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Three new alkaloids and a new iridoid glycoside from the roots of Rehmannia glutinosa
Phytochemistry Letters 21 (2017) 157–162
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Short communication
Three new alkaloids and a new iridoid glycoside from the roots of Rehmannia glutinosa
MARK
Meng Lia,b, Xiaolan Wanga,b, Zhiguang Zhanga,b, Jingke Zhanga,b, Xuan Zhaoa,b, Xiaoke Zhenga,b, ⁎ Weisheng Fenga,b, a b
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China Collaborative Innovation Center for Respiratory Disease Diagnosis And Treatment & Chinese Medicine Development of Henan Province, Zhengzhou 450046, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Rehmannia glutinosa Roots Alkaloids Iridoid glycosides The protective effects of cardiomyocytes
Three new alkaloids, rehmanalkaloid A-C (1-3), and a new iridoid glycoside, rehmaglutoside L (4), together with nine known compounds, including (8S)-7,8-dihydrogeniposid (5), diglycoside (6), monomelittoside (7), mussaenoside (8), darendoside A (9), syringing (10), phenyl-6-O-β-D-xylopyranosyl-O-β-D-glucopyranoside (11), (7R, 8S)-4,9-dihydroxy-3,3′-dimethoxy-7,8-dihydrobenzofuran-1′-propanalneolignan (12), and trans-liovil (13), were isolated from a 95% EtOH extract of dried roots from Rehmannia glutinosa. Their structures were determined by extensive spectroscopic (UV, IR, HR-ESI–MS, and 1D and 2D NMR) analyses. Additionally, compounds 4-8 (iridoid glycosides) were evaluated for their protective effects on H714444444444444444444466666666669c2 cardiocytes impaired by doxorubicin. Among them, compounds 4-8 exhibited protective effects against DOXinduced cardiotoxicity.
1. Introduction Rehmannia glutinosa, from the Scrophulariaceae family, has been used as a traditional Chinese herbal medicine for thousands of years. It was recorded in the Chinese medical classics ‘Shennong’s Herba’ and was thought to be ‘top-grade’ herb in China. Previous phytochemical studies on R. glutinosa dried and steamed roots have led to the isolation and identification of iridoid glycosides, ionone glycosides, phenethyl alcohol glycosides, and several other components (Liu et al., 2012, 2014a,b; Li et al., 2005; Sasaki et al., 1991). Pharmacological investigations with these compounds have demonstrated significant biological properties, including hypoglycemic effects (Zhang et al., 2004), anti-fatigue activity (Tan et al., 2012); prevention of bone loss (Lim and Kim, 2013) and immunoenhancement effects (Huang et al., 2013). Our group has also performed extensive phytochemical and pharmacological research on R. glutinosa (Zhang et al., 2013; Feng et al., 2013, 2015; Li et al., 2015; Zheng et al., 2013, 2012). Based on a bioassayguided isolation, further phytochemical study was undertaken to investigate the chemical constituents from a 95% EtOH extract from R. glutinosa dried roots, which led to the isolation of three new alkaloids, rehmanalkaloid A-C (1-3), and a new iridoid glycoside, rehmaglutoside L (4), together with nine known compounds, including (8S)-7,8-dihydrogeniposid (5), diglycoside e (8), darendoside A (9), syringing (10), phenyl-6-O-β-D-xylopyranosyl-O-β-D-glucopyranoside (11), (7R, 8S(6),
⁎
monomelittoside (7), mussaenosid)-4,9-dihydroxy-3,3′-dimethoxy-7,8dihydrobenzofuran-1′-propanalneolignan (12), and trans-liovil (13). The structures were unequivocally determined by extensive spectroscopic analysis (1D and 2D NMR spectroscopy and mass spectrometry) that was compared with the literature. 2. Results and discussion Compound 1 gave a molecular ion peak [M+Na]+ at m/z 208.0974 in the HR-ESI–MS, which is consistent with the molecular formula C11H14NO3Na (calcd. 208.0964). The IR spectrum showed the presence of a hydroxyl (3356 cm−1) and a carbonyl (1724 cm−1). In the 1H NMR spectrum (Table 1), proton signals at δH 8.87 (1 H, s, H-2) and 8.64 (1H, s, H-9) were recognized as belonging to the nitrogen atom-containing heterocyclic group; a methoxy group at δH 3.92 (3 H, s, OCH3, H-12) was also observed. In addition to the protons mentioned above, another 7 proton signals remained to be assigned in the 1H NMR spectrum. The 13 C NMR spectrum (Table 2) displayed a total of 11 carbons, which were classified from the DEPT and HSQC spectra as a methoxy carbon signal [δC 52.6 (C-12)], three methylene carbon signals [including one oxygenated methylene δC 65.7 (C-10) and two alkyl methylenes δC 33.5 (C-5) and 28.6 (C-6)], three methine carbon signals [including two Nvicinal methines δC 149.6 (C-2) and 149.3 (C-9) and a alkyl methane δC 46.8 (C-7)], as well as three quaternary carbon signals [δC 144.2 (C-8),
Corresponding author at: School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China. E-mail address: [email protected] (W. Feng).
