC. Spots were visualized by spraying 10% H2SO4 – EtOH followed by heating. Silica gel (Qingdao Haiyang Chemical Co.), polyamide (Jiangsu Changfeng Chemical Co., Jiangsu, China), Sephadex LH-20 (20 £ 100 mm; Pharmacia, Uppsala, Sweden), and ODS (40 – 63 mm, FuJi, Aichi, Japan) were used for open column chromatography. 3.2
Plant material
The aerial parts of G. inflata were collected in June 2013 from Alar, Xinjiang, and authenticated by Professor Wen-Juan Huang, College of Plant
Journal of Asian Natural Products Research Sciences, Tarim University. A voucher specimen (No. 2013060201) was deposited in the Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Group.
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3.3
Extraction and isolation
The dried materials (24.0 kg) were cut into pieces and extracted with 90% ethanol (EtOH) for three times at room temperature, and concentrated into an ointment after a simple filtration. The combined EtOH extract (682.0 g) was crudely separated on a silica gel using chloroform (CHCl3) –methanol (MeOH) solvent system (50:1, 0:1, v/v) to obtain the corresponding two fractions, I (236.5 g) and II (208.3 g). The fraction I (83 g) was chromatographed on another silica gel column [petroleum ether (PE):ethyl acetate (EtOAc), 30:1, 5:1, 1:1, and 1:3)] and separated into four fractions IA (0.98 g), IB (4.36 g), IC (39.1 g), and ID (10.9 g). Fraction IA was further separated by silica gel column using a gradient of PE –EtOAc as eluents, which afforded 92 fractions, and compounds 8 (6.2 mg) and 9 (70.5 mg) were, respectively, isolated from 58– 64 and 37– 56 fractions. Fraction IB was further subjected to silica gel column chromatography eluted with PE –EtOAc (3:1, 1:1, v/v) to afford two subfractions IBa (2.3 g) and IBb (1.0 g), then fraction IBb (1.03 g) was purified by passing over Sephadex LH-20 column (eluted with MeOH) to yield 7 (18.9 mg). Fraction IC was chromatographed over an ODS column eluted with MeOH – H2O gradient system (50%, 70%, and 100%) to provide three subfractions ICa (16.1 g), ICb (3.4 g), and ICc (1.3 g). Subfraction ICa was submitted to silica gel column with CHCl3 – MeOH gradient system (100: 1 ! 10:1) to obtain 4 (424.8 mg) and 3 (252.5 mg); subfraction ICb was further separated by silica gel column (eluted with PE:MeOH, 50:1, 30:1, and 0:1) and
5
Sephadex LH-20 (eluted with CH3OH) to afford 5 (64.3 mg) and 6 (164.4 mg). Fraction ID was subjected to polyamide column chromatography (60 – 90 mesh) with EtOH – H2O gradient system (30%, 50%, 70%, and 100%) to give four subfractions IDa (12.5 g), IDb (16.6 g), IDc (20.2 g), and IDd (0.86 g). Subfraction IDc was further chromatographed on silica gel column eluted with CHCl3 –MeOH gradient system (30:1, 20:1, and 1:1) to gain 2 (150.7 mg) and 1 (92.9 mg). 3.3.1
Compound 1
Yellow – orange oil; UV [MeOH – H2O (1:1)] lmax (log 1): 209 (4.53), 227 (4.27), 281 (3.69) nm; IR (KBr) nmax: 3422, 2972, 2926, 2859, 1614, 1512, 1450, 1382, 1338, 1134, 829, 543 cm – 1; for 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) spectral data, see Table 1; HR-TOF-MS: m/z 299.1658 [M þ H]þ (calcd for C19H23O3, 299.1647). 3.3.2
