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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new lignan with hypoglycemic activity from Tadehagi triquetrum ab
a
ab
ab
ab
Jiani Wu , Cai yun Zhang , Tingting Zhang , Dan Zhao , Ni An , a
c
b
a
Youbin Li , Nailiang Zhu , Shuai Wang , Feng Chen & Xiaopo Zhang
a
a
School of Pharmaceutical Science, Hainan Medical University, Haikou571101, P.R. China b
Research Center on Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin150076, P.R. China c
Click for updates
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing100193, P.R. China Published online: 22 Jan 2015.
To cite this article: Jiani Wu, Cai yun Zhang, Tingting Zhang, Dan Zhao, Ni An, Youbin Li, Nailiang Zhu, Shuai Wang, Feng Chen & Xiaopo Zhang (2015): A new lignan with hypoglycemic activity from Tadehagi triquetrum, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.1003136 To link to this article: http://dx.doi.org/10.1080/14786419.2014.1003136
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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Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2014.1003136
A new lignan with hypoglycemic activity from Tadehagi triquetrum Jiani Wuab1, Cai yun Zhanga1, Tingting Zhangab, Dan Zhaoab, Ni Anab, Youbin Lia, Nailiang Zhuc, Shuai Wangb, Feng Chena* and Xiaopo Zhanga*
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a School of Pharmaceutical Science, Hainan Medical University, Haikou 571101, P.R. China; bResearch Center on Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin 150076, P.R. China; cInstitute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, P.R. China
(Received 18 October 2014; final version received 24 December 2014)
A new lignan named tadehaginosin, together with a known compound 3,4-dihydro-4(40 -hydroxyphenyl)-5,7-dihydroxycoumarin, was isolated from the aerial part of Tadehagi triquetrum. The new structure was determined by various spectroscopic techniques (1H and 13C APT, HSQC, HMBC, 1H – 1HCOSY, NOESY and HR-ESIMS). The two isolates were evaluated for their hypoglycemic effects in vitro. Biological investigation showed that both of them possessed the capability to increase glucose consumption by HepG2 cells. Keywords: Tadehagi triquetrum; tadehaginosin; hypoglycemic effects
1. Introduction Tadehagi triquetrum is a member of the genus Tadehagim in the Leguminosae family, which is widely distributed in the subtropical and Pacific regions of the world (Kalyani et al. 2011a, 2011b). This plant with anti-inflammatory and hepatoprotective activities has been used as medicine in treating many chronic diseases worldwide (Kalyani et al. 2011a, 2011b; Tang et al. 2014). In China, it is commonly used as a traditional Chinese folk medicine in treating sore throat, bacillary dysentery and pregnant vomiting (Wang et al. 2007). Previously, chemical investigations have resulted in the isolation of flavonoids, phenolic compounds and terpenes (Gui et al. 2014). The phenoloids found in T. triquetrum had interesting chemical structures and biological activities. As an ongoing research, a new lignan, together with a known compound, was isolated from the aerial part of T. triquetrum. Moreover, the extract of T. triquetrum has been proved to display hypoglycemic activities (Wang et al. 2007). However, the bioactive constituents concerning its glucose lowering effects were unknown till now. Therefore, the
*Corresponding authors. Email: [email protected]; [email protected] q 2015 Taylor & Francis
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J. Wu et al.
effects of the isolates on consumption of glucose were assessed. In this paper, we reported the isolation, structural elucidation and the hypoglycemic effects of the new isolate in vitro.