http://dx.doi.org/10.1016/j.phytol.2017.06.010 Received 19 February 2017; Received in revised form 31 May 2017; Accepted 16 June 2017 1874-3900/ © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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11with H-10/12/13a/13b. Further analyses of the HMBC spectra in combination with chemical shifts and coupling patterns led to the identification of the planar structure of 2 due to the correlations (Fig. 2). In the NOESY spectrum, correlations between H-4 with H-5/H8 and H-6 with H-2/10/13a indicated that H-2/H-4/H-5/H-6/H-8/H10/H-13a were β-oriented. Moreover, the NOESY correlations between H-13b and H-11/H-12 confirmed that H-11/12/13b were α-oriented (Fig. 3). Based on the above analysis, 2 was identified as rehmanalkaloid B (Fig. 1). Compound 3 was obtained as a colorless crystalline powder and had a molecular formula of C9H13NO3 on the basis of the [M+Na]+ ion at m/z 206.0785 (calcd. 206.0793) in the HR-ESI–MS. It showed IR absorptions for a hydroxyl (3360 cm−1), methyl (2925 cm−1), carbonyl (1688 cm−1) and ether linkage (1201 and 1027 cm−1). The 1H NMR data for 3 (Table 1) indicated the presence of an aromatic proton [δ H 5.56 (1H, s, H-3)], a methoxy proton signal [δ H 3.69 (3H, s, H-11)], four methylene signals [δ H 3.32 (2H, t, J = 7.5 Hz, H-6), 2.59 (2H, t, J = 7.5 Hz, H-7), 2.50 (2H, t, J = 7.5 Hz, H-8), and 2.30 (2H, t, J = 7.5 Hz, H-5)]. The 13C NMR spectrum (Table 2) displayed a total of 9 carbons, which were classified from the DEPT and HSQC spectra as a methoxy carbon signal [δC 52.3 (C-11)], four methylene carbon signals [δC 38.6 (C-6), 31.0 (C-8), 30.8(C-7), and 26.8 (C-5)], one methine carbon signal [δC 116.6 (C-3)], as well as one sp2 carbon signal [δC 130.3 (C-4)] and two C]O units [δC 176.0 (C-9), 159.0 (C-2)]. Furthermore, the structure of 3 was confirmed by the HMBC and 1H–1H COSY correlations (Fig. 2). Hence, the structure of 3 was determined to be 1-methyl-3-[3′,4′-dihydropyridin-2′(1′H)-one] propanoate and it was named rehmanalkaloid C (Fig. 1). Compound 4 was isolated as a colorless crystalline powder with [α] 20 D −6.4° (c 0.8, MeOH). Its molecular formula was determined to be C31H48O18 by HR-ESI–MS at m/z 559.2007 [M + Na]+ (calcd. for C23H36O14Na, 559.2003). The IR spectrum showed the presence of a hydroxyl group (3371 cm−1), methyl groups (2925 cm−1), carbonyl groups (1679 and 1631 cm−1), and methoxyl groups (1140 and 1066 cm−1). In the 1H NMR spectrum, it showed protons of δ 7.46 (1H, d, J = 1.0 Hz, H-3) and 5.09 (1H, d, J = 7.0 Hz, H-1) at the low-field regime. Two anomeric proton signals of δ 4.73 (1H, d, J = 1.5 Hz, H1′′) and δ 4.64 (1H, d, J = 8.0 Hz, H-1′), a methoxy signal at 3.69 (3H, s, OCH3) and a methyl signal at 1.25 (3H, d, J = 6.5 Hz, H-6′′) were also observed. The 13C NMR and DEPT spectrum showed 23 carbon signals, 12 of which were assigned to two sugar moieties and the remaining eleven as a 4-substituted iridoid skeleton. The 1H and 13C NMR data displayed signals characteristic of an iridoid glycoside, which were similar to those reported for (8S)-7,8-dihydrogeniposid (Salama and Sticher, 1983). The only evident difference was that 4 showed resonances due to an additional rhamnopyranosyl unit. The C-6′ downfield shift (δ 68.0 from δ 62.0) suggested the attachment of the rhamnopyranosyl unit to the C-6′ carbon. The assumption was confirmed by the HMBC correlation of H-1′′ with the C-6′ carbon. In the NOESY spectrum, the correlations between H-5 and H-8/H-9 revealed that they were β-oriented (Fig. 4). In the acid hydrolysis of 4, d-glucose and lrhamnose were afforded and confirmed by TLC and optical rotations compared with the reference substances, respectively (Zhang et al., 2008, 2016). The configuration was determined by measuring the optical rotation value and the large 3JH1,H2 coupling constant. Based on the results described above, the structure of 4 was determined to be (8S)-7,8-dihydrogenin 1-O-α-L-rhamnopyranosyl (1 → 6)-β-D-glucopyranoside and it was named rehmaglutosides L (Fig. 1.). The known compounds were identified as (8S)-7,8-dihydrogeniposid (Salama and Sticher, 1983) (5), diglycoside (Zhang et al., 2008) (6), monomelittoside (Venditti et al., 2016) (7), mussaenoside (Venditti et al., 2013) (8), darendoside A (Nomura et al., 1983) (9), syringing (Wang et al., 2014) (10), phenyl-6-O-β-D-xylopyranosyl-O-βD-glucopyranoside (Chen et al., 2008) (11), (7R, 8S)-4,9-dihydroxy3,3′-dimethoxy-7,8-dihydrobenzofuran-1′-propanalneolignan (Yang et al., 2007) (12), and trans-liovil (Schottner et al., 1997) (13) by
Table 1 1 H NMR Spectroscopic Data of Compounds 1-3 (500 MHz). No.
1 (CD3OD)
2 (D2O)
2 3 4 5 6 7 8 9 10
8.87, s
4.41, d (14.5)
11 12 13
3 (D2O)
5.56, s 3.36, m; 3.25, m 2.29, m; 1.96, m 3.43, m
7.20, dd (1.0, 4.0) 6.22, dd (2.5, 4.0) 7.12, s 2.41, s
8.64, s 3.76, m 3.67 (1H, m)
3.57, m
3.92 (1H, m)
3.71, m 3.81, m 3.69, m; 3.49, m
2.30, 5.09, 2.59, 2.50,
t t t t
(7.5) (7.5 Hz) (7.5) (7.5 Hz)
3.69, s
Data assignment was based on HSQC and HMBC experiments. Table 2 13 C NMR Spectroscopic Data of Compounds 1-3 (125 MHz). No.
1 (CD3OD)
2 (D2O)
3 (D2O)
2 3 4 5 6 7 8 9 10 11 12 13
149.6 124.6 158.9 33.5 28.6 46.8 144.2 149.3 65.7 167.2 52.6
51.6 130.8 123.6 109.3 134.3 193.1 26.6 97.9 70.5 70.8 66.1 62.4
159.0 116.6 130.3 26.8 38.6 30.8 31.0 176.0 52.3
Data assignment was based on HSQC and HMBC experiments.
158.9 (C-4), and 124.6 (C-3)] and one C]O unit [δC 167.2 (C-11)]. By studying the 1H–1H COSY spectra, a large spin system −CH2(C-5)CH2(C-6)-CH(C-7)-CH2OH(C-10) group was established. In the HMBC spectrum, the methoxy carbon, which exhibited a strong correlation with the carbonyl carbon (δC 167.2), suggested the presence of a methyl nicotinate group. Moreover, the correlations of the H-5 with C-3/7/8 revealed the presence of a cyclopenta group (Fig. 2). Based on above NMR spectroscopic analysis, compound 1 was found to closely resemble isoplmrodorine (Gournenelis et al., 1989). In the NOESY spectrum, correlations between H-7 and H-8 indicated that H-7/8 were both β-oriented (Fig. 3). Thus, the structure of compound 1 was deduced as (3-methylcarboxylate-7-hydroxymethyl)-cyclopenta [5,9] pyridine and it was named rehmanalkaloid A (Fig. 1). Compound 2 was obtained as a colorless crystalline powder. The molecular formula, C12H17NO6, was determined by its HR-ESI–MS. The IR spectrum showed the presence of a hydroxyl (3355 cm−1) and a carbonyl (1617 cm−1). The 1H NMR spectrum showed three aromatic protons at δ 7.20 (1 H, dd, J = 1.0, 4.0 Hz, H-4), 7.12 (1 H, s, H-2), and 6.22 (1 H, dd, J = 2.5, 4.0 Hz, H-3) at the low-field regime. The presence of an acetyl group was observed at δ 2.41 (3 H, s, H-8). The 13C NMR spectrum (Table 2) displayed a total of 12 carbons, which were classified from the DEPT and HSQC spectra as a methyl carbon signal [δC 26.6 (C-8)], one methylene carbon signal [δC 62.4 (C-13)], six methine carbon signals {including three aromatic methines [δC 134.3 (C-6), 109.3 (C-5), and 123.6 (C-4)] and three oxygen-methines [δC 70.8 (C-11), 70.5 (C-10), and 66.1 (C-12)]} as well as three quaternary carbon signals [δC 130.8 (C-2) and 97.9 (C-9)] and one C]O unit [δC 193.1 (C-7)]. In the 1H–1H COSY spectra, the correlations of H-5 with H-4/6 provided evidence for a eCeCH]CH]CH- in compound 2. Moreover, a large spin system eCHOH(C-10)-CHOH(C-11)-CHOH(C12)-CH2OH(C-13) group was established by the correlations of H158
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Fig. 1. Structure of compounds 1-4.