Compound 2
Yellow – orange amorphous solid; UV [MeOH – H2O (1:1)] lmax (log 1): 204 (4.66), 290 (4.19), 340 (4.14) nm; IR (KBr) nmax: 3443, 1627, 1600, 1400, 1337, 1115, 539 cm – 1; for 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) spectral data, see Table 2; HRTOF-MS: m/z 373.1181 [M þ H]þ (calcd for C20H21O7, 373.1287); CD (MeOH): D1297 nm ¼ –12.5, D1336 nm ¼ þ 4.8. 3.4
Antimicrobial assay
S. aureus (ATCC 25923) and S. epidermidis (ATCC 35984) were used for testing antibacterial activity, and the MIC values were determined using the microbroth dilution method [12]. Briefly, an overnight culture of the strains in trypticase soy broth (TSB) was diluted 1:100 and incubated at 378C. Test
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6
B. Zhou and C.-X. Wan
compounds were dissolved into 2 mg/ml in acetone and then diluted in the medium and added to the test tubes, containing 2 ml sterile liquid TSB, at a series of concentrations (100, 50, 25, 12.5, 6.25, 3.13, 1.56 ml/tube), of which 100 ml per well was added in a 96-well plate. The same volume of acetone was used as a negative control. The plates were incubated at 378C overnight, and growth inhibition was determined by the change in OD590 at the end of the incubation period compared with the control. The MIC value was defined as the lowest concentration that prevented visible growth of the bacteria after incubation at 378C overnight. Acknowledgement This research work was financially supported by the Hi-Tech Project of Xinjiang Production and Construction Group (2006GJS19).
References [1] T.C. Kao, C.H. Wu, and G.C. Yen, J. Agric. Food Chem. 62, 542 (2014).
[2] K. Rajandeep, H. Kaur, and A.S. Dhindsa, Int. J. Pharm. Sci. Res. 4, 2470 (2013). [3] Q.Y. Zhang and M. Ye, J. Chromatogr. A 1216, 1954 (2009). [4] D.M. Biondi, C. Rocco, and G. Ruberto, J. Nat. Prod. 66, 477 (2003). [5] D.M. Biondi, C. Rocco, and G. Ruberto, J. Nat. Prod. 68, 1099 (2005). [6] C.X. Wan, P.H. Zhang, J.G. Luo, and L. Y. Kong, J. Nat. Prod. 74, 683 (2011). [7] L. Kraut, R. Mues, and H.D. Zinsmeister, Phytochemistry 45, 1249 (1997). [8] H. Hayashi, S.L. Zhang, T. Nakaizumi, K. Shimura, M. Yamaguchi, K. Inoue, K. Sarsenk, M. Ito, and G. Honda, Chem. Pharm. Bull. 51, 1147 (2003). [9] F. Bohlmann, C. Zdero, H. Robinson, and R.M. King, Phytochemistry 20, 2245 (1981). [10] J.F. Stevens, M. Ivancic, V.L. Hsu, and M.L. Deinzer, Phytochemistry 44, 1575 (1997). [11] F. Bohlmann and W.R. Abraham, Phytochemistry 18, 1851 (1979). [12] S. Inui, T. Hosoya, Y. Shimamura, S. Masuda, T. Ogawa, H. Kobayashi, K. Shirafuji, R.T. Moli, I. Kozone, K. Shin-Ya, and S. Kumazawa, J. Agric. Food Chem. 60, 11765 (2012).
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Phenolic constituents from the aerial parts of Glycyrrhiza inflata and their antibacterial activities a
Biao Zhou & Chuan-Xing Wan
a
a
Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Group, College of Life Science, Tarim University, Alar 843300, China Published online: 15 Oct 2014.