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2. Results and discussion Compound 1 was obtained as an optical active oil with ½a20 D 2 19.0 (c ¼ 0.01, CH3OH). Its molecular formula was determined as C20H20O8 by HR-ESI-MS at m/z 411.1060 [M þ Na] þ (calcd. 411.1056 [M þ Na] þ). The IR spectrum of 1 showed hydroxyl (3380 cm21) and carbonyl (1752, 1734 cm21) absorptions. The 1H NMR spectrum of 1 displayed the characteristic signals attributing to a para-substituted phenolic ring with two equivalent pairs of ortho-coupled protons at dH 6.72 (2H, d, J ¼ 8.4 Hz, H-3, 5), 7.23 (2H, d, J ¼ 8.4 Hz, H-2, 6) (Zhang et al. 2012). Furthermore, the 1H NMR spectrum showed the existence of a aromatic proton singlet at dH 7.36 (2H, s, H-20 , 60 ), one oxymethine at dH 5.12 (1H, d, J ¼ 5.4 Hz, H-7), two aliphatic methines at dH 3.38 (1H, t, J ¼ 5.4 Hz, H-8) and 4.38 (1H, m, H-80 ), one oxymethylene at dH 4.23 (1H, dd, J ¼ 9.0, 4.8 Hz, H-90 a) and 3.83 (1H, t, J ¼ 9.0 Hz, H-90 b), and two methoxyl groups at dH 3.91 (6H, s, 30 -OCH3, 50 -OCH3). Its 13C-APT spectrum indicated that 1 contained 14 sp2-hybridised carbon atoms corresponding to two carbonyl groups at dC 198.4 (C-70 ) and 178.6 (C-9), and two phenyl rings [dC 132.6 (C-1), 129.0 (C-2, 6), 116.3 (C-3, 5), 158.8 (C-4)] and [dC 127.4 (C-10 ), 108.2 (C-20 , 60 ), 143.4 (C-40 ), 149.3 (C-30 , 50 )]. The 13CAPT also displayed four sp3-hybridised carbon atoms including one oxymethine dC 73.7 (C-7), two methines at dC 53.7 (C-8) and 45.1 (C-80 ), and one oxymethylene dC 70.5 (C-90 ). In accordance with the 1H NMR data, two methoxyl groups at dC 57.1 (30 -OCH3, 50 -OCH3) were clearly figured out. The aforementioned NMR data showed that the carbon skeleton of 1 has 18 carbons and was assigned to be a lignan (Li et al. 2014). The 1H – 1H COSY spectrum suggested that all aliphatic methine and methylene protons were a contiguous spin system comprising H-7, H-8, H-80 and H-90 in the molecule (Figure S1). In the HMBC experiment, the correlations from dH 7.23 (H-2, 6) to dC 158.8 (C-4) and 73.7 (C7) suggested the presence of two hydroxyl groups at C-4 and C-7, respectively. The HMBC correlations between H-20 /60 and 198.4 (C-70 ) indicated the location of the keto group (C-70 ). Similarly, the position of another carbonyl group was confirmed to be at C-9 (178.6) since both dH 5.12 (H-7) and 3.38 (H-8) were all correlated with C-9. Furthermore, the HMBC correlations from dH 5.12 (H-7) to dC 132.6 (C-1), 129.0 (C-2, 6), 53.7 (C-8), 178.6 (C-9) and 45.1 (C-80 ) indicated linkage from C-1 to C-9 and C-8 to C-80 . In addition, the HMBC correlations between signal at dH 4.38 (H-80 ) and dC 73.7 (C-7), 53.7 (C-8), 178.6 (C-9), 198.4 (C-70 ) suggested the linkage from C-10 to C-90 . The connection between C-90 and C-9 through an oxygen atom was confirmed by the HMBC correlation from dH 3.83, 4.23 (H-90 ) to dC 178.6 (C-9). Furthermore, the correlations from dH 3.91 (6H, s, 30 -OCH3, 50 -OCH3) to dC 149.3 (C-30 , 50 ) confirmed that the methoxyl groups were located at C-30 , 50 . These data unambiguously showed that tadehaginosin (1) was a tetrahydrofuran lignan belonging to 9,90 -epoxy type (Wang et al. 2004; Gan et al. 2005). Thus, the planar structure of 1 was established. The relative stereochemistry of 1 was deduced from the NOESY experiment. In the NOESY spectrum, H-80 (dH 4.38) showed correlations with H-90 b (dH 3.83) and H-7 (dH 5.12), and no correlation between H-8 (dH 3.38) and H-80 , indicating that H-8 and H-80 were on the opposite side (Gan et al. 2005; Wang et al. 2004). Accordingly, 1 was established as 4,40 ,7-trihydroxy-30 ,50 dimethoxy-7,9-diketo-9,90 -epoxylignan, which was named tadehaginosin, as shown in Figure 1. Compound 2 was obtained as powder. Its chemical structure was established by analysing its 1D- and 2D NMR data. The absolute configuration of 1 was determined by comparing its positive optical rotation with reported data (Sun & Sneden 1999; Li et al. 2009). The extract of T. triquetrum has been proved to display hypoglycemic activities. However, the bioactive constituents concerning its glucose lowering effects were unknown till now.