and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm and 250 × 10 mm, 5 μm). Column chromatography was performed with a Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), Toyopearl HW-40, MCI gel CHP-20 (TOSOH Corp., Tokyo, Japan), Sephadex LH-20 (40–70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), Lichroprep RP-18 gel (40–63 μm, Merck, Darmstadt, Germany), and silica gel (160–200 mesh, Marine Chemical Industry, Qingdao, China). TLC was performed on self-made silica gel G (Qingdao Marine Chemical Industry) plates, CH2Cl2:MeOH:H2O (10:1:0.1, v/v), and CH2Cl2:MeOH:H2O (4:1:0.1, v/v) as the eluent, and spots were visualized by spraying with 10% H2SO4 in ethanol (v/v) followed by heating. The chemical reagents were supplied by Beijing Chemical Plant (Beijing, China) and Tianjin NO. 3 Reagent Plant (Tianjin, China).
comparing their physical and spectroscopic data with reported values in the literature. To investigate whether compounds 4-8 protect cells from DOX-induced cell death, H9c2 cells were treated with 50 μM DOX in the presence or absence of compounds 4-8 (50 μM) and the cell viability was assessed by the MTT assay. Among them, compounds 4-8 exhibited protective effects against DOX-induced cardiotoxicity at different concentrations (Fig. 5).
3. Experimental 3.1. General experimental procedures NMR spectra were recorded at room temperature in D2O or CD3OD using a Bruker Avance III 500 NMR spectrometer with TMS as an internal standard (500 MHz for 1H NMR and 125 MHz for 13C NMR). Optical rotation was measured with an AP-IV (Rudolph Research Analytical, USA). The IR spectrum was determined on a Nicolet iS10 Microscope Spectrometer (Thermo Scientific, USA). HR-ESI–MS spectra were recorded on a Bruker maxis HD mass spectrometer. UV spectra were recorded on a Shimadzu UV-2401PC apparatus. Preparative HPLC was conducted using a Saipuruisi LC-50 instrument with an UV200 detector (Beijing, China)
3.2. Plant materials Dried Rehmannia glutinosa roots were collected from Jiaozuo, Henan Province in China in November 2012. The plants were identified by Prof. Cheng-ming Dong of Henan University of TCM. A voucher specimen (No. 20101101A) has been stored at the Department of Natural Medicinal Chemistry, School of Pharmacy, Henan University of TCM, Zhengzhou, China.
Fig. 2. Key HMBC and 1H–1H COSY correlations of 1-3.
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Fig. 3. Key NOESY correlations of 1 and 2.
The 50% MeOH-H2O fraction was separated on a silica gel column and eluted with petroleum CH2Cl2-MeOH (0:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1, and 5:1) to afford Fr. 1–8. Fr. 7 was subjected to Toyopearl HW-40 and eluted with MeOH to yield Fr. 7.1-7.3. Fr. 7.2 was separated on MCI gel CHP-20 and eluted with 40% MeOH to afford Fr. 7.2.17.2.4. Fr. 7.2.1 was purified by preparative PHPLC (50% MeOH/H2O, 3 mL/min) to yield compound 1 (1.2 mg, tR = 24.4 min). The 10% MeOH-H2O fraction was isolated by column chromatography via multiple silica gel columns including a Toyopearl HW-40, RP-18, MCI gel CHP-20 and Sephadex LH-20 to yield compounds 7 (6.5 mg), 8 (11.2 mg) 10 (9.3 mg) and 11 (8.6 mg). The 50% MeOHH2O fraction was repeatedly separated on silica gel columns to yield compounds 12 (5.6 mg) and 13 (4.8 mg).
3.3. Extraction and isolation The dried R. glutinosa roots (20.0 kg) were cut into small pieces and extracted with 95% EtOH under heating reflux twice for 2 h. After concentration under reduced pressure in a vacuum evaporator, the concentrated solution (1500 mL) was centrifuged and subjected to Diaion HP-20 column chromatography, eluted with H2O and MeOH successively, and obtained in water and methanol extracts (101.6g). Next, the methanol extracts were dissolved in 10% MeOHeH2O (1000 mL), subjected to a Diaion HP-20 column again, and successively eluted with 10%, 30%, 50%, 70%, and 100% (v/v) MeOH-H2O (10 column volumes each). After removing the solvents, the 65.0 g, 16.6 g, 11.5 g, 4.2 g, and 2.0 g, respectively, were obtained from the extracts. The 30% MeOH-H2O fraction was subjected to Toyopearl HW-40 column chromatography, eluting with MeOH/H2O from 0% to 100% to receive Fr. 1–4. Fr. 3 (4.0 g) was separated over Sephadex LH-20 and eluted with MeOH as the mobile phase to give subfractions Fr. 3.1-3.3. Fr. 3.2 (0.8 g) was separated on MCI gel CHP-20 and eluted with 20% MeOH to afford Fr. 3.2.1-3.2.5. Fr. 3.2.4 (0.3 g) was further isolated by PHPLC (30% MeOH/H2O, 3 mL/min) to yield compounds 2 (2.6 mg, tR = 32.4 min), 4 (10.2 mg, tR = 62.4 min), 5 (11.6 mg, tR = 50.7 min) and 6 (18.3 mg, tR = 55.1 min). Compound 3 (0.7 mg, tR = 10.1 min) was obtained by PHPLC (33% MeOH/H2O, 3 mL/min) from Fr. 3.2.5 (10 mg). Fr. 3.3 (0.9 g) was further separated by repeated Sephadex LH-20 to afford compound 9 (8.8 mg).
3.3.1. Rehmanalkaloid A (1) Brown crystalline powder; [α]20 D 0.12 (c 0.60, CH3OH); UV (CH3OH) λmax (logε): 207 (2.98) nm, 275 (0.45) nm; IR (KBr) νmaxcm−1: 3356, 2950, 2872, 1721, 1579, 1436, 1299, 1211, 1141, 1125, and 1030 cm−1. HR-ESI–MS m/z: 208.0974 [M+ Na]+ (calcd. For C11H14NO3Na 208.0964). 1H NMR (500 MHz, CD3OD) spectral data see Table 1; 13C NMR (125 MHz, CD3OD) spectral data see Table 2. 3.3.2. Rehmanalkaloid B (2) Colorless crystalline powder; [α]20 D 0.05 (c 0.26, CH3OH); UV (CH3OH) λmax (logε): 201 (0.62) nm, 290 (1.33) nm; IR (KBr)
Fig. 4. Key HMBC, 1H–1H COSY and NOESY correlations of 4.
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Fig. 5. Protective effects of 4-8 on DOX-induced cytotoxicity in H9c2 cell (n = 3); * P < 0.05; ** P < 0.01 compared with the DOX group.
Table 3 1 H NMR (500 MHz) and
13
δ
No
δ
4.64, d (8.0)
153.4. 112.0 36.3 33.5
1′ 2′ 3′ 4′ 5′ 6′
100.6. 74.6. 77.9 71.6 77.0 68.0
26.4
1′
4.73, d (1.5)
102.2
44.3 44.3 66.5
2′ 3′ 4′
72.2 72.4 74.6
169.6 51.7
5′ 6′′
69.8 18.1
No.
