Fitoterapia 96 (2014) 138–145
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Hepatoprotective coumarins and secoiridoids from Hydrangea paniculata Jing Shi a,b, Chuang-Jun Li a, Jing-Zhi Yang a, Jie Ma a, Chao Wang a, Jia Tang a, Yan Li a, Hui Chen a, Dong-Ming Zhang a,⁎ 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 b Department of Pharmaceutics, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan 250117, China
a r t i c l e
i n f o
Article history: Received 2 March 2014 Accepted in revised form 19 April 2014 Accepted 21 April 2014 Available online 6 May 2014
a b s t r a c t Three new coumarin glucosides (1, 3, 4), and a new secoiridoid glucoside (2), together with one known secoiridoid glucoside (5), were isolated from the stems of Hydrangea paniculata. Their structures were elucidated on the basis of spectroscopic methods, including extensive NMR, MS and CD spectra. At 10 μM, compounds 1–5 showed hepatoprotective activities against DL-galactosamine-induced toxicity in HL-7702 cells. © 2014 Elsevier B.V. All rights reserved.
Keywords: Hydrangea paniculata Coumarins Secoiridoids Hepatoprotective activity
1. Introduction Hydrangea paniculata Sieb. (Saxifragaceae), widely distributed in southern China, is used to bring down fever, relieve sore throat and treat malaria in folk medicine [1]. Our previous study has shown that the effective fraction of H. paniculata is useful for prevention and/or treatment of renal insufficiency, hypertension and diabetic nephropathy [2,3]. As a part of our ongoing screening program for bioactive compounds, we studied the chemical constituents of the stems of H. paniculata systematically. In an earlier report, we described the isolation of coumarin glycosides, iridoid glucosides, and phenolic glycosides from the H2O extract of the stems of this plant [4,5]. During further investigation on active substances, we obtained three new coumarin glucosides, hydrangesides A, C and D (1, 3, 4), a new secoiridoid glucoside, hydrangeside B (2), along with one
⁎ Corresponding author at: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing 100050, China. Tel./fax: +86 10 6316 5227. E-mail address: [email protected] (D.-M. Zhang).
http://dx.doi.org/10.1016/j.fitote.2014.04.015 0367-326X/© 2014 Elsevier B.V. All rights reserved.
known secoiridoid glucoside (5). Herein, we report the isolation and structural elucidation of these compounds as well as their hepatoprotective activities against DL-galactosamine-induced toxicity in HL-7702 cells. 2. Experimental 2.1. General experimental procedures Optical rotations were measured on a Jasco P-2000 polarimeter. IR spectra were recorded on an IMPACT 400 spectrometer by a transmission microscope method. UV spectra were scanned by a Jasco V650 spectrophotometer. 1H NMR (400 or 500 MHz), 13C NMR (100 or 125 MHz), and 2D NMR spectra were run on Mercury-400 and Bruker AV500-III spectrometers 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. GC was conducted using an Agilent Technologies 7890A instrument. Reversed-phase silica MPLC was performed with pumps C-605 (Buchi), a UV photometer C-635 (Buchi), a fraction
J. Shi et al. / Fitoterapia 96 (2014) 138–145
collector C-660 (Buchi), and a ODS column (6 × 45 cm, 50 μm). Preparative HPLC was carried out on a Shimadzu LC-6AD instrument with a SPD-20A detector, using an YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Column chromatography was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China) and ODS (50 μm, YMC, Japan). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating.
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2.4. Hydrangeside A (1) White amorphous powder; [α]20 D − 108.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (4.59), 317 (4.10) nm; IR (microscope) νmax: 3368 (br), 2918, 1730, 1618, 1507, 1280, 1073 cm− 1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 1; ESI-MS: m/z 835 [M + Na]+, 811 [M − H]−; HR-ESI-MS: m/z 835.2432 [M + Na]+ (calcd. for C40H44O18Na, 835.2420).
2.2. Plant material 2.5. Hydrangeside B (2) 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. 2.3. Extraction and isolation The air-dried stems of H. paniculata (10.5 kg) were powdered and extracted with H2O (2 × 45 L, each for 2 h). The H2O extract was passed through macroporous resin (D101, 9 kg) column and eluted with H2O (12 L), 30% EtOH (18 L), 70% EtOH (18 L) and 95% EtOH (15 L). The 70% EtOH fraction (B, 108 g) was chromatographed on silica gel (200–300 mesh, 10 × 130 cm, 2.3 kg) with CHCl3–MeOH (9: 1, 4: 1, each 5 L) to give ten fractions (FrB.A–B.J). Fraction B.H (8.5 g) was separated by reversed-phase silica MPLC eluted with 22% CH3CN–H2O (25 mL/min, 3.5 h) to obtain 13 fractions (FrB.H-1– B.H-13). Fraction B.H-9 was further purified by preparative HPLC (CH3CN–H2O–CF3COOH, 24: 76: 0.05, 8 mL/min) to yield 5 (10 mg). Fraction B.H-13 was further purified by preparative HPLC (CH3CN–H2O–CF3COOH, 26:74:0.05, 8 mL/min) to afford 2 (10 mg). Fraction B.J (2.6 g) also was separated by reversed-phase silica MPLC with 5%–50% gradient MeOH–H2O (25 mL/min, 5 h) to obtain 17 fractions (FrB.J-1–B.J-17). Fraction B.J-10 was followed by repeatedly preparative HPLC (CH3CN–H2O, 20:80, 8 mL/min) to give 3 (6 mg) and 4 (5 mg). Fraction B.J-13 was further purified by preparative HPLC (CH3OH–H2O, 45: 55, 8 mL/min) to afford 1 (10 mg). The purities of these compounds were N 90%, as determined by HPLC. Table 1 1 H NMR and 13C NMR data of the half structure of compound 1 in DMSO-d6. Position
Position δHa
2 3 4 5 6 7 8 9 10 1′ 2′
δCb
160.0 6.29 d (9.5) 113.1 7.95 d (9.5) 144.0 7.58 d (8.5) 129.3 6.96 dd (8.5, 2.0) 113.3 159.9 7.01 d (2.0) 103.0 154.9 113.5 5.09 d (7.5) 99.4 3.72 m 73.7
δHa 3′ 4′ 5′ 6′a 6′b 1″ 2″a 2″b 3″ 4″ 5″
δCb
3.30 m 76.1 3.15 m 70.0 3.28 m 72.9 3.97 dd (10.5, 7.5) 63.5 4.31 brd (10.5) 5.13 brs 128.9 1.86 m 35.7 2.06 m 2.36 m 38.5 174.9 0.93 d (7.5) 15.8
Colorless oil; [α]20 D − 51.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε): 203 (3.37), 225 (3.13), 327 (2.89) nm; IR (microscope) νmax: 3372 (br), 2970, 2936, 1693, 1632, 1518, 1275, 1075 cm− 1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 2; CD (MeOH): 270 (Δε − 0.25), 281 (Δε − 0.23) nm; ESI-MS: m/z 731 [M + H]+, 753 [M + Na]+; HR-ESI-MS: m/z 731.2590 [M + H]+ (calcd. for C36H43O16, 731.2546).
2.6. Hydrangeside C (3) White amorphous powder; [α]20 D − 80.4 (c 0.08, MeOH); UV (MeOH) λmax (log ε): 203 (4.56), 330 (4.08) nm; IR (microscope) νmax: 3355 (br), 2920, 1701, 1615, 1510, 1258, 1063 cm−1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 3; ESI-MS: m/z 719 [M + Na]+, 695 [M − H]−; HR-ESI-MS: m/z 719.1940 [M + Na]+ (calcd. for C35H36O15Na, 719.1946).
Table 2 1 H NMR and
C NMR data of compound 2 in DMSO-d6.
