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Monoterpenes from the leaves of Hydrangea paniculata and their hepatoprotective activities ab
a
a
a
a
Jing Shi , Chuang-Jun Li , Jing-Zhi Yang , Jie Ma , Yan Li , Hui a
a
Chen & Dong-Ming Zhang a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Click for updates
b
Department of Pharmaceutics, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan 250117, China Published online: 22 May 2015.
To cite this article: Jing Shi, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma, Yan Li, Hui Chen & Dong-Ming Zhang (2015) Monoterpenes from the leaves of Hydrangea paniculata and their hepatoprotective activities, Journal of Asian Natural Products Research, 17:5, 512-518, DOI: 10.1080/10286020.2015.1042871 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1042871
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Journal of Asian Natural Products Research, 2015 Vol. 17, No. 5, 512–518, http://dx.doi.org/10.1080/10286020.2015.1042871
Monoterpenes from the leaves of Hydrangea paniculata and their hepatoprotective activities Jing Shiab, Chuang-Jun Lia, Jing-Zhi Yanga, Jie Maa, Yan Lia, Hui Chena and Dong-Ming Zhanga* a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China; bDepartment of Pharmaceutics, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan 250117, China
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(Received 23 December 2014; final version received 16 April 2015) Three new monoterpenes, hydrangines A– C (1 – 3), together with four known compounds, were isolated from the ethanol extract of the leaves of Hydrangea paniculata. The structures of new isolates were elucidated on the basis of extensive 1D and 2D NMR analyses, and their absolute configurations were determined by comparison of experimental and calculated electronic circular dichroism spectra. In in vitro bioassays at 10 mM, compounds 1 – 6 showed hepatoprotective activities against DL -galactosamine-induced toxicity in HL-7702 cells. Keywords: Saxifragaceae; monoterpenes; Hydrangea paniculata; ECD; hepatoprotective activities
1.
Introduction
Hydrangea paniculata Sieb. (Saxifragaceae), a folk herbal medicine, widely distributes in southern China. It has been locally used for curing malaria, sore throat, and fever [1]. In addition, the effective fraction of H. paniculata is used to make pharmaceutical compounds for the prevention and/or treatment of renal insufficiency, hypertension, and diabetic nephropathy [2,3]. In our previous research, a series of coumarin glycosides, iridoid glucosides, and phenolic glycosides were isolated from the stems of this plant which exhibited remarkable neuroprotective effects and hepatoprotective activities [4 – 6]. This prompted us to search for more bioactive compounds from H. paniculata. A chemical investigation of the leaves of this plant was thus carried out. Finally, three new monoterpenes, hydrangines A – C (1 – 3), were
isolated, together with four known compounds (Figure 1). The structures of three new monoterpenes were elucidated on the basis of extensive 1D and 2D NMR analyses, and their absolute configurations were determined by comparison of experimental and calculated electronic circular dichroism (ECD) spectra. All isolates were evaluated for their in vitro hepatoprotective activities against DL -galactosamine-induced toxicity in HL-7702 cells at 10 mM.
2.
Hydrangine A (1) was obtained as a colorless oil. The molecular formula was determined to be C11H16O4 with four degrees of unsaturation, as deduced by HR-ESI-MS at m/z 213.1118 [M þ H]þ. The IR absorption bands at 1677 and 1720 cm21 revealed the existence of a
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
Journal of Asian Natural Products Research O 11 OCH3 6 H 7
9
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OCH2CH3
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5
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1
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10 1
O
O
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O O COOCH3
O OCH3 O 4
OCH3
O
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COOCH3
O O
O
O 5
6
7
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Figure 1. Hydrangines A– C (1– 3) and four known compounds (4 – 7) from H. paniculata.
double bond and a carbonyl group. The 1H NMR spectrum showed one methyl signal at dH 1.26 (3H, d, J ¼ 6.5 Hz), one methoxyl signal at dH 3.71 (3H, br s), and one olefinic proton at dH 6.41 (1H, s). The 13C NMR spectrum displayed 11 carbon signals, which were categorized by the HSQC experiment into two methyls, three methylenes, four methines, and two quaternary carbons, including one carbonyl carbon [dC 171.5 (C, C-11)], one trisubstituted olefin [dC 117.6 (C, C-9), 138.3 (CH, C-1)], three oxygen-bearing carbons [dC 62.4 (CH2, C-3), 67.8 (CH2, C-7), 73.7 (CH, C-8)] (Table 1). The 1 H – 1H COSY spectrum of 1 showed correlations of H-4 at dH 3.07 with H2-3 at dH 3.81, 4.12, and H-5 at dH 2.55 –2.59; H2-6 at dH 1.65, 1.38 –1.42 with H2-7 at dH 4.08, 3.67, and H-5 at dH 2.55– 2.59; H-8 at dH 3.90 with H-10 at dH 1.26, which suggested two spin systems H2-3/H-4/H-5/ H2-6/H2-7 and H-8/H-10. In fact, the molecular formula of 1 accounted for four degrees of unsaturation. Apart from one double bond and one carbonyl group, the remaining two must be two rings. Furthermore, the key HMBC correlations from H-1 at dH 6.41 to C-3 at dC 62.4, C-5 at dC 33.9, C-8 at dC 73.7 and C-9 at dC 117.6, from H2-7 at dH 4.08, 3.67 to C-8 at dC 73.7, and from H-4 at dH 3.07 to C-11 at dC 171.5 were displayed (Figure 2).
