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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
Hepatoprotective benzofurans and furanolignans from Gymnema tingens ab
c
a
a
b
Jin Tian , Yan-Gai Wang , Jie Ma , Jian-Bo Yang , Ligang Zhou , a
a
a
Teng-Fei Ji , Ai-Guo Wang & Ya-Lun Su a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Material Medica, Beijing 100050, China b
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MOA Key Laboratory of Plant Pathology, Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China c
Department of Pharmacy, XuanWu Hospital of Capital Medical University, Beijing 100053, China Published online: 13 Mar 2015.
To cite this article: Jin Tian, Yan-Gai Wang, Jie Ma, Jian-Bo Yang, Ligang Zhou, Teng-Fei Ji, Ai-Guo Wang & Ya-Lun Su (2015) Hepatoprotective benzofurans and furanolignans from Gymnema tingens, Journal of Asian Natural Products Research, 17:3, 268-273, DOI: 10.1080/10286020.2015.1016002 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1016002
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Journal of Asian Natural Products Research, 2015 Vol. 17, No. 3, 268–273, http://dx.doi.org/10.1080/10286020.2015.1016002
Hepatoprotective benzofurans and furanolignans from Gymnema tingens Jin Tianab, Yan-Gai Wangc, Jie Maa, Jian-Bo Yanga, Ligang Zhoub, Teng-Fei Jia, Ai-Guo Wanga* and Ya-Lun Sua*
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a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Material Medica, Beijing 100050, China; bMOA Key Laboratory of Plant Pathology, Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China; c Department of Pharmacy, XuanWu Hospital of Capital Medical University, Beijing 100053, China (Received 4 December 2014; final version received 26 January 2015) Two new benzofurans, gymnefuranols A (1) and B (2), together with six known furanolignans (3 – 8), were isolated from Gymnema tingens. The structures of the new compounds were elucidated by comprehensive analysis of the NMR and HR-MS data. Compounds 1, 2, 6, and 7 showed hepatoprotective activities against D -galactosamineinduced HL-7702 cell damage. Keywords: Asclepiadaceae; Gymnema tingens; hepatoprotective; benzofuran; furanolignan; structure elucidation
1.
Introduction
The genus Gymnema belonging to the Asclepiadaceae family contains around 25 species worldwide and distributes in tropical and subtropical regions of Asia, southern Africa, and Oceania, while eight species are found in south and southwest China [1,2]. In Chinese folk medicine, the extract of Gymnema has the effect of detumescence, detoxicating, promoting granulation, and easing pain [3]. Secondary metabolites including saponins, steroids, alkaloids, phenolic glycosides, and peptides have been reported from Gymnema inodorum [4], Gymnema sylvestre [5], Gymnema yunnanense [6,7], Gymnema alternifolium [8–11], and Gymnema tingens [12], and showed interesting biological activities such as antisweet, antidiabetic, antiallergic, antiviral, hepatoprotective, and lipid lowering effects. To the best of our knowledge, phytochemical investigation on G. tingens had
been so far reported only by our group [12]. In a previous investigation of the hepatoprotective constituents of G. tingens, we had identified nine bioactive phenolic diglycosides [12]. In continuation of this work, two benzofurans (1– 2) and six furanoligans (3 –8) with hepatoprotective effect were isolated (Figure 1). In this paper, we describe the isolation and structure elucidation of new compounds, as well as the biological activities of the isolated compounds. 2. Results and discussion Compound 1 was isolated as a colorless oil. Its molecular formula was determined to be C13H14O4 as a prominent peak was observed at m/z 257.0783 [M þ Na]þ in the HR-ESI-MS, implying seven degrees of unsaturation. The IR spectrum showed characteristic bands of hydroxyl (3336 cm21), olefin (1658 cm21), and benzene groups (1619, 1601 cm21). The
*Corresponding authors. Emails: [email protected]; [email protected] q 2015 Taylor & Francis
Journal of Asian Natural Products Research 1 O 8 3 HO
OCH3
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OCH3 O
6 OH
9
4
10
11
OH
13 HO
1 OCH3
H3CO O
HO
2
OCH3
R2 O
HO
OH
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OH H3CO
O
OCH3
R1
OCH3 R3O
HO
4 R1 = H; R2 = OCH3; R3 = β-D-glucose
3
5 R1 = R2 = H; R3 = β-D-glucose 6 R1 = R2 = R3 = H 7 R1 = H; R2 = OCH3; R3 = H 8 R1 = R2 = OCH3; R3 = H
Figure 1. Structures of the isolated compounds 1 – 8. 1
H NMR spectrum exhibited signals for a pair of meta-coupling aromatic protons at dH 7.25 (d, J ¼ 1.1 Hz, H-4) and 7.02 (d, J ¼ 1.1 Hz, H-6), an isolate olefinic proton at dH 7.80 (s, H-2), and a transdisubstituted double bond at dH 6.61 (br. d, J ¼ 15.9 Hz, H-11) and 6.37 (dt, J ¼ 15.9 and 5.4 Hz, H-12) in the downfield region. The two triplets at dH 5.15 (OH-10) and 4.85 (OH-13) were D2Oexchangeable, and thus assigned to two hydroxyls, which each coupled to a methylene group at dH 4.59 (dd, J ¼ 5.6 and 0.8 Hz, H2 2 10), and 4.13 (td, J ¼ 5.4 and 1.6 Hz, H2 2 13), respectively. In addition, one methoxyl group was observed at dH 3.94. The 13C NMR
spectrum displayed signals for 10 sp2hybridized carbons that were assignable to one tetrasubstituted benzene ring (dC 144.9 C, 143.5 C, 132.8 C, 128.6 C, 110.5 CH, 104.7 CH), one trisubstituted double bond (dC 142.7 CH, 121.9 C), and one disubstituted double bond (dC 129.7 CH, 129.0 CH), and for three oxygenated sp3-hybridized carbons including one methoxyl (dC 55.8), and two hydroxymethyl groups (dC 53.8, 61.6), as revealed by the HSQC experiment. The connection of the aforementioned moieties was constructed by analysis of the HMBC spectrum (Figure 2). The correlations from both H-4 (dH 7.25, d) and H-6 (dH 7.02, d) to C-11 (dC 129.0,
O O
8
O O
6
2 9
HO
4 10
OH 13
OH HO
1
Figure 2. HMBC correlations (H ! C) of compounds 1 and 2.