Position
1 3 4 5 6
Position δHa
δCb
δHa
5.44 d (6.5) 7.43 s
95.4 151.5 109.9 29.8 28.7
7.26 brs
2″ 3″ 4″ 5″ 6″ 7″ 62.6 8″ 9″ 134.6 1‴
2.76 m 1.72 m 1.98 m 7 3.70 m 4.11 mc 8 5.70 ddd (17.5, 11.0, 9.5) 9 2.57 brdd (9.5, 6.5) 43.1 2‴ 10 5.23 brd (11.0) 118.6 3‴ 5.33 brd (17.5) 4‴ 11 167.8 5‴ 11-COOH 11.97 brs 1′ 4.52 d (8.0) 98.6 6‴ 2′ 2.96 m 73.0 7‴ 76.6 8‴ 3′ 3.13 mc 4′ 3.03 m 70.0 9‴ 77.2 5′ 3.14 mc 6′ 3.66 mc 61.1 3″-OCH3 3.41 m 3‴-OCH3 1″ 127.5 5‴-OH a 1
H NMR data (δH) were measured at 500 MHz. C NMR data (δC) were measured at 125 MHz. Overlapping signals.
a 1
b 13
b
c
H NMR data (δH) were measured at 500 MHz. 13 C NMR data (δC) were measured at 125 MHz.
13
δCb
112.2 143.9 149.9 129.8 7.25 brs 118.0 7.56 d (16.0) 144.9 6.48 d (16.0) 114.9 166.4 131.8 6.74 brsc 6.74 brsc
6.90 5.51 3.47 4.11 3.66 3.81 3.73 9.04
brs d (6.5) m mc mc s s s
118.6 147.5 115.3 146.5 110.3 87.9 52.5 62.3 55.8 55.6
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Table 3 1 H NMR and Position
13
C NMR data of compounds 3 and 4 in DMSO-d6.
3
4
δHa 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴a 9‴b 3″-OCH3 3‴-OCH3
6.24 7.87 7.57 6.99
c
δC d (9.5) d (9.5) d (8.5) dd (8.5, 2.0)
7.04 d (2.0)
5.13 3.31 3.33 3.26 3.78 4.21 4.40
d (7.0) m m m m dd (12.0, 6.5) brd (12.0)
7.27 brs
7.02 7.10 7.49 6.47
d (8.0) brd (8.0) d (16.0) d (16.0)
6.96 brs
6.67 6.75 4.70 4.38 3.57 3.26 3.78 3.72
d (8.0) brd (8.0) m m m m
160.1 113.1 144.0 129.4 113.5 159.9 103.1 155.0 113.3 99.5 73.0 76.2 69.8 73.7 63.2 126.6 111.0e 149.5 150.8 114.4 122.7 144.9 114.9 166.4 132.8 111.0e 147.0 145.5 114.6 119.0 70.9 83.8 60.1 55.6 55.4
δHb
δCd
6.25 d (9.6) 7.87 d (9.6) 7.57 d (8.8) 7.00e 7.07 d (2.0)
5.13 3.31 3.32 3.25 3.78 4.20 4.40
d (6.8) m m m m dd (12.0, 6.4) brd (12.0)
7.22 brs
6.99e 7.08 brd (8.4) 7.47 d (16.0) 6.46 d (16.0)
7.00e
6.66 6.77 4.69 4.42 3.60 3.60 3.76 3.72
d (8.0) brd (8.0) m m m m s s
160.1 113.1 144.0 129.4 113.5 159.9 103.1 155.0 113.3 99.5 73.0 76.2 69.8 73.7 63.2 126.5 111.0 149.6 150.5 114.4 122.7 144.9 114.9 166.4 133.0 111.4 146.9 145.5 114.5 119.5 71.5 83.3 60.2 55.4 55.3
a 1
H NMR data (δH) were measured at 500 MHz. H NMR data (δH) were measured at 400 MHz. C NMR data (δC) were measured at 125 MHz. 13 C NMR data (δC) were measured at 100 MHz. Overlapping signals.
b 1
c 13 d e
2.7. Hydrangeside D (4) White amorphous powder; [α]20 D − 102.5 (c 0.07, MeOH); UV (MeOH) λmax (log ε): 203 (4.50), 330 (4.03) nm; IR (microscope) νmax: 3429 (br), 2939, 1702, 1617, 1510, 1260, 1075 cm− 1; 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, see Table 3; ESI-MS: m/z 719 [M + Na]+, 695 [M − H]−; HR-ESI-MS: m/z 719.1941 [M + Na]+ (calcd. for C35H36O15Na, 719.1946). 2.8. Protective effect on cytotoxicity induced by DL-galactosamine in HL-7702 cells The hepatoprotective effects of compounds 1–5 were determined by a MTT colorimetric assay [6] in HL-7702 cells. Each cell suspension of 2 × 104 cells in 200 μL of RPMI 1640 containing fetal calf serum (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL) was placed in a 96-well microplate and precultured for 24 h at 37 °C under a 5% CO2 atmosphere. Fresh medium (100 μL) containing bicyclol and test samples
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 μL 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 μL 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 HL-7702 cells was evaluated. 2.9. Acid hydrolysis of compounds 1–4 Each compound (2 mg) was refluxed with 2 mL 2 M HCl–H2O at 80 °C for 5 h and then partitioned by EtOAc (1 mL × 3). The aqueous layer was evaporated in vacuo to furnish a neutral residue. The residue was dissolved in anhydrous pyridine (1 mL), to which 2 mg of L-cysteine methyl ester hydrochloride was added. The mixture was stirred at 60 °C for 2 h, and after evaporation in vacuo to dryness, 0.2 mL of N-trimethylsilylimidazole was added; the mixture was refluxed at 60 °C for another 2 h. The reaction mixture was partitioned between n-hexane and H2O (2 mL each), and the n-hexane extract was analyzed by GC under the following conditions: capillary column, HP-5 (30 m × 0.25 mm, with a 0.25 μm film, Dikma); detection, FID; detector temperature, 280 °C; injection temperature, 250 °C; initial temperature 160 °C, then raised to 280 °C at 5 °C/min, final temperature maintained for 10 min and carrier, N2 gas. The standard D-glucose was treated by the same reaction and gas chromatographic conditions. As a result, D-glucose [(tR(min): 19.0 min)] was detected from the acid hydrolysates of 1–4 [5,19]. 3. Results and discussion Compound 1 was obtained as a white amorphous powder. The absorption maxima at 202 and 317 nm in the UV spectrum indicated a coumarin skeleton. The IR spectrum showed absorption bands for hydroxyl (3367 cm− 1), carbonyl (1730 cm− 1), and aromatic ring (1618 and 1507 cm− 1) groups. The HR-ESI-MS showed a [M + Na]+ ion peek at m/z 835.2432 (calcd. for C40H44O18Na, 835.2420), consistent with the molecular formula C40H44O18. However, the 1 H and 13C NMR spectra of 1 exhibited resonances of 22 protons and 20 carbons (Table 1), which were only half the numbers of proton and carbon atoms expected from the molecular formula. These spectroscopic data suggested that 1 possessed a symmetric structure. Therefore, each signal in the NMR spectra of 1 represented a pair of overlapping resonances with a completely equivalent chemical and magnetic environment. For the half structural unit of 1, the 1H NMR spectrum displayed a pair of doublets at δH 7.95 (d, J = 9.5 Hz) and 6.29 (d, J = 9.5 Hz), and three ABX-type coupled aromatic protons at δH 7.58 (d, J = 8.5 Hz), 7.01 (d, J = 2.0 Hz), 6.96 (dd, J = 8.5, 2.0 Hz), and a sugar anomeric proton at δH 5.09 (d, J = 7.5 Hz, H-1′) suggesting the presence of one-substituted coumarin with a sugar moiety [4]. In addition, it exhibited one olefinic proton at δH 5.13 (brs, H-1″), one methyl group at δH 0.93 (3H, d, J = 7.5 Hz, H-5″), two methylene protons at δH 2.06, 1.86 (m each, H-2″) and one sp3 methine proton at δH 2.36 (m, H-3″). In the 13C
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141
Fig. 1. Chemical structures of compounds 1–5.
