Fitoterapia 93 (2014) 67–73
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Iridoid and phenylpropanoid glycosides from Scrophularia ningpoensis Hemsl. and their α-Glucosidase inhibitory activities Jing Hua 1, Jin Qi 1, Bo-Yang Yu ⁎ Department of Complex Prescription of Traditional Chinese Medicine, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 211198, PR China
a r t i c l e
i n f o
Article history: Received 28 August 2013 Accepted in revised form 26 November 2013 Available online 7 December 2013 Keywords: Scrophularia ningpoensis Hemsl. Iridoid glycosides Phenylpropanoid glycosides α-Glucosidase inhibitory activity Scrophulariaceae
a b s t r a c t A new phenylpropanoid glycoside, designated Scrophuside (1) and two new iridoid glycosides, respectively named Ningposide I (2) and Ningposide II (3), along with twelve known (4–15) iridoid and phenylpropanoid glycosides were obtained from the roots of Scrophularia ningpoensis Hemsl. by various chromatographic techniques and their structures were established through chemical methods and spectroscopic analyses. Most of the obtained compounds have been screened for α-Glucosidase inhibitory activity, in which compounds 4, 5, 7, 11, 12, 13, and 14 show significant activity. © 2013 Published by Elsevier B.V.
1. Introduction Scrophularia ningpoensis Hemsl. (family: Scrophulariaceae), a famous Chinese medicine named “Xuanshen”, has been used for hundreds of years to treat many diseases, including pharyngitis, neuritis, laryngitis and diabetes [1,2]. Phytochemical studies revealed that S. ningpoensis Hemsl. contains a variety of constituents such as iridoid glycosides and phenylpropanoid glycosides [3–6]. In addition, bioactivity investigation has shown that some of those constituents display antioxidant, antibacterial, anti-inflammatory, neuroprotective, antidiabetic activities, etc. In the proprietary literature some known iridoid glycosides from S. ningpoensis Hemsl. displayed the potential activity for the therapy of complicating diseases with disorders of glucose metabolism [7]. Additionally, some known phenylpropanoid
⁎ Corresponding author at: Department of Traditional Chinese Prescription, China Pharmaceutical University, Tongjia Xiang Street 24, Nanjing 210009, PR China. Tel./fax: +86 25 83271321. E-mail address: [email protected] (B.-Y. Yu). 1 These authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2013.11.011
glycosides such as acteoside indicated significant α-Glucosidase inhibitory activity [8]. These results prompted us to search additional new compounds with potential activities for treatment of diabetes from S. ningpoensis Hemsl. In this paper, we report the isolation and structure elucidation of a new phenylpropanoid glycoside (1) and two new iridoid glycosides (2–3), together with twelve known compounds (4–15), along with α-Glucosidase inhibitory activities of all isolated iridoid and phenylpropanoid glycosides. 2. Experimental 2.1. General Optical rotations were determined in CH3OH on a JASCO P-1020 digital polarimeter. Nuclear Magnetic Resonance (NMR) spectra were obtained on a Bruker AV-500 using MeOD with TMS spectrometer as internal standard. ESIMS and HRESITOFMS experiments were performed on Agilent 1100 Series MSD Trap mass spectrometer and an Agilent 6210 ESITOF spectrometer, respectively. Silica gel (200–300 mesh) for column chromatography and silica GF254 for TLC were produced by Qingdao