Natural Product Research
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OH 4'
OCH3 3'
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O
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OH
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HO 5
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7'
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O
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OH
OH
1' 4
5 6
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5'
3
OCH3 HO
7 8
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O
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Figure 1. Chemical structures of 1 and 2
In order to find novel compounds with hypoglycemic effects from this plant, the biological activity of 1 and 2 on consumption of glucose was assessed on HepG2 cells. As shown in Figure 2, treatment with insulin (100 nM) decreased the concentration of glucose obviously. Supplement with the two compounds (10 mM) significantly increased the consumption of glucose, respectively. Importantly, 1 (**p , 0.01) showed stronger activity than 2 (*p , 0.05) under the tested dose. This paper reported the structural elucidation of a new lignan and its hyperglycemic effects, providing scientific evidence for developing new chemical candidates in treating diabetes.
3. Experimental 3.1. General experimental procedures Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, CT, USA). UV spectra were recorded on Shimadzu UV2550 spectrometer and IR
Figure 2. Effects of 1 and 2 (10 mM) on glucose consumption by HepG2 cells. Cells were incubated in the serum-free high glucose DMEM (25 mM glucose contained) medium supplemented with 1 or 2 (10 mM) for 24 h. Then the glucose concentration in the medium was measured by a glucose quantification kit to evaluate the glucose consumption by cells. Values represent mean ^ SD. Results are representative of three different experiments with n ¼ 3. *p , 0.05, **p , 0.01.
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spectra were measured on FTIR-8400S spectrometer (Shimadzu, Kyoto, Japan). The NMR data were recorded on Bruker AV-600 (600 MHz for 1H and 150 MHz for 13C) in CD3OD with TMS as internal standard. The HR-ESI-MS data were performed on a LTQ-Obitrap XL spectrometer. Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and silica gel (100 –200 meshes, Qingdao Marine Chemical plant, Qingdao, P.R. China) were used for column chromatography, and precoated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, P.R. China) were used for TLC. Dulbecco’s modified Eagle’s medium (DMEM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were procured from SigmaAldrich, Inc. (St Louis, MO, USA). HepG2 cells, which originated from the American Type Culture Collection (ATCC) (Manassas, VA, USA), were obtained from the Peking Union Medical College (Beijing, China). 3.2. Plant material The whole plants of Tadehagi triquetrum were collected in July 2013 from Haikou, Hainan Province, China and identified by Prof. Jianping Tian at the School of Pharmaceutical Science, Hainan Medical University. A voucher specimen has been deposited there (Voucher specimen No. TT20130810). 3.3. Extraction and isolation The air-dried and powdered aerial part (5 kg) of T. triquetrum was extracted three times with solvent (CH3CH2OH: H2O ¼ 70:30) at 70 – 808C (50 L £ 2 h £ 3). After removal of the solvent under reduced pressure, the crude extract (500 g) was dissolved in distilled water, and was successively partitioned with petroleum ether, ethyl acetate and n-butanol, respectively. The EtOAc fraction (150 g) was subjected to silica gel column chromatography (80 cm £ 8 cm) eluted with CHCl3 – CH3COCH3 (1:0 to 2:1) to afford four fractions, Fr. 1– Fr. 4. Fr. 3 (15.5 g) was purified by Sephadex LH-20 column chromatography (120 cm £ 3 cm) eluting with CH3OH and the subfractions were further purified by semi-preparative HPLC using CH3OH – H2O (40:60) as the mobile phase (flow rate at 2.0 mL min21, wavelength at 210 nm, retention time at 22.125 min) to yield 1 (6.2 mg). Meanwhile, Fr. 4 (12.8 g) was purified by Sephadex LH-20 column chromatography (120 cm £ 3 cm) eluting with CH3OH and the subfractions were further purified by semi-preparative HPLC using CH3OH –H2O (47:53) as the mobile phase, (flow rate at 2.0 mL min21, wavelength at 210 nm, retention time at 27.125 min) to yield 2 (5.0 mg). 