- Email: [email protected]
The structural elucidation and antimicrobial activities of two isoflavane glycosides from Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao
Journal of Molecular Structure 1076 (2014) 535–538
Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
The structural elucidation and antimicrobial activities of two isoflavane glycosides from Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao Wang Qing-Hu ⇑, Han Na-ren-chao-ke-tu, Dai Na-yin-tai, Wang Xiu-lan, Ao Wu-Li-Ji College of Traditional Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A new isoflavane glycoside was
isolated from Astragalus membranaceus. Compound 1 is more active against Bacillus coagulans comparing to Streptomycin. Compound 2 is found to be similar than that of Griseofulvin against Fusarium oxysporum.
a r t i c l e
i n f o
Article history: Received 22 June 2014 Received in revised form 12 August 2014 Accepted 13 August 2014 Available online 23 August 2014 Keywords: Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao 20 ,50 -Dicarbonyl-30 ,40 dimethoxyisoflavanequinone-7-O-b-Dglucoside Spectroscopic methods Column chromatography Electrospray ionization mass spectrometry
a b s t r a c t Two isoflavane glycoside had been isolated from the EtOAc-soluble fraction of the roots of Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao. This is the first report on the structure elucidation of 20 ,50 -dicarbonyl-30 ,40 -dimethoxyisoflavanequinone-7-O-b-D-glucoside (1) based on spectroscopic methods including UV (Ultraviolet Spectrophotometry), IR (Infrared Absorption Spectroscopy), ESI-MS (Electrospray Ionization Mass Spectrometry), 1D NMR (Nuclear Magnetic Resonance Spectroscopy) and 2D NMR techniques. At the same time, antimicrobial activity of the two compounds was evaluated against various bacteria and fungi. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao (A. membranaceus), ‘‘Mengguhuangqi’’ in China, is a member
⇑ Corresponding author. Tel./fax: +86 0475 831424. E-mail address: [email protected] (Q.-H. Wang). http://dx.doi.org/10.1016/j.molstruc.2014.08.025 0022-2860/Ó 2014 Elsevier B.V. All rights reserved.
of the family Ranunculaceae, genus Paeonia and distributed throughout Inner Mongolia, Shanxi, Xinjiang of China. The roots of A. membranaceus is the most important tonic traditional Chinese medicine, and promotes the discharge of pus and the growth of new tissue [1,2]. Polysaccharide [3], saponins [4–6] and flavonoids [7–11] have been isolated from this plant. However, the secondary metabolites from A. membranaceus often differ when grown in
536
Q.-H. Wang et al. / Journal of Molecular Structure 1076 (2014) 535–538
different ecological environments. For aiming to discover structurally unique and bioactive compounds, two new compounds have been isolated from A. membranaceus (the plant indigenous to Xinganling, Inner Mongolia, China) in our previous study [12]. In continuation of our investigation for bioactive compounds, a new isoflavane glycoside, together with one known compound [13] (Fig. 1) from this plant for the first time, were isolated. The current paper describes the isolation and structure elucidation of two isoflavane glycosides and their antimicrobial activities are reported.
EtOAc crude extract (40 g) and n-BuOH crude extract (20 g). The EtOAc crude extract (40.0 g) was isolated by column chromatography on silica gel and gradiently eluted with CHCl3–CH3OH (60:1 to 10:1) to give 6 fractions (Fractions 1–6). Fraction 4 [300 mg, CHCl3–CH3OH(30:1)elute] was subjected to silica gel column chromatography using CHCl3–CH3OH with increasing polarity (40:1–10:1) to give 4 fractions (Fractions 4-1, 4-2, 4-3, 4-4). Fraction 4-2 was further chromatographed on Sephadex LH-20 column eluting with CH3OH, and then separated by semi-preparative HPLC (CH3OH–H2O, 31:69) yielding 1 (56 mg) and 2 (75 mg).