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Haiyang Chemical Co., Ltd. Macroporous Resin D101 (Cangzhou Bon Adsorber Technology Co., Ltd.), Sephadex LH20 (Pharmacia, Sweden), and ODS-A (12 nm, S-50 μm, YMC Co., Ltd., Japan) are also used for column chromatography. L-cysteine methyl ester hydrochloride, trimethylchlorosilane, hexamethyldisilazane, and standard D-glucose, standard D-galactose, and standard L-arabinose were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Flash Chromatography was purchased from Suzhou Lisure Science Co., Ltd. pNPG (p-Nitrophenyl-β-D-Glucopyranoside) and α-Glucosidase (Type 1, from baker yeast) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 2.2. Plant material The dried roots of S. ningpoensis Hemsl. were collected in November 2009 in Zhejiang province, People's Republic of China. The sample was botanically authenticated by associate Prof. Jin Qi. And its voucher specimen (Herbarium No. XS20091120) has been deposited at the herbarium of the Department of Complex Prescription of TCM, China Pharmaceutical University, China. 2.3. Extraction and isolation The pieces of dried roots of S. ningpoensis Hemsl. (31 kg) were extracted with 80% hot ethanol three times and the combined EtOH extract was concentrated under reduced pressure at 50 °C. Thereafter the residue was suspended in H2O and then partitioned successively with EtOAc and n-BuOH three times. The portion of n-BuOH layer residue (1 kg) was subjected to macro-porous resin D101 column chromatography (CC) on the (20 × 150 cm) eluted with H2O and 20%, 30%, 70% and 95% (v:v) EtOH. The 20% EtOH fraction (70 g) was subjected to silica gel CC (5.5 × 100 cm), eluted with a stepwise gradient of EtOAc–MeOH (95:5, 9:1, 85:15, 8:2) to yield fractions (Fr A–Fr E) according to their TLC profiles. Fr B (9.3 g) was subjected to silica gel CC (4 × 75 cm) gradient eluted with CHCl3–MeOH (95:5, 9:1, 85:15, 8:2) to afford subfraction (Fr B1–Fr B5). Further purification of Fr B2 (0.5 g) on Sephadex LH-20 CC (2 × 120 cm) eluted with CHCl3–MeOH (1:1) and then on ODS-A (2.8 × 60 cm) eluted with 20% MeOH afforded compound 4 (116 mg). And Fr B4 (4 g) was subjected to silica gel CC (4 × 75 cm) eluted with CHCl3– MeOH (85:15) to afford compound 5 (2 g). Fr C (4 g) was subjected to silica gel CC (3 × 50 cm) eluted with a stepwise gradient of EtOAc–MeOH (95:5, 9:1, 85:15, 8:2) to yield fractions (Fr C1–Fr C5). The further purification of Fr C3 (0.95 g) by silica gel CC (3 × 50 cm) eluted with CHCl3– MeOH (9:1), and then by ODS-A CC (2.8 × 60 cm) eluted with 25% MeOH to afford compound 6 (83 mg) and compound 11 (11.6 mg). Fr C4 (1.08 g) was subjected to silica gel CC (3 × 50 cm) eluted with CHCl3–MeOH (85:15), and then was further purified by ODS CC (2.8 × 60 cm) eluted with 25% MeOH to afford compound 7 (168 mg). The 30% EtOH fraction (84 g) was subjected to silica gel CC (8 × 60 cm) eluted with a stepwise gradient of CHCl3–MeOH (95:5, 9:1, 85:15, 8:2, 7:3) to yield fractions (Fr A–Fr D). Fr B (1.8 g) was subjected to silica gel CC (3 × 50 cm) eluted with CHCl3–MeOH (9:1), and then by Flash CC on silica gel (2.6 × 40 cm) eluted with CHCl3–MeOH (95:5) to afford
compound 12 (93 mg) and compound 13 (134 mg). Further purification of Fr C (31 g) on silica gel CC twice (4 × 75 cm) eluted with EtOAc–MeOH (9:1) to afford compound 8 (18 g). The 70% EtOH fraction (85 g) was subjected to silica gel CC (8 × 60 cm) eluted with a stepwise gradient of CHCl3–MeOH (95:5, 9:1, 85:15, 8:2, 7:3) to yield fractions (Fr A–Fr C). The Fr A (50 g) was subjected to silica gel CC (6 × 90 cm) gradient eluted with CHCl3–MeOH (9:1, 85:15, 8:2, 7:3) to yield fractions (A1–A5). Then the Fr A3 (1.47 g) was subjected to silica gel CC (2.5 × 70 cm) eluted with EtOAc–MeOH (9:1) to yield residue I (0.6 g) and residue II (0.13 g). Residue I was subjected to ODS CC (2.8 × 60 cm) with 35% MeOH to afford compound 9 (102 mg) and compound 10 (21 mg). And residue II was purified by silica gel CC (1.8 × 25 cm) with CHCl3–MeOH (9:1) to afford compound 1 (8.7 mg). The Fr B (45 g) was subjected to silica gel CC (5 × 80 cm) eluted with CHCl3–MeOH (9:1) to yield fractions (Fr B1–Fr B5). The Fr B1 (0.8 g) was purified by