By combining all this evidence and data, the planar structure of compound 1 was determined as shown in Figure 1. The relative configurations at C-4, C-5, and C-8 of 1 were deduced by analyses of the coupling constants (Table 1) and ROESY spectrum (Figure 2). The 3JH-3,H4 coupling constant was 11.0 and 3.5 Hz, and the 3JH-4,H-5 coupling constant was 6.5 Hz, which suggested b-orientations of H-4 and H-5 [7]. The ROESY spectrum gave diagnostic correlation of H-5 with H8, which indicated b-orientation of H-8. The H-4b, H-5b, and H-8b relative configurations of 1 indicated that there were only two possible structures, with the absolute configurations (4S, 5R, 8S) or (4R, 5S, 8R). The absolute configurations at C-4, C-5, and C-8 were established by theoretical calculations of its ECD using the time-dependent density functional theory (TD-DFT) method. Their optimized geometries were obtained, and then the ECD spectra were calculated at the B3LYP/6-31G(d) level with the TD-DFT/ PCM model in methanol solution. The result showed that the calculated ECD spectrum of (4S, 5R, 8S)-1 agreed well with the measured spectrum, while the enantiomer of (4R, 5S, 8R)-1 exhibited the opposite cotton effects (Figure 3). Therefore, the absolute configurations at C-4, C5, and C-8 were determined as 4S, 5R, 8S.
1
13
daH
1
3.71 s
1.26 d (6.5)
b 13
73.7 117.6 16.7 171.5 51.5
67.8
41.9 33.9 31.0
138.3 62.4
dbC
daH
2
3.72 s
1.35 d (6.5)
6.43 d (1.5) 4.11 brdd (11.0, 3.5) 3.84 t (11.0) 3.04 ddd (11.0,6.5,3.5) 2.80 – 2.84 m 1.61 ddd (17.0, 12.0, 5.0) 1.43 – 1.47 m 3.89 brdd (12.0, 3.0) 3.76 dd (12.0, 2.0) 4.28 q (6.5)
C NMR spectral data of compounds 1– 3 in CDCl3.
6.41 brs 4.12 ddd (11.0,3.5, 1.0) 3.81 t (11.0) 3.07 ddd (11.0, 6.5, 3.5) 2.55 –2.59 m 1.65 ddd (17.0, 12.5, 4.5) 1.38 –1.42 m 4.08 ddd (12.0, 4.5,2.0) 3.67 dd (12.0, 2.0) 3.90 q (6.5)
H NMR and
H NMR data (dH) were measured at 500 MHz. C NMR data (dC) were measured at 125 MHz.
a1
8 9 10 11 OCH3 OCH2CH3
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4 5 6
1 3
Position
Table 1.
71.5 116.9 17.9 171.5 51.5
60.6
41.9 28.0 31.1
138.9 62.3
dbC
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3
4.17 q (7.0) 1.27 t (7.0)
1.26 d (6.5)
6.41 brs 4.11 brdd (11.0, 3.5) 3.81 t (11.0) 3.04 ddd (11.0, 6.5, 3.5) 2.55– 2.59 m 1.67 ddd (17.0, 12.5, 4.5) 1.42, brd (12.5) 4.08 brdd (12.0, 4.5) 3.68 brt (12.0) 3.90 q (6.5)
daH
60.5 14.3
73.7 117.6 16.7 171.1
67.9
41.9 33.9 31.0
138.3 62.4
dbC
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Figure 2. Selected 1H– 1H COSY, HMBC and ROESY correlations of 1 – 2.
Thus, the structure of monoterpene 1 was established and termed hydrangine A. Hydrangine B (2) was obtained as a colorless oil. The HR-ESI-MS resulted in the same molecular formula as that of 1. The 1H and 13C NMR spectra of 2 were almost identical to those of 1, indicating it as a diastereomer of 1. In addition, the chemical shifts of C-5 (dC 28.0), C-7 (dC 60.6), and C-8 (dC 71.5) in 2 were different from the chemical shifts of C-5 (dC 33.9), C-7 (dC 67.8), and C-8 (dC 73.7) in 1, suggesting the possibility of different configurations at the chiral carbon C-5 and/or C-8 (Table 1). The 3JH-3,H-4 (11.0 and 3.5 Hz) and 3JH-4,H-5 (6.5 Hz) coupling constants of compound 2 suggested borientations of H-4 and H-5. The ROESY spectrum gave diagnostic correlation of H-5 with H-10, which indicated H-8a relative configuration. Therefore, the absolute configurations of 2 were limited to two enantiomers of (4S, 5R, 8R)-2 or (4R, 5S, 8S)-2 based on the above
established relative configurations, and were further determined by the TD-DFT calculated ECD spectra to be 4S, 5R, 8R (Figure 3). As a result, the structure of 2 was established and named as hydrangine B. Hydrangine C (3) was obtained as a colorless oil. The HR-ESI-MS showed an [M þ H]þ ion at m/z 227.1279, consistent with the molecular formula C12H18O4. The 1 H and 13C NMR data of 3 (Table 1) were excellently identical to those of 1, except for the difference of one ethoxyl substituent (dC 60.5, 14.3) at C-11 for 3 instead of one methoxyl substituent (dC 51.5) at C-11 for 1. The NMR data also suggested that compounds 3 and 1 had the same relative configurations at C-4, C-5, and C-8. The assumption was supported by coupling constants (Table 1), HMBC, and ROESY spectra (Figure 2). So the C-4, C5, and C-8 relative configurations of 3 indicated that there were only two possible structures, with the absolute configurations (4S, 5R, 8S) or (4R, 5S, 8R). The optical
Figure 3. Calculated and experimental CD spectra of 1 – 2.
