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Flavonoids from Heartwood of Dalbergia cochinchinensis
Liu RH et al. Chinese Herbal Medicines, 2016, 8(1): 89-93
89
Available online at SciVarse ScienceDirect
Chinese Herbal Medicines (CHM) ISSN 1674-6384
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E-mail: [email protected]
Original article
Flavonoids from Heartwood of Dalbergia cochinchinensis Rong-hua Liu*, Xin-chao Wen, Feng Shao, Pu-zhao Zhang, Hui-lian Huang, Shuang Zhang Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
ARTICLE INFO Article history Received: June 21, 2015 Revised: July 21, 2015 Accepted: August 19, 2015 Available online: January 15, 2016
DOI: 10.1016/S1674-6384(16)60014-X
ABSTRACT Objective
Methods
To study the flavonoids from the heartwood of Dalbergia cochinchinensis.
The chemical constituents were isolated and purified by combination of silica
gel, macroporous resin, Sephadex LH-20, and ODS column chromatography. Their structures were identified by means of spectral analysis. Results
Fifteen flavonoids were
isolated and identified as pinocembrin (1), liquiritigenin (2), galangin (3), 7-hydroxy6-methoxyflavone (4), naringenin (5), alpinetin (6),
2,3-dimethoxyxanthone (7),
6,4′-dihydroxy-7-methoxy-flavan (8), mucronulatol (9), 7,8-dihydroxyflavanone (10),
5,7,3′,5′-tetrahydroxyflavanone
(11),
4,2′,5′-trihydroxy-4′-methoxychalcone
(12),
isoliquiritigenin (13), butein (14), and 3′,5′,5,7-tetrahydroxy-6-C-β-D-glucopyranosyl-
flavanone (15), respectively. Conclusion Compounds 7, 8, 10, and 15 are isolated from the
plants of Dalbergia L. f. for the first time, and compounds 1, 3, 5, 6, 9, 11, 12, and 14 are
isolated from this plant for the first time.
Key words
Dalbergia cochinchinensis; 2,3-dimethoxyxanthone; 5,4′-dihydroxy-7-methoxyflavan;
7,8-dihydroxyflavanone; flavonoids
1. Introduction Dalbergia cochinchinensis Pierre ex Laness (Fabaceae), the Thailand rosewood or Siamese rosewood, is perennial non-climbing tree widely distributed in Indochina like Thailand, Vietnam, Laos, and Cambodia, etc. The plant is also cultivated in Xishuangbanna, Yunnan province, China (He et al, 2014). In Thailand, its core is used in folk medicine for the treatment of tumor and blood stasis (Palasuwan et al, 2005). Previous chemical investigations on this plant had led to the identification of a series of flavonoids, terpenes and benzoic acids (Donnelly et al, 1968; Pathak et al, 1997; Shirota et al, 2003; Zhong et al, 2013; Liu et al, 2015). Among all the constituents mentioned above, flavonoids are increasingly recognized as playing
© 2016 published by TIPR Press. All rights reserved.
potentially important roles in health including but not limited to their roles as antioxidants, free radical scavengers, metal chelators, and inhibiting lipid peroxidation (Peterson et al, 1998; Cook et al, 1996). Ten flavonoids have been isolated and identified from the heartwood of D. cochinchinensis so far (Pathak et al, 1997; Shirota et al, 2003). Further phytochemical investigation on D. cochinchinensis led to the isolation and characterization of fifteen flavonoids including seven flavanones (1, 2, 5, 6, 10, 11, and 15), a flavone (4), a flavonol (3), three chalcones (12, 13, and 14), a xanthone (7), a flavan (8), and an isoflavan (9). Among the flavonoids, compounds 7, 8, and 10 are reported for the first time from the plants of Dalbergia L. f., and compounds 1, 3, 5, 6, 9, 11, 12, and 14 are reported for the first time from this plant (Figure 1).
*Corresponding author: Liu RH
Tel: +86-791-8711 8918 E-mail: [email protected]
Fund: National Natural Science Foundation of China (81360629)
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Liu RH et al. Chinese Herbal Medicines, 2015, 8(1): 89-93
R6 R5 R2
O
R4
R3 R1 O 1 R1=R2=OH R3=R4=R5=H R3 O 2 R2=R5=OH R1=R3=R4=H 5 R1=R2=R5=OH R3=R4=H R2 R4 6 R1=OCH3 R2=OH R3=R4=R5=H R O 10 R2=R3=OH R1=R4=R5=H 1 11 R1=R2=R4=R6=OH R3=H 3 R1=R3=R4=OH R2=H 15 R1=R2=R4=R6=OH R3=Glc 4 R2=OCH3 R3=OH R1=R4=H
OH O
O
HO 8 Figure 1
Chemical structures of compounds 1-15 isolated from heartwood of Dalbergia cochinchinensis
2. Materials and methods
2.1 General The NMR spectra were recorded on a Bruker AVANCE III HD 400 MHz Instrument in deuterated chloroform and methanol with TMS as internal standard. ESI-MS was recorded on an Agilent 1100 Series LC/MSD Trap. Silica gel (100−200 and 200−300 mesh, Qingdao Marine Chemical, China), ODS gel (YMC, Japan), macroporous resin (D101, Tianjin Haiguang Chemical, China) and Sephadex LH-20 (GE Healthcare, Swiss) were used for column chromatography. TLC was performed on silica gel GF254 plates (Qingdao Marine Chemical Factory) and visualized with 10% sulphuric acid in ethanol.
2.2 Plant material The heartwood of Dalbergia cochinchinensis collected from Fangchenggang City of Guangxi Zhuang Autonomous Region, China, in June 2013, and identified by Prof. Ke-zhong Deng at Jiangxi University of Traditional Chinese Medicine. A voucher specimen (201306) was deposited at the Herbarium of Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine.