Experimental Results and discussion
General experimental procedures The UV spectra were recorded on a Shimadzu UV-2201 spectrometer. The IR spectra were recorded in KBr discs on a Thermo Nicolet 200 double beam spectrophotometer. The HR-ESI-MS spectra were measured on Bruker Daltonics Micro TOFQ. NMR spectra were measured on a Bruker AVAIVCE I—500 NMR spectrometer with tetramethylsilane (TMS) as the internal reference, and chemical shifts are expressed in d (ppm). Semi-preparative HPLC (High Performance Liquid Chromatography) was performed by using a Japanese liquid chromatograph (A Shimadzu LC20-AP pump, Shimadzu SPD-20A photodiode array detector, and Shimadzu CBM-20A software for data processing) equipped with a EZ0566 column (250 mm 10 mm, 10 lm). Column chromatography was performed by using silica gel (200–300 mesh, Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Fractions were monitored by TLC (silica gel GF25410–40 lm, Marine Chemical Factory, Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. Plant materials The roots of A. membranaceus, used as experimental material, were collected in Xinganling, Inner Mongolia of China, in August 2011, and identified by Prof. Buhebateer (Inner Mongolia University for Nationalities). A voucher (NO. 20110828) has been deposited in the School of Traditional Mongolian Medicine of Inner Mongolia University for Nationalities. Extraction and isolation The air dried roots of A. membranaceus, (3.0 kg) were powdered and extracted twice under reflux 95% EtOH (50 L). The combined extracts were concentrated under reduced pressure to afford a dark brown residue (484 g). the residue was suspended in distilled water (2 L) and partitioned with petroleum ether (PE), CHCl3, EtOAc and n-BuOH. Each fraction was concentrated under reduced pressure to give PE crude extract (38 g), CHCl3 crude extract (60 g),
Structure elucidation Compound 1, a red amorphous powder, was assigned the molecular formula C23H26O11 by HR-ESI-MS at m/z 477.1383 [M–H]. UV (MeOH) kmax (nm) (log e): 267 (3.96) and 386 (2.07); IR (KBr) mmax (cm1): 1724, 1713, 1601, 1490, 1211 and 1102 cm1. In the 1H NMR spectrum (Table 1), the signals of four aromatic protons at dH 6.87 (1H, d, J = 8.0 Hz, H-5), 6.31 (1H, dd, J = 8.5, 2.5 Hz, H-6), 6.19 (1H, d, J = 2.5 Hz, H-8), and 6.44 (1H, s, H-60 ) indicated the presence of an ABX system. Likewise a singlet resonance in the aromatic region at dH 6.44 (1H, s) was assigned to H-60 on the basis of long-range correlations of dC 183.4 (C-20 ) and 145.7 (C-50 ). In addition, the 1H NMR spectrum showed characteristic signals of two methoxyl at d 3.68 (3H, s, –OCH3), 3.74 (3H, s, –OCH3), and an anomeric protons at d 4.77 (1H, d, J = 7.5 Hz, H-100 ). The 13C NMR signals also proved the presence of the methoxyl groups (dC 60.7, 56.1), and aromatic rings (dC 157.1, 154.7, 146.7, 145.7, 144.9, 130.8, 130.6, 111.8, 108.9, 103.1) and two carbonyl groups (dC 184.3, 183.4). All protonated carbons were assigned by analysis of the HSQC (Heteronuclear Singular Quantum Correlation) and DEPT (Distortionless Enhancement by Polarization Transfer) spectrum. Further analysis of the data of 1 showed that it was similar to the known compound (Penduline) from A. membranaceus [14] except for the absence of the sugar moiety (dC 101.1, 77.4, 77.0, 73.7, 70.2, 61.2). As observed, the HMBC correlation from H-100 (dH 4.77) to C-7 (dc 157.1) suggested that the b-D-glucose was attached to C-7. The HMBC correlations (Fig. 2) from H-5 (dH 6.87) to C-7 (dc 157.1), C-9 (dc 154.7), and H-6 (dH 6.31) to C-8 (dc 103.1), C-10 (dc 111.8), and H-8 (dH 6.19) to C-6 (dc 108.9), C-10 (dc 111.8), and H-60 (dH 6.44) to C-20 (dc 183.4), C-40 (dc 145.7), and H-2 (dH 4.19, 3.96) to C-3 (dc 31.8), C-4 (dc 30.2), C-10 (dc 146.7), H-4 (dH 2.92, 2.81) to C-5 (dc 130.8), C-10 (dc 146.7) confirmed further the structure of 1. The relative configuration of C-3 in compound 1 was identified by comparison of these data with those of 3-(2-hydroxy-3,4-dimethoxy-phenyl)-chroman-4,7-diol7-O-b-D-glucopyranoside [15], of which the proton signal at dH 3.69 (H-2a) was b-oriented and 4.27 (H-2b) was a-oriented. In
Fig. 1. Structures of compounds 1 and 2.
537
Q.-H. Wang et al. / Journal of Molecular Structure 1076 (2014) 535–538 Table 1 H and 13C NMR data for compound 1.