NMR spectrum, 20 resonances were observed. Consistent with the 1H NMR spectrum data analysis, fifteen of the resonances were assigned to one-O-substituted coumarin skeleton and a glucose unit. The remaining five resonances were due to one methyl, one methylene, one sp3 methine, one sp2 methine, and one carbonyl. The HSQC spectrum of 1 led to the unambiguous assignment of resonances of protons and protonated carbons. On the basis of the HMBC correlations from H-4 (δH 7.95) to C-5 (δC 129.3) and C-9 (δC 154.9), from H-5 (δH 7.58) to C-7 (δC 159.9) and C-9 (δC 154.9), from H-6 (δH 6.96) to C-8 (δC 103.0) and C-10 (δC 113.5), and from H-8 (δH 7.01) to C-10 (δC 113.5), the 7-O-substituted coumarin skeleton was assigned. The presence of a prenyl moiety was proved by the HMBC correlations from H-1″
(δH 5.13) to C-2″ (δC 35.7), from H-2″ (δH 1.86, 2.06) to C-1″ (δC 128.9), from H-3″ (δH 2.36) to C-4″(δC 174.9) and from H-5″(δH 0.93) to C-2″(δC 35.7), C-3″(δC 38.5), and C-4″ (δC 174.9). The position of the ester group was confirmed by a HMBC correlation between H-6′ (δH 3.97) and the carbonyl carbon of the prenyl moiety (C-4″ δC 174.9). The glucose unit was obviously attached to C-7 due to the HMBC correlation between H-1′ (δH 5.09) and C-7 (δC 159.9). Moreover, only one sp2 methine (C-1″) was reasonably explained that a double bond connected the half structure to form the symmetrically dimeric structure. Finally, the glucose unit was identified by GC analysis as D-glucose after an acid hydrolysis of 1. Thus, the structure of 1 was determined as shown (Fig. 1) and named as hydrangeside A.
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Fig. 2. Key HMBC and 1H–1H COSY correlations of compounds 1–3.
The constituents of stems of H. paniculata have been previously investigated and shown to contain lots of skimmin (7). Biogenetically, as shown in Fig. 3, hydrangeside A may be derived from skimmin and isoprene via intermediate 2,7-dimethyl-4-octene-1,8-dioic acids (6).
Compound 2 was obtained as colorless oil. The molecular formula was C36H42O16, as deduced from its [M + H]+ ion peak at m/z 731.2590 (calcd. for C36H43O16, 731.2546). The absorption maxima were at 203, 225 and 327 nm in the UV spectrum. The IR spectrum showed absorption bands for
Fig. 3. Hypothesis for the biogenesis of 1.
J. Shi et al. / Fitoterapia 96 (2014) 138–145
hydroxyl (3372 cm−1), carbonyl (1693 cm−1), and aromatic ring (1632 and 1518 cm−1) groups. The proton signals at δH 7.43 (s, H-3), 5.70 (brdt, J = 17.5, 11.0 Hz, H-8), 5.44 (1H, d, J = 6.5 Hz, H-1), 5.33 (brd, J = 17.5 Hz, H-10), 5.23 (brd, J = 11.0 Hz, H-10), and δH 4.52 (1H, d, J = 8.0 Hz, H-1′) in the 1H NMR spectrum of 2 were characteristic of secoiridoid-type monoterpene glycoside [7,8].
143
Furthermore, the 1H NMR spectrum showed signals assignable to two trans-olefinic protons at δH 7.56 and 6.48 (1H and d each, J = 16.0 Hz, H-7″ and H-8″), five aromatic protons at δH 7.26 (1H, brs, H-2″), 7.25 (1H, brs, H-6″), 6.90 (1H, brs, H-6‴), 6.74 (2H, brs, H-2‴, H-4‴), two aromatic methoxyl at δH 3.81 (3H, s), 3.73 (3H, s), a phenolic hydroxy protons at δH 9.04(1H, brs, OH-5‴), a 2,3-disubstituted 2,3-dihydrobenzofuran moiety
Fig. 4. The CD and ΔδCD spectra of compounds 3 and 4 in MeOH.
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Table 4 Effects of compounds 1–5 on the survival rate of HL-7702 cells injured by DL-GalN (10 μM). Group
OD value
Survival rate (%)
Control Model Bicyclola 1 2 3 4 5
1.197 0.384 0.630 0.836 0.604 0.560 0.619 0.422
100.0 29%⁎⁎⁎⁎ 51%⁎⁎ 68%⁎⁎⁎ 48%⁎ 44%⁎⁎
± ± ± ± ± ± ± ±
0.030 0.028 0.064 0.031 0.085 0.059 0.152 0.004
50 32%⁎⁎
⁎ P b 0.05 vs. model. ⁎⁎ P b 0.01 vs. model. ⁎⁎⁎ P b 0.001 vs. model. ⁎⁎⁎⁎ P b 0.001 vs. control. a Positive control substance.
at δH 5.51 (1H, d, J = 6.5 Hz, H-7‴), 3.47 (1H, m, H-8‴), 4.11 and 3.66 (1H and m each, H-9‴). In the 13C NMR spectrum, 36 resonances were observed. Fifteen of the resonances were assigned to secoiridoid glycoside moiety. The HSQC spectrum of 2 led to the unambiguous assignment of resonances of protons and protonated carbons. The 1H–1H COSY spectrum (Fig. 2) further elucidated the spin system of H-7‴/H-8‴/H-9″ in 2,3-disubstituted 2,3-dihydrobenzofuran moiety. In the HMBC spectrum of 2, the correlations from H-2″ to C-4″, C-6″ and C-7″, from H-6″ to C-2″, C-4″ and C-7″, from H-7″ to C-2″, C-6″ and C-9″, from H-8″ to C-1″, from H-2‴ to C-1‴, C-3‴, C-6‴ and C-7‴, from H-4‴ to C-3‴ and C-6‴, from H-6‴ to C-4‴, C-5‴ and C-7‴, from H-7‴ to C-4″, C-1‴ and C-6‴, from H-8‴ to C-4″ and C-1‴, from OMe-3″ to C-3″, and from OMe-3‴ to C-3‴ were agreed with the presence of the cimicifugic acid moiety [9]. The connection of cimicifugic acid moiety and secoiridoid glycoside moiety was linked by an ester bond (C-7\O\C-9″), which was confirmed by the downfield chemical shifts of H-7 (δH 4.11) and C-7 (δC 62.6) [7,8,10]. The 7‴,8‴-trans configuration of the 2,3-disubstituted 2,3dihydrobenzofuran moiety in 2 was elucidated by the coupling constant (J7‴,8‴ = 6.5 Hz) [11]. The absolute stereostructure of the 2,3-disubstituted 2,3-dihydrobenzofuran moiety in 2 was elucidated by the circular dichroic (CD) spectroscopic analysis. Thus, the CD spectrum (in MeOH) of 2 showed negative cotton effects [270 (Δε − 0.25), 281 (Δε − 0.23) nm], which indicated the absolute configurations of the 7‴ and 8‴-positions to be 7‴R and 8‴S orientations [12–14]. Finally, the glucose unit was identified by GC analysis as D-glucose after an acid hydrolysis of 2. Thus, the structure of 2 was elucidated as shown (Fig. 1) and named as hydrangeside B. Compounds 3 and 4 were both obtained as white amorphous powder and displayed absorption bands for hydroxyl, carbonyl, and aromatic ring groups. Both of their molecular formulas were determined as C35H36O15 by HR-ESI-MS with an [M + Na]+ ion peaks at m/z 719.1940 and 719.1941, respectively (calcd. for C35H36O15Na, 719.1946). And the NMR spectra of 3 and 4 closely resembled each other and suggested that 3 and 4 had the same planar structure. The 1H NMR spectrum of 3 showed five aromatic proton signals at δH 7.87 (d, J = 9.5 Hz), 7.57 (d, J = 8.5 Hz), 7.04 (d, J = 2.0 Hz), 6.99 (dd, J = 8.5, 2.0 Hz), and 6.24 (d, J = 9.5 Hz), as well as a sugar anomeric signals at δH 5.13 (d, J = 7.0 Hz), suggesting the presence of 7-substituted coumarin with a sugar moiety. The proton