δ
1 2 3 4 5 6
5.09, d (8.0)
98.7
7.46, s.
7 8 9 10 11 OCH3
H
2.82, m 2.15, m 1.39,m 1.83, m 1.39, m 2.15, m 1.95, m 3.56, dd (6.5, 11.0) 3.50, dd (6.5, 11.0) 3.69, s
incubated in fresh medium with compounds 4-8 (50 μM) for an additional 24 h. The effects of compounds 4-8 on DOX-induced cytotoxicity were assessed using the MTT assay, as previously described (Han et al., 2008; Wang et al., 2012). The optical density of each well was then measured on a microplate spectrophotometer at a wavelength of 490 nm. The cell viability was determined as the percentage of surviving cells compared with that of the DOX-treated control. Experiments were performed in triplicate and the values are the averages of three (n = 4) independent experiments. Individual data were expressed as the mean ± standard deviation (SD). A post-hoc Dunnett’s test was used to obtain corrected p-values in the group comparisons. Statistical analyses were performed with one-way ANOVA (SPSS version 17.0). A p value less than or equal to 0.05 was considered significant.
C NMR (125 MHz) spectral data of compound 4 (in CD3OD). C
H
δ
C
Acknowledgement 1.25, d (6.0)
Our work was supported by the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-Year Plan Period (2011BAI06B02).
Data assignment was based on HSQC and HMBC experiments.
νmaxcm−1: 3355, 2925, 2854, 1617, 1464, 1409, 1088, and 1060 cm−1. HR-ESI–MS m/z: 294.0941 [M + Na]+ (calcd. For C12H17NO6Na 294.0954). 1H NMR (500 MHz, D2O) spectral data see Table 1; 13C NMR (125 MHz, D2O) spectral data see Table 2.
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3.3.3. Rehmanalkaloid C (3) Colorless crystalline powder; UVmax (CH3OH) λmax (logε): 204 (1.98) nm; IR (KBr) νmaxcm−1: 3360, 2925, 1688, 1614, 1437, 1201, 1135, and 1027 cm−1. HR-ESI–MS m/z: 206.0785 [M+Na]+ (calcd. For C9H13NO3Na 206.0793). 1H NMR (500 MHz, D2O) spectral data see Table 1; 13C NMR (125 MHz, D2O) spectral data see Table 2. 3.3.4. Frehmaglutoside G (4) colorless crystalline powder; [α]20 D −6.4° (c 0.80, CH3OH); UVmax (CH3OH) λmax (logε): 238.2 and 285.6 nm; IR (KBr) νmaxcm−1: 3371, 2926, 1679, 1631, 1440, 1187, 1146, and 1066. HR-ESI–MS m/z: 559.2007 [M+Na]+ (calcd. For C23H36O14Na 559.2003). 1H NMR (500 MHz, CD3OD) spectral data and 13C NMR (125 MHz, CD3OD) spectral data see Table 3. 3.4. Activity assay The rat cardiac H9c2 myocardial cells were spontaneously immortalized ventricular rat embryo myoblasts that were purchased from Biowit Technologies (Shenzhen, China). The cells were maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a water-saturated 5.0% CO2 incubator. The cells were split upon reaching a confluency of ∼80% using trypsinEDTA, and then seeded onto 96-well plates at a density of 1.0 × 104 cells L−1 (100 μL/well) and incubated for 24 h before treatment. Thereafter, the cells were exposed to DOX (50 μM) for 24 h and then 161
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J. Nat. Prod. 70, 521–525. Zhang, R.X., Zhou, J.H., Ha, Z.P., Zhang, Y.X., Gu, G.M., 2004. Hypoglycemic effect of Rehmannia glutinosa oligosaccharide in hyperglycemic and alloxan-induced diabetic rats and its mechanism. J. Ethnopharmacol. 90, 39–43. Zhang, Y.L., Gan, M.L., Lin, S., Liu, M.T., Song, W.X., Zi, J.C., Wang, S.J., Li, S., Yang, Y.C., Shi, J.G., 2008. Glycosides from the bark of Adina polycephala. J. Nat. Prod. 71, 905–909. Zhang, Y.L., Feng, W.S., Zheng, X.K., Cao, Y.G., Lv, Y.Y., Chen, H., Kuang, H.X., 2013. Three new ursane-type triterpenes from the leaves of Rehmannia glutinosa. Fitoterapia 89, 15–19. Zhang, C.L., Wang, Y., Liu, Y.F., Liang, D., Hao, Z.Y., Luo, H., Chen, R.Y., Cao, Z.Y., Yu, D.Q., 2016. Two new flavonoid glycosides from Iris tectorum. Phytochem. Lett. 15, 63–65. Zheng, X.K., Hou, W.W., Duan, P.F., Feng, Z.Y., Wang, X.L., Zhang, Y.L., Feng, W.S., 2012. Immunomodulatory effect of prepared Radix Rehmanniae extract in Vitro. Chin. Pharm. J. 47, 1995–2000. Zheng, X.K., Liu, Z.Y., Jiang, Y., Zhang, N., Feng, Z.Y., Su, C.F., Wang, X.L., Feng, W.S., 2013. Evaluation of the estrogenic effects of Rehmannia glutinosa Libosch. Chin. Pharm. J. 48, 1831–1836.
series glycosides from Euphrasia species. Planta Med. 47, 90–94. Sasaki, H., Nishimura, H., Morota, T., Katsuhara, T., Chin, M., Mitsuhashi, H., 1991. Norcarotenoids of Rehmannia glutinosa ver hueichingensis. Phytochemistry 30, 1997–2001. Schottner, M., Reiner, J., Tayman, F.S.K., 1997. (+)-Neo-olivil from roots of Urtica dioica. Phytochemistry 46, 1107–1109. Tan, W., Yu, K.Q., Liu, Y.Y., Yang, M.Z., Yan, M.H., Luo, R., Zhao, X.S., 2012. Anti-fatigue activity of polysaccharides extract from Radix Rehmanniae Preparata. Int. J. Biol. Macromol. 50, 59–62. Venditti, A., Serrilli, A.M., Bianco, A., 2013. Iridoids from bellardia trixago (L.) all. Nat. Prod. Res. 27, 1413–1416. Venditti, A., Frezza, C., Serafini, M., Bianco, A., 2016. Iridoids and phenylethanoid from pedicularis kerneri dalla torre growing in dolomites, Italy. Nat. Prod. Res. 30, 327–331. Wang, W.C., Uen, Y.H., Chang, M.L., Cheah, K.P., Li, J.S., Yu, W.Y., Lee, K.C., Choy, K.C., Hu, C.M., 2012. Protective effect of guggulsterone against cardiomyocyte injury induced by doxorubicin in vitro. BMC Complement. Altern. Med. 12, 138. Wang, S.Y., Ding, L.F., Wu, X.D., Wang, H.Y., Zhao, Q.S., Song, L.D., 2014. Chemical constituents of Eucommia ulmoides. J. Chin. Med. Mater. 37, 807–811. Yang, X.W., Zhao, P.J., Ma, Y.L., 2007. Mixed lignan-neolignans from Tarenna attenuate.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Short communication
Three new alkaloids and a new iridoid glycoside from the roots of Rehmannia glutinosa
MARK
Meng Lia,b, Xiaolan Wanga,b, Zhiguang Zhanga,b, Jingke Zhanga,b, Xuan Zhaoa,b, Xiaoke Zhenga,b, ⁎ Weisheng Fenga,b, a b
School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China Collaborative Innovation Center for Respiratory Disease Diagnosis And Treatment & Chinese Medicine Development of Henan Province, Zhengzhou 450046, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Rehmannia glutinosa Roots Alkaloids Iridoid glycosides The protective effects of cardiomyocytes
Three new alkaloids, rehmanalkaloid A-C (1-3), and a new iridoid glycoside, rehmaglutoside L (4), together with nine known compounds, including (8S)-7,8-dihydrogeniposid (5), diglycoside (6), monomelittoside (7), mussaenoside (8), darendoside A (9), syringing (10), phenyl-6-O-β-D-xylopyranosyl-O-β-D-glucopyranoside (11), (7R, 8S)-4,9-dihydroxy-3,3′-dimethoxy-7,8-dihydrobenzofuran-1′-propanalneolignan (12), and trans-liovil (13), were isolated from a 95% EtOH extract of dried roots from Rehmannia glutinosa. Their structures were determined by extensive spectroscopic (UV, IR, HR-ESI–MS, and 1D and 2D NMR) analyses. Additionally, compounds 4-8 (iridoid glycosides) were evaluated for their protective effects on H714444444444444444444466666666669c2 cardiocytes impaired by doxorubicin. Among them, compounds 4-8 exhibited protective effects against DOXinduced cardiotoxicity.