3.3.1. Tadehaginosin (1) Colourless oil, ½a20 D 2 19.0 (c ¼ 0.01, CH3OH); UV lmax (CH3OH) nm (log 1): 307 (4.08); IR (KBr) cm21 3380, 2918, 1752, 1734, 1646, 1610, 1512, 1113, 1019; 1H NMR (600 MHz, CD3OD) d: 7.23 (2H, d, J ¼ 8.4 Hz, H-2, 6), 6.72 (2H, d, J ¼ 8.4 Hz, H-3, 5), 5.12 (1H, d, J ¼ 5.4 Hz, H-7), 3.38 (1H, t, J ¼ 5.4 Hz, H-8), 7.36 (2H, s, H-20 , 60 ), 4.38 (1H, m, H-80 ), 4.23 (1H, dd, J ¼ 9.0, 4.8 Hz, H-90 a), 3.83 (1H, t, J ¼ 9.0 Hz, H-90 b), 3.91 (6H, s, 30 -OCH3, 50 OCH3); 13C NMR (150 MHz, CD3OD) d: 132.6 (C-1), 129.0 (C-2, 6), 116.3 (C-3, 5), 158.8 (C4), 73.7 (C-7), 53.7 (C-8), 178.6 (C-9), 127.4 (C-10 ), 108.2 (C-20 , 60 ), 143.4 (C-40 ), 149.3 (C-30 , 50 ), 198.4 (C-70 ), 45.1 (C-80 ), 70.5 (C-90 ), 57.1 (30 -OCH3, 50 -OCH3); HR-ESI-MS m/z 411.1060 [M þ Na] þ (calcd. for C20H20O8Na 411.1056). 3.4. Hypoglycemic evaluation and statistics analysis The biological activities of compounds 1 and 2 on consumption of glucose were assessed on HepG2 cells according to previous report (Supkamonseni et al. 2014). HepG2 cells were
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incubated with the serum-free DMEM containing different concentration of 1 and 2 for 24 h. Then the glucose concentration (mM L21) of medium was measured by glucose kit. The results were expressed as mean ^ SD. A one-way analysis of variance was done using the SPSS 13.0 software (Liu et al. 2013). Significance was accepted at p , 0.05. Values represent mean ^ SD. Results are representative of three different experiments with n ¼ 3. *p , 0.05, **p , 0.01. 4. Conclusion
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In this study, we reported the isolation, structural elucidation and hypoglycemic activity of a new lignan, tadehaginosin, from T. triquetrum. The result showed that the new isolate exhibited potent ability to increase glucose consumption in vitro. Supplementary material Supplementary material relating to this paper is available online, alongside Figures S1 –S8. Funding The financial assistance given by the Natural Sciences Foundation of China [grant number 81202994] is gratefully acknowledged.
Note 1. These authors contributed equally to this work.
References Gan LS, Yang SP, Fan CQ, Yue JM. 2005. Lignans and their degraded derivatives from Sarcostemma acidum. J Nat Prod. 68:221–225. Gui RC, Li YG, Zhang LB, Li LS, Zhang JJ. 2014. Advances in pharmacological functions of Tadehagi triquetrum (L.) DC. Jiang Xi J Trad Chin Med. 45:78–80. Kalyani GA, Ashok P, Taranalli AD, Ramesh CK, Krishna V, Swamy AH. 2011a. Anti-inflammatory and in vitro antioxidant activity of Desmodium triquetrum (L.). Indian J Pharmacol. 43:740–741. Kalyani GA, Ramesh CK, Krishna V. 2011b. Hepatoprotective and antioxidant activities of Desmodium triquetrum DC. Indian J Pharm Sci. 73:463–466. Li HF, Ma QY, Liu YQ, Qian JF, Zhou J, Zhao YX. 2009. Chemical constituents from Polygonum perfoliatum. Chin J Appl Environ Biol. 15:615–620. Liu Z, Wei F, Chen LJ, Xiong HR, Liu YY, Luo F, Hou W, Xiao H, Yang ZQ. 2013. In vitro and in vivo studies of the inhibitory effects of emodin isolated from Polygonum cuspidatum on Coxsakievirus B4. Molecules. 18:11842–11858. Li X, Li L, Wang J, Wang T, Wang L. 2014. Two new lignans with antioxidative activities from Jatropha curcas. Nat Prod Res. 28:1985–1991. Sun X, Sneden AT. 1999. Neoflavonoids from Polygonum perfoliatum. Planta Med. 65:671–673. Supkamonseni N, Thinkratok A, Meksuriyen D, Srisawat R. 2014. Hypolipidemic and hypoglycemic effects of Centella asiatica (L.) extract in vitro and in vivo. Indian J Exp Biol. 52:965–971. Tang AC, Chen XY, Lu QY, Zheng N, Wei YF, Wu XY. 2014. Anti-hepatotoxic effect of tadehaginoside, extracted from Tadehagi triquetrum (L.), against CCl4-lesioned rats through activating the Nrf2 signaling pathway and attenuating the inflammatory response. Inflammation. 37:1007–1013. Wang BG, Ebel R, Wang CY, Edrada RA, Wray V, Proksch P. 2004. Aglacins I-K, three highly methoxylated lignans from Aglaia cordata. J Nat Prod. 67:682–684. Wang YQ, Feng BM, Tang L, Shi LY, Shi HF. 2007. Research of Tadehagi triquetrum (L.) DC. J Dalian Univ. 28:49–51. Zhang XP, Wei N, Huang J, Tan YF, Jin DJ. 2012. A new feruloyl amide derivative from the fruits of Tribulus terrestris. Nat Prod Res. 26:1922–1925.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