signals at δH 7.49 (d, J = 16.0 Hz) and 6.47 (d, J = 16.0 Hz), together with ABX-type coupled aromatic protons at δH 7.27 (brs), 7.10 (brd, J = 8.0 Hz), and 7.02 (d, J = 8.0 Hz) suggested the presence of trans-feruloyl moiety. In addition, the ABX-type coupled aromatic protons at δH 6.96 (brs), 6.75 (brd, J = 8.0 Hz) and 6.67 (d, J = 8.0 Hz), along with two oxymethines at δH 4.70 (m), 4.38 (m), and an oxymethylene at δH 3.57 (m), and 3.26 (m) indicated the presence of 4‴-hydroxy3‴-methoxy-phenylglycerol-8‴-yl moiety. The 13C NMR spectrum of 3 showed 35 carbon signals corresponding to the above moieties. Fifteen of the carbon signals were consistent with the presence of a 7-O-substituted coumarin skeleton with a glucose moiety [4]. The remaining 20 carbon signals with the help of 2D NMR spectrum were attributable to trans-feruloyl moiety and 4‴-hydroxy-3‴-methoxy-phenylglycerol-8‴-yl moiety [15]. The linkages of the three moieties were confirmed by the correlations between δH 5.13 (H-1′) and δC 159.9 (C-7), between δH 4.21, 4.40 (H-6′) and δC 166.4 (C-9″), between δH 4.38 (H-8‴) and δC 150.8 (C-4″) in the HMBC spectrum. The small difference of the shifts of 7‴, 8‴ and 9‴ in 3 and 4 revealed that they were the erythro and threo 8‴-4″-oxyneolignane isomers. The ΔδC8‴-C7‴ values were applicable to differentiate threo and erythro phenylglycerol moiety since the NMR spectra 3 and 4 were obtained in DMSO-d6 [16,17]. The ΔδC8‴-C7‴ value of 4 (11.8 ppm) was smaller than that of 3 (12.9 ppm). This suggested that 3 possessed a threo relative configuration and 4 possessed an erythro relative configuration. According to the literature [11,12,17], the configuration of C-8‴ in phenylglycerol moiety was identification in the CD spectrum. A positive cotton effect at about 235 nm in the CD spectrum suggested the 8S configuration, while a negative cotton effect at about 235 nm suggested the 8R configuration. But there was no clear cotton effect at 235 nm in the CD spectrum of compounds 3 and 4, which perhaps was affected by the 7-O-glucosyl coumarin moiety (7, skimmin) in the structures of 3 and 4. So CD difference spectra were applied, the CD spectrum of skimmin subtracted from the CD spectra of 3 and 4 gave ΔδCD spectra of 3 and 4, respectively. The ΔδCD spectra of compounds 3 and 4 (Fig. 4) both showed negative cotton effect at 235 nm, which indicated the absolute configurations of 8‴-position of compound 3 and 4 to be 8‴R orientations. As a result, compound 3 had 7‴R,8‴R configuration and compound 4 had 7‴S,8‴R configuration on the basis of their relative configuration. Finally, the glucose unit was identified by GC analysis as D-glucose after an acid hydrolysis of 3 and 4. Hence, the structures of 3 and 4 were determined as shown (Fig. 1) and named as hydrangeside C and hydrangeside D, respectively. Additionally, one known secoiridoid glucoside named methylgrandifloroside was isolated [10,18]. Compounds 1–5 were bioassayed for their hepatoprotective activities against DL-galactosamine-induced toxicity in HL-7702 cells, using the hepatoprotective activity drug bicyclol as the positive control. At 10 μM, compounds 1–5 reduced DL-galactosamine (GalN)-induced HL-7702 cell damage by increasing the survival rate from 29% (P b 0.001) to 68% (P b 0.001), 48% (P b 0.05), 44% (P b 0.01), 50%, and 32% (P b 0.01), respectively, while the positive control bicyclol gave a 51.0% (P b 0.01) survival rate (Table 4). All the compounds
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displayed moderate hepatoprotective activities against DLgalactosamine-induced toxicity in HL-7702 cells. Conflict of interest There is no conflict of interest among all authors. Acknowledgment Financial support was provided by the National Natural Science Foundation of China (No. 21132009), the National Mega-project for Innovative Drugs (No. 2012ZX09101), and PCSIRT (No. IRT1007). References [1] Chinese Herbal Medicine Company. Chinese traditional medicine resource records. Beijing: Science Publishing House; 1994 480. [2] Zhang DM, Chen XG, Yang JZ, Li Y, Zheng XG. Chinese patent CN 1690069A; 2005. [3] Zhang DM, Chen XG, Yang JZ, Li Y. Chinese patent CN 1605343A; 2005. [4] Shi J, Yang JZ, Li CJ, Zhang DM. Chemical constituents from Hydrangea paniculata. China J Chin Mater Med 2010;35:3007–9. [5] Fu HZ, Li CJ, Yang JZ, Shen ZF, Zhang DM. Potential antiinflammatory constituents of the stems of Gordonia chrysandra. J Nat Prod 2011;74:1066–72. [6] Li F, Li CJ, Ma J, Yang JZ, Chen H, Liu XM, et al. Four new sesquiterpenes from the stems of Pogostemon cablin. Fitoterapia 2013;86:183–7. [7] Xu M, Wang D, Zhang YJ, Yang CR. Iridoidal glucosides from Gentiana rhodantha. J Asian Nat Prod Res 2008;6(10):491–8.