1
dH (DMSO-d6, 500 MHz) 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 600 30 -OCH3 40 -OCH3
dc (DMSO-d6, 500 MHz)
4.19 (brd, 1H, J = 9.5 Hz); 3.96 (t, 1H, J = 10.0 Hz) 3.31 (m, 1H) 2.92 (dd, 1H, J = 10.5, 15.5 Hz); 2.81 (dd, 1H, J = 4.0, 15.5 Hz) 6.87 (d, 1H, J = 8.5 Hz) 6.31 (dd, 1H, J = 8.5, 2.5 Hz) 6.19 (d, 1H, J = 2.5 Hz)
6.44 4.77 3.19 3.25 3.14 3.28 3.68 3.74 3.68
(s, 1H) (d, 1H, J = 7.5 Hz) (m, 1H) (m, 1H) (m, 1H) (m, 1H) (m, 1H), 3.46 (m, 1H) (s, 3H) (s, 3H)
69.7 31.8 30.2
(C-8), 156.7 (C-9), 114.6 (C-10), 118.7 (C-10 ), 105.8 (C-20 ), 141.6 (C-30 ), 147.9 (C-40 ), 93.7 (C-50 ), 154.1 (C-60 ), 100.7 (C-100 ), 73.6 (C200 ), 76.9 (C-300 ), 70.1 (C-400 ), 77.5 (C-500 ), 61.0 (C-600 ), 101.5 (–OCH2O–). Compound 2 was identified as trifolinhizin by comparison of the physical, 1H NMR and 13C NMR data with the reported data [16]. Antimicrobial activity
130.8 108.9 157.1 103.1 154.7 111.8 146.7 183.4 144.9 145.7 184.3 130.6 101.1 73.7 77.0 70.2 77.4 61.2 56.1 60.7
The antimicrobial activity of compounds 1 and 2 were determined by filter paper disc diffusion method [17]. The various bacterial species were first incubated at 45 °C for 48 h. The sterile filter paper discs (6 mm) were soaked with standard antibacterial agent and various test samples and were dried at 50 °C. The discs were then placed on soft nutrient agar (2%) petri plates previously seeded with suspension of each bacterial species. The diameter of zone of inhibitions were measured at 37 ± 1 °C after 24 h. For antifungal activity, Sabourauds broth media [18] with 4% agar was used for the preparation of plates and incubated with spores and mycelium suspension of fungi obtained from one week old culture. The diameter of zones of inhibition were measured at 28 ± 1 °C after 48 h. The results are recorded in Table 2. Antimicrobial activity of the above two compounds was evaluated against various bacteria and fungi. The results reported in Table 2 show that compound 1 is more active against Bacillus coagulans while being less active against Proteus vulgaris comparing to Streptomycin. Compound 2 shows antibacterial activity similar to Streptomycin against Escherichia coli and is less than that of Streptomycin against Staphylococcus aureus. In case of fungicidal activity, compound 1 behaves similarly to Griseofulvin against Aspergillus niger and less than that of Griseofulvin against Fusarium oxysporum. Compound 2 is found to be similar to Griseofulvin against F. oxysporum and less than that of Griseofulvin against A. niger. Conclusion
Fig. 2. Selected HMBC correlations for 1.
the NOESY experiment, the correlation from H-2 (a-H) to H-3 suggested the a-orientation of H-3. Thus, the structure of 1 was established as 20 ,50 -dicarbonyl-30 ,40 -dimethoxyisoflavanequinone7-O-b-D-glucoside. The key correlations of HMBC were shown in Fig. 2. Compound 2, a yellow amorphous powder, was assigned the molecular formula C22H22O10 by HR-ESI-MS at m/z 445.1129 [M–H]. UV (MeOH) kmax (nm) (log e): 261 (3.76) ; IR (KBr) mmax (cm1): 1587, 1491, 1234 and 1124 cm1. 1H NMR (DMSO-d6, 500 MHz) d: 4.27 (2H, dd, J = 6.0, 4.5 Hz, H-2), 3.62 (1H, m, H-3), 5.57 (1H, d, J = 7.5 Hz, H-4), 7.36 (1H, d, J = 8.5 Hz, H-5), 6.69 (1H, dd, J = 8.5, 2.5 Hz, H-6), 6.55 (1H, d, J = 2.5 Hz, H-8), 6.96 (1H, s, 20 -H), 6.54 (1H, s, 50 -H), 4.83 (1H, d, J = 7.5 Hz, H-100 ), 5.95 (2H, s, –OCH2O–). 13C NMR (DMSO-d6, 125 MHz) d: 66.3 (C-2), 40.2 (C-3), 78.1 (C-4), 132.4 (C-5), 110.8 (C-6), 158.5 (C-7), 104.5
Two isoflavane glycoside were isolated from the EtOAc-soluble fraction of the roots of A. membranaceus. Compound 1 is more active against B. coagulans comparing to Streptomycin and compound 2 shows similar than that of Streptomycin against E. coli. Compound 1 shows similar than that of Griseofulvin against Aspergillus niger and compound 2 is found to be similar than that of Griseofulvin against F. oxysporum. Acknowledgement The authors thank Ning Xu and Narenchaoketu for the measurements of NMR spectra. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molstruc.2014.08. 025.