2
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CH), and from H-11 (dH 6.61, br.d) to C-4 (dC 110.5, CH), C-5 (dC 132.8, C), and C-6 (dC 104.7, CH), indicated that the disubstituted double bond was connected to C-5 of the aromatic ring. Additional correlations from the olefinic protons H-11 and H-12 (dH 6.37, dt) to C-13 (dC 61.6, CH2), and from H2-13 (dH 4.13, td) to C-11 and C-12 (dC 129.7, CH) suggested that one hydroxymethyl group was connected to the other end of this disubstituted double bond. The second hydroxymethyl group (dH 4.59 dd, H2-10; dC 53.8, CH2-10) showed correlations to C-2 (dC 142.7, CH), C-3 (dC 121.9, C), and C-9 (dC 128.6, C), thus allowing the assignment of this group to C-3 of the trisubstituted double bond, and the connection between C-3 and C-9 of the benzene ring. Further HMBC correlations from H-2 (dH 7.80, s) to C-8 (dC 143.5, C), and taking into consideration of the chemical shifts of C-2 (dC 142.7, CH) and C-8, suggested that C-2 was connected to C-8 via an oxygen bridge, thus furnishing a benzofuran skeleton. The correlation from the methoxyl group (dH 3.94 s; dC 55.8 CH3) to C-7 (dC 144.9, C) indicated that this group was substituted at C-7 of the benzene ring. Therefore, compound 1 was elucidated as (E)-3-(3-(hydroxymethyl)-7-methoxybenzofuran-5-yl)prop-2-en-1-ol, and named gymnefuranol A. Compound 2 was also obtained as a colorless oil. The molecular formula of 2 was deduced as C13H16O4 by HR-ESI-MS, which bears two more protons than 1, and accordingly one less degree of unsaturation than 1. The NMR data were similar for both compounds, indicating they are congeners. The only differences were attributed to the side chain, in which the signals for the trans-disubstituted double bond in 1 were replaced by those for two methylene groups [dH 2.67 (t, J ¼ 7.8 Hz, H2-11), dC 32.0 (CH2-11); dH 1.76 (tt, J ¼ 7.8, 6.5 Hz, H2-12), dC 34.8 (CH2-12)] in 2. This was further corroborated by analysis of the HSQC and HMBC spectra.
Therefore, compound 2 was established as the dihydro derivative of 1, and named 3(3-(hydroxymethyl)-7-methoxybenzofuran-5-yl)propan-1-ol. The trivial name gymnefuranol B was given for this compound. The known compounds were identified by comparing their spectroscopic data with those in the literature, and included (70 S,8S,80 R)-4,40 -dihydroxy-3,3 0 ,5,50 -tetramethoxy-70 ,9-epoxylignan-9 0 -ol-7-one (3) [13], alangilignoside D (4) [14], (– )lariciresinol 9-O-b-D -glucopyranoside (5) [15], (– )-lariciresinol (6) [16], 50 -methoxylariciresinol (7) [17], and 5,50 -dimethoxylariciresinol (8) [18]. The hepatoprotective effect of compounds 1 –8 were evaluated against Dgalactosamine-induced toxicity in HL7702 cells in vitro. Compounds 1, 2, 6, and 7 showed better cell survival rates than the control at 10 mM, while 1 and 6 exhibited better hepatoprotective effect than the positive control bicyclol by comparison of their cell survival rates and inhibition rates (Table 2). Among them, compound 6 displayed the most significant protective effect against the D galactosamine-induced cell damage (97.7% cell survival rate after treatment). Thus, this compound could be a promising agent for the treatment of hepatic disease. Hepatoprotective compounds of various structural types were documented in the literature, such as sesquiterpene glycosides [19], phenolic glycosides [12,20], iridoid glycosides [21,22], lignans [23], triterpenes [24], and triterpene saponins [25]. The identification of two new hepatoprotective benzofurans (1 –2) in this study has enriched the chemical diversity of the hepatoprotective compounds. 3.