silica gel CC (1.8 × 28 cm) with Petroleum ether–EtOAc (3:1) to yield compound 14 (40 mg). The Fr B4 (10 g) was subjected to silica gel (4 × 75 cm) eluted with CHCl3–MeOH (9:1) to yield compound 15 (5 g). The Fr B5 (5.6 g) was subjected to silica gel CC (4 × 75 cm) eluted with CHCl3–MeOH (85:15) to yield residue I and residue II. The residue I (0.9 g) was purified by silica gel CC (1.8 × 28 cm) with CHCl3–MeOH (9:1), and then by ODS with 45% MeOH to obtain compound 2 (19 mg). The residue II (1.2 g) was purified by silica gel CC (1.8 × 28 cm) with CHCl3–MeOH (9:1) frequently to obtain compound 3 (13 mg). Scrophuside (1): Colorless amorphous powder; [α]25 D : −22 (c 0.10, MeOH); UV (MeOH): λmax (log ε) = 326 (2.08), 288 (1.93), 292 (1.95), 262 (1.60) nm; IR (KBr): νmax = 3422, 2934, 1702, 1631, 1516, 1432, 1378, 1272, 1158, 1131, 1067, 1028, 812 cm−1; 1H and 13C NMR spectroscopic data was shown in Table 1; HR-ESI–FTMS (negative-ion mode): m/z = 637.2104 [M−H]− (calcd. for C30H37O15: 637.2137). Ningposide I (2): Colorless amorphous powder; [α]25 D : − 10.8 (c 0.10, MeOH); UV (MeOH): λmax (log ε) = 278 (2.40), 237 (1.88) nm; νmax = 3418, 2919, 1693, 1637, 1336, 1238, 1186, 1119, 1075, 1014, 988, 770, 686, 543 cm−1; 1H and 13 C NMR spectroscopic data was shown in Table 1; HR-ESI– FTMS (negative-ion mode): m/z =655.2251 [M−H]− (calcd. for C30H39O16: 655.2243). Ningposide II (3): Colorless amorphous powder; [α] 25 D : − 98.4 (c 0.10, MeOH); UV (MeOH): λmax (log ε) = 279 (2.42), 239 (1.92) nm; νmax = 3423, 2930, 1695, 1638, 1338, 1239, 1188, 1120, 1077, 1016, 990, 772, 687, 544 cm−1; 1 H and 13C NMR spectroscopic data was shown in Table 1; HR-EI–MS (negative-ion mode): m/z = 655.2248 [M−H]− (calcd. for C30H39O16: 655.2243). 2.4. Acid hydrolysis and sugar analysis Each compound (2 mg of 1–3) was refluxed with 2 mL 2 M HCl (dioxane–H2O) at 100 °C for 4 h. After the dioxane was removed, the solution was diluted with H2O and extracted with EtOAc (1 mL × 3). The aqueous layer was evaporated under vacuum, diluted repeatedly with H2O, and then evaporated under vacuum to obtain a neutral residue. Then the residue was dissolved in pyridine (300 μL) and 4 mg of L-cysteine methyl ester hydrochloride was added. The mixture was heated in an oil
J. Hua et al. / Fitoterapia 93 (2014) 67–73
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Table 1 NMR data for compounds 1–3 (MeOD, 500 MHz) (δ in ppm, J in Hz). 1 δC Aglycone 1 2 3 4 5 6 7 8 O\CH3 Feruloyl 1′ 2′ 3′ 4′ 5′ 6′ β α C_O O\CH3 Glucose 1″ 2″ 3″ 4″ 5″ 6″ 1‴ Arabinose 1‴ 2‴ 3‴ 4‴ 5‴
133.6 117.6 148.1 147.9 113.5 121.7 72.6
2 δH (HSQC)
37.1 57.1
\ 6.75 \ \ 6.82 6.69 3.75 4.04 2.82 3.88
128.2 112.4 149.9 151.3 117.1 124.8 148.2 115.6 169.1 57.0
\ 7.18 \ \ 6.81 7.08 7.65 6.38 \ 3.80
(3H, s)
104.8 75.7 76.3 72.9 75.6 69.7 105.2
4.37 3.30 3.62 4.89 3.72 3.60 3.86
(d, 7.5) (m) (t, 9.5) (t, 9.5) (m) (d, 11.5) (dd, 2, 11.5)
105.6 74.6 72.9 70.0 67.2
4.24 3.47 3.56 3.75 3.46 3.81
(d, 6.5) (m) (d, 8.5) (m) (d, 12.5) (dd, 2, 12.5)
(d, 2)
(d, 8) (dd, 2, 8) (d, 7) (dd, 2.5, 7) (2H, t, 7.5) (3H, s)
(d, 2)
(d, 8) (dd, 2, 8) (d, 16) (d, 16)
Aglycone 1 2 3 4 5 6 7 8 9 10 Cinnamoyl 1′ 2′, 6′ 3′, 5′ 4′ β α C_O Glucose 1″ 2″ 3″ 4″ 5″ 6″ Galactose 1‴ 3‴ 5‴ 6‴
bath at 60 °C for 1.5 h, and then 300 μL of HMDS–TMCS (hexamethyldisilazane–trimethylchlorosilane, 2:1) was added. Then the mixture was heated in an oil bath at 60 °C for another
3
δC
δH (HSQC)
δC
δH (HSQC)
95.1 \ 144.3 107.6 73.9 78.2 46.8
6.16 (d, 1.5)
95.3 \ 144.3 107.6 74.0 78.2 46.8
6.20 (d, 1.5)
89.2 56.1 23.2
6.40 4.94 \ 3.75 2.01 2.26 \ 2.93 1.53
(d, 6.5) (d, 1.5, 6.5)
89.2 56.1 23.2
6.41 4.95 \ 3.77 2.02 2.28 \ 2.95 1.54
136.3 129.7 130.5 132.0 146.6 120.7 169.2
\ 7.58 7.39 7.39 7.66 6.50 \
(2H, m) (2H, m) (m) (d, 16) (d, 16)
136.3 129.7 130.5 132.0 146.6 120.7 169.2