1. Introduction Rehmannia glutinosa, from the Scrophulariaceae family, has been used as a traditional Chinese herbal medicine for thousands of years. It was recorded in the Chinese medical classics ‘Shennong’s Herba’ and was thought to be ‘top-grade’ herb in China. Previous phytochemical studies on R. glutinosa dried and steamed roots have led to the isolation and identification of iridoid glycosides, ionone glycosides, phenethyl alcohol glycosides, and several other components (Liu et al., 2012, 2014a,b; Li et al., 2005; Sasaki et al., 1991). Pharmacological investigations with these compounds have demonstrated significant biological properties, including hypoglycemic effects (Zhang et al., 2004), anti-fatigue activity (Tan et al., 2012); prevention of bone loss (Lim and Kim, 2013) and immunoenhancement effects (Huang et al., 2013). Our group has also performed extensive phytochemical and pharmacological research on R. glutinosa (Zhang et al., 2013; Feng et al., 2013, 2015; Li et al., 2015; Zheng et al., 2013, 2012). Based on a bioassayguided isolation, further phytochemical study was undertaken to investigate the chemical constituents from a 95% EtOH extract from R. glutinosa dried roots, which led to the isolation of three new alkaloids, rehmanalkaloid A-C (1-3), and a new iridoid glycoside, rehmaglutoside L (4), together with nine known compounds, including (8S)-7,8-dihydrogeniposid (5), diglycoside e (8), darendoside A (9), syringing (10), phenyl-6-O-β-D-xylopyranosyl-O-β-D-glucopyranoside (11), (7R, 8S(6),
⁎
monomelittoside (7), mussaenosid)-4,9-dihydroxy-3,3′-dimethoxy-7,8dihydrobenzofuran-1′-propanalneolignan (12), and trans-liovil (13). The structures were unequivocally determined by extensive spectroscopic analysis (1D and 2D NMR spectroscopy and mass spectrometry) that was compared with the literature. 2. Results and discussion Compound 1 gave a molecular ion peak [M+Na]+ at m/z 208.0974 in the HR-ESI–MS, which is consistent with the molecular formula C11H14NO3Na (calcd. 208.0964). The IR spectrum showed the presence of a hydroxyl (3356 cm−1) and a carbonyl (1724 cm−1). In the 1H NMR spectrum (Table 1), proton signals at δH 8.87 (1 H, s, H-2) and 8.64 (1H, s, H-9) were recognized as belonging to the nitrogen atom-containing heterocyclic group; a methoxy group at δH 3.92 (3 H, s, OCH3, H-12) was also observed. In addition to the protons mentioned above, another 7 proton signals remained to be assigned in the 1H NMR spectrum. The 13 C NMR spectrum (Table 2) displayed a total of 11 carbons, which were classified from the DEPT and HSQC spectra as a methoxy carbon signal [δC 52.6 (C-12)], three methylene carbon signals [including one oxygenated methylene δC 65.7 (C-10) and two alkyl methylenes δC 33.5 (C-5) and 28.6 (C-6)], three methine carbon signals [including two Nvicinal methines δC 149.6 (C-2) and 149.3 (C-9) and a alkyl methane δC 46.8 (C-7)], as well as three quaternary carbon signals [δC 144.2 (C-8),
Corresponding author at: School of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China. E-mail address: [email protected] (W. Feng).
http://dx.doi.org/10.1016/j.phytol.2017.06.010 Received 19 February 2017; Received in revised form 31 May 2017; Accepted 16 June 2017 1874-3900/ © 2017 Published by Elsevier Ltd on behalf of Phytochemical Society of Europe.