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rotation values of the enantiomers were equal but opposite. So the fact that the optical rotation value of 3 (þ 80.6) was close to that of 1 (þ 84.5) could determine the absolute configurations at C-4, C-5, and C-8 of 3 as 4S, 5R, 8S, also the same as those of 1. Thus, the structure of 3 was elucidated and named as hydrangine C. Compounds 4 –7 were identified as triohima A (4) [8], triohima B (5) [8], sarracenin (6) [9], and 8-episarracenin (7) [10] by comparison of their spectroscopic data with literature values. All isolates were bioassayed for their hepatoprotective activities against DL galactosamine-induced toxicity in HL7702 cells, using the hepatoprotective activity drug bicyclol as the positive control. At 10 mM, compounds 1 – 6 reduced DL -galactosamine (GalN)induced HL-7702 cells damage by increasing the survival rate from 33% ( p , 0.001) to 69% ( p , 0.001), 63% ( p , 0.01), 51% ( p , 0.01), 55% ( p , 0.05), 54% ( p , 0.01), and 60% ( p , 0.01), respectively, while the positive control bicyclol gave a 61% ( p , 0.05) survival rate (Table 2). Compounds 1 –6 displayed moderate hepatoprotective activities against DL galactosamine-induced toxicity in HL-
Table 2. Effects of compounds 1 – 7 on the survival rate of HL-7702 cells injured by DLGalN. Group Control Model Bicyclola 1 2 3 4 5 6 7
OD value
Survival rate (%)
1.125 ^ 0.020 0.411 ^ 0.037 0.710 ^ 0.048 0.836 ^ 0.041 0.730 ^ 0.006 0.638 ^ 0.071 0.655 ^ 0.091 0.637 ^ 0.007 0.701 ^ 0.013 0.506 ^ 0.057
100.0 33%### 61%* 69%*** 63%** 51%** 55%* 54%** 60%** 42%
Note: ###p , 0.001 vs. control, *p , 0.05 vs. model, **p , 0.01 vs. model, ***p , 0.001 vs. model. a Positive control substance.
7702 cells. This information may facilitate identification of other hepatoprotective activity lead compounds from monoterpenes. In previous studies, mainly coumarin glucosides and secoiridoid glucosides from the stems of H. paniculata have been reported for their potent hepatoprotective activities. So, H. paniculata might be a potent hepatoprotective agent. These provide support for further studies. 3.
Experimental
3.1 General experimental procedures Optical rotations were measured on a Jasco P-2000 polarimeter (JASCO, Tokyo, Japan). IR spectra were measured on a Nicolet 5700 spectrometer using an FT-IR microscope transmission method (Thermo Electron, Madison, WI, USA). UV spectra were scanned by a Jasco V650 spectro1 photometer (JASCO). H NMR 13 (500 MHz), C NMR (125 MHz), and 2D NMR spectra were run on Bruker AV500-III spectrometers (Bruker, Billerica, MA, USA) with TMS as internal standard. HR-ESI-MS were performed on a Finnigan LTQFT mass spectrometer and ESI-MS on an Agilent 1100 series LC/ MSD Trap-SL mass spectrometer (Agilent, Waldbronn, Germany). Reversedphase silica MPLC was performed with pumps C-605, a UV photometer C-635, a fraction collector C-660 (Buchi, Flawil, Switzerland), and an ODS column (6 cm £ 45 cm, 50 mm, YMC, Kyoto, Japan). Preparative HPLC was carried out on a Shimadzu LC-6AD instrument with a SPD-20A detector (Shimadzu, Kyoto, Japan), using an YMC-Pack ODS-A column (250 mm £ 20 mm, 5 mm, YMC). Column chromatography was performed with silica gel (200 – 300 mesh, Qingdao Marine Chemical Company, Qingdao, China) and ODS (50 mm, YMC). TLC was carried out with glass precoated with silica gel GF254 plates (Qingdao Marine Chemical Company). Spots were visual-
Journal of Asian Natural Products Research ized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating.