A new lignan with hypoglycemic activity from Tadehagi triquetrum ab
a
ab
ab
ab
Jiani Wu , Cai yun Zhang , Tingting Zhang , Dan Zhao , Ni An , a
c
b
a
Youbin Li , Nailiang Zhu , Shuai Wang , Feng Chen & Xiaopo Zhang
a
a
School of Pharmaceutical Science, Hainan Medical University, Haikou571101, P.R. China b
Research Center on Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin150076, P.R. China c
Click for updates
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing100193, P.R. China Published online: 22 Jan 2015.
To cite this article: Jiani Wu, Cai yun Zhang, Tingting Zhang, Dan Zhao, Ni An, Youbin Li, Nailiang Zhu, Shuai Wang, Feng Chen & Xiaopo Zhang (2015): A new lignan with hypoglycemic activity from Tadehagi triquetrum, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2014.1003136 To link to this article: http://dx.doi.org/10.1080/14786419.2014.1003136
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.
Downloaded by [New York University] at 08:15 15 July 2015
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
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2014.1003136
A new lignan with hypoglycemic activity from Tadehagi triquetrum Jiani Wuab1, Cai yun Zhanga1, Tingting Zhangab, Dan Zhaoab, Ni Anab, Youbin Lia, Nailiang Zhuc, Shuai Wangb, Feng Chena* and Xiaopo Zhanga*
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a School of Pharmaceutical Science, Hainan Medical University, Haikou 571101, P.R. China; bResearch Center on Life Sciences and Environmental Sciences, Harbin University of Commerce, Harbin 150076, P.R. China; cInstitute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, P.R. China
(Received 18 October 2014; final version received 24 December 2014)
A new lignan named tadehaginosin, together with a known compound 3,4-dihydro-4(40 -hydroxyphenyl)-5,7-dihydroxycoumarin, was isolated from the aerial part of Tadehagi triquetrum. The new structure was determined by various spectroscopic techniques (1H and 13C APT, HSQC, HMBC, 1H – 1HCOSY, NOESY and HR-ESIMS). The two isolates were evaluated for their hypoglycemic effects in vitro. Biological investigation showed that both of them possessed the capability to increase glucose consumption by HepG2 cells. Keywords: Tadehagi triquetrum; tadehaginosin; hypoglycemic effects
1. Introduction Tadehagi triquetrum is a member of the genus Tadehagim in the Leguminosae family, which is widely distributed in the subtropical and Pacific regions of the world (Kalyani et al. 2011a, 2011b). This plant with anti-inflammatory and hepatoprotective activities has been used as medicine in treating many chronic diseases worldwide (Kalyani et al. 2011a, 2011b; Tang et al. 2014). In China, it is commonly used as a traditional Chinese folk medicine in treating sore throat, bacillary dysentery and pregnant vomiting (Wang et al. 2007). Previously, chemical investigations have resulted in the isolation of flavonoids, phenolic compounds and terpenes (Gui et al. 2014). The phenoloids found in T. triquetrum had interesting chemical structures and biological activities. As an ongoing research, a new lignan, together with a known compound, was isolated from the aerial part of T. triquetrum. Moreover, the extract of T. triquetrum has been proved to display hypoglycemic activities (Wang et al. 2007). However, the bioactive constituents concerning its glucose lowering effects were unknown till now. Therefore, the
*Corresponding authors. Email: [email protected]; [email protected] q 2015 Taylor & Francis
2
J. Wu et al.
effects of the isolates on consumption of glucose were assessed. In this paper, we reported the isolation, structural elucidation and the hypoglycemic effects of the new isolate in vitro.