To cite this article: Biao Zhou & Chuan-Xing Wan (2014): Phenolic constituents from the aerial parts of Glycyrrhiza inflata and their antibacterial activities, Journal of Asian Natural Products Research, DOI: 10.1080/10286020.2014.966095 To link to this article: http://dx.doi.org/10.1080/10286020.2014.966095
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Southern Queensland] at 10:52 16 October 2014
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2014 http://dx.doi.org/10.1080/10286020.2014.966095
Phenolic constituents from the aerial parts of Glycyrrhiza inflata and their antibacterial activities Biao Zhou and Chuan-Xing Wan*
Downloaded by [University of Southern Queensland] at 10:52 16 October 2014
Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Group, College of Life Science, Tarim University, Alar 843300, China (Received 18 April 2014; final version received 11 September 2014) Chemical investigation on 90% ethanol extracts of the aerial parts of Glycyrrhiza inflata afforded two new phenolic constituents, 2-(3-methyl-2-butenyl)-3,5,40 trihydroxy-bibenzyl (1) and (2S)-6-[(E)-3-hydroxymethyl-2-butenyl]-30 ,40 ,5,7-tetrahydroxy-dihydroflavanone (2) along with seven known dihydroflavanones (3 – 9). Compounds 1 –9 were tested for their minimum inhibitory concentration (MIC) values of inhibiting Staphylococcus aureus and Staphylococcus epidermidis. Compound 1 showed moderate antibacterial activities against both S. aureus (MIC of 50.00 mg/ml) and S. epidermidis (MIC of 12.50 mg/ml). The analysis of structure – activity relationships revealed that the antibacterial activity of dihydroflavanones (2 – 9) was significantly affected by the position of prenyl group. Keywords: Glycyrrhiza inflata; phenolic constituents; antibacterial activity
1.
Introduction
Licorice, a popular Chinese herbal medicine derived from Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata, is widely used in the food and tobacco industries as a sweetener, traditional Chinese medicine, and complementary medicine [1], which shows a variety of pharmacological functions such as antiulcer, antiviral, antioxidant, antibacterial, antifungal, antimalarial, antihepatotoxic, antispasmodic, and antiinflammatory activities [2]. Phytochemical investigation of licorice has shown the presence of triterpene saponins, flavonoids, and other phenolic compounds [3]. Moreover, new dihydrostilbene derivatives in the leaves of G. glabra have also been reported [4,5]. In this paper, two new phenolic constituents, 2-(3-methyl-2-butenyl)-3,5,40 -trihydroxy-bibenzyl (1) and isolicoleafol (2) along with seven known
dihydroflavanones (3– 9), were reported from the aerial parts of G. inflata, together with their antibacterial activities against Staphylococcus aureus and Staphylococcus epidermidis (Figure 1).
2.
Results and discussion
Compound 1 was obtained as a yellow – orange oil. UV absorptions at 212, 231, and 280 nm, ascribable to aromatic ring chromophores, were indicative of stilbenoid compounds [4]. The signals at dH 1.65, dH 1.73 (each 3H, s), 3.30 (2H, d, J ¼ 6.6 Hz) and one triplet partially overlapped at dH 5.11 (1H, t, J ¼ 6.6 Hz) in the 1H NMR spectrum (Table 1) of 1 showed the presence of one prenyl (3methyl-2-butenyl) substituent [6]. This prenyl moiety is located at C-2 based on the HMBC correlations (Figure 2) of H-7 at dH 3.30 (2H, d, J ¼ 6.6 Hz) to C-2 (dC