Experimental
3.1 General experimental procedures The optical rotations were measured on a Jasco P-2000 polarimeter (JASCO Corp.,
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Journal of Asian Natural Products Research Tokyo, Japan). The UV spectra were obtained with a Jasco V650 spectrophotometer (JASCO Corp.). IR spectra were recorded using a Nicolet 5700 FT-IR spectrometer (Thermo Electron Scientific Instrument Crop., Madison, WI, USA). 1H, 13 C NMR, and 2D NMR spectra were measured on a Varian Unity INOVA 600 spectrometer (Varian, Inc., Palo Alto, CA, USA). The chemical shifts were referred to the solvent residual peak of DMSO-d6 (dH 2.50 for 1H, and dC 39.5 for 13C). ESI-MS and HR-ESI-MS data were obtained using an Agilent 1100 series LC/MSD Trap SL mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Column chromatography was performed using RP-18 (50 mm; YMC Co., Ltd, Kyoto, Japan), and silica gel (160 – 200 mesh, and 200 –300 mesh; Qingdao Marine Chemical, Inc. Co., Ltd, Qingdao, China). Preparative HPLC was conducted using a Shimadzu LC-6AD instrument with an SPD-20A detector (SHIMADZU, Corp., Tokyo, Japan) and an YMC Pack ODS-A column (250 £ 20 mm, 10 mm; YMC Co., Ltd). HPLC – DAD analysis was performed using an Agilent 1200 series system (Agilent Technologies) with an analytic C18 column (250 mm £ 4.6 mm, 5 mm; YMC Co., Ltd). Precoated silica gel GF-254 plates (Yantai Jiangyou Silica Gel Exploitation Co., Ltd, Yantai, China) were used for analytical TLC. Spots were visualized under UV light (254 or 365 nm) or by spraying with 10% H2SO4 in 95% EtOH followed by heating. 3.2 Plant material The stems and leaves of G. tingens Roxb. ex Spreng. (Asclepiadaceae) were collected in Dai Autonomous Prefecture of Xishuangbanna, Yunnan Province, China, in August 2010. The plant material was identified by Prof. Jing-yun Cui (Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences). A voucher specimen (No. 035413) has been deposited at the Herbar-
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ium of Xishuangbanna tropical botanical garden (Chinese Academy of Sciences, Xishuangbanna, Yunnan, China). 3.3 Extraction and isolation As reported previously, the EtOAc extract of the ethanol extract of G. tingens was subjected to vacuum liquid chromatography over silica gel to yield 22 fractions, among which fraction 9 was found to be a pool of nine phenolic diglycosides [12]. The less polar fraction 6 (15 g) was subjected to chromatography over ODS eluting with a gradient of methanol – water (from 60% to 100%) to obtain 12 subfractions (F6-1 – F6-12). F6-6 (400 mg) was purified by semi-preparative HPLC using 18% ACN – H2O as eluent (6 ml/min) to afford compounds 4 (5 mg, Rt 29 min) and 5 (2 mg, Rt 32 min). Similarly, compound 3 (14 mg, Rt 42 min) was obtained from subfraction F6-12 (100 mg) by semi-preparative HPLC using 21% ACN – H2O as mobile phase (6 ml/min). Fraction 7 (13 g) was subjected to MPLC eluting with a gradient of MeOH– H2O to yield eight subfractions (F7-1– F7-8), among which F7-8 (1.1 g) was subjected to column chromatography over silica gel (200–300 mesh) eluting with CHCl3 –MeOH (40:1) to obtain a fraction containing compound 8 (5 mg), which was purified by a subsequent RP-HPLC separation using 45% MeOH–H2O as eluent (Rt 13 min for 8, 6 ml/min). Fraction 8 (7 g) was chromatographed over silica gel (200 –300 mesh) eluting with petroleum ether – acetone (3:1) to yield five subfractions (F8-1 –F8-5). Subfraction F8-2 (0.6 g) was separated by RPHPLC using 35% MeOH – H2O as mobile phase to afford compound 6 (13 mg, Rt 18.5 min, 6 ml/min). Subfraction F8-4 (1.5 g) was subjected to size exclusion chromatography over Sephadex LH-20 using methanol as eluent to obtain four further subfractions (F8-4-1 – F8-4-4),
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among which F-8-4-1 (410 mg), F-8-4-2 (380 mg), and F-8-4-3 (360 mg) were purified by RP-HPLC using 40% MeOH – H2O as eluent (6 ml/min) to afford compounds 2 (12 mg, Rt 42 min), 1 (5 mg, Rt 41 min), and 7 (23 mg, Rt 39 min), respectively.