2.3 Extraction and isolation The dried sliced heartwood of D. cochinchinensis (20 kg) was extracted with 70% EtOH (reflux, 2 h, three times). After the removal of ethanol by concentrated under reduced
pressure, the extract (4.2 kg, 5.4 kg in total) was suspended in water and partitioned sequentially with petroleum ether (60−90 oC), chloroform, EtOAc, and n-BuOH, respectively. The petroleum ether extract (381.2 g) was separated into nine fractions (Frs. 1−9) by applying silica gel column, eluted with petroleum ether-EtOAc (50:1 to 1:1). Fr. 5 was purified by repeated silica gel and Sephadex LH-20 with CH2Cl2MeOH (1:1) to give compound 1 (932.2 mg). The chloroform extract (902.6 g) was separated into 9 fractions (Frs. 1−9) by adopting silica gel column and eluted with gradient mixture of petroleum ether- EtOAc (50:1 to 1:1). Fr. 4 was subjected to silica gel using CH2Cl2-MeOH (100:1 to 1:1), followed by Sephadex LH-20 with CH2Cl2-MeOH (1:1) to give compounds 2 (529.2 mg), 3 (11.9 mg), and 4 (421.5 mg). Fr. 5 was separated over silica gel with CH2Cl2-MeOH (50:1 to 1:1), purified by Sephadex LH-20 using CH2Cl2-MeOH (1:1) as an eluent to afford compounds 5 (57.9 mg) and 6 (14.2 mg). Compounds 7 (1.5 mg), 8 (576.4 mg), and 9 (35.5 mg) were obtained by applying silica gel, eluted with CH2Cl2-MeOH (50:1 to 1:1) and Sephadex LH-20 with CH2Cl2-MeOH (1:1). The EtOAc extract (1397.7 g) was separated into 23 fractions (Fr. 1-23) by the method of silica gel column using CH2Cl2-MeOH (100:0 to 1:1) as eluent. Fr. 6 was subjected to silica gel eluted with gradient mixture of CH2Cl2-MeOH (80:1 to 1:1), purified by Sephadex LH-20 using CH2Cl2MeOH (1:1) to give compound 10 (88.9 mg). Fr. 10 was separated by the means of silica gel with CH2Cl2-MeOH (50:1 to 1:1), followed by Sephadex LH-20 using CH2Cl2-MeOH (1:1) and ODS gel eluted with 50% MeOH to give compounds 11 (10.9 mg) and 12 (10.9 mg). Fr. 16 was
Liu RH et al. Chinese Herbal Medicines, 2015, 8(1): 89-93 separated over silica gel, eluted with CH2Cl2-MeOH (50:1 to 1:1), next ODS gel using 65% MeOH as an eluent to obtain compounds 13 (90.4 mg) and 14 (6.9 mg). The n-BuOH extract (462.7 g) was separated into 10 fractions (Fr. 1-10) via macroporous resin column, eluted with EtOH-water (0:1 to 1:0). Fr. 4 was purified subsequently by Sephadex LH-20 with CH2Cl2-MeOH (1:1) and ODS gel using 40% MeOH to yield compound 15 (8.4 mg).
3. Results Compound 1: white needle crystal. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CDCl3, 400 MHz) δ: 12.04 (1H, s, OH), 7.55−7.33(5H, m, B ring), 6.01 (2H, s, H-6/8), 5.42 (1H, dd, J = 13.0, 3.0 Hz, H-2), 3.10 (1H, dd, J = 17.2, 13.0 Hz, H-3α), 2.82 (1H, dd, J = 17.2, 3.0 Hz, H-3β); 13C-NMR (CDCl3, 100 MHz) δ: 195.82 (C-4), 164.46 (C-5), 164.32 (C-9), 163.15 (C-7), 138.22 (C-1′), 128.92 (C-3′/5′), 128.89 (C-4′), 126.13 (C-2′/6′), 103.22 (C-10), 96.75 (C-6), 95.48 (C-8), 79.23 (C-2), 43.32 (C-3). Compound 1 was identified as pinocembrin by comparison of the physical, 1H-NMR, and 13 C-NMR data with the reported data (Aboushoer et al, 2010). Compound 2: yellow powder. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.74 (1H, d, J = 8.7 Hz, H-5), 7.32 (2H, d, J = 8.5 Hz, H-2′/6′), 6.81 (2H, d, J = 8.5 Hz, H-3′/5′), 6.49 (1H, dd, J = 8.7, 2.3 Hz, H-6), 6.35 (1H, d, J = 2.3 Hz, H-8), 5.38 (1H, dd, J = 13.1, 2.9 Hz, H-2), 3.05 (1H, dd, J = 16.9, 13.1 Hz, H-3ɑ), 2.69 (1H, dd, J = 16.9, 2.9 Hz, H-3β); 13C-NMR (CD3OD, 100 MHz) δ: 193.54 (C-4), 166.94 (C-7), 165.59 (C-9), 158.98 (C-4′), 131.35 (C-1′), 129.85 (C-5), 129.01 (C-2′/6′), 116.29 (C-3′,5′), 114.91 (C-10), 111.80 (C-6), 103.82 (C-8), 81.05 (C-2), 44.96 (C-3). Compound 2 was identified as liquiritigenin by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Zhao et al, 2011). Compound 3: yellow needle crystal. ESI-MS m/z: 271 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 8.16 (2H, d, J = 7.6 Hz, H-2′/6′), 7.46 (3H, dt, J = 14.5, 7.6 Hz, H-3′/4′/5′), 6.39 (1H, d, J =2.0 Hz, H-8), 6.17 (1H, d, J = 2.0 Hz, H-6); 13 C-NMR (CD3OD, 100 MHz) δ: 177.65 (C-4), 165.89 (C-7), 162.57 (C-5), 158.41 (C-9), 146.88 (C-2), 138.50 (C-3), 132.60 (C-1′), 130.87 (C-4′), 129.42 (C-2′/6′), 128.73 (C-3′/5′), 104.66 (C-10), 99.36 (C-6), 94.52 (C-8). Compound 3 was identified as galangin by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Kim et al, 2006). Compound 4: yellow needle crystal. ESI-MS m/z: 269 [M + H]+; 1H-NMR (CDCl3, 400 MHz) δ: 7.55 ~ 7.34 (5H, m, B ring), 6.98 (1H, s, H-8), 6.89 (1H, s, H-5), 6.24 (1H, s, H-3), 5.64 (1H, s, OH), 3.98 (3H, s, OCH3-6); 13C-NMR (CDCl3, 100 MHz) δ: 161.52 (C-4), 155.79 (C-2), 150.06 (C-6), 149.30 (C-9), 142.40 (C-7), 135.54 (C-1′), 129.56 (C-4′), 128.81 (C-3′,5′), 128.30 (C-2′,6′), 112.50 (C-3), 112.29 (C-10), 110.49 (C-8), 99.56 (C-5), 56.45 (OCH3-4′). Compound 4 was identified as 7-hydroxy-6-methoxyflavone by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Pathak et al, 1997).