Table 2 Antibacterial and antifungal activity of compounds. Compd.
1 2 Streptomycin
Antibacterial activity (zone of inhibition in mm)
Antifungal activity (zone of inhibition in mm)
E. coli
S. aureus
B. coagulans
P. vulgaris
P. digitatum
F. oxysporum
A. niger
15.32 19.32 19.61
16.89 11.35 18.54
21.10 15.21 19.69
12.74 17.25 18.96
13.37 15.36 17.61
12.86 19.04 20.91
15.24 13.02 16.02
Griseofulvin
538
Q.-H. Wang et al. / Journal of Molecular Structure 1076 (2014) 535–538
References [1] P.C. State, Chinese Pharmacopoeia, China Medical Pharmaceutical Science and Technology Publishing House, Beijing, 2010. p. 283. [2] W.S.T. Neimenggu, Standard of Traditional Mongolian Medicinal Materials, Inner Mongolia Science and Technology Press, Chifeng, 1987. p. 245. [3] Q.S. Huang, G.B. Lu, Y.C. Li, J.H. Guo, R.X. Wang, Acta. Pharm. Sin. 17 (1982) 200. [4] Y.Y. Bian, J. Guan, Z.M. Bi, Y. Song, P. Li, Chin. Pharm. J. 41 (2006) 1217. [5] Z. Luo, M.Z. Su, M. Yan, G.B. Shi, Q.C. Zhao, Chin. Trad. Herb. Drug. 43 (2012) 458. [6] Y.H. Wen, L. Cheng, D. Zheng, X.S. Huang, L. Han, Prac. Pharm. Clin. Rem. 13 (2010) 115. [7] M. Zhao, J.A. Duan, W.Z. Huang, R.H. Zhou, Z.T. Che, J. Chin. Pharm. Univ. 33 (2002) 274. [8] J. Sun, L. Zhang, X.L. Zhang, R.Q. Yan, X. Chai, Y.F. Wang, Drugs Clinic 28 (2013) 138.
[9] Z.M. Bi, Q.T. Yu, P. Li, Y. Lin, X.D. Gao, Chin. J. Nat. Med. 15 (2007) 263. [10] R.F. Li, Y.Z. Zhou, L. Qiao, H.W. Fu, Y.H. Fei, J. Shen, Pharm. Univ. 24 (2007) 20. [11] J.L. Wang, H.M. Xu, W.H. Li, Z. Hua, S.J. Zhang, Chin. J. Chin. Mater. Med. 33 (2008) 414. [12] Q.H. Wang, N.R.C.K.T. Han, N.Y.T. Dai, X.L. Wang, W.L.J. Ao, J. Mol. Struct. 1074 (2014) 284. [13] W. Liu, J. Chen, W.J. Zuo, X. Li, J.H. Wang, Chin. Chem. Lett. 18 (2007) 1092. [14] M. Zhao, J.A. Duan, W.Z. Huang, R.H. Zhou, Z.T. Che, J. Chin. Pharm. Univ. 33 (2002) 274. [15] W. Liu, J. Chen, W.J. ZUO, X. Li, J.H. Wang, Chin. Chem. Lett. 18 (2007) 1092. [16] N. Yang, B.H. Liang, K. Srivastava, J. Zneg, J.X. Zhan, L. Brown, H. Sampson, J. Goldfarb, C. Emala, X.M. Li, Phytochemistry 95 (2013) 259. [17] P.P. Deohate, B.N. Berad, Indian J. Chem. 44 (2005) 638. [18] S. Maddila, L. Palakondu, V.R. Chunduri, Der. Pharm. Lett. 2 (2010) 393.
Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
The structural elucidation and antimicrobial activities of two isoflavane glycosides from Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao Wang Qing-Hu ⇑, Han Na-ren-chao-ke-tu, Dai Na-yin-tai, Wang Xiu-lan, Ao Wu-Li-Ji College of Traditional Mongolian Medicine, Inner Mongolia University for Nationalities, Tongliao 028000, China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
A new isoflavane glycoside was
isolated from Astragalus membranaceus. Compound 1 is more active against Bacillus coagulans comparing to Streptomycin. Compound 2 is found to be similar than that of Griseofulvin against Fusarium oxysporum.
a r t i c l e
i n f o
Article history: Received 22 June 2014 Received in revised form 12 August 2014 Accepted 13 August 2014 Available online 23 August 2014 Keywords: Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao 20 ,50 -Dicarbonyl-30 ,40 dimethoxyisoflavanequinone-7-O-b-Dglucoside Spectroscopic methods Column chromatography Electrospray ionization mass spectrometry
a b s t r a c t Two isoflavane glycoside had been isolated from the EtOAc-soluble fraction of the roots of Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao. This is the first report on the structure elucidation of 20 ,50 -dicarbonyl-30 ,40 -dimethoxyisoflavanequinone-7-O-b-D-glucoside (1) based on spectroscopic methods including UV (Ultraviolet Spectrophotometry), IR (Infrared Absorption Spectroscopy), ESI-MS (Electrospray Ionization Mass Spectrometry), 1D NMR (Nuclear Magnetic Resonance Spectroscopy) and 2D NMR techniques. At the same time, antimicrobial activity of the two compounds was evaluated against various bacteria and fungi. Ó 2014 Elsevier B.V. All rights reserved.
Introduction Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao (A. membranaceus), ‘‘Mengguhuangqi’’ in China, is a member
⇑ Corresponding author. Tel./fax: +86 0475 831424. E-mail address: [email protected] (Q.-H. Wang). http://dx.doi.org/10.1016/j.molstruc.2014.08.025 0022-2860/Ó 2014 Elsevier B.V. All rights reserved.
of the family Ranunculaceae, genus Paeonia and distributed throughout Inner Mongolia, Shanxi, Xinjiang of China. The roots of A. membranaceus is the most important tonic traditional Chinese medicine, and promotes the discharge of pus and the growth of new tissue [1,2]. Polysaccharide [3], saponins [4–6] and flavonoids [7–11] have been isolated from this plant. However, the secondary metabolites from A. membranaceus often differ when grown in
536
Q.-H. Wang et al. / Journal of Molecular Structure 1076 (2014) 535–538
different ecological environments. For aiming to discover structurally unique and bioactive compounds, two new compounds have been isolated from A. membranaceus (the plant indigenous to Xinganling, Inner Mongolia, China) in our previous study [12]. In continuation of our investigation for bioactive compounds, a new isoflavane glycoside, together with one known compound [13] (Fig. 1) from this plant for the first time, were isolated. The current paper describes the isolation and structure elucidation of two isoflavane glycosides and their antimicrobial activities are reported.
EtOAc crude extract (40 g) and n-BuOH crude extract (20 g). The EtOAc crude extract (40.0 g) was isolated by column chromatography on silica gel and gradiently eluted with CHCl3–CH3OH (60:1 to 10:1) to give 6 fractions (Fractions 1–6). Fraction 4 [300 mg, CHCl3–CH3OH(30:1)elute] was subjected to silica gel column chromatography using CHCl3–CH3OH with increasing polarity (40:1–10:1) to give 4 fractions (Fractions 4-1, 4-2, 4-3, 4-4). Fraction 4-2 was further chromatographed on Sephadex LH-20 column eluting with CH3OH, and then separated by semi-preparative HPLC (CH3OH–H2O, 31:69) yielding 1 (56 mg) and 2 (75 mg).