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3.3.1
Gymnefuranol A (1)
Colorless oil; UV (MeOH) lmax (log 1) 206 (3.63), 236 (sh) (3.11) nm; IR nmax 3336, 3283, 1658, 1619, 1601, 1371, 1086, 972, 806 cm21; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectral data, see Table 1; HRESI-MS: m/z 235.0960 [M þ H]þ (calcd for C13H 15O 4, 235.0965), 257.0783 [M þ Na] þ (calcd for C 13H 14O 4Na, 257.0784).
3.3.2
Gymnefuranol B (2)
Colorless oil; UV (MeOH) lmax (log 1) 207 (3.55) nm; IR nmax 3428, 1656, 1370, 1230, 1049, 1027, 1006, 826, 764 cm21; 1 H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectral data, see Table 1; HR-ESI-MS: m/z 259.0940 Table 1.
1
[M þ Na] þ (calcd for C13H 16O 4Na, 259.0941) (Table 2).
3.4 Hepatoprotective effect assay The hepatoprotective effect of the isolated compounds was determined by an 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in HL-7702 cells as described previously [12]. The cells were suspended in Roswell Park Memorial Institute 1640 medium (200 ml) containing fetal calf serum (10%), penicillin (100 U/ml), and streptomycin (100 mg/ml), and pre-cultured for 24 h at 378C under a 5% CO2 atmosphere. Then, fresh medium (100 ml) containing bicyclol (a hepatoprotective drug, positive control) and test samples was added, and the cells were cultured for 1 h before exposing to 25 mM D -galactosamine for an additional 24 h. After that, 100 ml of 0.5 mg/ml MTT was added to each well after withdrawal of the culture medium, and incubated for a further 4 h. The resulting formazan was dissolved in 150 ml of DMSO after aspiration of the culture medium. The optical density (OD) of the formazan solution was measured at 492 nm. Inhibition (%) was calculated according to the
H (600 MHz) and 13C (150 MHz) NMR spectral data of 1 and 2 (DMSO-d6). 1
Position 2 3 4 5 6 7 8 9 10 11 12 13 7-OMe 10-OH 13-OH
1 H 7.80 s – 7.25 d (1.1) – 7.02 d (1.1) – – – 4.59 dd (5.6, 0.8) 6.61 br.d (15.9) 6.37 dt (15.9,5.4) 4.13 td (5.4, 1.6) 3.94 s 5.15 t (5.6) 4.85 t (5.54)
2 13 C 142.7 CH 121.9 C 110.5 CH 132.8 C 104.7 CH 144.9 C 143.5 C 128.6 C 53.8 CH2 129.0 CH 129.7 CH 61.6 CH2 55.8 CH3 – –
1 H 7.76 s – 7.04 s – 6.76 s – – – 4.57 d (5.4) 2.67 t (7.8) 1.76 tt (7.8, 6.5) 3.43 td (6.5, 5.1) 3.90 s 5.10 t (5.54) 4.46 t (5.1)
13 C 142.4 CH 121.6 C 111.3 CH 137.6 C 107.5 CH 144.5 C 142.5 C 128.4 C 53.9 CH2 32.0 CH2 34.8 CH2 60.1 CH2 55.7 CH3 – –
Journal of Asian Natural Products Research
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Table 2. Hepatoprotective activities of 1, 2, 6, and 7 (10 mM).
Compound
Cell survival rate (% of normal)a
Inhibition (% of control)
Normal Control Bicyclolc 1 2 6 7
100.0 ^ 1.0 67.9 ^ 2.1b 86.0 ^ 1.1d 87.0 ^ 3.6 85.2 ^ 0.7 97.7 ^ 3.2d 83.7 ^ 4.8
56.4 59.5 53.9 92.8 49.2
a Results are expressed as means ^ SD (n ¼ 3; for normal and control, n ¼ 6). b p , 0.001 (compared to the normal). c Positive control. d p , 0.05 (compared to the control).
following formula: ðODðsampleÞ 2 ODðcontrolÞ Þ inhibitionð%Þ ¼ ODðnormalÞ 2 ODðcontrolÞ £ 100: Acknowledgements We thank Professor W.Y. She for measuring the NMR spectra.
Funding This work was financially supported by the National Science and Technology Project of China [grant number 2012ZX09301002-002] and PCSIRT [grant number IRT1007].
Disclosure Statement No potential conflict of interest was reported by the authors.
References [1] Iconographia Cormophytorum Sinicorum (Science Press, Beijing, 1983), Vol. 3. [2] Flora of China (Science Press, Beijing, 1977), Vol. 63. [3] Y. Xia, X. Liu, and G. Yu, Chin. Wild Plant Resour 19, 1 (2000). [4] K. Shimizu, M. Ozeki, A. Iino, S. Nakajyo, N. Urakawa, and M. Atsuchi, Jpn. J. Pharmacol 86, 223 (2001). [5] E. Porchezhian and R.M. Dobriyal, Pharmazie 58, 5 (2003).