\ 7.59 7.40 7.40 7.67 6.50 \
(2H, m) (2H, m) (m) (d, 16) (d, 16)
100.4 74.8 76.6 81.3 77.3 62.5
4.65 3.28 3.56 3.59 3.48 3.90 3.99
(d, 8) (m) (m) (t, 8.5) (m) (d,12) (dd, 2.5, 12)
100.0 74.6 87.6 70.7 78.4 63.4
4.68 3.42 3.62 3.44 3.29 3.75 3.95
(d, 8) (m) (t, 8.5) (m) (m) (d, 12) (dd, 2.5, 12)
105.2 75.4 78.4 71.9 78.6 63.0
4.41 3.23 3.38 3.32 3.34 3.66 3.87
(d, 7.5) (t, 8) (m) (m) (m) (d, 12) (dd, 2, 12)
105.7 76.1 78.3 72.1 78.7 63.2
4.59 3.27 3.38 3.29 3.32 3.64 3.89
(d, 8) (t, 8) (m) (m) (m) (d, 12) (dd, 2, 12)
(d, 4) (d, 15) (dd, 4, 15) (s) (3H, s)
(d, 6) (d, 1.5,6) (d, 4) (d, 15) (dd, 4, 15) (s) (3H, s)
30 min. After centrifugation, the supernatant was analyzed by GC under the following conditions: capillary column, CP-SIL 5CB (0.32 mm × 30 mm); detection, FID; detector temperature,
Fig. 1. The chemical structure of the three new compounds.
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a)
b)
Fig. 2. Selected HMBC correlations for 1–3. a. Selected HMBC correlations for 1. b. Selected HMBC correlations for 2 and 3.
280 °C; injection temperature, 250 °C; initial temperature, 140 °C, and then raised to 280 °C at 10 °C/min; carrier, N2 gas. The standard monosaccharides were subjected to the same
reaction and GC–MS analysis. Under these conditions, the derivatives of L-arabinose, D-glucose and D-galactose were detected at 18.22, 19.75 and 19.91 min, respectively.
Fig. 3. The structure of the isolated phenylpropanoid glycosides.
J. Hua et al. / Fitoterapia 93 (2014) 67–73
2.5. Assay procedure for α-Glucosidase inhibitory activity A microplate-based screening method was adopted and modified with reference to previous literature [10,11]. a total of 100 μL of reaction mixture contained 25 μL of 0.1 mol/L phosphate buffer (pH 6.8), 25 μL of substrate solution (2.5 mmol/L pNPG in 0.1 mol/L phosphate buffer), 25 μL varying concentration of experimental drugs, and 25 μL of α-Glucosidase solution (0.2 U/mL α-Glucosidase in 0.1 mol/L phosphate buffer). After incubation of the plates at 37 °C for 15 min, 25 μL of Na2CO3 (0.2 mol/L) was added to each well to stop the reaction. The absorption was measured at 405 nm using Multiskan plate reader (Thermo Labsystems, UK). The inhibitory rate (%) was calculated according to the formula: [[1 − (ODtest − ODblank)] / (control ODtest − control ODblank)] × 100%. 3. Results and discussion Compound 1 was obtained as amorphous white powder. A molecular formula of C30H38O15 is assigned for 1 based on
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the deprotonated molecular ion determined at [M−H]− m/z 637.2104 (calcd. for C30H37O15: 637.2137) in the HR-ESI–MS. The 1 H NMR signals of 1 at δH 7.18 (d, J = 2 Hz), δH 6.81 (d, J = 8 Hz), δH 7.08 (dd, J = 2, 8 Hz) and δH 6.75 (d, J =2 Hz), δH 6.82 (d, J = 8 Hz), δH 6.69 (dd, J = 2, 8 Hz) suggested the presence of two 1,3,4-trisubstituted phenyl groups. In addition, the signals at δH 7.65 (d, J = 16 Hz) and δH 6.38 (d, J = 16 Hz) of two trans olefinic protons indicated that one of these two might be assigned to a feruloyl group. And the signals of a broad triplet signal at δH 2.82 (2H, t, J =7.5 Hz) and two non-equivalent protons at δH 3.75 (d, J =7 Hz) and δH 4.04 (dd, J = 2.5, 7 Hz) suggested that another phenyl group was assigned to a 1,3,4-trisubstituted phenylethanol moiety. Besides, the signals for two anomeric protons at δH 4.37 (d, J = 7.5 Hz) and δH 4.24 (d, J = 6.5 Hz) suggesting that 1 was a phenylpropanoid diglycoside. Compound 1 was then hydrolyzed with hydrochloric acid to yield a D-glucose and a L-arabinose, which was consistent with the above conclusion. The NMR data of 1 were similar to that of the known phenylpropanoid glycoside, 2-(3-hydroxy-4-methoxyphenyl) ethyl-6-O-α-L-arabinopyranosyl-β-D-Glucopyranoside (6) [3]
Fig. 4. The structure of the isolated iridoids.