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Compound 5: light yellow powder. ESI-MS m/z: 273 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.30 (2H, d, J = 8.5 Hz, H-2′/6′), 6.81 (2H, d, J = 8.5 Hz, H-3′/5′), 5.88 (2H, d, J = 2.1 Hz, H-6/8), 5.30 (1H, dd, J = 13.0, 3.0 Hz, H-2), 3.09 (1H, dd, J = 17.1, 13.0 Hz, H-3α), 2.67 (1H, dd, J = 17.1, 3.0 Hz, H-3β); 13C-NMR (CD3OD, 100 MHz) δ: 197.75 (C-4), 168.29 (C-7), 165.40 (C-5), 164.83 (C-9), 158.95 (C-4′), 131.02 (C-1′), 129.03 (C-2′, 6′), 116.29 (C-3′, 5′), 103.30 (C-10), 97.03 (C-6), 96.15 (C-8), 80.43 (C-2), 43.98 (C-3). Compound 5 was identified as naringenin by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Tang et al, 2012). Compound 6: colorless needle crystal. ESI-MS m/z: 271 [M + H]+; 1H-NMR (DMSO-d6, 400 MHz) δ: 10.58 (1H, br s, OH), 7.64−7.20 (5H, m, B ring), 6.07 (1H, d, J = 2.2 Hz, H-8), 6.01 (1H, d, J = 2.2 Hz, H-6), 5.48 (1H, dd, J = 12.4, 2.9 Hz, H-2), 3.74 (3H, s, OCH3-5), 2.97 (1H, dd, J = 16.3, 12.4 Hz, H-3α), 2.60 (1H, dd, J = 16.3, 2.9 Hz, H-3β); 13C-NMR (DMSO-d6, 100 MHz) δ: 187.37 (C-4), 164.44 (C-7), 164.05 (C-5), 162.22 (C-9), 139.19 (C-1′), 128.53 (C-3′/5′), 128.35 (C-4′), 126.46 (C-2′/6′), 104.47 (C-10), 95.66 (C-6), 93.37 (C-8), 78.05 (C-2), 55.65 (OCH3-7), 44.87 (C-3). Compound 6 was identified as alpinetin by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Li et al, 2004). Compound 7: light brown powder. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CDCl3, 400 MHz) δ: 8.35(1H, dd, J = 8.0, 1.7 Hz, H-8), 7.73−7.69 (1H, m, H-6), 7.68 (1H, s, H-1), 7.47 (1H, d, J =8.4 Hz, H-5), 7.38 (1H, t, J = 8.0 Hz, H-7), 6.94 (1H, s, H-4), 4.02 (3H, s, OCH3-3), 4.00 (3H, s, OCH3-2); 13 C-NMR (CDCl3, 100 MHz) δ: 176.13 (C-9), 156.05 (C-5a), 155.51 (C-4a), 152.45 (C-3), 146.63 (C-2), 133.99 (C-6), 126.54 (C-8), 123.77 (C-7), 121.50 (C-8a), 117.66 (C-5), 114.90 (C-1a), 105.36 (C-1), 99.62 (C-4), 56.38 (OCH3-3), 55.49 (OCH3-2). Compound 7 was identified as 2, 3-dimethoxyxanthone by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Dubrovskiy et al, 2013; Ali et al, 2014). Compound 8: light yellow powder. ESI-MS m/z: 273 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.20 (2H, d, J = 8.5 Hz, H-2′/H-6′), 6.77 (2H, d, J = 8.5 Hz, H-3′/H-5′), 6.50 (lH, s, H-8), 6.38 (1H, s, H-5), 4.80 (1H, d, J = 10.2 Hz, H-2), 3.75 (3H, s, 7-OCH3), 2.82 (1H, dddt, J = 16.3, 11.5, 6.0, 1.5 Hz, H-4α), 2.58 (1H, ddt, J = 16.3, 5.2, 2.2 Hz, H-4β), 2.03 (lH, ddq, J = 13.5, 6.0, 2.2 Hz, H-3α), 1.95 (1H, ddddd, J = 13.5, 11.5, 10.2, 5.2, 1.5 Hz, H-3β); 13C-NMR (CD3OD, 100 MHz) δ: 158.04 (C-4′), 149.61 (C-7), 148.04 (C-9), 140.97 (C-6), 134.33 (C-1′), 128.49 (C-2′/6′), 116.16 (C-5), 116.03 (C-3′/5′), 114.41 (C-10), 101.73 (C-8), 78.84 (C-2), 56.31 (OCH3-7), 31.25 (C-3), 25.64 (C-4). Compound 8 was identified as 6,4′-dihydroxy-7-methoxyflavan by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Pathak et al, 1997). Compound 9: white powder. ESI-MS m/z: 303 [M + H]+; 1 H-NMR (CD3OD, 400 MHz) δ: 6.86 (1H, d, J = 8.2 Hz, H-5), 6.69 (1H, d, J = 8.6 Hz, H-6′), 6.59 (1H, d, J = 8.6 Hz, H-5′), 6.31 (1H, dd, J = 8.2, 2.5 Hz, H-6), 6.23 (1H, d, J = 2.5 Hz,
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H-8), 4.17 (1H, ddd, J = 10.3, 3.5, 2.0 Hz, H-2α), 3.91 (1H, t, J = 10.3 Hz, H-2β), 3.83 (3H, s, OCH3-4′), 3.82 (1H, s, OCH3-2′), 3.42 (1H, tdd, J = 10.6, 5.4, 3.5 Hz, H-3), 2.88 (1H, dd, J = 15.5, 10.6 Hz, H-4α), 2.77 (1H, ddd, J = 15.5, 5.4, 2.0 Hz, H-4β); 13C-NMR (CD3OD, 100 MHz) δ: 156.18 (C-7), 154.86 (C-9), 147.66 (C-4′), 145.82 (C-2′), 139.23 (C-3′), 129.72 (C-5), 127.17 (C-6′), 116.25 (C-1′), 113.27 (C-10), 107.64 (C-6), 106.97 (C-5′), 102.37 (C-8), 70.18 (C-2), 55.20 (OCH3), 31.77 (C-3), 31.10 (C-4). Compound 9 was identified as mucronulatol by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Hamburger et al, 1987). Compound 10: light yellow powder. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.51−7.34 (5H, m, B ring), 5.91 (2H, dd, J = 12.0, 2.1 Hz, H-5/6), 5.44 (1H, dd, J = 12.8, 3.0 Hz, H-2), 3.07 (1H, dd, J = 17.1, 12.8 Hz, H-3ɑ), 2.75 (1H, dd, J = 17.1, 3.0Hz, H-3β); 13C-NMR (CD3OD, 100 MHz) δ: 195.90 (C-4), 166.98 (C-7), 164.04 (C-8), 163.23 (C-9), 138.97 (C-1′), 128.28 (C-3′, 5′), 128.21 (C-4′), 125.92 (C-2′, 6′), 101.93 (C-10), 95.73 (C-6), 94.79 (C-5), 79.02 (C-2), 42.76 (C-3). Compound 10 was identified as 7,8-dihydroxyflavanone by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Hahm et al, 2003). Compound 11: white powder. ESI-MS m/z: 289 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 12.05(1H, s, OH), 8.01(1H, s, OH), 6.92(1H, s, H-4′), 6.79 (1H, s, H-2′), 6.77 (1H, s, H-6′), 5.89 (1H, d, J = 2.2 Hz, H-6), 5.87 (1H, d, J = 2.2 Hz, H-8), 5.26 (1H, dd, J = 12.8, 3.0 Hz, H-2), 3.06 (1H, dd, J = 17.1, 12.8 Hz, H-3β), 2.68 (1H, dd, J = 17.1, 3.0 Hz, H-3α); 13C-NMR (CD3OD, 100 MHz) δ: 197.77 (C-4), 168.34 (C-7), 165.43 (C-5), 164.83 (C-9), 146.87 (C-4′), 146.49 (C-3′), 131.75 (C-1′), 119.25 (C-6′), 116.23 (C-5′), 114.69 (C-2′), 103.33 (C-10), 97.01 (C-6), 96.15 (C-8), 80.50 (C-2), 44.09 (C-3). Compound 11 was identified as 5,7,3′,5′tetrahydroxyflavanone by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Zheng et al, 2008). Compound 12: dark yellow powder. ESI-MS m/z: 287 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.78 (1H, d, J = 15.4 Hz, H-β), 7.61 (2H, d, J = 8.6 Hz, H-2/6), 7.52 (1H, d, J = 15.4 Hz, H-α), 7.42 (1H, s, H-6′), 6.84 (2H, d, J = 8.6 Hz, H-3/5), 6.50 (1H, s, H-3′), 3.91 (3H, s, -OCH3); 13C-NMR (CD3OD, 100 MHz) δ: 193.50 (C=O), 161.65 (C-4), 161.20 (C-2′), 156.92 (C-4′), 145.58 (C-β), 140.13 (C-5′), 131.87 (C-2/6), 127.78 (C-1), 118.33 (C-α), 116.95 (C-3/5), 115.01 (C-6′), 113.7 (C-1′), 101.00 (C-3′), 56.55 (OCH3-7). Compound 12 was identified as 4,2′,5′-trihydroxy-4′methoxychalcone by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (An et al, 2008). Compound 13: yellow powder. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.91 (1H, d, J = 8.9 Hz, H-6′), 7.74 (1H, d, J = 15.4 Hz, H-β), 7.56 (2H, d, J = 8.6 Hz, H-2/6), 7.54 (1H, d, J = 15.4 Hz, H-α), 6.83 (2H, d, J = 8.6 Hz, H-3/5), 6.40 (1H, dd, J = 8.9, 2.4 Hz, H-5′), 6.29 (1H, d, J = 2.4 Hz, H-3′); 13C-NMR (CD3OD, 100 MHz) δ: 193.41 (C=O), 167.34 (C-4′), 166.20 (C-2′), 161.37 (C-4), 145.60
(C-β), 133.30 (C-6′), 131.78 (C-2/6), 127.74 (C-1), 118.17 (C-α), 116.84 (C-3/5), 114.64 (C-1′), 109.12 (C-5′), 103.78 (C-3′). Compound 13 was identified as isoliquiritigenin by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Yang et al, 2009). Compound 14: yellow powder. ESI-MS m/z: 273 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.95 (1H, d, J = 8.9 Hz, H-6′), 7.73 (1H, d, J = 15.3 Hz, H-α), 7.54 (1H, d, J = 15.3 Hz, H-β), 7.19 (1H, d, J = 2.0 Hz, H-2), 7.12 (1H, dd, J = 8.2, 2.0 Hz, H-6), 6.82 (1H, d, J = 8.2 Hz, H-5), 6.42 (1H, dd, J = 8.9, 2.4 Hz, H-5′), 6.29 (1H, d, J = 2.4 Hz, H-3′); 13C-NMR (CD3OD, 100 MHz) δ: 193.47 (C=O), 167.49 (C-2′), 166.35 (C-4′), 149.94 (C-4), 146.84 (C-β), 146.09 (C-3), 133.29 (C-6′), 128.39 (C-1), 123.63 (C-6), 118.25 (C-α), 116.60 (C-2), 115.78 (C-5), 114.69 (C-1′), 109.14 (C-5′), 103.80 (C-3′). Compound 14 was identified as butein by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Júnior et al, 2008). Compound 15: yellow powder. ESI-MS m/z: 449 [M − H]－; 1H-NMR (CD3OD, 400 MHz) δ: 6.90 (1H, s, H-2′), 6.78 (2H, s, H-4′/6′), 5.96 (1H, s, H-8), 5.30 (1H, dd, J = 12.3, 3.0 Hz, H-2), 4.78 (1H, d, J = 9.9 Hz, H-1′′), 4.15 ~ 3.35 (5H, m, sugar-H), 3.08 (1H, dd, J = 17.1, 12.3 Hz, H-3α), 2.75 (1H, dd, J = 17.1, 3.0 Hz, H-3β); 13C-NMR (CD3OD, 125 MHz) δ: 198.03 (C-4), 167.29 (C-7), 164.23 (C-5), 164.15 (C-9), 146.92 (C-4′), 146.52 (C-3′), 131.59 (C-1′), 119.27 (C-6′), 116.23 (C-5′), 114.72 (C-2′), 105.94 (C-8), 103.26 (C-10), 96.38 (C-6), 82.54 (C-5′′), 80.38 (C-2), 80.19 (C-3′′), 75.16 (C-1′′), 72.54 (C-2′′), 71.82 (C-4′′), 62.91 (C-6′′), 43.91 (C-3). Compound 15 was identified as 3′,5′,5,7-tetrahydroxy-6-Cβ-D-glucopyranosyl-flavanone by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Chen et al, 2015).
References Aboushoer MI, Fathy HM, Abdel-Kader MS, Goetz G, Omar AA, 2010. Terpenes and flavonoids from an Egyptian collection of Cleome droserifolia. Nat Prod Res 24(7): 687-696. Ali M, Latif A, Zaman K, Arfan M, Maitland D, Ahmad H, Ahmad M, 2014. Anti-ulcer xanthones from the roots of Hypericum oblongifolium Wall. Fitoterapia 95: 258-265. An R, Jeong G, Kim Y, 2008. Flavonoids from the heartwood of Dalbergia odorifera and their protective effect on glutamateinduced oxidative injury in HT22 cells. Chem Pharm Bull 56(12): 1722-1724. Cook NC, Samman S, 1996. Flavonoids—Chemistry, metabolism, cardioprotective effects, and dietary sources. J Nutr Biochem 7(2): 66-76. Chen YJ, Xie GY, Xu GK, Dai YQ, Shi L, Qin MJ, 2015. Chemical constituents of Pyrrosia calvata. Nat Prod Commun 10(7): 1191-1193. Donnelly DMX, Nangle BJ, Prendergast JP, O′Sullivan AM, 1968. Dalbergia species V. Isolation of R-5-O-methyllatifolin from Dalbergia cochinchinensis, Pierre. Phytochemistry 7(4): 647-649. Dubrovskiy AV, Larock RC, 2013. Intermolecular C–O addition of carboxylic acids to arynes: synthesis of O-hydroxyaryl ketones, xanthones, 4-chromanones, and flavones. Tetrahedron 69(13): 2789-2798.