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3.2 Plant material The stems of H. paniculata were collected in the County of Jinxiu, Guangxi Zhuang Autonomous Region, China, in May 2009 and identified by Mr Guangri Long (Liuzhou Forestry Bureau of Guangxi). A voucher specimen (ID-4645) was deposited at the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing. 3.3
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column chromatography over silica gel (CHCl3 – MeOH 20:1, 8 ml/min, 210 nm) to afford 4 (9.7 mg, tR:64 min) and 5 (23.0 mg, tR:72 min). 3.3.1
Hydrangenine A (1)
A colorless oil; ½a20 D þ 84.5 (c ¼ 0.12, CHCl3); UV (MeOH) lmax (log 1): 203 (3.74) nm; IR (microscope) nmax: 1720, 1677, 1588, 1431 and 1366 cm21; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data, see Table 1; ESI-MS: m/z 213 [M þ H]þ, 235 [M þ Na] þ ; HR-ESI-MS: m/z 213.1118 [M þ H]þ (calcd for C11H17O4, 213.1121).
Extraction and isolation
Air-dried leaves of H. paniculata (5.0 kg) were extracted with 95% EtOH, yielding a semi-dry residue (550.0 g). The residue was passed through a silica gel column and eluted with petroleum ether, CHCl3, EtOAc, acetone, and MeOH, successively. Thus the petroleum ether fraction (41.5 g), CHCl3 fraction (62.0 g), EtOAc fraction (71.9 g), acetone fraction (145.1 g), and MeOH fraction (168.5 g) were obtained after removing the respective solvent. The CHCl3 fraction (62.0 g) was applied to silica gel column chromatography (200 – 300 mesh, 600.0 g) and eluted with CHCl3 – MeOH gradients (100:0 –100:5) to obtain eight fractions (Fr E-1 –E-8). Fraction E-5 (24.3 g) was further fractionated into seven subfractions E-5.1 to E5.7 by silica gel column chromatography (200 – 300 mesh, 350.0 g) according to their TLC profiles. Subfraction E-5.2 (9.4 g) was separated by reversed-phase silica MPLC (50 mm, 800.0 g) with 20– 100% aqueous MeOH, followed by repeated reversed-phase HPLC (250 mm £ 20 mm, 5 mm, CH3OH – H2O, 35:65, 8 ml/min, 210 nm) to give 1 (10.0 mg, tR: 57 min), 2 (2.3 mg, tR:62 min), 3 (8.6 mg, tR:65 min), 6 (2.6 mg, tR:72 min), and 7 (5.8 mg, tR: 80 min). Subfraction E-5.5 (5.1 g) was further subjected to repeated
3.3.2
Hydrangenine B (2)
A colorless oil; ½a20 D þ 35.5 (c ¼ 0.10, CHCl3); UV (MeOH) lmax (log 1): 203 (3.65) nm; IR (microscope) nmax: 1725, 1685, 1600, 1470, and 1385 cm21; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data, see Table 1; ESI-MS: m/z 213 [M þ H]þ, 235 [M þ Na] þ ; HR-ESI-MS: m/z 213.1124 [M þ H]þ (calcd for C11H17O4, 213.1121). 3.3.3 Hydrangenine C (3) A colorless oil; ½a20 D þ 80.6 (c ¼ 0.10, CHCl3); UV (MeOH) lmax (log 1): 203 (3.67) nm; IR (microscope) nmax: 1727, 1684, 1615, 1499 and 1373 cm21; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data, see Table 1; ESI-MS: m/z 227 [M þ H]þ, 249 [M þ Na] þ ; HR-ESI-MS: m/z 227.1279 [M þ H]þ (calcd for C12H19O4, 227.1278). 3.4 Protective effect on cytotoxicity induced by DL -galactosaminein HL-7702 cells The hepatoprotective effects of compounds 1 – 7 were determined by an MTT
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colorimetric assay in HL-7702 cells [11]. Each cell suspension of 2 £ 104 cells in 200 ml of RPMI (Roswell Park Memorial Institute) 1640 containing fetal calf serum (10%), penicillin (100 U/ml), and streptomycin (100 mg/ml) was placed in a 96-well microplate and precultured for 24 h at 378C under a 5% CO2 atmosphere Fresh medium (100 ml) containing bicyclol (10 mM) and test samples (10 mM) was added, and the cells were cultured for 1 h. Then, the cultured cells were exposed to 25 mM DL -galactosamine for 24 h. Then, 100 ml of 0.5 mg/ml MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 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 on a microplate reader at 492 nm. The survival rate of HL7702 cells was evaluated. Disclosure statement There is no conflict of interest among all authors.
Funding This work was financially supported by the National Mega-project for Innovative Drugs [grant number 2012ZX09101].