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11with H-10/12/13a/13b. Further analyses of the HMBC spectra in combination with chemical shifts and coupling patterns led to the identification of the planar structure of 2 due to the correlations (Fig. 2). In the NOESY spectrum, correlations between H-4 with H-5/H8 and H-6 with H-2/10/13a indicated that H-2/H-4/H-5/H-6/H-8/H10/H-13a were β-oriented. Moreover, the NOESY correlations between H-13b and H-11/H-12 confirmed that H-11/12/13b were α-oriented (Fig. 3). Based on the above analysis, 2 was identified as rehmanalkaloid B (Fig. 1). Compound 3 was obtained as a colorless crystalline powder and had a molecular formula of C9H13NO3 on the basis of the [M+Na]+ ion at m/z 206.0785 (calcd. 206.0793) in the HR-ESI–MS. It showed IR absorptions for a hydroxyl (3360 cm−1), methyl (2925 cm−1), carbonyl (1688 cm−1) and ether linkage (1201 and 1027 cm−1). The 1H NMR data for 3 (Table 1) indicated the presence of an aromatic proton [δ H 5.56 (1H, s, H-3)], a methoxy proton signal [δ H 3.69 (3H, s, H-11)], four methylene signals [δ H 3.32 (2H, t, J = 7.5 Hz, H-6), 2.59 (2H, t, J = 7.5 Hz, H-7), 2.50 (2H, t, J = 7.5 Hz, H-8), and 2.30 (2H, t, J = 7.5 Hz, H-5)]. The 13C NMR spectrum (Table 2) displayed a total of 9 carbons, which were classified from the DEPT and HSQC spectra as a methoxy carbon signal [δC 52.3 (C-11)], four methylene carbon signals [δC 38.6 (C-6), 31.0 (C-8), 30.8(C-7), and 26.8 (C-5)], one methine carbon signal [δC 116.6 (C-3)], as well as one sp2 carbon signal [δC 130.3 (C-4)] and two C]O units [δC 176.0 (C-9), 159.0 (C-2)]. Furthermore, the structure of 3 was confirmed by the HMBC and 1H–1H COSY correlations (Fig. 2). Hence, the structure of 3 was determined to be 1-methyl-3-[3′,4′-dihydropyridin-2′(1′H)-one] propanoate and it was named rehmanalkaloid C (Fig. 1). Compound 4 was isolated as a colorless crystalline powder with [α] 20 D −6.4° (c 0.8, MeOH). Its molecular formula was determined to be C31H48O18 by HR-ESI–MS at m/z 559.2007 [M + Na]+ (calcd. for C23H36O14Na, 559.2003). The IR spectrum showed the presence of a hydroxyl group (3371 cm−1), methyl groups (2925 cm−1), carbonyl groups (1679 and 1631 cm−1), and methoxyl groups (1140 and 1066 cm−1). In the 1H NMR spectrum, it showed protons of δ 7.46 (1H, d, J = 1.0 Hz, H-3) and 5.09 (1H, d, J = 7.0 Hz, H-1) at the low-field regime. Two anomeric proton signals of δ 4.73 (1H, d, J = 1.5 Hz, H1′′) and δ 4.64 (1H, d, J = 8.0 Hz, H-1′), a methoxy signal at 3.69 (3H, s, OCH3) and a methyl signal at 1.25 (3H, d, J = 6.5 Hz, H-6′′) were also observed. The 13C NMR and DEPT spectrum showed 23 carbon signals, 12 of which were assigned to two sugar moieties and the remaining eleven as a 4-substituted iridoid skeleton. The 1H and 13C NMR data displayed signals characteristic of an iridoid glycoside, which were similar to those reported for (8S)-7,8-dihydrogeniposid (Salama and Sticher, 1983). The only evident difference was that 4 showed resonances due to an additional rhamnopyranosyl unit. The C-6′ downfield shift (δ 68.0 from δ 62.0) suggested the attachment of the rhamnopyranosyl unit to the C-6′ carbon. The assumption was confirmed by the HMBC correlation of H-1′′ with the C-6′ carbon. In the NOESY spectrum, the correlations between H-5 and H-8/H-9 revealed that they were β-oriented (Fig. 4). In the acid hydrolysis of 4, d-glucose and lrhamnose were afforded and confirmed by TLC and optical rotations compared with the reference substances, respectively (Zhang et al., 2008, 2016). The configuration was determined by measuring the optical rotation value and the large 3JH1,H2 coupling constant. Based on the results described above, the structure of 4 was determined to be (8S)-7,8-dihydrogenin 1-O-α-L-rhamnopyranosyl (1 → 6)-β-D-glucopyranoside and it was named rehmaglutosides L (Fig. 1.). The known compounds were identified as (8S)-7,8-dihydrogeniposid (Salama and Sticher, 1983) (5), diglycoside (Zhang et al., 2008) (6), monomelittoside (Venditti et al., 2016) (7), mussaenoside (Venditti et al., 2013) (8), darendoside A (Nomura et al., 1983) (9), syringing (Wang et al., 2014) (10), phenyl-6-O-β-D-xylopyranosyl-O-βD-glucopyranoside (Chen et al., 2008) (11), (7R, 8S)-4,9-dihydroxy3,3′-dimethoxy-7,8-dihydrobenzofuran-1′-propanalneolignan (Yang et al., 2007) (12), and trans-liovil (Schottner et al., 1997) (13) by
Table 1 1 H NMR Spectroscopic Data of Compounds 1-3 (500 MHz). No.
1 (CD3OD)
2 (D2O)
2 3 4 5 6 7 8 9 10
8.87, s
4.41, d (14.5)
11 12 13
3 (D2O)
5.56, s 3.36, m; 3.25, m 2.29, m; 1.96, m 3.43, m
7.20, dd (1.0, 4.0) 6.22, dd (2.5, 4.0) 7.12, s 2.41, s
8.64, s 3.76, m 3.67 (1H, m)
3.57, m
3.92 (1H, m)
3.71, m 3.81, m 3.69, m; 3.49, m
2.30, 5.09, 2.59, 2.50,
t t t t
(7.5) (7.5 Hz) (7.5) (7.5 Hz)
3.69, s
Data assignment was based on HSQC and HMBC experiments. Table 2 13 C NMR Spectroscopic Data of Compounds 1-3 (125 MHz). No.
1 (CD3OD)
2 (D2O)
3 (D2O)
2 3 4 5 6 7 8 9 10 11 12 13
149.6 124.6 158.9 33.5 28.6 46.8 144.2 149.3 65.7 167.2 52.6
51.6 130.8 123.6 109.3 134.3 193.1 26.6 97.9 70.5 70.8 66.1 62.4
159.0 116.6 130.3 26.8 38.6 30.8 31.0 176.0 52.3
Data assignment was based on HSQC and HMBC experiments.
158.9 (C-4), and 124.6 (C-3)] and one C]O unit [δC 167.2 (C-11)]. By studying the 1H–1H COSY spectra, a large spin system −CH2(C-5)CH2(C-6)-CH(C-7)-CH2OH(C-10) group was established. In the HMBC spectrum, the methoxy carbon, which exhibited a strong correlation with the carbonyl carbon (δC 167.2), suggested the presence of a methyl nicotinate group. Moreover, the correlations of the H-5 with C-3/7/8 revealed the presence of a cyclopenta group (Fig. 2). Based on above NMR spectroscopic analysis, compound 1 was found to closely resemble isoplmrodorine (Gournenelis et al., 1989). In the NOESY spectrum, correlations between H-7 and H-8 indicated that H-7/8 were both β-oriented (Fig. 3). Thus, the structure of compound 1 was deduced as (3-methylcarboxylate-7-hydroxymethyl)-cyclopenta [5,9] pyridine and it was named rehmanalkaloid A (Fig. 1). Compound 2 was obtained as a colorless crystalline powder. The molecular formula, C12H17NO6, was determined by its HR-ESI–MS. The IR spectrum showed the presence of a hydroxyl (3355 cm−1) and a carbonyl (1617 cm−1). The 1H NMR spectrum showed three aromatic protons at δ 7.20 (1 H, dd, J = 1.0, 4.0 Hz, H-4), 7.12 (1 H, s, H-2), and 6.22 (1 H, dd, J = 2.5, 4.0 Hz, H-3) at the low-field regime. The presence of an acetyl group was observed at δ 2.41 (3 H, s, H-8). The 13C NMR spectrum (Table 2) displayed a total of 12 carbons, which were classified from the DEPT and HSQC spectra as a methyl carbon signal [δC 26.6 (C-8)], one methylene carbon signal [δC 62.4 (C-13)], six methine carbon signals {including three aromatic methines [δC 134.3 (C-6), 109.3 (C-5), and 123.6 (C-4)] and three oxygen-methines [δC 70.8 (C-11), 70.5 (C-10), and 66.1 (C-12)]} as well as three quaternary carbon signals [δC 130.8 (C-2) and 97.9 (C-9)] and one C]O unit [δC 193.1 (C-7)]. In the 1H–1H COSY spectra, the correlations of H-5 with H-4/6 provided evidence for a eCeCH]CH]CH- in compound 2. Moreover, a large spin system eCHOH(C-10)-CHOH(C-11)-CHOH(C12)-CH2OH(C-13) group was established by the correlations of H158
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Fig. 1. Structure of compounds 1-4.