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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
Hepatoprotective benzofurans and furanolignans from Gymnema tingens ab
c
a
a
b
Jin Tian , Yan-Gai Wang , Jie Ma , Jian-Bo Yang , Ligang Zhou , a
a
a
Teng-Fei Ji , Ai-Guo Wang & Ya-Lun Su a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Material Medica, Beijing 100050, China b
Click for updates
MOA Key Laboratory of Plant Pathology, Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China c
Department of Pharmacy, XuanWu Hospital of Capital Medical University, Beijing 100053, China Published online: 13 Mar 2015.
To cite this article: Jin Tian, Yan-Gai Wang, Jie Ma, Jian-Bo Yang, Ligang Zhou, Teng-Fei Ji, Ai-Guo Wang & Ya-Lun Su (2015) Hepatoprotective benzofurans and furanolignans from Gymnema tingens, Journal of Asian Natural Products Research, 17:3, 268-273, DOI: 10.1080/10286020.2015.1016002 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1016002
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.
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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 & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Journal of Asian Natural Products Research, 2015 Vol. 17, No. 3, 268–273, http://dx.doi.org/10.1080/10286020.2015.1016002
Hepatoprotective benzofurans and furanolignans from Gymnema tingens Jin Tianab, Yan-Gai Wangc, Jie Maa, Jian-Bo Yanga, Ligang Zhoub, Teng-Fei Jia, Ai-Guo Wanga* and Ya-Lun Sua*
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a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Chinese Academy of Medical Sciences and Peking Union Medical College, Institute of Material Medica, Beijing 100050, China; bMOA Key Laboratory of Plant Pathology, Department of Plant Pathology, College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China; c Department of Pharmacy, XuanWu Hospital of Capital Medical University, Beijing 100053, China (Received 4 December 2014; final version received 26 January 2015) Two new benzofurans, gymnefuranols A (1) and B (2), together with six known furanolignans (3 – 8), were isolated from Gymnema tingens. The structures of the new compounds were elucidated by comprehensive analysis of the NMR and HR-MS data. Compounds 1, 2, 6, and 7 showed hepatoprotective activities against D -galactosamineinduced HL-7702 cell damage. Keywords: Asclepiadaceae; Gymnema tingens; hepatoprotective; benzofuran; furanolignan; structure elucidation
1.
Introduction
The genus Gymnema belonging to the Asclepiadaceae family contains around 25 species worldwide and distributes in tropical and subtropical regions of Asia, southern Africa, and Oceania, while eight species are found in south and southwest China [1,2]. In Chinese folk medicine, the extract of Gymnema has the effect of detumescence, detoxicating, promoting granulation, and easing pain [3]. Secondary metabolites including saponins, steroids, alkaloids, phenolic glycosides, and peptides have been reported from Gymnema inodorum [4], Gymnema sylvestre [5], Gymnema yunnanense [6,7], Gymnema alternifolium [8–11], and Gymnema tingens [12], and showed interesting biological activities such as antisweet, antidiabetic, antiallergic, antiviral, hepatoprotective, and lipid lowering effects. To the best of our knowledge, phytochemical investigation on G. tingens had
been so far reported only by our group [12]. In a previous investigation of the hepatoprotective constituents of G. tingens, we had identified nine bioactive phenolic diglycosides [12]. In continuation of this work, two benzofurans (1– 2) and six furanoligans (3 –8) with hepatoprotective effect were isolated (Figure 1). In this paper, we describe the isolation and structure elucidation of new compounds, as well as the biological activities of the isolated compounds. 2. Results and discussion Compound 1 was isolated as a colorless oil. Its molecular formula was determined to be C13H14O4 as a prominent peak was observed at m/z 257.0783 [M þ Na]þ in the HR-ESI-MS, implying seven degrees of unsaturation. The IR spectrum showed characteristic bands of hydroxyl (3336 cm21), olefin (1658 cm21), and benzene groups (1619, 1601 cm21). The
*Corresponding authors. Emails: [email protected]; [email protected] q 2015 Taylor & Francis
Journal of Asian Natural Products Research 1 O 8 3 HO
OCH3
269
OCH3 O
6 OH
9
4
10
11
OH
13 HO
1 OCH3
H3CO O
HO
2
OCH3
R2 O
HO
OH
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OH H3CO
O
OCH3
R1
OCH3 R3O
HO
4 R1 = H; R2 = OCH3; R3 = β-D-glucose
3
5 R1 = R2 = H; R3 = β-D-glucose 6 R1 = R2 = R3 = H 7 R1 = H; R2 = OCH3; R3 = H 8 R1 = R2 = OCH3; R3 = H
Figure 1. Structures of the isolated compounds 1 – 8. 1