References [1] Chinese Herbal Medicine Company, Chinese Traditional Medicine Resource Records (Beijing Science Press, Beijing, 1994). [2] D.M. Zhang, X.G. Chen, J.Z. Yang, Y. Li, X.G. Chinese Patent CN 1690069A Zheng, (2005). [3] D.M. Zhang, X.G. Chen, J.Z. Yang, Y. Chinese Patent CN 1605343A Li, (2005). [4] J. Shi, J.Z. Yang, C.J. Li, and D.M. Zhang, J. Chin. Mater. Med. 35, 3007 (2010). [5] J. Shi, C.J. Li, J.Z. Yang, Y.H. Yuan, N.H. Chen, and D.M. Zhang, Planta Med. 78, 1844 (2012). doi:10.1055/s-00321315394. [6] J. Shi, C.J. Li, J.Z. Yang, J. Ma, C. Wang, J. Tang, Y. Li, H. Chen, and D.M. Zhang, Fitoterapia. 96, 138 (2014). doi:10.1016/ j.fitote.2014.04.015. [7] I. Kubo, I. Miura, and K. Nakanishi, J. Am. Chem. Soc. 98, 6704 (1976). doi:10.1021/ja00437a049. [8] Z.M. Li, J.J. Chen, Y. Li, K. Gao, J. Chang, and X.J. Yao, Tetrahedron Lett. 50, 4132 (2009). doi:10.1016/j.tetlet. 2009.04.111. [9] D.H. Miles, U. Kokpol, J. Bhattacharyya, J.L. Atwood, K.E. Stone, T.A. Bryson, and C. Wilson, J. Am. Chem. Soc. 98, 1569 (1976). doi:10.1021/ja00422a048. [10] J.K. Whitesell, R.S. Matthews, M.A. Minton, and A.M. Helbling, J. Am. Chem. Soc. 103, 3468 (1981). doi:10. 1021/ja00402a035. [11] F.M. Xu, T. Morikawa, H. Matsuda, K. Ninomiya, and M. Yoshikawa, J. Nat. Prod. 67, 569 (2004). doi:10.1021/ np030368k.
Journal of Asian Natural Products Research Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ganp20
Monoterpenes from the leaves of Hydrangea paniculata and their hepatoprotective activities ab
a
a
a
a
Jing Shi , Chuang-Jun Li , Jing-Zhi Yang , Jie Ma , Yan Li , Hui a
a
Chen & Dong-Ming Zhang a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Click for updates
b
Department of Pharmaceutics, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan 250117, China Published online: 22 May 2015.
To cite this article: Jing Shi, Chuang-Jun Li, Jing-Zhi Yang, Jie Ma, Yan Li, Hui Chen & Dong-Ming Zhang (2015) Monoterpenes from the leaves of Hydrangea paniculata and their hepatoprotective activities, Journal of Asian Natural Products Research, 17:5, 512-518, DOI: 10.1080/10286020.2015.1042871 To link to this article: http://dx.doi.org/10.1080/10286020.2015.1042871
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. 5, 512–518, http://dx.doi.org/10.1080/10286020.2015.1042871
Monoterpenes from the leaves of Hydrangea paniculata and their hepatoprotective activities Jing Shiab, Chuang-Jun Lia, Jing-Zhi Yanga, Jie Maa, Yan Lia, Hui Chena and Dong-Ming Zhanga* a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China; bDepartment of Pharmaceutics, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan 250117, China
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(Received 23 December 2014; final version received 16 April 2015) Three new monoterpenes, hydrangines A– C (1 – 3), together with four known compounds, were isolated from the ethanol extract of the leaves of Hydrangea paniculata. The structures of new isolates were elucidated on the basis of extensive 1D and 2D NMR analyses, and their absolute configurations were determined by comparison of experimental and calculated electronic circular dichroism spectra. In in vitro bioassays at 10 mM, compounds 1 – 6 showed hepatoprotective activities against DL -galactosamine-induced toxicity in HL-7702 cells. Keywords: Saxifragaceae; monoterpenes; Hydrangea paniculata; ECD; hepatoprotective activities
1.
Introduction
Hydrangea paniculata Sieb. (Saxifragaceae), a folk herbal medicine, widely distributes in southern China. It has been locally used for curing malaria, sore throat, and fever [1]. In addition, the effective fraction of H. paniculata is used to make pharmaceutical compounds for the prevention and/or treatment of renal insufficiency, hypertension, and diabetic nephropathy [2,3]. In our previous research, a series of coumarin glycosides, iridoid glucosides, and phenolic glycosides were isolated from the stems of this plant which exhibited remarkable neuroprotective effects and hepatoprotective activities [4 – 6]. This prompted us to search for more bioactive compounds from H. paniculata. A chemical investigation of the leaves of this plant was thus carried out. Finally, three new monoterpenes, hydrangines A – C (1 – 3), were
isolated, together with four known compounds (Figure 1). The structures of three new monoterpenes were elucidated on the basis of extensive 1D and 2D NMR analyses, and their absolute configurations were determined by comparison of experimental and calculated electronic circular dichroism (ECD) spectra. All isolates were evaluated for their in vitro hepatoprotective activities against DL -galactosamine-induced toxicity in HL-7702 cells at 10 mM.
2.