Downloaded by [New York University] at 08:15 15 July 2015
2. Results and discussion Compound 1 was obtained as an optical active oil with ½a20 D 2 19.0 (c ¼ 0.01, CH3OH). Its molecular formula was determined as C20H20O8 by HR-ESI-MS at m/z 411.1060 [M þ Na] þ (calcd. 411.1056 [M þ Na] þ). The IR spectrum of 1 showed hydroxyl (3380 cm21) and carbonyl (1752, 1734 cm21) absorptions. The 1H NMR spectrum of 1 displayed the characteristic signals attributing to a para-substituted phenolic ring with two equivalent pairs of ortho-coupled protons at dH 6.72 (2H, d, J ¼ 8.4 Hz, H-3, 5), 7.23 (2H, d, J ¼ 8.4 Hz, H-2, 6) (Zhang et al. 2012). Furthermore, the 1H NMR spectrum showed the existence of a aromatic proton singlet at dH 7.36 (2H, s, H-20 , 60 ), one oxymethine at dH 5.12 (1H, d, J ¼ 5.4 Hz, H-7), two aliphatic methines at dH 3.38 (1H, t, J ¼ 5.4 Hz, H-8) and 4.38 (1H, m, H-80 ), one oxymethylene at dH 4.23 (1H, dd, J ¼ 9.0, 4.8 Hz, H-90 a) and 3.83 (1H, t, J ¼ 9.0 Hz, H-90 b), and two methoxyl groups at dH 3.91 (6H, s, 30 -OCH3, 50 -OCH3). Its 13C-APT spectrum indicated that 1 contained 14 sp2-hybridised carbon atoms corresponding to two carbonyl groups at dC 198.4 (C-70 ) and 178.6 (C-9), and two phenyl rings [dC 132.6 (C-1), 129.0 (C-2, 6), 116.3 (C-3, 5), 158.8 (C-4)] and [dC 127.4 (C-10 ), 108.2 (C-20 , 60 ), 143.4 (C-40 ), 149.3 (C-30 , 50 )]. The 13CAPT also displayed four sp3-hybridised carbon atoms including one oxymethine dC 73.7 (C-7), two methines at dC 53.7 (C-8) and 45.1 (C-80 ), and one oxymethylene dC 70.5 (C-90 ). In accordance with the 1H NMR data, two methoxyl groups at dC 57.1 (30 -OCH3, 50 -OCH3) were clearly figured out. The aforementioned NMR data showed that the carbon skeleton of 1 has 18 carbons and was assigned to be a lignan (Li et al. 2014). The 1H – 1H COSY spectrum suggested that all aliphatic methine and methylene protons were a contiguous spin system comprising H-7, H-8, H-80 and H-90 in the molecule (Figure S1). In the HMBC experiment, the correlations from dH 7.23 (H-2, 6) to dC 158.8 (C-4) and 73.7 (C7) suggested the presence of two hydroxyl groups at C-4 and C-7, respectively. The HMBC correlations between H-20 /60 and 198.4 (C-70 ) indicated the location of the keto group (C-70 ). Similarly, the position of another carbonyl group was confirmed to be at C-9 (178.6) since both dH 5.12 (H-7) and 3.38 (H-8) were all correlated with C-9. Furthermore, the HMBC correlations from dH 5.12 (H-7) to dC 132.6 (C-1), 129.0 (C-2, 6), 53.7 (C-8), 178.6 (C-9) and 45.1 (C-80 ) indicated linkage from C-1 to C-9 and C-8 to C-80 . In addition, the HMBC correlations between signal at dH 4.38 (H-80 ) and dC 73.7 (C-7), 53.7 (C-8), 178.6 (C-9), 198.4 (C-70 ) suggested the linkage from C-10 to C-90 . The connection between C-90 and C-9 through an oxygen atom was confirmed by the HMBC correlation from dH 3.83, 4.23 (H-90 ) to dC 178.6 (C-9). Furthermore, the