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
2
B. Zhou and C.-X. Wan OH 5 β
6' 5' HO 4'
6 α 1 8
1' 2' 3'
5'
4 3 2 OH
OH 4''
7
9 11
HO 7 2'' 6 3'' 1''
5''
10
1 9 O 2
8
5 10 4 OH O
6' 1'
2'
4' OH HO 3' OH
OH HO
O
OH
1
OH
3
2
O 3
OH OH
Downloaded by [University of Southern Queensland] at 10:52 16 October 2014
HO
O
HO
O
HO
OH OH
OH
OH OH O
O
O
OH
4
5
O 6
OH HO
HO
O HO
OH
O
O OH
O OH 7
O 8
O
9
Figure 1. Structures of compounds 1 –9. Table 1. 1H and 13C NMR spectroscopic data for compound 1 (acetone-d6). 1 Position 1 2 3 4 5 6 7 8 9 10 11 A B 10 20 30 40 50 60 3-OH 5-OH 40 -OH
dC 143.0 118.1 156.7 101.3 156.7 108.5 25.0 125.7 130.1 18.1 25.9 36.5 37.6 133.8 130.0 115.9 156.4 115.9 130.0
dH (J in Hz)
6.28 d (2.0) 6.24 d (2.0) 3.30 d (6.6) 5.11 t (6.6) 1.73 br 1.65 br 2.73 br 2.73 br
s s s s
7.06 d (8.2) 6.76 d (8.2) 6.76 d (8.2) 7.06 d (8.2) 7.97 br s 7.83 br s 8.08 br s
118.1), C-1 (dC 143.0), and C-3 (dC 156.7), as well as H-8 at dH 5.11 (1H, t, J ¼ 6.6 Hz) to C-2 (dC 118.1). In particular, the analysis of the aromatic region of the spectrum led us to establish the substitution pattern of the aromatic rings. Two meta-coupled signals at dH 6.24 (1H, d, J ¼ 2.0 Hz) and dH 6.28 (1H, d, J ¼ 2.0 Hz), characteristics of a 1,2,3,5-tetrasubstituted benzene ring, were attributed to the A ring, whereas a 10 ,40 para-substitution pattern was assigned to the aromatic B ring owing to the presence of the signals of four aromatic protons at dH 6.76 (2H, d, J ¼ 8.2 Hz) and 7.06 (2H, d, J ¼ 8.2 Hz). Finally, the 1H NMR spectrum was very similar to that of 2-(3-methyl-2butenyl)-3,5-dihydroxy-bibenzyl [7], except for the appearance of an additional hydroxy signal in B ring. The HMBC correlations of OH-3 at dH 7.97 (1H, br s) to C-2 (dC 118.1), C-3 (dC 156.7), and C-4 (dC 101.3); OH-5 at dH 7.83 (1H, s) to C-4 (dC 101.3), C-5 (dC 156.7), and C-6 (dC 108.5); and OH-40 at dH
Journal of Asian Natural Products Research
3
OH OH HO
OH
HO
O OH
HO
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OH 1
HMBC
ROESY
O
2
Figure 2. Selected HMBC and ROESY correlations of compounds 1 and 2.
8.08(1H, s) to C-30 (dC 115.9), C-40 (dC 156.4), and C-50 (dC 115.9) suggested that the signals at dH 7.97, 7.83, and 8.08 were assigned to C-3 (dC 156.7), C-5 (dC 56.7), and C-40 (dC 156.4), respectively. That was also confirmed by the NOESY experiment (Figure 2) showing the cross-peak from OH-3 (dH 7.97) to H-4 (dH 6.28), from OH-5 (dH 7.83) to H-6 (dH 6.24), and from OH-40 (dH 8.08) to H-30 /50 (dH 6.76). On the basis of the above analysis, the structure of 1 was characterized as 2(3-methyl-2-butenyl)-3,5,40 -trihydroxybibenzyl. Compound 2 was achieved as a yellow – orange amorphous solid. Three double-doublets at dH 5.37 (2H, dd, J ¼ 13.0, 3.0 Hz), 3.13 (1H, dd, J ¼ 17.0, 13.0 Hz), and 2.75 (1H, dd, J ¼ 17.0, 3.0 Hz) in its 1H NMR spectrum (Table 2) suggested the characteristic signals of a dihydroflavanone [8]. In addition, the 1H NMR spectrum showed a signal due to one isolated aromatic proton at dH 6.04 (s) and three aromatic protons at dH 6.86 (2H, s), and 7.03 (1H, s). These signals were similar to those of licoleafol [8], except that the prenyl (3-hydroxymethyl-2-butenyl) was substituted at C-6 in 2, not C-8. The position of prenyl was further confirmed by the HMBC correlations (Figure 2) of H-100 at dH 3.29 (2H, d, J ¼ 7.0 Hz) to C-5 (dC 162.3), C-6 (dC