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Table 2 α-Glucosidase inhibition assay results of compounds 1–15. Compd IC50, mM
Acarbose 0.37 ± 0.01
1 2 3 4 ND ND ND 5.51 ± 1.12
5 1.62 ± 0.29
6 7 ND 12.01 ± 0.633
with the exception of the signals of an additional feruloyl group. In the HMBC spectra, the correlation between δH 4.37 (Glc, H-1″) and δC 72.6 (C-7), and the δH 4.89 (Glc, H-4″) and δC 169.1 (C_O), which indicated that the feruloyl group and the phenylethanol moiety were linked to C-1″ and C-4″ of the glucosyl, respectively. Moreover, the clear correlation of δH 4.24 (ara, H-1‴) to δC 69.7 (C-6″) indicated that the arabinose should be attached to C-6″ of glucosyl. Therefore, the structure of 1 was elucidated as 2-(3-hydroxy-4-methoxyphenyl)ethyl-4O-feruloyl-6-O-α-L-arabinopyranosyl-β-D-Glucopyranoside. It is a new phenylpropanoid glycoside, and therefore a trivial name, Scrophuside, was designated for it. Compound 2 exhibited a deprotonated molecular ion [M− H]− at m/z 655.2251 (calcd. for C30H39O16: 655.2243) in HR-ESI–MS, suggesting a molecular formula of C30H40O16. The 1H NMR data of 2 at δH 6.16 (d, J = 1.5 Hz), δH 6.40 (d, J = 6.5 Hz), δH 4.94 (d, J = 1.5, 6.5 Hz), δH 2.26 (dd, J = 4, 15 Hz), δH 2.01 (d, J = 15 Hz) and δH 1.53 (3H, s) were the easily distinguishable signals of iridoid aglycone. And the signals of five aromatic protons [δH 7.58 (2H, m), 7.39 (3H, m)], together with two trans olefinic protons [δH 7.66 (d, J = 16 Hz), 6.50 (d, J = 16 Hz)] indicated the presence of the trans-cinnamoyl moiety. Two methine proton signals at δH 4.65 (d, J = 8 Hz) and δH 4.41 (d, J = 7.5 Hz) indicated that 2 should be an iridoid diglycoside. Moreover, compound 2 was hydrolyzed with hydrochloric acid to yield a D-glucose and a D-galactose, which is consistent with the deduction. The NMR spectral data of 2 were similar to those reported for harpagoside (15) [5], except for the signals of an additional hexose group and a different chemical shift at C-4″ position [δC 81.3 (about + 9.3 ppm)] [9]. In the HMBC spectrum, the long range correlations between δH 4.65 (Glc, H-1″) and δC 95.1 (C-1), δH 4.41 (d, J = 7.5 Hz) and δC 81.3 (Glc C-4″) revealed that the glucose was attached to C-1 of iridoid aglycone and the galactose was linked at C-4″ of glucose. Therefore, the structure of 2 was elucidated as 4″-O-β-D-galactopyranosylharpagoside, and named it as Ningposide I (Figs. 1 and 2, Table 1). Compound 3 was obtained as a white amorphous powder with the molecular formula C30H40O16 based on a deprotonated molecular ion [M−H]− at m/z 655.2248 (calcd. for C30H39O16: 655.2243), which indicated that 2 and 3 are isomers. The spectroscopic data of 3 were highly similar to 2, except for some NMR spectral data of the glucose group. Acid hydrolysis of compound 3 produced a D-glucose and a D-galactose same as 2 indicating that the different NMR data may be caused by different substituted locations of the galactose group. In the 13 C NMR spectra, the C-3″ (δC 87.6) of 3 displayed about 6.3 ppm downfield from that of 2 due to the known effects of O-glycosylation, which suggested that the galactose was linked at the C-3″ of glucose in 3 [9]. The HMBC correlation between δH 4.59 (d, J = 8 Hz) and δC 87.6 (Glc C-3″) further
8 9 10 11 ND ND ND 2.16 ± 0.13
12 3.02 ± 0.16
13 3.09 ± 0.16
14 1.54 ± 0.31
15 ND
confirmed the deduction. Consequently, the structure of 3 was determined to be 3″-O-β-D-galactopyranosylharpagoside, and named it as Ningposide II (Figs. 1 and 2, Table 1). The structures of the known compounds were identified as darendoside B (4), acteoside (5), 2-(3-hydroxy4-methoxyphenyl)ethyl-6-O-α- L -arabinopyranosyl-β-DGlucopyranoside (6), 2-(3-hydroxy-4-methoxyphenyl) ethyl-O-α-L -arabinopyranosyl-(1 → 6)-O-[6-deoxy-α-Lmannopyranosyl-(1 → 3)]-β-D-Glucopyranoside (7), angoroside C (8), cistanoside D (9), trans-cistanoside D (10), 6-O-αD-galactopyranmosylharpagoside (11), 8-O-feruloylharpagide (12), 8-O-(coumaroyl)harpagide (13), ninpogenin (14), and harpagoside (15), by comparing the