H NMR spectrum exhibited signals for a pair of meta-coupling aromatic protons at dH 7.25 (d, J ¼ 1.1 Hz, H-4) and 7.02 (d, J ¼ 1.1 Hz, H-6), an isolate olefinic proton at dH 7.80 (s, H-2), and a transdisubstituted double bond at dH 6.61 (br. d, J ¼ 15.9 Hz, H-11) and 6.37 (dt, J ¼ 15.9 and 5.4 Hz, H-12) in the downfield region. The two triplets at dH 5.15 (OH-10) and 4.85 (OH-13) were D2Oexchangeable, and thus assigned to two hydroxyls, which each coupled to a methylene group at dH 4.59 (dd, J ¼ 5.6 and 0.8 Hz, H2 2 10), and 4.13 (td, J ¼ 5.4 and 1.6 Hz, H2 2 13), respectively. In addition, one methoxyl group was observed at dH 3.94. The 13C NMR
spectrum displayed signals for 10 sp2hybridized carbons that were assignable to one tetrasubstituted benzene ring (dC 144.9 C, 143.5 C, 132.8 C, 128.6 C, 110.5 CH, 104.7 CH), one trisubstituted double bond (dC 142.7 CH, 121.9 C), and one disubstituted double bond (dC 129.7 CH, 129.0 CH), and for three oxygenated sp3-hybridized carbons including one methoxyl (dC 55.8), and two hydroxymethyl groups (dC 53.8, 61.6), as revealed by the HSQC experiment. The connection of the aforementioned moieties was constructed by analysis of the HMBC spectrum (Figure 2). The correlations from both H-4 (dH 7.25, d) and H-6 (dH 7.02, d) to C-11 (dC 129.0,
O O
8
O O
6
2 9
HO
4 10
OH 13
OH HO
1
Figure 2. HMBC correlations (H ! C) of compounds 1 and 2.
2
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CH), and from H-11 (dH 6.61, br.d) to C-4 (dC 110.5, CH), C-5 (dC 132.8, C), and C-6 (dC 104.7, CH), indicated that the disubstituted double bond was connected to C-5 of the aromatic ring. Additional correlations from the olefinic protons H-11 and H-12 (dH 6.37, dt) to C-13 (dC 61.6, CH2), and from H2-13 (dH 4.13, td) to C-11 and C-12 (dC 129.7, CH) suggested that one hydroxymethyl group was connected to the other end of this disubstituted double bond. The second hydroxymethyl group (dH 4.59 dd, H2-10; dC 53.8, CH2-10) showed correlations to C-2 (dC 142.7, CH), C-3 (dC 121.9, C), and C-9 (dC 128.6, C), thus allowing the assignment of this group to C-3 of the trisubstituted double bond, and the connection between C-3 and C-9 of the benzene ring. Further HMBC correlations from H-2 (dH 7.80, s) to C-8 (dC 143.5, C), and taking into consideration of the chemical shifts of C-2 (dC 142.7, CH) and C-8, suggested that C-2 was connected to C-8 via an oxygen bridge, thus furnishing a benzofuran skeleton. The correlation from the methoxyl group (dH 3.94 s; dC 55.8 CH3) to C-7 (dC 144.9, C) indicated that this group was substituted at C-7 of the benzene ring. Therefore, compound 1 was elucidated as (E)-3-(3-(hydroxymethyl)-7-methoxybenzofuran-5-yl)prop-2-en-1-ol, and named gymnefuranol A. Compound 2 was also obtained as a colorless oil. The molecular formula of 2 was deduced as C13H16O4 by HR-ESI-MS, which bears two more protons than 1, and accordingly one less degree of unsaturation than 1. The NMR data were similar for both compounds, indicating they are congeners. The only differences were attributed to the side chain, in which the signals for the trans-disubstituted double bond in 1 were replaced by those for two methylene groups [dH 2.67 (t, J ¼ 7.8 Hz, H2-11), dC 32.0 (CH2-11); dH 1.76 (tt, J ¼ 7.8, 6.5 Hz, H2-12), dC 34.8 (CH2-12)] in 2. This was further corroborated by analysis of the HSQC and HMBC spectra.
Therefore, compound 2 was established as the dihydro derivative of 1, and named 3(3-(hydroxymethyl)-7-methoxybenzofuran-5-yl)propan-1-ol. The trivial name gymnefuranol B was given for this compound. The known compounds were identified by comparing their spectroscopic data with those in the literature, and included (70 S,8S,80 R)-4,40 -dihydroxy-3,3 0 ,5,50 -tetramethoxy-70 ,9-epoxylignan-9 0 -ol-7-one (3) [13], alangilignoside D (4) [14], (– )lariciresinol 9-O-b-D -glucopyranoside (5) [15], (– )-lariciresinol (6) [16], 50 -methoxylariciresinol (7) [17], and 5,50 -dimethoxylariciresinol (8) [18]. The hepatoprotective effect of compounds 1 –8 were evaluated against Dgalactosamine-induced toxicity in HL7702 cells in vitro. Compounds 1, 2, 6, and 7 showed better cell survival rates than the control at 10 mM, while 1 and 6 exhibited better hepatoprotective effect than the positive control bicyclol by comparison of their cell survival rates and inhibition rates (Table 2). Among them, compound 6 displayed the most significant protective effect against the D galactosamine-induced cell damage (97.7% cell survival rate after treatment). Thus, this compound could be a promising agent for the treatment of hepatic disease. Hepatoprotective compounds of various structural types were documented in the literature, such as sesquiterpene glycosides [19], phenolic glycosides [12,20], iridoid glycosides [21,22], lignans [23], triterpenes [24], and triterpene saponins [25]. The identification of two new hepatoprotective benzofurans (1 –2) in this study has enriched the chemical diversity of the hepatoprotective compounds. 3.