108.7), and C-7 (dC 164.8), as well as H-200 at dH 5.49 (1H, t, J ¼ 7.0 Hz) to C-6 (dC 108.7). Furthermore, the geometry configuration of this substituent was concluded to be E by comparison of the 13C NMR spectrum of 2 with that of the prenyl Table 2. 1H and 13C NMR spectroscopic data for compound 2 (acetone-d6). 2 Position
dC
dH (J in Hz)
2 3
79.9 43.7
5.37 dd (13.0, 3.0) 2.75 dd (17.0, 3.0) 3.13 dd (17.0, 13.0)
4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 5-OH 7-OH 30 -OH 40 -OH
197.3 162.3 108.7 164.8 95.3 162.0 103.2 131.7 114.7 146.4 146.0 116.0 119.2 21.2 123.1 135.9 68.4 13.8
6.04 s
7.03 s 6.86 s 6.86 s 3.29 d (7.0) 5.49 t (7.0) 3.89 s 1.77 s 12.47 br s 9.74 br s 8.09 s 8.14 s
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B. Zhou and C.-X. Wan
group in licoleafol [(S)-8-[(E)-3-hydroxymethyl-2-butenyl]-30 ,40 ,5,7-tetrahydroxyflavanone] [8]. This was also confirmed by the ROESY correlation from H-100 at dH 3.29 (1H, d, J ¼ 7.0 Hz) to H-500 at dH 1.77 (1H, s). The absolute configuration at C-2 of 2 was assigned to be S by the negative Cotton effect of CD spectrum at 297 nm (D1 ¼ – 12.5) and the positive Cotton effect at 336 nm (D1 ¼ þ 4.8) [8]. Accordingly, the structure of 2 (isolicoleafol) was established to be (2S)-6-[(E)-3-hydroxymethyl-2-butenyl]-30 ,40 ,5,7-tetrahydroxydihydroflavanone. In addition, the known compounds, licoleafol (3) [8], 5,7,30 ,40 -tetrahydroxy-8(30 ,30 -dimethylallyl)-flavanone (4), 5,7,30 , 40 -tetrahydroxy-6-(30 ,30 -dimethylallyl)-flavanone (5) [9], 8-prenylnaringenin (6), 6-prenylnaringenin (7) [10], 5,7-dihydroxy-8-(3 0 ,3 0 -dimethylallyl)-flavanone (8), and 5,7-dihydroxy-6-(30 ,30 -dimethylallyl)-flavanone (9) [11], were identified by comparing their spectral data with those previously reported. Compounds 1– 9 were investigated on their antibacterial activities against S. aureus and S. epidermidis (Table 3). Compound 1 showed moderate antibacterial activities against both S. aureus (MIC of 50.00 mg/ml) and S. epidermidis (MIC of 12.50 mg/ml). MIC of other compounds ranged from 12.50 to 100.00 mg/ml. The Table 3. Minimum inhibitory concentration (MIC) values of compounds 1 – 9 against S. aureus and S. epidermidis. MIC (mg/ml) Compounds
S. aureus
S. epidermidis
1 2 3 4 5 6 7 8 9
50.00 25.00 100.00 12.50 12.50 12.50 . 100.00 25.00 . 100.00
12.50 25.00 .100.00 25.00 50.00 12.50 .100.00 25.00 .100.00
structure –activity analysis revealed that the bioactivity was strongly affected by the position of prenyl (3-methyl-2-butenyl) group. Comparing the MICs of 4 and 5, 6 and 7, and 8 and 9 revealed that the dihydroflavanones with a prenyl group substituted at C-8 position showed stronger activity than those of C-6 substitutions (Table 3). However, if the prenyl moiety possessed a hydroxyl group, the results were reversed, indicated by the MICs of 2 and 3. 3.