Hydrangine A (1) was obtained as a colorless oil. The molecular formula was determined to be C11H16O4 with four degrees of unsaturation, as deduced by HR-ESI-MS at m/z 213.1118 [M þ H]þ. The IR absorption bands at 1677 and 1720 cm21 revealed the existence of a
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
Results and discussion
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Figure 1. Hydrangines A– C (1– 3) and four known compounds (4 – 7) from H. paniculata.
double bond and a carbonyl group. The 1H NMR spectrum showed one methyl signal at dH 1.26 (3H, d, J ¼ 6.5 Hz), one methoxyl signal at dH 3.71 (3H, br s), and one olefinic proton at dH 6.41 (1H, s). The 13C NMR spectrum displayed 11 carbon signals, which were categorized by the HSQC experiment into two methyls, three methylenes, four methines, and two quaternary carbons, including one carbonyl carbon [dC 171.5 (C, C-11)], one trisubstituted olefin [dC 117.6 (C, C-9), 138.3 (CH, C-1)], three oxygen-bearing carbons [dC 62.4 (CH2, C-3), 67.8 (CH2, C-7), 73.7 (CH, C-8)] (Table 1). The 1 H – 1H COSY spectrum of 1 showed correlations of H-4 at dH 3.07 with H2-3 at dH 3.81, 4.12, and H-5 at dH 2.55 –2.59; H2-6 at dH 1.65, 1.38 –1.42 with H2-7 at dH 4.08, 3.67, and H-5 at dH 2.55– 2.59; H-8 at dH 3.90 with H-10 at dH 1.26, which suggested two spin systems H2-3/H-4/H-5/ H2-6/H2-7 and H-8/H-10. In fact, the molecular formula of 1 accounted for four degrees of unsaturation. Apart from one double bond and one carbonyl group, the remaining two must be two rings. Furthermore, the key HMBC correlations from H-1 at dH 6.41 to C-3 at dC 62.4, C-5 at dC 33.9, C-8 at dC 73.7 and C-9 at dC 117.6, from H2-7 at dH 4.08, 3.67 to C-8 at dC 73.7, and from H-4 at dH 3.07 to C-11 at dC 171.5 were displayed (Figure 2).
By combining all this evidence and data, the planar structure of compound 1 was determined as shown in Figure 1. The relative configurations at C-4, C-5, and C-8 of 1 were deduced by analyses of the coupling constants (Table 1) and ROESY spectrum (Figure 2). The 3JH-3,H4 coupling constant was 11.0 and 3.5 Hz, and the 3JH-4,H-5 coupling constant was 6.5 Hz, which suggested b-orientations of H-4 and H-5 [7]. The ROESY spectrum gave diagnostic correlation of H-5 with H8, which indicated b-orientation of H-8. The H-4b, H-5b, and H-8b relative configurations of 1 indicated that there were only two possible structures, with the absolute configurations (4S, 5R, 8S) or (4R, 5S, 8R). The absolute configurations at C-4, C-5, and C-8 were established by theoretical calculations of its ECD using the time-dependent density functional theory (TD-DFT) method. Their optimized geometries were obtained, and then the ECD spectra were calculated at the B3LYP/6-31G(d) level with the TD-DFT/ PCM model in methanol solution. The result showed that the calculated ECD spectrum of (4S, 5R, 8S)-1 agreed well with the measured spectrum, while the enantiomer of (4R, 5S, 8R)-1 exhibited the opposite cotton effects (Figure 3). Therefore, the absolute configurations at C-4, C5, and C-8 were determined as 4S, 5R, 8S.
1
13
daH
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73.7 117.6 16.7 171.5 51.5
67.8
41.9 33.9 31.0
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6.43 d (1.5) 4.11 brdd (11.0, 3.5) 3.84 t (11.0) 3.04 ddd (11.0,6.5,3.5) 2.80 – 2.84 m 1.61 ddd (17.0, 12.0, 5.0) 1.43 – 1.47 m 3.89 brdd (12.0, 3.0) 3.76 dd (12.0, 2.0) 4.28 q (6.5)
C NMR spectral data of compounds 1– 3 in CDCl3.
6.41 brs 4.12 ddd (11.0,3.5, 1.0) 3.81 t (11.0) 3.07 ddd (11.0, 6.5, 3.5) 2.55 –2.59 m 1.65 ddd (17.0, 12.5, 4.5) 1.38 –1.42 m 4.08 ddd (12.0, 4.5,2.0) 3.67 dd (12.0, 2.0) 3.90 q (6.5)
H NMR and
H NMR data (dH) were measured at 500 MHz. C NMR data (dC) were measured at 125 MHz.
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Table 1.
71.5 116.9 17.9 171.5 51.5
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1.26 d (6.5)
6.41 brs 4.11 brdd (11.0, 3.5) 3.81 t (11.0) 3.04 ddd (11.0, 6.5, 3.5) 2.55– 2.59 m 1.67 ddd (17.0, 12.5, 4.5) 1.42, brd (12.5) 4.08 brdd (12.0, 4.5) 3.68 brt (12.0) 3.90 q (6.5)
daH
60.5 14.3
73.7 117.6 16.7 171.1
67.9
41.9 33.9 31.0
138.3 62.4
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Figure 2. Selected 1H– 1H COSY, HMBC and ROESY correlations of 1 – 2.