Experimental
3.1 General experimental procedures The optical rotations were measured on a Jasco P-2000 polarimeter (JASCO Corp.,
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Journal of Asian Natural Products Research Tokyo, Japan). The UV spectra were obtained with a Jasco V650 spectrophotometer (JASCO Corp.). IR spectra were recorded using a Nicolet 5700 FT-IR spectrometer (Thermo Electron Scientific Instrument Crop., Madison, WI, USA). 1H, 13 C NMR, and 2D NMR spectra were measured on a Varian Unity INOVA 600 spectrometer (Varian, Inc., Palo Alto, CA, USA). The chemical shifts were referred to the solvent residual peak of DMSO-d6 (dH 2.50 for 1H, and dC 39.5 for 13C). ESI-MS and HR-ESI-MS data were obtained using an Agilent 1100 series LC/MSD Trap SL mass spectrometer (Agilent Technologies, Santa Clara, CA, USA). Column chromatography was performed using RP-18 (50 mm; YMC Co., Ltd, Kyoto, Japan), and silica gel (160 – 200 mesh, and 200 –300 mesh; Qingdao Marine Chemical, Inc. Co., Ltd, Qingdao, China). Preparative HPLC was conducted using a Shimadzu LC-6AD instrument with an SPD-20A detector (SHIMADZU, Corp., Tokyo, Japan) and an YMC Pack ODS-A column (250 £ 20 mm, 10 mm; YMC Co., Ltd). HPLC – DAD analysis was performed using an Agilent 1200 series system (Agilent Technologies) with an analytic C18 column (250 mm £ 4.6 mm, 5 mm; YMC Co., Ltd). Precoated silica gel GF-254 plates (Yantai Jiangyou Silica Gel Exploitation Co., Ltd, Yantai, China) were used for analytical TLC. Spots were visualized under UV light (254 or 365 nm) or by spraying with 10% H2SO4 in 95% EtOH followed by heating. 3.2 Plant material The stems and leaves of G. tingens Roxb. ex Spreng. (Asclepiadaceae) were collected in Dai Autonomous Prefecture of Xishuangbanna, Yunnan Province, China, in August 2010. The plant material was identified by Prof. Jing-yun Cui (Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences). A voucher specimen (No. 035413) has been deposited at the Herbar-
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ium of Xishuangbanna tropical botanical garden (Chinese Academy of Sciences, Xishuangbanna, Yunnan, China). 3.3 Extraction and isolation As reported previously, the EtOAc extract of the ethanol extract of G. tingens was subjected to vacuum liquid chromatography over silica gel to yield 22 fractions, among which fraction 9 was found to be a pool of nine phenolic diglycosides [12]. The less polar fraction 6 (15 g) was subjected to chromatography over ODS eluting with a gradient of methanol – water (from 60% to 100%) to obtain 12 subfractions (F6-1 – F6-12). F6-6 (400 mg) was purified by semi-preparative HPLC using 18% ACN – H2O as eluent (6 ml/min) to afford compounds 4 (5 mg, Rt 29 min) and 5 (2 mg, Rt 32 min). Similarly, compound 3 (14 mg, Rt 42 min) was obtained from subfraction F6-12 (100 mg) by semi-preparative HPLC using 21% ACN – H2O as mobile phase (6 ml/min). Fraction 7 (13 g) was subjected to MPLC eluting with a gradient of MeOH– H2O to yield eight subfractions (F7-1– F7-8), among which F7-8 (1.1 g) was subjected to column chromatography over silica gel (200–300 mesh) eluting with CHCl3 –MeOH (40:1) to obtain a fraction containing compound 8 (5 mg), which was purified by a subsequent RP-HPLC separation using 45% MeOH–H2O as eluent (Rt 13 min for 8, 6 ml/min). Fraction 8 (7 g) was chromatographed over silica gel (200 –300 mesh) eluting with petroleum ether – acetone (3:1) to yield five subfractions (F8-1 –F8-5). Subfraction F8-2 (0.6 g) was separated by RPHPLC using 35% MeOH – H2O as mobile phase to afford compound 6 (13 mg, Rt 18.5 min, 6 ml/min). Subfraction F8-4 (1.5 g) was subjected to size exclusion chromatography over Sephadex LH-20 using methanol as eluent to obtain four further subfractions (F8-4-1 – F8-4-4),
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among which F-8-4-1 (410 mg), F-8-4-2 (380 mg), and F-8-4-3 (360 mg) were purified by RP-HPLC using 40% MeOH – H2O as eluent (6 ml/min) to afford compounds 2 (12 mg, Rt 42 min), 1 (5 mg, Rt 41 min), and 7 (23 mg, Rt 39 min), respectively.