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[8] Kitajima M, Fujii N, Yoshino F, Sudo H, Saito K, Aimi N, et al. Camptothecins and two new monoterpene glucosides from Ophiorrhiza liukiuensis. Chem Pharm Bull 2005;53(10):1355–8. [9] Zhao XH, Chen DH, Si JY, Pan RL, Shen LG. Studies on the phenolic acid constituents from Chinese medicine “sheng-ma”, rhizome of Cimicifuga foetida L. Acta Pharm Sin 2002;37(7):535–8. [10] Rasoanaivo P, Nicoletti M, Multari G, Palazzino G, Galeffi C. Research on African medicinal plants parts XXXIII. Secoiridoids and related monoterpenes of Anthocleista amplexicaulis. Fitoterapia 1994;65(1):38–43. [11] Kim KH, Moon E, Kim SY, Lee KR. Lignans from the tuber-barks of Colocasiaantiquorum var. esculenta and their antimelanogenic activity. J Agric Food Chem 2010;58:4779–85. [12] Xiong L, Zhu CG, Li YR, Tian Y, Lin S, Yuan SP, et al. Lignans and neolignans from Sinocalamus affinis and their absolute configurations. J Nat Prod 2011;74:1188–200. [13] Antus S, Kurtan T, Juhasz L, Kiss L, Hollo M, Majer ZS, et al. Chiroptical properties of 2,3-dihydrobenzo[b]furan and chromane chromophores in naturally occurring O-heterocycles. Chirality 2001;13:493–506. [14] Yoshikawa M, Morikawa T, Xu FM, Ando S, Matsuda H. (7R,8S) And (7S,8R) 8-5 linked neolignans from egyptian herbal medicine anastatica hierochuntica and inhibitory activities of lignans on nitric oxide production. Heterocycles 2003;60:1787–92. [15] Sun Y, Ding Y, Lin WH. Isolation and identification of compounds from marine mangrove plant Avicennia marina. J Peking Univ (Health Sci) 2009;41(2):221–5. [16] Bardet M, Robert D, Lundquist K, Unge SV. Distribution of erythro and threo forms of different types of β-O-4 structures in aspen lignin by 13 C NMR using the 2D INADEQUATE experiment. Magn Reson Chem 1998;36:597–600. [17] Gan ML, Zhang YL, Lin S, Liu MT, Song WX, Zi JC, et al. Glycosides from the root of Iodescirrhosa. J Nat Prod 2008;71:647–54. [18] Chapelle JP. Grandifloroside et methylgrandifloroside, glucosides iridoides nouveaux d'Anthocleista grandiflora. Phytochemistry 1976;15:1305–7. [19] Lin S, Wang SJ, Liu MT, Gan ML, Li S, Yang YC, et al. Glycosides from the stem bark of Fraxinus sieboldiana. J Nat Prod 2007;70:817–23.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Hepatoprotective coumarins and secoiridoids from Hydrangea paniculata Jing Shi a,b, Chuang-Jun Li a, Jing-Zhi Yang a, Jie Ma a, Chao Wang a, Jia Tang a, Yan Li a, Hui Chen a, Dong-Ming Zhang a,⁎ 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 b Department of Pharmaceutics, Shandong Cancer Hospital and Institute, Shandong Academy of Medical Sciences, Jinan 250117, China
a r t i c l e
i n f o
Article history: Received 2 March 2014 Accepted in revised form 19 April 2014 Accepted 21 April 2014 Available online 6 May 2014
a b s t r a c t Three new coumarin glucosides (1, 3, 4), and a new secoiridoid glucoside (2), together with one known secoiridoid glucoside (5), were isolated from the stems of Hydrangea paniculata. Their structures were elucidated on the basis of spectroscopic methods, including extensive NMR, MS and CD spectra. At 10 μM, compounds 1–5 showed hepatoprotective activities against DL-galactosamine-induced toxicity in HL-7702 cells. © 2014 Elsevier B.V. All rights reserved.
Keywords: Hydrangea paniculata Coumarins Secoiridoids Hepatoprotective activity
1. Introduction Hydrangea paniculata Sieb. (Saxifragaceae), widely distributed in southern China, is used to bring down fever, relieve sore throat and treat malaria in folk medicine [1]. Our previous study has shown that the effective fraction of H. paniculata is useful for prevention and/or treatment of renal insufficiency, hypertension and diabetic nephropathy [2,3]. As a part of our ongoing screening program for bioactive compounds, we studied the chemical constituents of the stems of H. paniculata systematically. In an earlier report, we described the isolation of coumarin glycosides, iridoid glucosides, and phenolic glycosides from the H2O extract of the stems of this plant [4,5]. During further investigation on active substances, we obtained three new coumarin glucosides, hydrangesides A, C and D (1, 3, 4), a new secoiridoid glucoside, hydrangeside B (2), along with one
⁎ Corresponding author at: Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street, Beijing 100050, China. Tel./fax: +86 10 6316 5227. E-mail address: [email protected] (D.-M. Zhang).
http://dx.doi.org/10.1016/j.fitote.2014.04.015 0367-326X/© 2014 Elsevier B.V. All rights reserved.
known secoiridoid glucoside (5). Herein, we report the isolation and structural elucidation of these compounds as well as their hepatoprotective activities against DL-galactosamine-induced toxicity in HL-7702 cells. 2. Experimental 2.1. General experimental procedures Optical rotations were measured on a Jasco P-2000 polarimeter. IR spectra were recorded on an IMPACT 400 spectrometer by a transmission microscope method. UV spectra were scanned by a Jasco V650 spectrophotometer. 1H NMR (400 or 500 MHz), 13C NMR (100 or 125 MHz), and 2D NMR spectra were run on Mercury-400 and Bruker AV500-III spectrometers 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. GC was conducted using an Agilent Technologies 7890A instrument. Reversed-phase silica MPLC was performed with pumps C-605 (Buchi), a UV photometer C-635 (Buchi), a fraction
J. Shi et al. / Fitoterapia 96 (2014) 138–145
collector C-660 (Buchi), and a ODS column (6 × 45 cm, 50 μm). Preparative HPLC was carried out on a Shimadzu LC-6AD instrument with a SPD-20A detector, using an YMC-Pack ODS-A column (250 × 20 mm, 5 μm). Column chromatography was performed with silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China) and ODS (50 μm, YMC, Japan). TLC was carried out with glass precoated silica gel GF254 plates. Spots were visualized under UV light or by spraying with 10% sulfuric acid in EtOH followed by heating.
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2.4. Hydrangeside A (1) White amorphous powder; [α]20 D − 108.1 (c 0.1, MeOH); UV (MeOH) λmax (log ε): 202 (4.59), 317 (4.10) nm; IR (microscope) νmax: 3368 (br), 2918, 1730, 1618, 1507, 1280, 1073 cm− 1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 1; ESI-MS: m/z 835 [M + Na]+, 811 [M − H]−; HR-ESI-MS: m/z 835.2432 [M + Na]+ (calcd. for C40H44O18Na, 835.2420).
2.2. Plant material 2.5. Hydrangeside B (2) 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. 2.3. Extraction and isolation The air-dried stems of H. paniculata (10.5 kg) were powdered and extracted with H2O (2 × 45 L, each for 2 h). The H2O extract was passed through macroporous resin (D101, 9 kg) column and eluted with H2O (12 L), 30% EtOH (18 L), 70% EtOH (18 L) and 95% EtOH (15 L). The 70% EtOH fraction (B, 108 g) was chromatographed on silica gel (200–300 mesh, 10 × 130 cm, 2.3 kg) with CHCl3–MeOH (9: 1, 4: 1, each 5 L) to give ten fractions (FrB.A–B.J). Fraction B.H (8.5 g) was separated by reversed-phase silica MPLC eluted with 22% CH3CN–H2O (25 mL/min, 3.5 h) to obtain 13 fractions (FrB.H-1– B.H-13). Fraction B.H-9 was further purified by preparative HPLC (CH3CN–H2O–CF3COOH, 24: 76: 0.05, 8 mL/min) to yield 5 (10 mg). Fraction B.H-13 was further purified by preparative HPLC (CH3CN–H2O–CF3COOH, 26:74:0.05, 8 mL/min) to afford 2 (10 mg). Fraction B.J (2.6 g) also was separated by reversed-phase silica MPLC with 5%–50% gradient MeOH–H2O (25 mL/min, 5 h) to obtain 17 fractions (FrB.J-1–B.J-17). Fraction B.J-10 was followed by repeatedly preparative HPLC (CH3CN–H2O, 20:80, 8 mL/min) to give 3 (6 mg) and 4 (5 mg). Fraction B.J-13 was further purified by preparative HPLC (CH3OH–H2O, 45: 55, 8 mL/min) to afford 1 (10 mg). The purities of these compounds were N 90%, as determined by HPLC. Table 1 1 H NMR and 13C NMR data of the half structure of compound 1 in DMSO-d6. Position
Position δHa
2 3 4 5 6 7 8 9 10 1′ 2′
δCb
160.0 6.29 d (9.5) 113.1 7.95 d (9.5) 144.0 7.58 d (8.5) 129.3 6.96 dd (8.5, 2.0) 113.3 159.9 7.01 d (2.0) 103.0 154.9 113.5 5.09 d (7.5) 99.4 3.72 m 73.7
δHa 3′ 4′ 5′ 6′a 6′b 1″ 2″a 2″b 3″ 4″ 5″
δCb
3.30 m 76.1 3.15 m 70.0 3.28 m 72.9 3.97 dd (10.5, 7.5) 63.5 4.31 brd (10.5) 5.13 brs 128.9 1.86 m 35.7 2.06 m 2.36 m 38.5 174.9 0.93 d (7.5) 15.8
Colorless oil; [α]20 D − 51.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε): 203 (3.37), 225 (3.13), 327 (2.89) nm; IR (microscope) νmax: 3372 (br), 2970, 2936, 1693, 1632, 1518, 1275, 1075 cm− 1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 2; CD (MeOH): 270 (Δε − 0.25), 281 (Δε − 0.23) nm; ESI-MS: m/z 731 [M + H]+, 753 [M + Na]+; HR-ESI-MS: m/z 731.2590 [M + H]+ (calcd. for C36H43O16, 731.2546).