Liu RH et al. Chinese Herbal Medicines, 2015, 8(1): 89-93 Hahm E, Park S, Yang C, 2003. 7, 8-Dihydroxyflavanone as an inhibitor for Jun-Fos-DNA complex formation and its cytotoxic effect on cultured human cancer cells. Nat Prod Res 17(6): 431-436. Hamburger M, Cordell G, Tantivatana P, Ruangrungsi N, 1987. Traditional medicinal plants of Thailand, VIII. Isoflavonoids of Dalbergia candenatensis. J Nat Prod 50(4): 696-699. He L, 2014. Present situation on cultivation and utilization of precious and rare timber tree species in Xishuangbanna. Trop Agric Sci Technol 37(3): 40-42. Jnior GMV, De M. Sousa CM, Cavalheiro AJ, Lago JOHG, Chaves MH, 2008. Phenolic derivatives from fruits of Dipteryx lacunifera Ducke and evaluation of their antiradical activities. Helv Chim Acta 91(11): 2159-2167. Kim H, Yoo M, Kim H, Lee B, Oh K, Seo H, Yon G, 2006. Vasorelaxation effect of the flavonoids from the Rhizome extract of Alpinia offcinarum on isolated rat thoracic aorta. Korean J Pharmacogn 37(1): 56-59. Li Y, Du G, 2004. Effects of alpinetin on rat vascular smooth muscle cells. J Asian Nat Prod Res 6(2): 87-92. Palasuwan A, Soogarun S, Lertlum T, Pradniwat P, Wiwanitkit V, 2005. Inhibition of Heinz body induction in an in vitro model and total antioxidant activity of medicinal Thai plants. Asian Pac J
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Cancer Prev 6(4): 458. Pathak V, Shirota O, Sekita S, Hirayama Y, Hakamata Y, Hayashi T, Yanagawa T, Satake M, 1997. Antiandrogenic phenolic constituents from Dalbergia cochinchinensis. Phytochemistry 46(7): 1219-1223. Peterson J, Dwyer J, 1998. Flavonoids: Dietary occurrence and biochemical activity. Nutr Res 18(12): 1995-2018. Shirota O, Pathak V, Sekita S, Satake M, Nagashima Y, Hirayama Y, Hakamata Y, Hayashi T, 2003. Phenolic constituents from Dalbergia cochinchinensis. J Nat Prod 66(8): 1128-1131. Tang R, Qu X, Guan S, Xu P, Shi Y, Guo D, 2012. Chemical constituents of Spatholobus suberectus. Chin J Nat Med 10(1): 32-35. Yang H, Wang D, Tong L, Cai B, 2009. Flavonoid aglycones of Oxytropis falcata. Chem Nat Compd 45(2): 239-241. Zhao X, Mei W, Gong M, Zuo W, Bai H, Dai H, 2011. Antibacterial activity of the flavonoids from Dalbergia odorifera on Ralstonia solanacearum. Molecules 16(12): 9775-9782. Zheng ZP, Cheng KW, Chao J, Wu J, Wang M, 2008. Tyrosinase inhibitors from paper mulberry (Broussonetia papyrifera). Food Chem 106: 529-535. Zhong YX, Huang RM, Zhou XJ, Zhu YH, Xu ZF, Qiu SX, 2013. Chemical constituents from the heartwood of Dalbergia cochinchinensis. Nat Prod Res Dev 25(11): 1515-1518.
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Available online at SciVarse ScienceDirect
Chinese Herbal Medicines (CHM) ISSN 1674-6384
Journal homepage: www.tiprpress.com
E-mail: [email protected]
Original article
Flavonoids from Heartwood of Dalbergia cochinchinensis Rong-hua Liu*, Xin-chao Wen, Feng Shao, Pu-zhao Zhang, Hui-lian Huang, Shuang Zhang Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, China
ARTICLE INFO Article history Received: June 21, 2015 Revised: July 21, 2015 Accepted: August 19, 2015 Available online: January 15, 2016
DOI: 10.1016/S1674-6384(16)60014-X
ABSTRACT Objective
Methods
To study the flavonoids from the heartwood of Dalbergia cochinchinensis.
The chemical constituents were isolated and purified by combination of silica
gel, macroporous resin, Sephadex LH-20, and ODS column chromatography. Their structures were identified by means of spectral analysis. Results
Fifteen flavonoids were
isolated and identified as pinocembrin (1), liquiritigenin (2), galangin (3), 7-hydroxy6-methoxyflavone (4), naringenin (5), alpinetin (6),
2,3-dimethoxyxanthone (7),
6,4′-dihydroxy-7-methoxy-flavan (8), mucronulatol (9), 7,8-dihydroxyflavanone (10),
5,7,3′,5′-tetrahydroxyflavanone
(11),
4,2′,5′-trihydroxy-4′-methoxychalcone
(12),
isoliquiritigenin (13), butein (14), and 3′,5′,5,7-tetrahydroxy-6-C-β-D-glucopyranosyl-
flavanone (15), respectively. Conclusion Compounds 7, 8, 10, and 15 are isolated from the
plants of Dalbergia L. f. for the first time, and compounds 1, 3, 5, 6, 9, 11, 12, and 14 are
isolated from this plant for the first time.
Key words
Dalbergia cochinchinensis; 2,3-dimethoxyxanthone; 5,4′-dihydroxy-7-methoxyflavan;
7,8-dihydroxyflavanone; flavonoids
1. Introduction Dalbergia cochinchinensis Pierre ex Laness (Fabaceae), the Thailand rosewood or Siamese rosewood, is perennial non-climbing tree widely distributed in Indochina like Thailand, Vietnam, Laos, and Cambodia, etc. The plant is also cultivated in Xishuangbanna, Yunnan province, China (He et al, 2014). In Thailand, its core is used in folk medicine for the treatment of tumor and blood stasis (Palasuwan et al, 2005). Previous chemical investigations on this plant had led to the identification of a series of flavonoids, terpenes and benzoic acids (Donnelly et al, 1968; Pathak et al, 1997; Shirota et al, 2003; Zhong et al, 2013; Liu et al, 2015). Among all the constituents mentioned above, flavonoids are increasingly recognized as playing
© 2016 published by TIPR Press. All rights reserved.
potentially important roles in health including but not limited to their roles as antioxidants, free radical scavengers, metal chelators, and inhibiting lipid peroxidation (Peterson et al, 1998; Cook et al, 1996). Ten flavonoids have been isolated and identified from the heartwood of D. cochinchinensis so far (Pathak et al, 1997; Shirota et al, 2003). Further phytochemical investigation on D. cochinchinensis led to the isolation and characterization of fifteen flavonoids including seven flavanones (1, 2, 5, 6, 10, 11, and 15), a flavone (4), a flavonol (3), three chalcones (12, 13, and 14), a xanthone (7), a flavan (8), and an isoflavan (9). Among the flavonoids, compounds 7, 8, and 10 are reported for the first time from the plants of Dalbergia L. f., and compounds 1, 3, 5, 6, 9, 11, 12, and 14 are reported for the first time from this plant (Figure 1).
*Corresponding author: Liu RH
Tel: +86-791-8711 8918 E-mail: [email protected]
Fund: National Natural Science Foundation of China (81360629)
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Liu RH et al. Chinese Herbal Medicines, 2015, 8(1): 89-93
R6 R5 R2
O
R4
R3 R1 O 1 R1=R2=OH R3=R4=R5=H R3 O 2 R2=R5=OH R1=R3=R4=H 5 R1=R2=R5=OH R3=R4=H R2 R4 6 R1=OCH3 R2=OH R3=R4=R5=H R O 10 R2=R3=OH R1=R4=R5=H 1 11 R1=R2=R4=R6=OH R3=H 3 R1=R3=R4=OH R2=H 15 R1=R2=R4=R6=OH R3=Glc 4 R2=OCH3 R3=OH R1=R4=H
OH O
O
HO 8 Figure 1
Chemical structures of compounds 1-15 isolated from heartwood of Dalbergia cochinchinensis
2. Materials and methods
2.1 General The NMR spectra were recorded on a Bruker AVANCE III HD 400 MHz Instrument in deuterated chloroform and methanol with TMS as internal standard. ESI-MS was recorded on an Agilent 1100 Series LC/MSD Trap. Silica gel (100−200 and 200−300 mesh, Qingdao Marine Chemical, China), ODS gel (YMC, Japan), macroporous resin (D101, Tianjin Haiguang Chemical, China) and Sephadex LH-20 (GE Healthcare, Swiss) were used for column chromatography. TLC was performed on silica gel GF254 plates (Qingdao Marine Chemical Factory) and visualized with 10% sulphuric acid in ethanol.