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3.3.1
Gymnefuranol A (1)
Colorless oil; UV (MeOH) lmax (log 1) 206 (3.63), 236 (sh) (3.11) nm; IR nmax 3336, 3283, 1658, 1619, 1601, 1371, 1086, 972, 806 cm21; 1H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectral data, see Table 1; HRESI-MS: m/z 235.0960 [M þ H]þ (calcd for C13H 15O 4, 235.0965), 257.0783 [M þ Na] þ (calcd for C 13H 14O 4Na, 257.0784).
3.3.2
Gymnefuranol B (2)
Colorless oil; UV (MeOH) lmax (log 1) 207 (3.55) nm; IR nmax 3428, 1656, 1370, 1230, 1049, 1027, 1006, 826, 764 cm21; 1 H NMR (DMSO-d6, 600 MHz) and 13C NMR (DMSO-d6, 150 MHz) spectral data, see Table 1; HR-ESI-MS: m/z 259.0940 Table 1.
1
[M þ Na] þ (calcd for C13H 16O 4Na, 259.0941) (Table 2).
3.4 Hepatoprotective effect assay The hepatoprotective effect of the isolated compounds was determined by an 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay in HL-7702 cells as described previously [12]. The cells were suspended in Roswell Park Memorial Institute 1640 medium (200 ml) containing fetal calf serum (10%), penicillin (100 U/ml), and streptomycin (100 mg/ml), and pre-cultured for 24 h at 378C under a 5% CO2 atmosphere. Then, fresh medium (100 ml) containing bicyclol (a hepatoprotective drug, positive control) and test samples was added, and the cells were cultured for 1 h before exposing to 25 mM D -galactosamine for an additional 24 h. After that, 100 ml of 0.5 mg/ml MTT was added to each well after withdrawal of the culture medium, and incubated for a further 4 h. The resulting formazan was dissolved in 150 ml of DMSO after aspiration of the culture medium. The optical density (OD) of the formazan solution was measured at 492 nm. Inhibition (%) was calculated according to the
H (600 MHz) and 13C (150 MHz) NMR spectral data of 1 and 2 (DMSO-d6). 1
Position 2 3 4 5 6 7 8 9 10 11 12 13 7-OMe 10-OH 13-OH
1 H 7.80 s – 7.25 d (1.1) – 7.02 d (1.1) – – – 4.59 dd (5.6, 0.8) 6.61 br.d (15.9) 6.37 dt (15.9,5.4) 4.13 td (5.4, 1.6) 3.94 s 5.15 t (5.6) 4.85 t (5.54)
2 13 C 142.7 CH 121.9 C 110.5 CH 132.8 C 104.7 CH 144.9 C 143.5 C 128.6 C 53.8 CH2 129.0 CH 129.7 CH 61.6 CH2 55.8 CH3 – –
1 H 7.76 s – 7.04 s – 6.76 s – – – 4.57 d (5.4) 2.67 t (7.8) 1.76 tt (7.8, 6.5) 3.43 td (6.5, 5.1) 3.90 s 5.10 t (5.54) 4.46 t (5.1)
13 C 142.4 CH 121.6 C 111.3 CH 137.6 C 107.5 CH 144.5 C 142.5 C 128.4 C 53.9 CH2 32.0 CH2 34.8 CH2 60.1 CH2 55.7 CH3 – –
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Table 2. Hepatoprotective activities of 1, 2, 6, and 7 (10 mM).
Compound
Cell survival rate (% of normal)a
Inhibition (% of control)
Normal Control Bicyclolc 1 2 6 7
100.0 ^ 1.0 67.9 ^ 2.1b 86.0 ^ 1.1d 87.0 ^ 3.6 85.2 ^ 0.7 97.7 ^ 3.2d 83.7 ^ 4.8
56.4 59.5 53.9 92.8 49.2
a Results are expressed as means ^ SD (n ¼ 3; for normal and control, n ¼ 6). b p , 0.001 (compared to the normal). c Positive control. d p , 0.05 (compared to the control).
following formula: ðODðsampleÞ 2 ODðcontrolÞ Þ inhibitionð%Þ ¼ ODðnormalÞ 2 ODðcontrolÞ £ 100: Acknowledgements We thank Professor W.Y. She for measuring the NMR spectra.
Funding This work was financially supported by the National Science and Technology Project of China [grant number 2012ZX09301002-002] and PCSIRT [grant number IRT1007].
Disclosure Statement No potential conflict of interest was reported by the authors.
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