NMR data with that reported in the literature [3–6,12–16]. In which, compound 10 was first found in this plant (Figs. 3 and 4). Compounds 1–15 were screened for α-Glucosidase inhibitory activity according to methods described by Li Ting et al. [10,11]. Acarbose (from Bayer Schering Pharma) was used as positive control with IC50 values of 0.37 ± 0.01 mM (n = 3). Compounds 4, 5, 7, 11, 12, 13, and 14 exhibited moderate α-Glucosidase inhibitory activity, with the IC50 values of the 5.51 ± 1.12, 1.62 ± 0.29, 12.01 ± 0.63, 2.16 ± 0.13, 3.02 ± 0.16, 3.09 ± 0.16, and 1.54 ± 0.31 mM (n = 3) respectively (Table 2). The result of compound 5 is in accordance with the previous report [17]. Our findings could provide more scientific support for the medicinal use of this species. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research was financially funded by the National Natural Science Foundation of China (No. 30901956), the 2011' Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References [1] Sciences Flora of China Editorial Committee of Chinese Academy of. Flora of China. 1997;67:46–83. [2] Miyazawa M, Okuno Y. Volatile components from the roots of Scrophularia ningpoensis Hemsl. Flavour Frag J 2003;18:398–400. [3] Kajimoto T, Hidaka M, Shoyama K, Nohara T. Iridoids from Scrophularia ningpoensis. Phytochemistry 1989;28:2701–4. [4] Li YM, Jiang SH, Gao WY, Zhu DY. Phenylpropanoid glycosides from Scrophularia ningpoensis. Phytochemistry 2000;54:923–5. [5] Li YM, Jiang SH, Gao WY, Zhu DY. Iridoid glycosides from Scrophularia ningpoensis. Phytochemistry 1999;50:101–4. [6] Qian J, Hunkler D, Rimpler H. Iridoid-related aglycone and its glycosides from Scrophularia ningpoensis. Phytochemistry 1992;31:905–11. [7] Qi J, Song XC, Yu BY. New medical applications of harpagoside and its derivatives. Chinese Patent No. ZL 201110104810.8. 18/09/2012.
J. Hua et al. / Fitoterapia 93 (2014) 67–73 [8] KANEBO Cosmetics INC (Kane). Alpha glucosidase inhibitor useful as e.g. tablet, drink formulation, solid agent, jelly, chewing gum and candy for treating diabetes and obesity originating by chronic hyperglycemia, contains acteoside as active ingredient. Japan Patent No. JP2005082546-A. 25/08/2005. [9] Machida K, Kaneko A, Hosogai T, Kakuda R, Yaoita Y, Kikuchi M. Studies on the constituents of Syringa species. X. Five new iridoid glycosides from the leaves of Syringa reticulata (Blume) Hara. Chem Pharm Bull 2002;50:493–7. [10] Li T, Zhang XD, Song YW. A microplate-based screening method for alphaglucosidase inhibitors. Chin J Clin Pharmacol Ther 2005;10:1128–34. [11] Habtemariam S. α-Glucosidase inhibitory activity of kaempferol3-O-rutinoside. Nat Prod Commun 2011;6(2):201–3. [12] Houghton PJ. Phenylpropanoid glycosides in Buddleja davidii. J Nat Prod 1985;48(6):1005–6.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Iridoid and phenylpropanoid glycosides from Scrophularia ningpoensis Hemsl. and their α-Glucosidase inhibitory activities Jing Hua 1, Jin Qi 1, Bo-Yang Yu ⁎ Department of Complex Prescription of Traditional Chinese Medicine, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 211198, PR China
a r t i c l e
i n f o
Article history: Received 28 August 2013 Accepted in revised form 26 November 2013 Available online 7 December 2013 Keywords: Scrophularia ningpoensis Hemsl. Iridoid glycosides Phenylpropanoid glycosides α-Glucosidase inhibitory activity Scrophulariaceae
a b s t r a c t A new phenylpropanoid glycoside, designated Scrophuside (1) and two new iridoid glycosides, respectively named Ningposide I (2) and Ningposide II (3), along with twelve known (4–15) iridoid and phenylpropanoid glycosides were obtained from the roots of Scrophularia ningpoensis Hemsl. by various chromatographic techniques and their structures were established through chemical methods and spectroscopic analyses. Most of the obtained compounds have been screened for α-Glucosidase inhibitory activity, in which compounds 4, 5, 7, 11, 12, 13, and 14 show significant activity. © 2013 Published by Elsevier B.V.