and a YMC-Pack ODS-A column (250 × 20 mm, 5 μm and 250 × 10 mm, 5 μm). Column chromatography was performed with a Diaion HP-20 (Mitsubishi Chemical Corporation, Tokyo, Japan), Toyopearl HW-40, MCI gel CHP-20 (TOSOH Corp., Tokyo, Japan), Sephadex LH-20 (40–70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), Lichroprep RP-18 gel (40–63 μm, Merck, Darmstadt, Germany), and silica gel (160–200 mesh, Marine Chemical Industry, Qingdao, China). TLC was performed on self-made silica gel G (Qingdao Marine Chemical Industry) plates, CH2Cl2:MeOH:H2O (10:1:0.1, v/v), and CH2Cl2:MeOH:H2O (4:1:0.1, v/v) as the eluent, and spots were visualized by spraying with 10% H2SO4 in ethanol (v/v) followed by heating. The chemical reagents were supplied by Beijing Chemical Plant (Beijing, China) and Tianjin NO. 3 Reagent Plant (Tianjin, China).
comparing their physical and spectroscopic data with reported values in the literature. To investigate whether compounds 4-8 protect cells from DOX-induced cell death, H9c2 cells were treated with 50 μM DOX in the presence or absence of compounds 4-8 (50 μM) and the cell viability was assessed by the MTT assay. Among them, compounds 4-8 exhibited protective effects against DOX-induced cardiotoxicity at different concentrations (Fig. 5).
3. Experimental 3.1. General experimental procedures NMR spectra were recorded at room temperature in D2O or CD3OD using a Bruker Avance III 500 NMR spectrometer with TMS as an internal standard (500 MHz for 1H NMR and 125 MHz for 13C NMR). Optical rotation was measured with an AP-IV (Rudolph Research Analytical, USA). The IR spectrum was determined on a Nicolet iS10 Microscope Spectrometer (Thermo Scientific, USA). HR-ESI–MS spectra were recorded on a Bruker maxis HD mass spectrometer. UV spectra were recorded on a Shimadzu UV-2401PC apparatus. Preparative HPLC was conducted using a Saipuruisi LC-50 instrument with an UV200 detector (Beijing, China)
3.2. Plant materials Dried Rehmannia glutinosa roots were collected from Jiaozuo, Henan Province in China in November 2012. The plants were identified by Prof. Cheng-ming Dong of Henan University of TCM. A voucher specimen (No. 20101101A) has been stored at the Department of Natural Medicinal Chemistry, School of Pharmacy, Henan University of TCM, Zhengzhou, China.
Fig. 2. Key HMBC and 1H–1H COSY correlations of 1-3.
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Fig. 3. Key NOESY correlations of 1 and 2.
The 50% MeOH-H2O fraction was separated on a silica gel column and eluted with petroleum CH2Cl2-MeOH (0:1, 80:1, 60:1, 40:1, 30:1, 20:1, 10:1, and 5:1) to afford Fr. 1–8. Fr. 7 was subjected to Toyopearl HW-40 and eluted with MeOH to yield Fr. 7.1-7.3. Fr. 7.2 was separated on MCI gel CHP-20 and eluted with 40% MeOH to afford Fr. 7.2.17.2.4. Fr. 7.2.1 was purified by preparative PHPLC (50% MeOH/H2O, 3 mL/min) to yield compound 1 (1.2 mg, tR = 24.4 min). The 10% MeOH-H2O fraction was isolated by column chromatography via multiple silica gel columns including a Toyopearl HW-40, RP-18, MCI gel CHP-20 and Sephadex LH-20 to yield compounds 7 (6.5 mg), 8 (11.2 mg) 10 (9.3 mg) and 11 (8.6 mg). The 50% MeOHH2O fraction was repeatedly separated on silica gel columns to yield compounds 12 (5.6 mg) and 13 (4.8 mg).
3.3. Extraction and isolation The dried R. glutinosa roots (20.0 kg) were cut into small pieces and extracted with 95% EtOH under heating reflux twice for 2 h. After concentration under reduced pressure in a vacuum evaporator, the concentrated solution (1500 mL) was centrifuged and subjected to Diaion HP-20 column chromatography, eluted with H2O and MeOH successively, and obtained in water and methanol extracts (101.6g). Next, the methanol extracts were dissolved in 10% MeOHeH2O (1000 mL), subjected to a Diaion HP-20 column again, and successively eluted with 10%, 30%, 50%, 70%, and 100% (v/v) MeOH-H2O (10 column volumes each). After removing the solvents, the 65.0 g, 16.6 g, 11.5 g, 4.2 g, and 2.0 g, respectively, were obtained from the extracts. The 30% MeOH-H2O fraction was subjected to Toyopearl HW-40 column chromatography, eluting with MeOH/H2O from 0% to 100% to receive Fr. 1–4. Fr. 3 (4.0 g) was separated over Sephadex LH-20 and eluted with MeOH as the mobile phase to give subfractions Fr. 3.1-3.3. Fr. 3.2 (0.8 g) was separated on MCI gel CHP-20 and eluted with 20% MeOH to afford Fr. 3.2.1-3.2.5. Fr. 3.2.4 (0.3 g) was further isolated by PHPLC (30% MeOH/H2O, 3 mL/min) to yield compounds 2 (2.6 mg, tR = 32.4 min), 4 (10.2 mg, tR = 62.4 min), 5 (11.6 mg, tR = 50.7 min) and 6 (18.3 mg, tR = 55.1 min). Compound 3 (0.7 mg, tR = 10.1 min) was obtained by PHPLC (33% MeOH/H2O, 3 mL/min) from Fr. 3.2.5 (10 mg). Fr. 3.3 (0.9 g) was further separated by repeated Sephadex LH-20 to afford compound 9 (8.8 mg).
3.3.1. Rehmanalkaloid A (1) Brown crystalline powder; [α]20 D 0.12 (c 0.60, CH3OH); UV (CH3OH) λmax (logε): 207 (2.98) nm, 275 (0.45) nm; IR (KBr) νmaxcm−1: 3356, 2950, 2872, 1721, 1579, 1436, 1299, 1211, 1141, 1125, and 1030 cm−1. HR-ESI–MS m/z: 208.0974 [M+ Na]+ (calcd. For C11H14NO3Na 208.0964). 1H NMR (500 MHz, CD3OD) spectral data see Table 1; 13C NMR (125 MHz, CD3OD) spectral data see Table 2. 3.3.2. Rehmanalkaloid B (2) Colorless crystalline powder; [α]20 D 0.05 (c 0.26, CH3OH); UV (CH3OH) λmax (logε): 201 (0.62) nm, 290 (1.33) nm; IR (KBr)
Fig. 4. Key HMBC, 1H–1H COSY and NOESY correlations of 4.
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Fig. 5. Protective effects of 4-8 on DOX-induced cytotoxicity in H9c2 cell (n = 3); * P < 0.05; ** P < 0.01 compared with the DOX group.
Table 3 1 H NMR (500 MHz) and
13
δ
No
δ
4.64, d (8.0)
153.4. 112.0 36.3 33.5
1′ 2′ 3′ 4′ 5′ 6′
100.6. 74.6. 77.9 71.6 77.0 68.0
26.4
1′
4.73, d (1.5)
102.2
44.3 44.3 66.5
2′ 3′ 4′
72.2 72.4 74.6
169.6 51.7
5′ 6′′
69.8 18.1
No.