2.6. Hydrangeside C (3) White amorphous powder; [α]20 D − 80.4 (c 0.08, MeOH); UV (MeOH) λmax (log ε): 203 (4.56), 330 (4.08) nm; IR (microscope) νmax: 3355 (br), 2920, 1701, 1615, 1510, 1258, 1063 cm−1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz, DMSO-d6) data, see Table 3; ESI-MS: m/z 719 [M + Na]+, 695 [M − H]−; HR-ESI-MS: m/z 719.1940 [M + Na]+ (calcd. for C35H36O15Na, 719.1946).
Table 2 1 H NMR and
C NMR data of compound 2 in DMSO-d6.
Position
1 3 4 5 6
Position δHa
δCb
δHa
5.44 d (6.5) 7.43 s
95.4 151.5 109.9 29.8 28.7
7.26 brs
2″ 3″ 4″ 5″ 6″ 7″ 62.6 8″ 9″ 134.6 1‴
2.76 m 1.72 m 1.98 m 7 3.70 m 4.11 mc 8 5.70 ddd (17.5, 11.0, 9.5) 9 2.57 brdd (9.5, 6.5) 43.1 2‴ 10 5.23 brd (11.0) 118.6 3‴ 5.33 brd (17.5) 4‴ 11 167.8 5‴ 11-COOH 11.97 brs 1′ 4.52 d (8.0) 98.6 6‴ 2′ 2.96 m 73.0 7‴ 76.6 8‴ 3′ 3.13 mc 4′ 3.03 m 70.0 9‴ 77.2 5′ 3.14 mc 6′ 3.66 mc 61.1 3″-OCH3 3.41 m 3‴-OCH3 1″ 127.5 5‴-OH a 1
H NMR data (δH) were measured at 500 MHz. C NMR data (δC) were measured at 125 MHz. Overlapping signals.
a 1
b 13
b
c
H NMR data (δH) were measured at 500 MHz. 13 C NMR data (δC) were measured at 125 MHz.
13
δCb
112.2 143.9 149.9 129.8 7.25 brs 118.0 7.56 d (16.0) 144.9 6.48 d (16.0) 114.9 166.4 131.8 6.74 brsc 6.74 brsc
6.90 5.51 3.47 4.11 3.66 3.81 3.73 9.04
brs d (6.5) m mc mc s s s
118.6 147.5 115.3 146.5 110.3 87.9 52.5 62.3 55.8 55.6
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J. Shi et al. / Fitoterapia 96 (2014) 138–145
Table 3 1 H NMR and Position
13
C NMR data of compounds 3 and 4 in DMSO-d6.
3
4
δHa 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′a 6′b 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ 9″ 1‴ 2‴ 3‴ 4‴ 5‴ 6‴ 7‴ 8‴ 9‴a 9‴b 3″-OCH3 3‴-OCH3
6.24 7.87 7.57 6.99
c
δC d (9.5) d (9.5) d (8.5) dd (8.5, 2.0)
7.04 d (2.0)
5.13 3.31 3.33 3.26 3.78 4.21 4.40
d (7.0) m m m m dd (12.0, 6.5) brd (12.0)
7.27 brs
7.02 7.10 7.49 6.47
d (8.0) brd (8.0) d (16.0) d (16.0)
6.96 brs
6.67 6.75 4.70 4.38 3.57 3.26 3.78 3.72
d (8.0) brd (8.0) m m m m
160.1 113.1 144.0 129.4 113.5 159.9 103.1 155.0 113.3 99.5 73.0 76.2 69.8 73.7 63.2 126.6 111.0e 149.5 150.8 114.4 122.7 144.9 114.9 166.4 132.8 111.0e 147.0 145.5 114.6 119.0 70.9 83.8 60.1 55.6 55.4
δHb
δCd
6.25 d (9.6) 7.87 d (9.6) 7.57 d (8.8) 7.00e 7.07 d (2.0)
5.13 3.31 3.32 3.25 3.78 4.20 4.40
d (6.8) m m m m dd (12.0, 6.4) brd (12.0)
7.22 brs
6.99e 7.08 brd (8.4) 7.47 d (16.0) 6.46 d (16.0)
7.00e
6.66 6.77 4.69 4.42 3.60 3.60 3.76 3.72
d (8.0) brd (8.0) m m m m s s
160.1 113.1 144.0 129.4 113.5 159.9 103.1 155.0 113.3 99.5 73.0 76.2 69.8 73.7 63.2 126.5 111.0 149.6 150.5 114.4 122.7 144.9 114.9 166.4 133.0 111.4 146.9 145.5 114.5 119.5 71.5 83.3 60.2 55.4 55.3
a 1
H NMR data (δH) were measured at 500 MHz. H NMR data (δH) were measured at 400 MHz. C NMR data (δC) were measured at 125 MHz. 13 C NMR data (δC) were measured at 100 MHz. Overlapping signals.
b 1
c 13 d e
2.7. Hydrangeside D (4) White amorphous powder; [α]20 D − 102.5 (c 0.07, MeOH); UV (MeOH) λmax (log ε): 203 (4.50), 330 (4.03) nm; IR (microscope) νmax: 3429 (br), 2939, 1702, 1617, 1510, 1260, 1075 cm− 1; 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) data, see Table 3; ESI-MS: m/z 719 [M + Na]+, 695 [M − H]−; HR-ESI-MS: m/z 719.1941 [M + Na]+ (calcd. for C35H36O15Na, 719.1946). 2.8. Protective effect on cytotoxicity induced by DL-galactosamine in HL-7702 cells The hepatoprotective effects of compounds 1–5 were determined by a MTT colorimetric assay [6] in HL-7702 cells. Each cell suspension of 2 × 104 cells in 200 μL of RPMI 1640 containing fetal calf serum (10%), penicillin (100 U/mL), and streptomycin (100 μg/mL) was placed in a 96-well microplate and precultured for 24 h at 37 °C under a 5% CO2 atmosphere. Fresh medium (100 μL) containing bicyclol and test samples
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 μL 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 μL 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 HL-7702 cells was evaluated. 2.9. Acid hydrolysis of compounds 1–4 Each compound (2 mg) was refluxed with 2 mL 2 M HCl–H2O at 80 °C for 5 h and then partitioned by EtOAc (1 mL × 3). The aqueous layer was evaporated in vacuo to furnish a neutral residue. The residue was dissolved in anhydrous pyridine (1 mL), to which 2 mg of L-cysteine methyl ester hydrochloride was added. The mixture was stirred at 60 °C for 2 h, and after evaporation in vacuo to dryness, 0.2 mL of N-trimethylsilylimidazole was added; the mixture was refluxed at 60 °C for another 2 h. The reaction mixture was partitioned between n-hexane and H2O (2 mL each), and the n-hexane extract was analyzed by GC under the following conditions: capillary column, HP-5 (30 m × 0.25 mm, with a 0.25 μm film, Dikma); detection, FID; detector temperature, 280 °C; injection temperature, 250 °C; initial temperature 160 °C, then raised to 280 °C at 5 °C/min, final temperature maintained for 10 min and carrier, N2 gas. The standard D-glucose was treated by the same reaction and gas chromatographic conditions. As a result, D-glucose [(tR(min): 19.0 min)] was detected from the acid hydrolysates of 1–4 [5,19]. 3. Results and discussion Compound 1 was obtained as a white amorphous powder. The absorption maxima at 202 and 317 nm in the UV spectrum indicated a coumarin skeleton. The IR spectrum showed absorption bands for hydroxyl (3367 cm− 1), carbonyl (1730 cm− 1), and aromatic ring (1618 and 1507 cm− 1) groups. The HR-ESI-MS showed a [M + Na]+ ion peek at m/z 835.2432 (calcd. for C40H44O18Na, 835.2420), consistent with the molecular formula C40H44O18. However, the 1 H and 13C NMR spectra of 1 exhibited resonances of 22 protons and 20 carbons (Table 1), which were only half the numbers of proton and carbon atoms expected from the molecular formula. These spectroscopic data suggested that 1 possessed a symmetric structure. Therefore, each signal in the NMR spectra of 1 represented a pair of overlapping resonances with a completely equivalent chemical and magnetic environment. For the half structural unit of 1, the 1H NMR spectrum displayed a pair of doublets at δH 7.95 (d, J = 9.5 Hz) and 6.29 (d, J = 9.5 Hz), and three ABX-type coupled aromatic protons at δH 7.58 (d, J = 8.5 Hz), 7.01 (d, J = 2.0 Hz), 6.96 (dd, J = 8.5, 2.0 Hz), and a sugar anomeric proton at δH 5.09 (d, J = 7.5 Hz, H-1′) suggesting the presence of one-substituted coumarin with a sugar moiety [4]. In addition, it exhibited one olefinic proton at δH 5.13 (brs, H-1″), one methyl group at δH 0.93 (3H, d, J = 7.5 Hz, H-5″), two methylene protons at δH 2.06, 1.86 (m each, H-2″) and one sp3 methine proton at δH 2.36 (m, H-3″). In the 13C
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Fig. 1. Chemical structures of compounds 1–5.