Thus, the structure of monoterpene 1 was established and termed hydrangine A. Hydrangine B (2) was obtained as a colorless oil. The HR-ESI-MS resulted in the same molecular formula as that of 1. The 1H and 13C NMR spectra of 2 were almost identical to those of 1, indicating it as a diastereomer of 1. In addition, the chemical shifts of C-5 (dC 28.0), C-7 (dC 60.6), and C-8 (dC 71.5) in 2 were different from the chemical shifts of C-5 (dC 33.9), C-7 (dC 67.8), and C-8 (dC 73.7) in 1, suggesting the possibility of different configurations at the chiral carbon C-5 and/or C-8 (Table 1). The 3JH-3,H-4 (11.0 and 3.5 Hz) and 3JH-4,H-5 (6.5 Hz) coupling constants of compound 2 suggested borientations of H-4 and H-5. The ROESY spectrum gave diagnostic correlation of H-5 with H-10, which indicated H-8a relative configuration. Therefore, the absolute configurations of 2 were limited to two enantiomers of (4S, 5R, 8R)-2 or (4R, 5S, 8S)-2 based on the above
established relative configurations, and were further determined by the TD-DFT calculated ECD spectra to be 4S, 5R, 8R (Figure 3). As a result, the structure of 2 was established and named as hydrangine B. Hydrangine C (3) was obtained as a colorless oil. The HR-ESI-MS showed an [M þ H]þ ion at m/z 227.1279, consistent with the molecular formula C12H18O4. The 1 H and 13C NMR data of 3 (Table 1) were excellently identical to those of 1, except for the difference of one ethoxyl substituent (dC 60.5, 14.3) at C-11 for 3 instead of one methoxyl substituent (dC 51.5) at C-11 for 1. The NMR data also suggested that compounds 3 and 1 had the same relative configurations at C-4, C-5, and C-8. The assumption was supported by coupling constants (Table 1), HMBC, and ROESY spectra (Figure 2). So the C-4, C5, and C-8 relative configurations of 3 indicated that there were only two possible structures, with the absolute configurations (4S, 5R, 8S) or (4R, 5S, 8R). The optical
Figure 3. Calculated and experimental CD spectra of 1 – 2.
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rotation values of the enantiomers were equal but opposite. So the fact that the optical rotation value of 3 (þ 80.6) was close to that of 1 (þ 84.5) could determine the absolute configurations at C-4, C-5, and C-8 of 3 as 4S, 5R, 8S, also the same as those of 1. Thus, the structure of 3 was elucidated and named as hydrangine C. Compounds 4 –7 were identified as triohima A (4) [8], triohima B (5) [8], sarracenin (6) [9], and 8-episarracenin (7) [10] by comparison of their spectroscopic data with literature values. All isolates were bioassayed for their hepatoprotective activities against DL galactosamine-induced toxicity in HL7702 cells, using the hepatoprotective activity drug bicyclol as the positive control. At 10 mM, compounds 1 – 6 reduced DL -galactosamine (GalN)induced HL-7702 cells damage by increasing the survival rate from 33% ( p , 0.001) to 69% ( p , 0.001), 63% ( p , 0.01), 51% ( p , 0.01), 55% ( p , 0.05), 54% ( p , 0.01), and 60% ( p , 0.01), respectively, while the positive control bicyclol gave a 61% ( p , 0.05) survival rate (Table 2). Compounds 1 –6 displayed moderate hepatoprotective activities against DL galactosamine-induced toxicity in HL-
Table 2. Effects of compounds 1 – 7 on the survival rate of HL-7702 cells injured by DLGalN. Group Control Model Bicyclola 1 2 3 4 5 6 7
OD value
Survival rate (%)
1.125 ^ 0.020 0.411 ^ 0.037 0.710 ^ 0.048 0.836 ^ 0.041 0.730 ^ 0.006 0.638 ^ 0.071 0.655 ^ 0.091 0.637 ^ 0.007 0.701 ^ 0.013 0.506 ^ 0.057
100.0 33%### 61%* 69%*** 63%** 51%** 55%* 54%** 60%** 42%
Note: ###p , 0.001 vs. control, *p , 0.05 vs. model, **p , 0.01 vs. model, ***p , 0.001 vs. model. a Positive control substance.
7702 cells. This information may facilitate identification of other hepatoprotective activity lead compounds from monoterpenes. In previous studies, mainly coumarin glucosides and secoiridoid glucosides from the stems of H. paniculata have been reported for their potent hepatoprotective activities. So, H. paniculata might be a potent hepatoprotective agent. These provide support for further studies. 3.
Experimental
3.1 General experimental procedures Optical rotations were measured on a Jasco P-2000 polarimeter (JASCO, Tokyo, Japan). IR spectra were measured on a Nicolet 5700 spectrometer using an FT-IR microscope transmission method (Thermo Electron, Madison, WI, USA). UV spectra were scanned by a Jasco V650 spectro1 photometer (JASCO). H NMR 13 (500 MHz), C NMR (125 MHz), and 2D NMR spectra were run on Bruker AV500-III spectrometers (Bruker, Billerica, MA, USA) with TMS as internal standard. HR-ESI-MS were performed on a Finnigan LTQFT mass spectrometer and ESI-MS on an Agilent 1100 series LC/ MSD Trap-SL mass spectrometer (Agilent, Waldbronn, Germany). Reversedphase silica MPLC was performed with pumps C-605, a UV photometer C-635, a fraction collector C-660 (Buchi, Flawil, Switzerland), and an ODS column (6 cm £ 45 cm, 50 mm, YMC, Kyoto, Japan). Preparative HPLC was carried out on a Shimadzu LC-6AD instrument with a SPD-20A detector (Shimadzu, Kyoto, Japan), using an YMC-Pack ODS-A column (250 mm £ 20 mm, 5 mm, YMC). Column chromatography was performed with silica gel (200 – 300 mesh, Qingdao Marine Chemical Company, Qingdao, China) and ODS (50 mm, YMC). TLC was carried out with glass precoated with silica gel GF254 plates (Qingdao Marine Chemical Company). Spots were visual-
Journal of Asian Natural Products Research ized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating.