Experimental
3.1 General experimental procedures UV spectra were recorded using a UV2450 UV – visible spectrophotometer (Shimadzu Corporation, Kyoto, Japan). IR spectra were performed on a Nicolot 380 spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). CD spectra were determined with a JASCO P-1020 digital polarimeter (Shimadzu). NMR spectra were measured in acetone-d6 on a Varian 400M NMR spectrometer (Varian, Palo Alto, CA, USA) with TMS as the internal standard. HR-ESI-MS data were obtained on a G1969A TOF-MS instrument (Agilent Technologies, Inc., Palo Alto, CA, USA). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Qingdao, China) were employed for TLC. Spots were visualized by spraying 10% H2SO4 – EtOH followed by heating. Silica gel (Qingdao Haiyang Chemical Co.), polyamide (Jiangsu Changfeng Chemical Co., Jiangsu, China), Sephadex LH-20 (20 £ 100 mm; Pharmacia, Uppsala, Sweden), and ODS (40 – 63 mm, FuJi, Aichi, Japan) were used for open column chromatography. 3.2
Plant material
The aerial parts of G. inflata were collected in June 2013 from Alar, Xinjiang, and authenticated by Professor Wen-Juan Huang, College of Plant
Journal of Asian Natural Products Research Sciences, Tarim University. A voucher specimen (No. 2013060201) was deposited in the Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production & Construction Group.
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3.3
Extraction and isolation
The dried materials (24.0 kg) were cut into pieces and extracted with 90% ethanol (EtOH) for three times at room temperature, and concentrated into an ointment after a simple filtration. The combined EtOH extract (682.0 g) was crudely separated on a silica gel using chloroform (CHCl3) –methanol (MeOH) solvent system (50:1, 0:1, v/v) to obtain the corresponding two fractions, I (236.5 g) and II (208.3 g). The fraction I (83 g) was chromatographed on another silica gel column [petroleum ether (PE):ethyl acetate (EtOAc), 30:1, 5:1, 1:1, and 1:3)] and separated into four fractions IA (0.98 g), IB (4.36 g), IC (39.1 g), and ID (10.9 g). Fraction IA was further separated by silica gel column using a gradient of PE –EtOAc as eluents, which afforded 92 fractions, and compounds 8 (6.2 mg) and 9 (70.5 mg) were, respectively, isolated from 58– 64 and 37– 56 fractions. Fraction IB was further subjected to silica gel column chromatography eluted with PE –EtOAc (3:1, 1:1, v/v) to afford two subfractions IBa (2.3 g) and IBb (1.0 g), then fraction IBb (1.03 g) was purified by passing over Sephadex LH-20 column (eluted with MeOH) to yield 7 (18.9 mg). Fraction IC was chromatographed over an ODS column eluted with MeOH – H2O gradient system (50%, 70%, and 100%) to provide three subfractions ICa (16.1 g), ICb (3.4 g), and ICc (1.3 g). Subfraction ICa was submitted to silica gel column with CHCl3 – MeOH gradient system (100: 1 ! 10:1) to obtain 4 (424.8 mg) and 3 (252.5 mg); subfraction ICb was further separated by silica gel column (eluted with PE:MeOH, 50:1, 30:1, and 0:1) and
5