δ
1 2 3 4 5 6
5.09, d (8.0)
98.7
7.46, s.
7 8 9 10 11 OCH3
H
2.82, m 2.15, m 1.39,m 1.83, m 1.39, m 2.15, m 1.95, m 3.56, dd (6.5, 11.0) 3.50, dd (6.5, 11.0) 3.69, s
incubated in fresh medium with compounds 4-8 (50 μM) for an additional 24 h. The effects of compounds 4-8 on DOX-induced cytotoxicity were assessed using the MTT assay, as previously described (Han et al., 2008; Wang et al., 2012). The optical density of each well was then measured on a microplate spectrophotometer at a wavelength of 490 nm. The cell viability was determined as the percentage of surviving cells compared with that of the DOX-treated control. Experiments were performed in triplicate and the values are the averages of three (n = 4) independent experiments. Individual data were expressed as the mean ± standard deviation (SD). A post-hoc Dunnett’s test was used to obtain corrected p-values in the group comparisons. Statistical analyses were performed with one-way ANOVA (SPSS version 17.0). A p value less than or equal to 0.05 was considered significant.
C NMR (125 MHz) spectral data of compound 4 (in CD3OD). C
H
δ
C
Acknowledgement 1.25, d (6.0)
Our work was supported by the Key Projects in the National Science & Technology Pillar Program during the Twelfth Five-Year Plan Period (2011BAI06B02).
Data assignment was based on HSQC and HMBC experiments.
νmaxcm−1: 3355, 2925, 2854, 1617, 1464, 1409, 1088, and 1060 cm−1. HR-ESI–MS m/z: 294.0941 [M + Na]+ (calcd. For C12H17NO6Na 294.0954). 1H NMR (500 MHz, D2O) spectral data see Table 1; 13C NMR (125 MHz, D2O) spectral data see Table 2.
References Chen, B., Liu, Y., Liu, H.W., Wang, N.L., Yang, B.F., Yao, X.S., 2008. Iridoid and aromatic glycosides fro: Scrophularia ningpoensis Hemsl. and their inhibition of [Ca2+] increase induced by KCl. Chem. Biodivers. 5, 1723–1735. Feng, W.S., Lv, Y.Y., Zheng, X.K., Zhang, Y.L., Cao, Y.G., Pei, Y.Y., 2013. A new megastigmane from fresh roots of Rehmannia glutinosa. Acta. Pharm. Sin. B 3, 333–336. Feng, W.S., Li, M., Zheng, X.K., Zhang, N., Song, K., Wang, J.C., Kuang, H.X., 2015. Two new ionone glycosides from the roots of Rehmannia glutinosa Libosch. Nat. Prod. Res. 29, 59–63. Gournenelis, D., Skaltsounis, A.L., Tillequin, F., Koch, M., 1989. Plantes de nowelleCaledonie, cxxi: iridoides et alcaloides de Plectronza odorata. J. Nat. Prod. 52, 306–316. Han, X., Ren, D., Fan, P., Shen, T., Lou, H., 2008. Protective effects of naringenin-7-Oglucoside on doxorubicin-induced apoptosis in H9c2 cells. Eur. J. Pharmacol. 581, 47–53. Huang, Y., Jiang, C.M., Hu, Y.L., Zhao, X.J., Shi, C., Yu, Y., Liu, C., Tao, Y., Pan, H.R., Feng, Y.B., 2013. Immunoenhancement effect of Rehmannia glutinosa polysaccharide on lymphocyte proliferation and dendritic cell. Carbohydr. Polym. 96, 516–521. Li, Y.S., Chen, Z.J., Zhu, D.Y., 2005. A novel bis-furan derivative, two new natural furan derivatives from Rehmannia glutinosa and their bioactivity. Nat. Prod. Res. 19, 165–170. Li, M., Wang, X.L., Zheng, X.K., Wang, J.C., Zhao, W., Song, K., Zhao, X., Zhang, Y.L., Kuang, H.X., Feng, W.S., 2015. A new ionone glycoside and three new rhemaneolignans from the roots of Rehmannia glutinosa. Molecules 20, 15192–15201. Lim, D.W., Kim, Y.T., 2013. Dried root of Rehmannia glutinosa prevents bone loss in ovariectomized rats. Molecules 18, 5804–5813. Liu, Y.F., Liang, D., Luo, H., Hao, Z.Y., Wang, Y., Zhang, C.L., Zhang, Q.J., Chen, R.Y., Yu, D.Q., 2012. Hepatoprotective iridoid glycosides from the roots of Rehmannia glutinosa. J. Nat. Prod. 75, 1625–1631. Liu, Y.F., Liang, D., Luo, H., Hao, Z.Y., Wang, Y., Zhang, C.L., Zhang, Q.J., Chen, R.Y., Yu, D.Q., 2014a. Chemical constituents from root tubers of Rehmannia glutinosa. Tradit. Herb. Drugs. 45, 16–22. Liu, Y.F., Liang, D., Luo, H., Hao, Z.Y., Wang, Y., Zhang, C.L., Ni, G., Chen, R.Y., Yu, D.Q., 2014b. Ionone glycosides from the roots of Rehmannia glutinosa. J. Asian Nat. Prod. Res. 16, 11–19. Nomura, T., Fukai, T., Shimada, T., Chen, L.S., 1983. Components of root bark of Morus australis. Planta Med. 49, 90–94. Salama, O., Sticher, O., 1983. Iridoid glucosides from euphrasia rostkoviana part 4 in the
3.3.3. Rehmanalkaloid C (3) Colorless crystalline powder; UVmax (CH3OH) λmax (logε): 204 (1.98) nm; IR (KBr) νmaxcm−1: 3360, 2925, 1688, 1614, 1437, 1201, 1135, and 1027 cm−1. HR-ESI–MS m/z: 206.0785 [M+Na]+ (calcd. For C9H13NO3Na 206.0793). 1H NMR (500 MHz, D2O) spectral data see Table 1; 13C NMR (125 MHz, D2O) spectral data see Table 2. 3.3.4. Frehmaglutoside G (4) colorless crystalline powder; [α]20 D −6.4° (c 0.80, CH3OH); UVmax (CH3OH) λmax (logε): 238.2 and 285.6 nm; IR (KBr) νmaxcm−1: 3371, 2926, 1679, 1631, 1440, 1187, 1146, and 1066. HR-ESI–MS m/z: 559.2007 [M+Na]+ (calcd. For C23H36O14Na 559.2003). 1H NMR (500 MHz, CD3OD) spectral data and 13C NMR (125 MHz, CD3OD) spectral data see Table 3. 3.4. Activity assay The rat cardiac H9c2 myocardial cells were spontaneously immortalized ventricular rat embryo myoblasts that were purchased from Biowit Technologies (Shenzhen, China). The cells were maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a water-saturated 5.0% CO2 incubator. The cells were split upon reaching a confluency of ∼80% using trypsinEDTA, and then seeded onto 96-well plates at a density of 1.0 × 104 cells L−1 (100 μL/well) and incubated for 24 h before treatment. Thereafter, the cells were exposed to DOX (50 μM) for 24 h and then 161
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