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3.2 Plant material The stems of H. paniculata were collected in the County of Jinxiu, Guangxi Zhuang Autonomous Region, China, in May 2009 and identified by Mr Guangri Long (Liuzhou Forestry Bureau of Guangxi). A voucher specimen (ID-4645) was deposited at the Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing. 3.3
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column chromatography over silica gel (CHCl3 – MeOH 20:1, 8 ml/min, 210 nm) to afford 4 (9.7 mg, tR:64 min) and 5 (23.0 mg, tR:72 min). 3.3.1
Hydrangenine A (1)
A colorless oil; ½a20 D þ 84.5 (c ¼ 0.12, CHCl3); UV (MeOH) lmax (log 1): 203 (3.74) nm; IR (microscope) nmax: 1720, 1677, 1588, 1431 and 1366 cm21; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data, see Table 1; ESI-MS: m/z 213 [M þ H]þ, 235 [M þ Na] þ ; HR-ESI-MS: m/z 213.1118 [M þ H]þ (calcd for C11H17O4, 213.1121).
Extraction and isolation
Air-dried leaves of H. paniculata (5.0 kg) were extracted with 95% EtOH, yielding a semi-dry residue (550.0 g). The residue was passed through a silica gel column and eluted with petroleum ether, CHCl3, EtOAc, acetone, and MeOH, successively. Thus the petroleum ether fraction (41.5 g), CHCl3 fraction (62.0 g), EtOAc fraction (71.9 g), acetone fraction (145.1 g), and MeOH fraction (168.5 g) were obtained after removing the respective solvent. The CHCl3 fraction (62.0 g) was applied to silica gel column chromatography (200 – 300 mesh, 600.0 g) and eluted with CHCl3 – MeOH gradients (100:0 –100:5) to obtain eight fractions (Fr E-1 –E-8). Fraction E-5 (24.3 g) was further fractionated into seven subfractions E-5.1 to E5.7 by silica gel column chromatography (200 – 300 mesh, 350.0 g) according to their TLC profiles. Subfraction E-5.2 (9.4 g) was separated by reversed-phase silica MPLC (50 mm, 800.0 g) with 20– 100% aqueous MeOH, followed by repeated reversed-phase HPLC (250 mm £ 20 mm, 5 mm, CH3OH – H2O, 35:65, 8 ml/min, 210 nm) to give 1 (10.0 mg, tR: 57 min), 2 (2.3 mg, tR:62 min), 3 (8.6 mg, tR:65 min), 6 (2.6 mg, tR:72 min), and 7 (5.8 mg, tR: 80 min). Subfraction E-5.5 (5.1 g) was further subjected to repeated
3.3.2
Hydrangenine B (2)
A colorless oil; ½a20 D þ 35.5 (c ¼ 0.10, CHCl3); UV (MeOH) lmax (log 1): 203 (3.65) nm; IR (microscope) nmax: 1725, 1685, 1600, 1470, and 1385 cm21; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data, see Table 1; ESI-MS: m/z 213 [M þ H]þ, 235 [M þ Na] þ ; HR-ESI-MS: m/z 213.1124 [M þ H]þ (calcd for C11H17O4, 213.1121). 3.3.3 Hydrangenine C (3) A colorless oil; ½a20 D þ 80.6 (c ¼ 0.10, CHCl3); UV (MeOH) lmax (log 1): 203 (3.67) nm; IR (microscope) nmax: 1727, 1684, 1615, 1499 and 1373 cm21; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) spectral data, see Table 1; ESI-MS: m/z 227 [M þ H]þ, 249 [M þ Na] þ ; HR-ESI-MS: m/z 227.1279 [M þ H]þ (calcd for C12H19O4, 227.1278). 3.4 Protective effect on cytotoxicity induced by DL -galactosaminein HL-7702 cells The hepatoprotective effects of compounds 1 – 7 were determined by an MTT
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colorimetric assay in HL-7702 cells [11]. Each cell suspension of 2 £ 104 cells in 200 ml of RPMI (Roswell Park Memorial Institute) 1640 containing fetal calf serum (10%), penicillin (100 U/ml), and streptomycin (100 mg/ml) was placed in a 96-well microplate and precultured for 24 h at 378C under a 5% CO2 atmosphere Fresh medium (100 ml) containing bicyclol (10 mM) and test samples (10 mM) was added, and the cells were cultured for 1 h. Then, the cultured cells were exposed to 25 mM DL -galactosamine for 24 h. Then, 100 ml of 0.5 mg/ml MTT was added to each well after the withdrawal of the culture medium and incubated for an additional 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 on a microplate reader at 492 nm. The survival rate of HL7702 cells was evaluated. Disclosure statement There is no conflict of interest among all authors.
Funding This work was financially supported by the National Mega-project for Innovative Drugs [grant number 2012ZX09101].
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