Experimental Results and discussion
General experimental procedures The UV spectra were recorded on a Shimadzu UV-2201 spectrometer. The IR spectra were recorded in KBr discs on a Thermo Nicolet 200 double beam spectrophotometer. The HR-ESI-MS spectra were measured on Bruker Daltonics Micro TOFQ. NMR spectra were measured on a Bruker AVAIVCE I—500 NMR spectrometer with tetramethylsilane (TMS) as the internal reference, and chemical shifts are expressed in d (ppm). Semi-preparative HPLC (High Performance Liquid Chromatography) was performed by using a Japanese liquid chromatograph (A Shimadzu LC20-AP pump, Shimadzu SPD-20A photodiode array detector, and Shimadzu CBM-20A software for data processing) equipped with a EZ0566 column (250 mm 10 mm, 10 lm). Column chromatography was performed by using silica gel (200–300 mesh, Marine Chemical Factory, Qingdao, China) and Sephadex LH-20 (Pharmacia, Uppsala, Sweden). Fractions were monitored by TLC (silica gel GF25410–40 lm, Marine Chemical Factory, Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H2SO4 in EtOH. Plant materials The roots of A. membranaceus, used as experimental material, were collected in Xinganling, Inner Mongolia of China, in August 2011, and identified by Prof. Buhebateer (Inner Mongolia University for Nationalities). A voucher (NO. 20110828) has been deposited in the School of Traditional Mongolian Medicine of Inner Mongolia University for Nationalities. Extraction and isolation The air dried roots of A. membranaceus, (3.0 kg) were powdered and extracted twice under reflux 95% EtOH (50 L). The combined extracts were concentrated under reduced pressure to afford a dark brown residue (484 g). the residue was suspended in distilled water (2 L) and partitioned with petroleum ether (PE), CHCl3, EtOAc and n-BuOH. Each fraction was concentrated under reduced pressure to give PE crude extract (38 g), CHCl3 crude extract (60 g),
Structure elucidation Compound 1, a red amorphous powder, was assigned the molecular formula C23H26O11 by HR-ESI-MS at m/z 477.1383 [M–H]. UV (MeOH) kmax (nm) (log e): 267 (3.96) and 386 (2.07); IR (KBr) mmax (cm1): 1724, 1713, 1601, 1490, 1211 and 1102 cm1. In the 1H NMR spectrum (Table 1), the signals of four aromatic protons at dH 6.87 (1H, d, J = 8.0 Hz, H-5), 6.31 (1H, dd, J = 8.5, 2.5 Hz, H-6), 6.19 (1H, d, J = 2.5 Hz, H-8), and 6.44 (1H, s, H-60 ) indicated the presence of an ABX system. Likewise a singlet resonance in the aromatic region at dH 6.44 (1H, s) was assigned to H-60 on the basis of long-range correlations of dC 183.4 (C-20 ) and 145.7 (C-50 ). In addition, the 1H NMR spectrum showed characteristic signals of two methoxyl at d 3.68 (3H, s, –OCH3), 3.74 (3H, s, –OCH3), and an anomeric protons at d 4.77 (1H, d, J = 7.5 Hz, H-100 ). The 13C NMR signals also proved the presence of the methoxyl groups (dC 60.7, 56.1), and aromatic rings (dC 157.1, 154.7, 146.7, 145.7, 144.9, 130.8, 130.6, 111.8, 108.9, 103.1) and two carbonyl groups (dC 184.3, 183.4). All protonated carbons were assigned by analysis of the HSQC (Heteronuclear Singular Quantum Correlation) and DEPT (Distortionless Enhancement by Polarization Transfer) spectrum. Further analysis of the data of 1 showed that it was similar to the known compound (Penduline) from A. membranaceus [14] except for the absence of the sugar moiety (dC 101.1, 77.4, 77.0, 73.7, 70.2, 61.2). As observed, the HMBC correlation from H-100 (dH 4.77) to C-7 (dc 157.1) suggested that the b-D-glucose was attached to C-7. The HMBC correlations (Fig. 2) from H-5 (dH 6.87) to C-7 (dc 157.1), C-9 (dc 154.7), and H-6 (dH 6.31) to C-8 (dc 103.1), C-10 (dc 111.8), and H-8 (dH 6.19) to C-6 (dc 108.9), C-10 (dc 111.8), and H-60 (dH 6.44) to C-20 (dc 183.4), C-40 (dc 145.7), and H-2 (dH 4.19, 3.96) to C-3 (dc 31.8), C-4 (dc 30.2), C-10 (dc 146.7), H-4 (dH 2.92, 2.81) to C-5 (dc 130.8), C-10 (dc 146.7) confirmed further the structure of 1. The relative configuration of C-3 in compound 1 was identified by comparison of these data with those of 3-(2-hydroxy-3,4-dimethoxy-phenyl)-chroman-4,7-diol7-O-b-D-glucopyranoside [15], of which the proton signal at dH 3.69 (H-2a) was b-oriented and 4.27 (H-2b) was a-oriented. In
Fig. 1. Structures of compounds 1 and 2.
537
Q.-H. Wang et al. / Journal of Molecular Structure 1076 (2014) 535–538 Table 1 H and 13C NMR data for compound 1.