2.2 Plant material The heartwood of Dalbergia cochinchinensis collected from Fangchenggang City of Guangxi Zhuang Autonomous Region, China, in June 2013, and identified by Prof. Ke-zhong Deng at Jiangxi University of Traditional Chinese Medicine. A voucher specimen (201306) was deposited at the Herbarium of Key Laboratory of Modern Preparation of TCM, Ministry of Education, Jiangxi University of Traditional Chinese Medicine.
2.3 Extraction and isolation The dried sliced heartwood of D. cochinchinensis (20 kg) was extracted with 70% EtOH (reflux, 2 h, three times). After the removal of ethanol by concentrated under reduced
pressure, the extract (4.2 kg, 5.4 kg in total) was suspended in water and partitioned sequentially with petroleum ether (60−90 oC), chloroform, EtOAc, and n-BuOH, respectively. The petroleum ether extract (381.2 g) was separated into nine fractions (Frs. 1−9) by applying silica gel column, eluted with petroleum ether-EtOAc (50:1 to 1:1). Fr. 5 was purified by repeated silica gel and Sephadex LH-20 with CH2Cl2MeOH (1:1) to give compound 1 (932.2 mg). The chloroform extract (902.6 g) was separated into 9 fractions (Frs. 1−9) by adopting silica gel column and eluted with gradient mixture of petroleum ether- EtOAc (50:1 to 1:1). Fr. 4 was subjected to silica gel using CH2Cl2-MeOH (100:1 to 1:1), followed by Sephadex LH-20 with CH2Cl2-MeOH (1:1) to give compounds 2 (529.2 mg), 3 (11.9 mg), and 4 (421.5 mg). Fr. 5 was separated over silica gel with CH2Cl2-MeOH (50:1 to 1:1), purified by Sephadex LH-20 using CH2Cl2-MeOH (1:1) as an eluent to afford compounds 5 (57.9 mg) and 6 (14.2 mg). Compounds 7 (1.5 mg), 8 (576.4 mg), and 9 (35.5 mg) were obtained by applying silica gel, eluted with CH2Cl2-MeOH (50:1 to 1:1) and Sephadex LH-20 with CH2Cl2-MeOH (1:1). The EtOAc extract (1397.7 g) was separated into 23 fractions (Fr. 1-23) by the method of silica gel column using CH2Cl2-MeOH (100:0 to 1:1) as eluent. Fr. 6 was subjected to silica gel eluted with gradient mixture of CH2Cl2-MeOH (80:1 to 1:1), purified by Sephadex LH-20 using CH2Cl2MeOH (1:1) to give compound 10 (88.9 mg). Fr. 10 was separated by the means of silica gel with CH2Cl2-MeOH (50:1 to 1:1), followed by Sephadex LH-20 using CH2Cl2-MeOH (1:1) and ODS gel eluted with 50% MeOH to give compounds 11 (10.9 mg) and 12 (10.9 mg). Fr. 16 was
Liu RH et al. Chinese Herbal Medicines, 2015, 8(1): 89-93 separated over silica gel, eluted with CH2Cl2-MeOH (50:1 to 1:1), next ODS gel using 65% MeOH as an eluent to obtain compounds 13 (90.4 mg) and 14 (6.9 mg). The n-BuOH extract (462.7 g) was separated into 10 fractions (Fr. 1-10) via macroporous resin column, eluted with EtOH-water (0:1 to 1:0). Fr. 4 was purified subsequently by Sephadex LH-20 with CH2Cl2-MeOH (1:1) and ODS gel using 40% MeOH to yield compound 15 (8.4 mg).
3. Results Compound 1: white needle crystal. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CDCl3, 400 MHz) δ: 12.04 (1H, s, OH), 7.55−7.33(5H, m, B ring), 6.01 (2H, s, H-6/8), 5.42 (1H, dd, J = 13.0, 3.0 Hz, H-2), 3.10 (1H, dd, J = 17.2, 13.0 Hz, H-3α), 2.82 (1H, dd, J = 17.2, 3.0 Hz, H-3β); 13C-NMR (CDCl3, 100 MHz) δ: 195.82 (C-4), 164.46 (C-5), 164.32 (C-9), 163.15 (C-7), 138.22 (C-1′), 128.92 (C-3′/5′), 128.89 (C-4′), 126.13 (C-2′/6′), 103.22 (C-10), 96.75 (C-6), 95.48 (C-8), 79.23 (C-2), 43.32 (C-3). Compound 1 was identified as pinocembrin by comparison of the physical, 1H-NMR, and 13 C-NMR data with the reported data (Aboushoer et al, 2010). Compound 2: yellow powder. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.74 (1H, d, J = 8.7 Hz, H-5), 7.32 (2H, d, J = 8.5 Hz, H-2′/6′), 6.81 (2H, d, J = 8.5 Hz, H-3′/5′), 6.49 (1H, dd, J = 8.7, 2.3 Hz, H-6), 6.35 (1H, d, J = 2.3 Hz, H-8), 5.38 (1H, dd, J = 13.1, 2.9 Hz, H-2), 3.05 (1H, dd, J = 16.9, 13.1 Hz, H-3ɑ), 2.69 (1H, dd, J = 16.9, 2.9 Hz, H-3β); 13C-NMR (CD3OD, 100 MHz) δ: 193.54 (C-4), 166.94 (C-7), 165.59 (C-9), 158.98 (C-4′), 131.35 (C-1′), 129.85 (C-5), 129.01 (C-2′/6′), 116.29 (C-3′,5′), 114.91 (C-10), 111.80 (C-6), 103.82 (C-8), 81.05 (C-2), 44.96 (C-3). Compound 2 was identified as liquiritigenin by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Zhao et al, 2011). Compound 3: yellow needle crystal. ESI-MS m/z: 271 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 8.16 (2H, d, J = 7.6 Hz, H-2′/6′), 7.46 (3H, dt, J = 14.5, 7.6 Hz, H-3′/4′/5′), 6.39 (1H, d, J =2.0 Hz, H-8), 6.17 (1H, d, J = 2.0 Hz, H-6); 13 C-NMR (CD3OD, 100 MHz) δ: 177.65 (C-4), 165.89 (C-7), 162.57 (C-5), 158.41 (C-9), 146.88 (C-2), 138.50 (C-3), 132.60 (C-1′), 130.87 (C-4′), 129.42 (C-2′/6′), 128.73 (C-3′/5′), 104.66 (C-10), 99.36 (C-6), 94.52 (C-8). Compound 3 was identified as galangin by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Kim et al, 2006). Compound 4: yellow needle crystal. ESI-MS m/z: 269 [M + H]+; 1H-NMR (CDCl3, 400 MHz) δ: 7.55 ~ 7.34 (5H, m, B ring), 6.98 (1H, s, H-8), 6.89 (1H, s, H-5), 6.24 (1H, s, H-3), 5.64 (1H, s, OH), 3.98 (3H, s, OCH3-6); 13C-NMR (CDCl3, 100 MHz) δ: 161.52 (C-4), 155.79 (C-2), 150.06 (C-6), 149.30 (C-9), 142.40 (C-7), 135.54 (C-1′), 129.56 (C-4′), 128.81 (C-3′,5′), 128.30 (C-2′,6′), 112.50 (C-3), 112.29 (C-10), 110.49 (C-8), 99.56 (C-5), 56.45 (OCH3-4′). Compound 4 was identified as 7-hydroxy-6-methoxyflavone by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Pathak et al, 1997).