1. Introduction Scrophularia ningpoensis Hemsl. (family: Scrophulariaceae), a famous Chinese medicine named “Xuanshen”, has been used for hundreds of years to treat many diseases, including pharyngitis, neuritis, laryngitis and diabetes [1,2]. Phytochemical studies revealed that S. ningpoensis Hemsl. contains a variety of constituents such as iridoid glycosides and phenylpropanoid glycosides [3–6]. In addition, bioactivity investigation has shown that some of those constituents display antioxidant, antibacterial, anti-inflammatory, neuroprotective, antidiabetic activities, etc. In the proprietary literature some known iridoid glycosides from S. ningpoensis Hemsl. displayed the potential activity for the therapy of complicating diseases with disorders of glucose metabolism [7]. Additionally, some known phenylpropanoid
⁎ Corresponding author at: Department of Traditional Chinese Prescription, China Pharmaceutical University, Tongjia Xiang Street 24, Nanjing 210009, PR China. Tel./fax: +86 25 83271321. E-mail address: [email protected] (B.-Y. Yu). 1 These authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.fitote.2013.11.011
glycosides such as acteoside indicated significant α-Glucosidase inhibitory activity [8]. These results prompted us to search additional new compounds with potential activities for treatment of diabetes from S. ningpoensis Hemsl. In this paper, we report the isolation and structure elucidation of a new phenylpropanoid glycoside (1) and two new iridoid glycosides (2–3), together with twelve known compounds (4–15), along with α-Glucosidase inhibitory activities of all isolated iridoid and phenylpropanoid glycosides. 2. Experimental 2.1. General Optical rotations were determined in CH3OH on a JASCO P-1020 digital polarimeter. Nuclear Magnetic Resonance (NMR) spectra were obtained on a Bruker AV-500 using MeOD with TMS spectrometer as internal standard. ESIMS and HRESITOFMS experiments were performed on Agilent 1100 Series MSD Trap mass spectrometer and an Agilent 6210 ESITOF spectrometer, respectively. Silica gel (200–300 mesh) for column chromatography and silica GF254 for TLC were produced by Qingdao
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Haiyang Chemical Co., Ltd. Macroporous Resin D101 (Cangzhou Bon Adsorber Technology Co., Ltd.), Sephadex LH20 (Pharmacia, Sweden), and ODS-A (12 nm, S-50 μm, YMC Co., Ltd., Japan) are also used for column chromatography. L-cysteine methyl ester hydrochloride, trimethylchlorosilane, hexamethyldisilazane, and standard D-glucose, standard D-galactose, and standard L-arabinose were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Flash Chromatography was purchased from Suzhou Lisure Science Co., Ltd. pNPG (p-Nitrophenyl-β-D-Glucopyranoside) and α-Glucosidase (Type 1, from baker yeast) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 2.2. Plant material The dried roots of S. ningpoensis Hemsl. were collected in November 2009 in Zhejiang province, People's Republic of China. The sample was botanically authenticated by associate Prof. Jin Qi. And its voucher specimen (Herbarium No. XS20091120) has been deposited at the herbarium of the Department of Complex Prescription of TCM, China Pharmaceutical University, China. 