correlations from dH 3.91 (6H, s, 30 -OCH3, 50 -OCH3) to dC 149.3 (C-30 , 50 ) confirmed that the methoxyl groups were located at C-30 , 50 . These data unambiguously showed that tadehaginosin (1) was a tetrahydrofuran lignan belonging to 9,90 -epoxy type (Wang et al. 2004; Gan et al. 2005). Thus, the planar structure of 1 was established. The relative stereochemistry of 1 was deduced from the NOESY experiment. In the NOESY spectrum, H-80 (dH 4.38) showed correlations with H-90 b (dH 3.83) and H-7 (dH 5.12), and no correlation between H-8 (dH 3.38) and H-80 , indicating that H-8 and H-80 were on the opposite side (Gan et al. 2005; Wang et al. 2004). Accordingly, 1 was established as 4,40 ,7-trihydroxy-30 ,50 dimethoxy-7,9-diketo-9,90 -epoxylignan, which was named tadehaginosin, as shown in Figure 1. Compound 2 was obtained as powder. Its chemical structure was established by analysing its 1D- and 2D NMR data. The absolute configuration of 1 was determined by comparing its positive optical rotation with reported data (Sun & Sneden 1999; Li et al. 2009). The extract of T. triquetrum has been proved to display hypoglycemic activities. However, the bioactive constituents concerning its glucose lowering effects were unknown till now.
Natural Product Research
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OH 4'
OCH3 3'
3
2
O
1
4
6'
OH
2'
HO 5
5'
3'
7'
7
6
8'
8
9'
O
9
4'
1'
OH
OH
1' 4
5 6
6'
5'
3
OCH3 HO
7 8
O
2
O
1
O
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Figure 1. Chemical structures of 1 and 2
In order to find novel compounds with hypoglycemic effects from this plant, the biological activity of 1 and 2 on consumption of glucose was assessed on HepG2 cells. As shown in Figure 2, treatment with insulin (100 nM) decreased the concentration of glucose obviously. Supplement with the two compounds (10 mM) significantly increased the consumption of glucose, respectively. Importantly, 1 (**p , 0.01) showed stronger activity than 2 (*p , 0.05) under the tested dose. This paper reported the structural elucidation of a new lignan and its hyperglycemic effects, providing scientific evidence for developing new chemical candidates in treating diabetes.
3. Experimental 3.1. General experimental procedures Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, CT, USA). UV spectra were recorded on Shimadzu UV2550 spectrometer and IR
Figure 2. Effects of 1 and 2 (10 mM) on glucose consumption by HepG2 cells. Cells were incubated in the serum-free high glucose DMEM (25 mM glucose contained) medium supplemented with 1 or 2 (10 mM) for 24 h. Then the glucose concentration in the medium was measured by a glucose quantification kit to evaluate the glucose consumption by cells. Values represent mean ^ SD. Results are representative of three different experiments with n ¼ 3. *p , 0.05, **p , 0.01.
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J. Wu et al.