1
dH (DMSO-d6, 500 MHz) 2 3 4 5 6 7 8 9 10 10 20 30 40 50 60 100 200 300 400 500 600 30 -OCH3 40 -OCH3
dc (DMSO-d6, 500 MHz)
4.19 (brd, 1H, J = 9.5 Hz); 3.96 (t, 1H, J = 10.0 Hz) 3.31 (m, 1H) 2.92 (dd, 1H, J = 10.5, 15.5 Hz); 2.81 (dd, 1H, J = 4.0, 15.5 Hz) 6.87 (d, 1H, J = 8.5 Hz) 6.31 (dd, 1H, J = 8.5, 2.5 Hz) 6.19 (d, 1H, J = 2.5 Hz)
6.44 4.77 3.19 3.25 3.14 3.28 3.68 3.74 3.68
(s, 1H) (d, 1H, J = 7.5 Hz) (m, 1H) (m, 1H) (m, 1H) (m, 1H) (m, 1H), 3.46 (m, 1H) (s, 3H) (s, 3H)
69.7 31.8 30.2
(C-8), 156.7 (C-9), 114.6 (C-10), 118.7 (C-10 ), 105.8 (C-20 ), 141.6 (C-30 ), 147.9 (C-40 ), 93.7 (C-50 ), 154.1 (C-60 ), 100.7 (C-100 ), 73.6 (C200 ), 76.9 (C-300 ), 70.1 (C-400 ), 77.5 (C-500 ), 61.0 (C-600 ), 101.5 (–OCH2O–). Compound 2 was identified as trifolinhizin by comparison of the physical, 1H NMR and 13C NMR data with the reported data [16]. Antimicrobial activity
130.8 108.9 157.1 103.1 154.7 111.8 146.7 183.4 144.9 145.7 184.3 130.6 101.1 73.7 77.0 70.2 77.4 61.2 56.1 60.7
The antimicrobial activity of compounds 1 and 2 were determined by filter paper disc diffusion method [17]. The various bacterial species were first incubated at 45 °C for 48 h. The sterile filter paper discs (6 mm) were soaked with standard antibacterial agent and various test samples and were dried at 50 °C. The discs were then placed on soft nutrient agar (2%) petri plates previously seeded with suspension of each bacterial species. The diameter of zone of inhibitions were measured at 37 ± 1 °C after 24 h. For antifungal activity, Sabourauds broth media [18] with 4% agar was used for the preparation of plates and incubated with spores and mycelium suspension of fungi obtained from one week old culture. The diameter of zones of inhibition were measured at 28 ± 1 °C after 48 h. The results are recorded in Table 2. Antimicrobial activity of the above two compounds was evaluated against various bacteria and fungi. The results reported in Table 2 show that compound 1 is more active against Bacillus coagulans while being less active against Proteus vulgaris comparing to Streptomycin. Compound 2 shows antibacterial activity similar to Streptomycin against Escherichia coli and is less than that of Streptomycin against Staphylococcus aureus. In case of fungicidal activity, compound 1 behaves similarly to Griseofulvin against Aspergillus niger and less than that of Griseofulvin against Fusarium oxysporum. Compound 2 is found to be similar to Griseofulvin against F. oxysporum and less than that of Griseofulvin against A. niger. Conclusion
Fig. 2. Selected HMBC correlations for 1.
the NOESY experiment, the correlation from H-2 (a-H) to H-3 suggested the a-orientation of H-3. Thus, the structure of 1 was established as 20 ,50 -dicarbonyl-30 ,40 -dimethoxyisoflavanequinone7-O-b-D-glucoside. The key correlations of HMBC were shown in Fig. 2. Compound 2, a yellow amorphous powder, was assigned the molecular formula C22H22O10 by HR-ESI-MS at m/z 445.1129 [M–H]. UV (MeOH) kmax (nm) (log e): 261 (3.76) ; IR (KBr) mmax (cm1): 1587, 1491, 1234 and 1124 cm1. 1H NMR (DMSO-d6, 500 MHz) d: 4.27 (2H, dd, J = 6.0, 4.5 Hz, H-2), 3.62 (1H, m, H-3), 5.57 (1H, d, J = 7.5 Hz, H-4), 7.36 (1H, d, J = 8.5 Hz, H-5), 6.69 (1H, dd, J = 8.5, 2.5 Hz, H-6), 6.55 (1H, d, J = 2.5 Hz, H-8), 6.96 (1H, s, 20 -H), 6.54 (1H, s, 50 -H), 4.83 (1H, d, J = 7.5 Hz, H-100 ), 5.95 (2H, s, –OCH2O–). 13C NMR (DMSO-d6, 125 MHz) d: 66.3 (C-2), 40.2 (C-3), 78.1 (C-4), 132.4 (C-5), 110.8 (C-6), 158.5 (C-7), 104.5
Two isoflavane glycoside were isolated from the EtOAc-soluble fraction of the roots of A. membranaceus. Compound 1 is more active against B. coagulans comparing to Streptomycin and compound 2 shows similar than that of Streptomycin against E. coli. Compound 1 shows similar than that of Griseofulvin against Aspergillus niger and compound 2 is found to be similar than that of Griseofulvin against F. oxysporum. Acknowledgement The authors thank Ning Xu and Narenchaoketu for the measurements of NMR spectra. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molstruc.2014.08. 025.