NMR spectrum, 20 resonances were observed. Consistent with the 1H NMR spectrum data analysis, fifteen of the resonances were assigned to one-O-substituted coumarin skeleton and a glucose unit. The remaining five resonances were due to one methyl, one methylene, one sp3 methine, one sp2 methine, and one carbonyl. The HSQC spectrum of 1 led to the unambiguous assignment of resonances of protons and protonated carbons. On the basis of the HMBC correlations from H-4 (δH 7.95) to C-5 (δC 129.3) and C-9 (δC 154.9), from H-5 (δH 7.58) to C-7 (δC 159.9) and C-9 (δC 154.9), from H-6 (δH 6.96) to C-8 (δC 103.0) and C-10 (δC 113.5), and from H-8 (δH 7.01) to C-10 (δC 113.5), the 7-O-substituted coumarin skeleton was assigned. The presence of a prenyl moiety was proved by the HMBC correlations from H-1″
(δH 5.13) to C-2″ (δC 35.7), from H-2″ (δH 1.86, 2.06) to C-1″ (δC 128.9), from H-3″ (δH 2.36) to C-4″(δC 174.9) and from H-5″(δH 0.93) to C-2″(δC 35.7), C-3″(δC 38.5), and C-4″ (δC 174.9). The position of the ester group was confirmed by a HMBC correlation between H-6′ (δH 3.97) and the carbonyl carbon of the prenyl moiety (C-4″ δC 174.9). The glucose unit was obviously attached to C-7 due to the HMBC correlation between H-1′ (δH 5.09) and C-7 (δC 159.9). Moreover, only one sp2 methine (C-1″) was reasonably explained that a double bond connected the half structure to form the symmetrically dimeric structure. Finally, the glucose unit was identified by GC analysis as D-glucose after an acid hydrolysis of 1. Thus, the structure of 1 was determined as shown (Fig. 1) and named as hydrangeside A.
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Fig. 2. Key HMBC and 1H–1H COSY correlations of compounds 1–3.
The constituents of stems of H. paniculata have been previously investigated and shown to contain lots of skimmin (7). Biogenetically, as shown in Fig. 3, hydrangeside A may be derived from skimmin and isoprene via intermediate 2,7-dimethyl-4-octene-1,8-dioic acids (6).
Compound 2 was obtained as colorless oil. The molecular formula was C36H42O16, as deduced from its [M + H]+ ion peak at m/z 731.2590 (calcd. for C36H43O16, 731.2546). The absorption maxima were at 203, 225 and 327 nm in the UV spectrum. The IR spectrum showed absorption bands for
Fig. 3. Hypothesis for the biogenesis of 1.
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hydroxyl (3372 cm−1), carbonyl (1693 cm−1), and aromatic ring (1632 and 1518 cm−1) groups. The proton signals at δH 7.43 (s, H-3), 5.70 (brdt, J = 17.5, 11.0 Hz, H-8), 5.44 (1H, d, J = 6.5 Hz, H-1), 5.33 (brd, J = 17.5 Hz, H-10), 5.23 (brd, J = 11.0 Hz, H-10), and δH 4.52 (1H, d, J = 8.0 Hz, H-1′) in the 1H NMR spectrum of 2 were characteristic of secoiridoid-type monoterpene glycoside [7,8].
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Furthermore, the 1H NMR spectrum showed signals assignable to two trans-olefinic protons at δH 7.56 and 6.48 (1H and d each, J = 16.0 Hz, H-7″ and H-8″), five aromatic protons at δH 7.26 (1H, brs, H-2″), 7.25 (1H, brs, H-6″), 6.90 (1H, brs, H-6‴), 6.74 (2H, brs, H-2‴, H-4‴), two aromatic methoxyl at δH 3.81 (3H, s), 3.73 (3H, s), a phenolic hydroxy protons at δH 9.04(1H, brs, OH-5‴), a 2,3-disubstituted 2,3-dihydrobenzofuran moiety
Fig. 4. The CD and ΔδCD spectra of compounds 3 and 4 in MeOH.
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Table 4 Effects of compounds 1–5 on the survival rate of HL-7702 cells injured by DL-GalN (10 μM). Group
OD value
Survival rate (%)
Control Model Bicyclola 1 2 3 4 5
1.197 0.384 0.630 0.836 0.604 0.560 0.619 0.422
100.0 29%⁎⁎⁎⁎ 51%⁎⁎ 68%⁎⁎⁎ 48%⁎ 44%⁎⁎
± ± ± ± ± ± ± ±
0.030 0.028 0.064 0.031 0.085 0.059 0.152 0.004
50 32%⁎⁎
⁎ P b 0.05 vs. model. ⁎⁎ P b 0.01 vs. model. ⁎⁎⁎ P b 0.001 vs. model. ⁎⁎⁎⁎ P b 0.001 vs. control. a Positive control substance.