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spectra were measured on FTIR-8400S spectrometer (Shimadzu, Kyoto, Japan). The NMR data were recorded on Bruker AV-600 (600 MHz for 1H and 150 MHz for 13C) in CD3OD with TMS as internal standard. The HR-ESI-MS data were performed on a LTQ-Obitrap XL spectrometer. Sephadex LH-20 (Pharmacia, Uppsala, Sweden) and silica gel (100 –200 meshes, Qingdao Marine Chemical plant, Qingdao, P.R. China) were used for column chromatography, and precoated silica gel GF254 plates (Zhi Fu Huang Wu Pilot Plant of Silica Gel Development, Yantai, P.R. China) were used for TLC. Dulbecco’s modified Eagle’s medium (DMEM) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were procured from SigmaAldrich, Inc. (St Louis, MO, USA). HepG2 cells, which originated from the American Type Culture Collection (ATCC) (Manassas, VA, USA), were obtained from the Peking Union Medical College (Beijing, China). 3.2. Plant material The whole plants of Tadehagi triquetrum were collected in July 2013 from Haikou, Hainan Province, China and identified by Prof. Jianping Tian at the School of Pharmaceutical Science, Hainan Medical University. A voucher specimen has been deposited there (Voucher specimen No. TT20130810). 3.3. Extraction and isolation The air-dried and powdered aerial part (5 kg) of T. triquetrum was extracted three times with solvent (CH3CH2OH: H2O ¼ 70:30) at 70 – 808C (50 L £ 2 h £ 3). After removal of the solvent under reduced pressure, the crude extract (500 g) was dissolved in distilled water, and was successively partitioned with petroleum ether, ethyl acetate and n-butanol, respectively. The EtOAc fraction (150 g) was subjected to silica gel column chromatography (80 cm £ 8 cm) eluted with CHCl3 – CH3COCH3 (1:0 to 2:1) to afford four fractions, Fr. 1– Fr. 4. Fr. 3 (15.5 g) was purified by Sephadex LH-20 column chromatography (120 cm £ 3 cm) eluting with CH3OH and the subfractions were further purified by semi-preparative HPLC using CH3OH – H2O (40:60) as the mobile phase (flow rate at 2.0 mL min21, wavelength at 210 nm, retention time at 22.125 min) to yield 1 (6.2 mg). Meanwhile, Fr. 4 (12.8 g) was purified by Sephadex LH-20 column chromatography (120 cm £ 3 cm) eluting with CH3OH and the subfractions were further purified by semi-preparative HPLC using CH3OH –H2O (47:53) as the mobile phase, (flow rate at 2.0 mL min21, wavelength at 210 nm, retention time at 27.125 min) to yield 2 (5.0 mg). 3.3.1. Tadehaginosin (1) Colourless oil, ½a20 D 2 19.0 (c ¼ 0.01, CH3OH); UV lmax (CH3OH) nm (log 1): 307 (4.08); IR (KBr) cm21 3380, 2918, 1752, 1734, 1646, 1610, 1512, 1113, 1019; 1H NMR (600 MHz, CD3OD) d: 7.23 (2H, d, J ¼ 8.4 Hz, H-2, 6), 6.72 (2H, d, J ¼ 8.4 Hz, H-3, 5), 5.12 (1H, d, J ¼ 5.4 Hz, H-7), 3.38 (1H, t, J ¼ 5.4 Hz, H-8), 7.36 (2H, s, H-20 , 60 ), 4.38 (1H, m, H-80 ), 4.23 (1H, dd, J ¼ 9.0, 4.8 Hz, H-90 a), 3.83 (1H, t, J ¼ 9.0 Hz, H-90 b), 3.91 (6H, s, 30 -OCH3, 50 OCH3); 13C NMR (150 MHz, CD3OD) d: 132.6 (C-1), 129.0 (C-2, 6), 116.3 (C-3, 5), 158.8 (C4), 73.7 (C-7), 53.7 (C-8), 178.6 (C-9), 127.4 (C-10 ), 108.2 (C-20 , 60 ), 143.4 (C-40 ), 149.3 (C-30 , 50 ), 198.4 (C-70 ), 45.1 (C-80 ), 70.5 (C-90 ), 57.1 (30 -OCH3, 50 -OCH3); HR-ESI-MS m/z 411.1060 [M þ Na] þ (calcd. for C20H20O8Na 411.1056). 3.4. Hypoglycemic evaluation and statistics analysis The biological activities of compounds 1 and 2 on consumption of glucose were assessed on HepG2 cells according to previous report (Supkamonseni et al. 2014). HepG2 cells were
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incubated with the serum-free DMEM containing different concentration of 1 and 2 for 24 h. Then the glucose concentration (mM L21) of medium was measured by glucose kit. The results were expressed as mean ^ SD. A one-way analysis of variance was done using the SPSS 13.0 software (Liu et al. 2013). Significance was accepted at p , 0.05. Values represent mean ^ SD. Results are representative of three different experiments with n ¼ 3. *p , 0.05, **p , 0.01. 4. Conclusion
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In this study, we reported the isolation, structural elucidation and hypoglycemic activity of a new lignan, tadehaginosin, from T. triquetrum. The result showed that the new isolate exhibited potent ability to increase glucose consumption in vitro. Supplementary material Supplementary material relating to this paper is available online, alongside Figures S1 –S8. Funding The financial assistance given by the Natural Sciences Foundation of China [grant number 81202994] is gratefully acknowledged.
Note 1. These authors contributed equally to this work.
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