Table 2 Antibacterial and antifungal activity of compounds. Compd.
1 2 Streptomycin
Antibacterial activity (zone of inhibition in mm)
Antifungal activity (zone of inhibition in mm)
E. coli
S. aureus
B. coagulans
P. vulgaris
P. digitatum
F. oxysporum
A. niger
15.32 19.32 19.61
16.89 11.35 18.54
21.10 15.21 19.69
12.74 17.25 18.96
13.37 15.36 17.61
12.86 19.04 20.91
15.24 13.02 16.02
Griseofulvin
538
Q.-H. Wang et al. / Journal of Molecular Structure 1076 (2014) 535–538
References [1] P.C. State, Chinese Pharmacopoeia, China Medical Pharmaceutical Science and Technology Publishing House, Beijing, 2010. p. 283. [2] W.S.T. Neimenggu, Standard of Traditional Mongolian Medicinal Materials, Inner Mongolia Science and Technology Press, Chifeng, 1987. p. 245. [3] Q.S. Huang, G.B. Lu, Y.C. Li, J.H. Guo, R.X. Wang, Acta. Pharm. Sin. 17 (1982) 200. [4] Y.Y. Bian, J. Guan, Z.M. Bi, Y. Song, P. Li, Chin. Pharm. J. 41 (2006) 1217. [5] Z. Luo, M.Z. Su, M. Yan, G.B. Shi, Q.C. Zhao, Chin. Trad. Herb. Drug. 43 (2012) 458. [6] Y.H. Wen, L. Cheng, D. Zheng, X.S. Huang, L. Han, Prac. Pharm. Clin. Rem. 13 (2010) 115. [7] M. Zhao, J.A. Duan, W.Z. Huang, R.H. Zhou, Z.T. Che, J. Chin. Pharm. Univ. 33 (2002) 274. [8] J. Sun, L. Zhang, X.L. Zhang, R.Q. Yan, X. Chai, Y.F. Wang, Drugs Clinic 28 (2013) 138.
[9] Z.M. Bi, Q.T. Yu, P. Li, Y. Lin, X.D. Gao, Chin. J. Nat. Med. 15 (2007) 263. [10] R.F. Li, Y.Z. Zhou, L. Qiao, H.W. Fu, Y.H. Fei, J. Shen, Pharm. Univ. 24 (2007) 20. [11] J.L. Wang, H.M. Xu, W.H. Li, Z. Hua, S.J. Zhang, Chin. J. Chin. Mater. Med. 33 (2008) 414. [12] Q.H. Wang, N.R.C.K.T. Han, N.Y.T. Dai, X.L. Wang, W.L.J. Ao, J. Mol. Struct. 1074 (2014) 284. [13] W. Liu, J. Chen, W.J. Zuo, X. Li, J.H. Wang, Chin. Chem. Lett. 18 (2007) 1092. [14] M. Zhao, J.A. Duan, W.Z. Huang, R.H. Zhou, Z.T. Che, J. Chin. Pharm. Univ. 33 (2002) 274. [15] W. Liu, J. Chen, W.J. ZUO, X. Li, J.H. Wang, Chin. Chem. Lett. 18 (2007) 1092. [16] N. Yang, B.H. Liang, K. Srivastava, J. Zneg, J.X. Zhan, L. Brown, H. Sampson, J. Goldfarb, C. Emala, X.M. Li, Phytochemistry 95 (2013) 259. [17] P.P. Deohate, B.N. Berad, Indian J. Chem. 44 (2005) 638. [18] S. Maddila, L. Palakondu, V.R. Chunduri, Der. Pharm. Lett. 2 (2010) 393.