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Compound 5: light yellow powder. ESI-MS m/z: 273 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.30 (2H, d, J = 8.5 Hz, H-2′/6′), 6.81 (2H, d, J = 8.5 Hz, H-3′/5′), 5.88 (2H, d, J = 2.1 Hz, H-6/8), 5.30 (1H, dd, J = 13.0, 3.0 Hz, H-2), 3.09 (1H, dd, J = 17.1, 13.0 Hz, H-3α), 2.67 (1H, dd, J = 17.1, 3.0 Hz, H-3β); 13C-NMR (CD3OD, 100 MHz) δ: 197.75 (C-4), 168.29 (C-7), 165.40 (C-5), 164.83 (C-9), 158.95 (C-4′), 131.02 (C-1′), 129.03 (C-2′, 6′), 116.29 (C-3′, 5′), 103.30 (C-10), 97.03 (C-6), 96.15 (C-8), 80.43 (C-2), 43.98 (C-3). Compound 5 was identified as naringenin by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Tang et al, 2012). Compound 6: colorless needle crystal. ESI-MS m/z: 271 [M + H]+; 1H-NMR (DMSO-d6, 400 MHz) δ: 10.58 (1H, br s, OH), 7.64−7.20 (5H, m, B ring), 6.07 (1H, d, J = 2.2 Hz, H-8), 6.01 (1H, d, J = 2.2 Hz, H-6), 5.48 (1H, dd, J = 12.4, 2.9 Hz, H-2), 3.74 (3H, s, OCH3-5), 2.97 (1H, dd, J = 16.3, 12.4 Hz, H-3α), 2.60 (1H, dd, J = 16.3, 2.9 Hz, H-3β); 13C-NMR (DMSO-d6, 100 MHz) δ: 187.37 (C-4), 164.44 (C-7), 164.05 (C-5), 162.22 (C-9), 139.19 (C-1′), 128.53 (C-3′/5′), 128.35 (C-4′), 126.46 (C-2′/6′), 104.47 (C-10), 95.66 (C-6), 93.37 (C-8), 78.05 (C-2), 55.65 (OCH3-7), 44.87 (C-3). Compound 6 was identified as alpinetin by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Li et al, 2004). Compound 7: light brown powder. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CDCl3, 400 MHz) δ: 8.35(1H, dd, J = 8.0, 1.7 Hz, H-8), 7.73−7.69 (1H, m, H-6), 7.68 (1H, s, H-1), 7.47 (1H, d, J =8.4 Hz, H-5), 7.38 (1H, t, J = 8.0 Hz, H-7), 6.94 (1H, s, H-4), 4.02 (3H, s, OCH3-3), 4.00 (3H, s, OCH3-2); 13 C-NMR (CDCl3, 100 MHz) δ: 176.13 (C-9), 156.05 (C-5a), 155.51 (C-4a), 152.45 (C-3), 146.63 (C-2), 133.99 (C-6), 126.54 (C-8), 123.77 (C-7), 121.50 (C-8a), 117.66 (C-5), 114.90 (C-1a), 105.36 (C-1), 99.62 (C-4), 56.38 (OCH3-3), 55.49 (OCH3-2). Compound 7 was identified as 2, 3-dimethoxyxanthone by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Dubrovskiy et al, 2013; Ali et al, 2014). Compound 8: light yellow powder. ESI-MS m/z: 273 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.20 (2H, d, J = 8.5 Hz, H-2′/H-6′), 6.77 (2H, d, J = 8.5 Hz, H-3′/H-5′), 6.50 (lH, s, H-8), 6.38 (1H, s, H-5), 4.80 (1H, d, J = 10.2 Hz, H-2), 3.75 (3H, s, 7-OCH3), 2.82 (1H, dddt, J = 16.3, 11.5, 6.0, 1.5 Hz, H-4α), 2.58 (1H, ddt, J = 16.3, 5.2, 2.2 Hz, H-4β), 2.03 (lH, ddq, J = 13.5, 6.0, 2.2 Hz, H-3α), 1.95 (1H, ddddd, J = 13.5, 11.5, 10.2, 5.2, 1.5 Hz, H-3β); 13C-NMR (CD3OD, 100 MHz) δ: 158.04 (C-4′), 149.61 (C-7), 148.04 (C-9), 140.97 (C-6), 134.33 (C-1′), 128.49 (C-2′/6′), 116.16 (C-5), 116.03 (C-3′/5′), 114.41 (C-10), 101.73 (C-8), 78.84 (C-2), 56.31 (OCH3-7), 31.25 (C-3), 25.64 (C-4). Compound 8 was identified as 6,4′-dihydroxy-7-methoxyflavan by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Pathak et al, 1997). Compound 9: white powder. ESI-MS m/z: 303 [M + H]+; 1 H-NMR (CD3OD, 400 MHz) δ: 6.86 (1H, d, J = 8.2 Hz, H-5), 6.69 (1H, d, J = 8.6 Hz, H-6′), 6.59 (1H, d, J = 8.6 Hz, H-5′), 6.31 (1H, dd, J = 8.2, 2.5 Hz, H-6), 6.23 (1H, d, J = 2.5 Hz,
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H-8), 4.17 (1H, ddd, J = 10.3, 3.5, 2.0 Hz, H-2α), 3.91 (1H, t, J = 10.3 Hz, H-2β), 3.83 (3H, s, OCH3-4′), 3.82 (1H, s, OCH3-2′), 3.42 (1H, tdd, J = 10.6, 5.4, 3.5 Hz, H-3), 2.88 (1H, dd, J = 15.5, 10.6 Hz, H-4α), 2.77 (1H, ddd, J = 15.5, 5.4, 2.0 Hz, H-4β); 13C-NMR (CD3OD, 100 MHz) δ: 156.18 (C-7), 154.86 (C-9), 147.66 (C-4′), 145.82 (C-2′), 139.23 (C-3′), 129.72 (C-5), 127.17 (C-6′), 116.25 (C-1′), 113.27 (C-10), 107.64 (C-6), 106.97 (C-5′), 102.37 (C-8), 70.18 (C-2), 55.20 (OCH3), 31.77 (C-3), 31.10 (C-4). Compound 9 was identified as mucronulatol by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Hamburger et al, 1987). Compound 10: light yellow powder. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.51−7.34 (5H, m, B ring), 5.91 (2H, dd, J = 12.0, 2.1 Hz, H-5/6), 5.44 (1H, dd, J = 12.8, 3.0 Hz, H-2), 3.07 (1H, dd, J = 17.1, 12.8 Hz, H-3ɑ), 2.75 (1H, dd, J = 17.1, 3.0Hz, H-3β); 13C-NMR (CD3OD, 100 MHz) δ: 195.90 (C-4), 166.98 (C-7), 164.04 (C-8), 163.23 (C-9), 138.97 (C-1′), 128.28 (C-3′, 5′), 128.21 (C-4′), 125.92 (C-2′, 6′), 101.93 (C-10), 95.73 (C-6), 94.79 (C-5), 79.02 (C-2), 42.76 (C-3). Compound 10 was identified as 7,8-dihydroxyflavanone by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Hahm et al, 2003). Compound 11: white powder. ESI-MS m/z: 289 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 12.05(1H, s, OH), 8.01(1H, s, OH), 6.92(1H, s, H-4′), 6.79 (1H, s, H-2′), 6.77 (1H, s, H-6′), 5.89 (1H, d, J = 2.2 Hz, H-6), 5.87 (1H, d, J = 2.2 Hz, H-8), 5.26 (1H, dd, J = 12.8, 3.0 Hz, H-2), 3.06 (1H, dd, J = 17.1, 12.8 Hz, H-3β), 2.68 (1H, dd, J = 17.1, 3.0 Hz, H-3α); 13C-NMR (CD3OD, 100 MHz) δ: 197.77 (C-4), 168.34 (C-7), 165.43 (C-5), 164.83 (C-9), 146.87 (C-4′), 146.49 (C-3′), 131.75 (C-1′), 119.25 (C-6′), 116.23 (C-5′), 114.69 (C-2′), 103.33 (C-10), 97.01 (C-6), 96.15 (C-8), 80.50 (C-2), 44.09 (C-3). Compound 11 was identified as 5,7,3′,5′tetrahydroxyflavanone by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Zheng et al, 2008). Compound 12: dark yellow powder. ESI-MS m/z: 287 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.78 (1H, d, J = 15.4 Hz, H-β), 7.61 (2H, d, J = 8.6 Hz, H-2/6), 7.52 (1H, d, J = 15.4 Hz, H-α), 7.42 (1H, s, H-6′), 6.84 (2H, d, J = 8.6 Hz, H-3/5), 6.50 (1H, s, H-3′), 3.91 (3H, s, -OCH3); 13C-NMR (CD3OD, 100 MHz) δ: 193.50 (C=O), 161.65 (C-4), 161.20 (C-2′), 156.92 (C-4′), 145.58 (C-β), 140.13 (C-5′), 131.87 (C-2/6), 127.78 (C-1), 118.33 (C-α), 116.95 (C-3/5), 115.01 (C-6′), 113.7 (C-1′), 101.00 (C-3′), 56.55 (OCH3-7). Compound 12 was identified as 4,2′,5′-trihydroxy-4′methoxychalcone by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (An et al, 2008). Compound 13: yellow powder. ESI-MS m/z: 257 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.91 (1H, d, J = 8.9 Hz, H-6′), 7.74 (1H, d, J = 15.4 Hz, H-β), 7.56 (2H, d, J = 8.6 Hz, H-2/6), 7.54 (1H, d, J = 15.4 Hz, H-α), 6.83 (2H, d, J = 8.6 Hz, H-3/5), 6.40 (1H, dd, J = 8.9, 2.4 Hz, H-5′), 6.29 (1H, d, J = 2.4 Hz, H-3′); 13C-NMR (CD3OD, 100 MHz) δ: 193.41 (C=O), 167.34 (C-4′), 166.20 (C-2′), 161.37 (C-4), 145.60
(C-β), 133.30 (C-6′), 131.78 (C-2/6), 127.74 (C-1), 118.17 (C-α), 116.84 (C-3/5), 114.64 (C-1′), 109.12 (C-5′), 103.78 (C-3′). Compound 13 was identified as isoliquiritigenin by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Yang et al, 2009). Compound 14: yellow powder. ESI-MS m/z: 273 [M + H]+; 1H-NMR (CD3OD, 400 MHz) δ: 7.95 (1H, d, J = 8.9 Hz, H-6′), 7.73 (1H, d, J = 15.3 Hz, H-α), 7.54 (1H, d, J = 15.3 Hz, H-β), 7.19 (1H, d, J = 2.0 Hz, H-2), 7.12 (1H, dd, J = 8.2, 2.0 Hz, H-6), 6.82 (1H, d, J = 8.2 Hz, H-5), 6.42 (1H, dd, J = 8.9, 2.4 Hz, H-5′), 6.29 (1H, d, J = 2.4 Hz, H-3′); 13C-NMR (CD3OD, 100 MHz) δ: 193.47 (C=O), 167.49 (C-2′), 166.35 (C-4′), 149.94 (C-4), 146.84 (C-β), 146.09 (C-3), 133.29 (C-6′), 128.39 (C-1), 123.63 (C-6), 118.25 (C-α), 116.60 (C-2), 115.78 (C-5), 114.69 (C-1′), 109.14 (C-5′), 103.80 (C-3′). Compound 14 was identified as butein by comparison of the physical, 1H-NMR, and 13C-NMR data with the reported data (Júnior et al, 2008). Compound 15: yellow powder. ESI-MS m/z: 449 [M − H]－; 1H-NMR (CD3OD, 400 MHz) δ: 6.90 (1H, s, H-2′), 6.78 (2H, s, H-4′/6′), 5.96 (1H, s, H-8), 5.30 (1H, dd, J = 12.3, 3.0 Hz, H-2), 4.78 (1H, d, J = 9.9 Hz, H-1′′), 4.15 ~ 3.35 (5H, m, sugar-H), 3.08 (1H, dd, J = 17.1, 12.3 Hz, H-3α), 2.75 (1H, dd, J = 17.1, 3.0 Hz, H-3β); 13C-NMR (CD3OD, 125 MHz) δ: 198.03 (C-4), 167.29 (C-7), 164.23 (C-5), 164.15 (C-9), 146.92 (C-4′), 146.52 (C-3′), 131.59 (C-1′), 119.27 (C-6′), 116.23 (C-5′), 114.72 (C-2′), 105.94 (C-8), 103.26 (C-10), 96.38 (C-6), 82.54 (C-5′′), 80.38 (C-2), 80.19 (C-3′′), 75.16 (C-1′′), 72.54 (C-2′′), 71.82 (C-4′′), 62.91 (C-6′′), 43.91 (C-3). Compound 15 was identified as 3′,5′,5,7-tetrahydroxy-6-Cβ-D-glucopyranosyl-flavanone by comparison of the physical, 1 H-NMR, and 13C-NMR data with the reported data (Chen et al, 2015).
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