Sephadex LH-20 (eluted with CH3OH) to afford 5 (64.3 mg) and 6 (164.4 mg). Fraction ID was subjected to polyamide column chromatography (60 – 90 mesh) with EtOH – H2O gradient system (30%, 50%, 70%, and 100%) to give four subfractions IDa (12.5 g), IDb (16.6 g), IDc (20.2 g), and IDd (0.86 g). Subfraction IDc was further chromatographed on silica gel column eluted with CHCl3 –MeOH gradient system (30:1, 20:1, and 1:1) to gain 2 (150.7 mg) and 1 (92.9 mg). 3.3.1
Compound 1
Yellow – orange oil; UV [MeOH – H2O (1:1)] lmax (log 1): 209 (4.53), 227 (4.27), 281 (3.69) nm; IR (KBr) nmax: 3422, 2972, 2926, 2859, 1614, 1512, 1450, 1382, 1338, 1134, 829, 543 cm – 1; for 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) spectral data, see Table 1; HR-TOF-MS: m/z 299.1658 [M þ H]þ (calcd for C19H23O3, 299.1647). 3.3.2
Compound 2
Yellow – orange amorphous solid; UV [MeOH – H2O (1:1)] lmax (log 1): 204 (4.66), 290 (4.19), 340 (4.14) nm; IR (KBr) nmax: 3443, 1627, 1600, 1400, 1337, 1115, 539 cm – 1; for 1H NMR (acetone-d6, 400 MHz) and 13C NMR (acetone-d6, 100 MHz) spectral data, see Table 2; HRTOF-MS: m/z 373.1181 [M þ H]þ (calcd for C20H21O7, 373.1287); CD (MeOH): D1297 nm ¼ –12.5, D1336 nm ¼ þ 4.8. 3.4
Antimicrobial assay
S. aureus (ATCC 25923) and S. epidermidis (ATCC 35984) were used for testing antibacterial activity, and the MIC values were determined using the microbroth dilution method [12]. Briefly, an overnight culture of the strains in trypticase soy broth (TSB) was diluted 1:100 and incubated at 378C. Test
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B. Zhou and C.-X. Wan
compounds were dissolved into 2 mg/ml in acetone and then diluted in the medium and added to the test tubes, containing 2 ml sterile liquid TSB, at a series of concentrations (100, 50, 25, 12.5, 6.25, 3.13, 1.56 ml/tube), of which 100 ml per well was added in a 96-well plate. The same volume of acetone was used as a negative control. The plates were incubated at 378C overnight, and growth inhibition was determined by the change in OD590 at the end of the incubation period compared with the control. The MIC value was defined as the lowest concentration that prevented visible growth of the bacteria after incubation at 378C overnight. Acknowledgement This research work was financially supported by the Hi-Tech Project of Xinjiang Production and Construction Group (2006GJS19).
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[2] K. Rajandeep, H. Kaur, and A.S. Dhindsa, Int. J. Pharm. Sci. Res. 4, 2470 (2013). [3] Q.Y. Zhang and M. Ye, J. Chromatogr. A 1216, 1954 (2009). [4] D.M. Biondi, C. Rocco, and G. Ruberto, J. Nat. Prod. 66, 477 (2003). [5] D.M. Biondi, C. Rocco, and G. Ruberto, J. Nat. Prod. 68, 1099 (2005). [6] C.X. Wan, P.H. Zhang, J.G. Luo, and L. Y. Kong, J. Nat. Prod. 74, 683 (2011). [7] L. Kraut, R. Mues, and H.D. Zinsmeister, Phytochemistry 45, 1249 (1997). [8] H. Hayashi, S.L. Zhang, T. Nakaizumi, K. Shimura, M. Yamaguchi, K. Inoue, K. Sarsenk, M. Ito, and G. Honda, Chem. Pharm. Bull. 51, 1147 (2003). [9] F. Bohlmann, C. Zdero, H. Robinson, and R.M. King, Phytochemistry 20, 2245 (1981). [10] J.F. Stevens, M. Ivancic, V.L. Hsu, and M.L. Deinzer, Phytochemistry 44, 1575 (1997). [11] F. Bohlmann and W.R. Abraham, Phytochemistry 18, 1851 (1979). [12] S. Inui, T. Hosoya, Y. Shimamura, S. Masuda, T. Ogawa, H. Kobayashi, K. Shirafuji, R.T. Moli, I. Kozone, K. Shin-Ya, and S. Kumazawa, J. Agric. Food Chem. 60, 11765 (2012).