at δH 5.51 (1H, d, J = 6.5 Hz, H-7‴), 3.47 (1H, m, H-8‴), 4.11 and 3.66 (1H and m each, H-9‴). In the 13C NMR spectrum, 36 resonances were observed. Fifteen of the resonances were assigned to secoiridoid glycoside moiety. The HSQC spectrum of 2 led to the unambiguous assignment of resonances of protons and protonated carbons. The 1H–1H COSY spectrum (Fig. 2) further elucidated the spin system of H-7‴/H-8‴/H-9″ in 2,3-disubstituted 2,3-dihydrobenzofuran moiety. In the HMBC spectrum of 2, the correlations from H-2″ to C-4″, C-6″ and C-7″, from H-6″ to C-2″, C-4″ and C-7″, from H-7″ to C-2″, C-6″ and C-9″, from H-8″ to C-1″, from H-2‴ to C-1‴, C-3‴, C-6‴ and C-7‴, from H-4‴ to C-3‴ and C-6‴, from H-6‴ to C-4‴, C-5‴ and C-7‴, from H-7‴ to C-4″, C-1‴ and C-6‴, from H-8‴ to C-4″ and C-1‴, from OMe-3″ to C-3″, and from OMe-3‴ to C-3‴ were agreed with the presence of the cimicifugic acid moiety [9]. The connection of cimicifugic acid moiety and secoiridoid glycoside moiety was linked by an ester bond (C-7\O\C-9″), which was confirmed by the downfield chemical shifts of H-7 (δH 4.11) and C-7 (δC 62.6) [7,8,10]. The 7‴,8‴-trans configuration of the 2,3-disubstituted 2,3dihydrobenzofuran moiety in 2 was elucidated by the coupling constant (J7‴,8‴ = 6.5 Hz) [11]. The absolute stereostructure of the 2,3-disubstituted 2,3-dihydrobenzofuran moiety in 2 was elucidated by the circular dichroic (CD) spectroscopic analysis. Thus, the CD spectrum (in MeOH) of 2 showed negative cotton effects [270 (Δε − 0.25), 281 (Δε − 0.23) nm], which indicated the absolute configurations of the 7‴ and 8‴-positions to be 7‴R and 8‴S orientations [12–14]. Finally, the glucose unit was identified by GC analysis as D-glucose after an acid hydrolysis of 2. Thus, the structure of 2 was elucidated as shown (Fig. 1) and named as hydrangeside B. Compounds 3 and 4 were both obtained as white amorphous powder and displayed absorption bands for hydroxyl, carbonyl, and aromatic ring groups. Both of their molecular formulas were determined as C35H36O15 by HR-ESI-MS with an [M + Na]+ ion peaks at m/z 719.1940 and 719.1941, respectively (calcd. for C35H36O15Na, 719.1946). And the NMR spectra of 3 and 4 closely resembled each other and suggested that 3 and 4 had the same planar structure. The 1H NMR spectrum of 3 showed five aromatic proton signals at δH 7.87 (d, J = 9.5 Hz), 7.57 (d, J = 8.5 Hz), 7.04 (d, J = 2.0 Hz), 6.99 (dd, J = 8.5, 2.0 Hz), and 6.24 (d, J = 9.5 Hz), as well as a sugar anomeric signals at δH 5.13 (d, J = 7.0 Hz), suggesting the presence of 7-substituted coumarin with a sugar moiety. The proton
signals at δH 7.49 (d, J = 16.0 Hz) and 6.47 (d, J = 16.0 Hz), together with ABX-type coupled aromatic protons at δH 7.27 (brs), 7.10 (brd, J = 8.0 Hz), and 7.02 (d, J = 8.0 Hz) suggested the presence of trans-feruloyl moiety. In addition, the ABX-type coupled aromatic protons at δH 6.96 (brs), 6.75 (brd, J = 8.0 Hz) and 6.67 (d, J = 8.0 Hz), along with two oxymethines at δH 4.70 (m), 4.38 (m), and an oxymethylene at δH 3.57 (m), and 3.26 (m) indicated the presence of 4‴-hydroxy3‴-methoxy-phenylglycerol-8‴-yl moiety. The 13C NMR spectrum of 3 showed 35 carbon signals corresponding to the above moieties. Fifteen of the carbon signals were consistent with the presence of a 7-O-substituted coumarin skeleton with a glucose moiety [4]. The remaining 20 carbon signals with the help of 2D NMR spectrum were attributable to trans-feruloyl moiety and 4‴-hydroxy-3‴-methoxy-phenylglycerol-8‴-yl moiety [15]. The linkages of the three moieties were confirmed by the correlations between δH 5.13 (H-1′) and δC 159.9 (C-7), between δH 4.21, 4.40 (H-6′) and δC 166.4 (C-9″), between δH 4.38 (H-8‴) and δC 150.8 (C-4″) in the HMBC spectrum. The small difference of the shifts of 7‴, 8‴ and 9‴ in 3 and 4 revealed that they were the erythro and threo 8‴-4″-oxyneolignane isomers. The ΔδC8‴-C7‴ values were applicable to differentiate threo and erythro phenylglycerol moiety since the NMR spectra 3 and 4 were obtained in DMSO-d6 [16,17]. The ΔδC8‴-C7‴ value of 4 (11.8 ppm) was smaller than that of 3 (12.9 ppm). This suggested that 3 possessed a threo relative configuration and 4 possessed an erythro relative configuration. According to the literature [11,12,17], the configuration of C-8‴ in phenylglycerol moiety was identification in the CD spectrum. A positive cotton effect at about 235 nm in the CD spectrum suggested the 8S configuration, while a negative cotton effect at about 235 nm suggested the 8R configuration. But there was no clear cotton effect at 235 nm in the CD spectrum of compounds 3 and 4, which perhaps was affected by the 7-O-glucosyl coumarin moiety (7, skimmin) in the structures of 3 and 4. So CD difference spectra were applied, the CD spectrum of skimmin subtracted from the CD spectra of 3 and 4 gave ΔδCD spectra of 3 and 4, respectively. The ΔδCD spectra of compounds 3 and 4 (Fig. 4) both showed negative cotton effect at 235 nm, which indicated the absolute configurations of 8‴-position of compound 3 and 4 to be 8‴R orientations. As a result, compound 3 had 7‴R,8‴R configuration and compound 4 had 7‴S,8‴R configuration on the basis of their relative configuration. Finally, the glucose unit was identified by GC analysis as D-glucose after an acid hydrolysis of 3 and 4. Hence, the structures of 3 and 4 were determined as shown (Fig. 1) and named as hydrangeside C and hydrangeside D, respectively. Additionally, one known secoiridoid glucoside named methylgrandifloroside was isolated [10,18]. Compounds 1–5 were bioassayed for their hepatoprotective activities against DL-galactosamine-induced toxicity in HL-7702 cells, using the hepatoprotective activity drug bicyclol as the positive control. At 10 μM, compounds 1–5 reduced DL-galactosamine (GalN)-induced HL-7702 cell damage by increasing the survival rate from 29% (P b 0.001) to 68% (P b 0.001), 48% (P b 0.05), 44% (P b 0.01), 50%, and 32% (P b 0.01), respectively, while the positive control bicyclol gave a 51.0% (P b 0.01) survival rate (Table 4). All the compounds
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displayed moderate hepatoprotective activities against DLgalactosamine-induced toxicity in HL-7702 cells. Conflict of interest There is no conflict of interest among all authors. Acknowledgment Financial support was provided by the National Natural Science Foundation of China (No. 21132009), the National Mega-project for Innovative Drugs (No. 2012ZX09101), and PCSIRT (No. IRT1007). References [1] Chinese Herbal Medicine Company. Chinese traditional medicine resource records. Beijing: Science Publishing House; 1994 480. [2] Zhang DM, Chen XG, Yang JZ, Li Y, Zheng XG. Chinese patent CN 1690069A; 2005. [3] Zhang DM, Chen XG, Yang JZ, Li Y. Chinese patent CN 1605343A; 2005. [4] Shi J, Yang JZ, Li CJ, Zhang DM. Chemical constituents from Hydrangea paniculata. China J Chin Mater Med 2010;35:3007–9. [5] Fu HZ, Li CJ, Yang JZ, Shen ZF, Zhang DM. Potential antiinflammatory constituents of the stems of Gordonia chrysandra. J Nat Prod 2011;74:1066–72. [6] Li F, Li CJ, Ma J, Yang JZ, Chen H, Liu XM, et al. Four new sesquiterpenes from the stems of Pogostemon cablin. Fitoterapia 2013;86:183–7. [7] Xu M, Wang D, Zhang YJ, Yang CR. Iridoidal glucosides from Gentiana rhodantha. J Asian Nat Prod Res 2008;6(10):491–8.
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