2.3. Extraction and isolation The pieces of dried roots of S. ningpoensis Hemsl. (31 kg) were extracted with 80% hot ethanol three times and the combined EtOH extract was concentrated under reduced pressure at 50 °C. Thereafter the residue was suspended in H2O and then partitioned successively with EtOAc and n-BuOH three times. The portion of n-BuOH layer residue (1 kg) was subjected to macro-porous resin D101 column chromatography (CC) on the (20 × 150 cm) eluted with H2O and 20%, 30%, 70% and 95% (v:v) EtOH. The 20% EtOH fraction (70 g) was subjected to silica gel CC (5.5 × 100 cm), eluted with a stepwise gradient of EtOAc–MeOH (95:5, 9:1, 85:15, 8:2) to yield fractions (Fr A–Fr E) according to their TLC profiles. Fr B (9.3 g) was subjected to silica gel CC (4 × 75 cm) gradient eluted with CHCl3–MeOH (95:5, 9:1, 85:15, 8:2) to afford subfraction (Fr B1–Fr B5). Further purification of Fr B2 (0.5 g) on Sephadex LH-20 CC (2 × 120 cm) eluted with CHCl3–MeOH (1:1) and then on ODS-A (2.8 × 60 cm) eluted with 20% MeOH afforded compound 4 (116 mg). And Fr B4 (4 g) was subjected to silica gel CC (4 × 75 cm) eluted with CHCl3– MeOH (85:15) to afford compound 5 (2 g). Fr C (4 g) was subjected to silica gel CC (3 × 50 cm) eluted with a stepwise gradient of EtOAc–MeOH (95:5, 9:1, 85:15, 8:2) to yield fractions (Fr C1–Fr C5). The further purification of Fr C3 (0.95 g) by silica gel CC (3 × 50 cm) eluted with CHCl3– MeOH (9:1), and then by ODS-A CC (2.8 × 60 cm) eluted with 25% MeOH to afford compound 6 (83 mg) and compound 11 (11.6 mg). Fr C4 (1.08 g) was subjected to silica gel CC (3 × 50 cm) eluted with CHCl3–MeOH (85:15), and then was further purified by ODS CC (2.8 × 60 cm) eluted with 25% MeOH to afford compound 7 (168 mg). The 30% EtOH fraction (84 g) was subjected to silica gel CC (8 × 60 cm) eluted with a stepwise gradient of CHCl3–MeOH (95:5, 9:1, 85:15, 8:2, 7:3) to yield fractions (Fr A–Fr D). Fr B (1.8 g) was subjected to silica gel CC (3 × 50 cm) eluted with CHCl3–MeOH (9:1), and then by Flash CC on silica gel (2.6 × 40 cm) eluted with CHCl3–MeOH (95:5) to afford
compound 12 (93 mg) and compound 13 (134 mg). Further purification of Fr C (31 g) on silica gel CC twice (4 × 75 cm) eluted with EtOAc–MeOH (9:1) to afford compound 8 (18 g). The 70% EtOH fraction (85 g) was subjected to silica gel CC (8 × 60 cm) eluted with a stepwise gradient of CHCl3–MeOH (95:5, 9:1, 85:15, 8:2, 7:3) to yield fractions (Fr A–Fr C). The Fr A (50 g) was subjected to silica gel CC (6 × 90 cm) gradient eluted with CHCl3–MeOH (9:1, 85:15, 8:2, 7:3) to yield fractions (A1–A5). Then the Fr A3 (1.47 g) was subjected to silica gel CC (2.5 × 70 cm) eluted with EtOAc–MeOH (9:1) to yield residue I (0.6 g) and residue II (0.13 g). Residue I was subjected to ODS CC (2.8 × 60 cm) with 35% MeOH to afford compound 9 (102 mg) and compound 10 (21 mg). And residue II was purified by silica gel CC (1.8 × 25 cm) with CHCl3–MeOH (9:1) to afford compound 1 (8.7 mg). The Fr B (45 g) was subjected to silica gel CC (5 × 80 cm) eluted with CHCl3–MeOH (9:1) to yield fractions (Fr B1–Fr B5). The Fr B1 (0.8 g) was purified by silica gel CC (1.8 × 28 cm) with Petroleum ether–EtOAc (3:1) to yield compound 14 (40 mg). The Fr B4 (10 g) was subjected to silica gel (4 × 75 cm) eluted with CHCl3–MeOH (9:1) to yield compound 15 (5 g). The Fr B5 (5.6 g) was subjected to silica gel CC (4 × 75 cm) eluted with CHCl3–MeOH (85:15) to yield residue I and residue II. The residue I (0.9 g) was purified by silica gel CC (1.8 × 28 cm) with CHCl3–MeOH (9:1), and then by ODS with 45% MeOH to obtain compound 2 (19 mg). The residue II (1.2 g) was purified by silica gel CC (1.8 × 28 cm) with CHCl3–MeOH (9:1) frequently to obtain compound 3 (13 mg). Scrophuside (1): Colorless amorphous powder; [α]25 D : −22 (c 0.10, MeOH); UV (MeOH): λmax (log ε) = 326 (2.08), 288 (1.93), 292 (1.95), 262 (1.60) nm; IR (KBr): νmax = 3422, 2934, 1702, 1631, 1516, 1432, 1378, 1272, 1158, 1131, 1067, 1028, 812 cm−1; 1H and 13C NMR spectroscopic data was shown in Table 1; HR-ESI–FTMS (negative-ion mode): m/z = 637.2104 [M−H]− (calcd. for C30H37O15: 637.2137). Ningposide I (2): Colorless amorphous powder; [α]25 D : − 10.8 (c 0.10, MeOH); UV (MeOH): λmax (log ε) = 278 (2.40), 237 (1.88) nm; νmax = 3418, 2919, 1693, 1637, 1336, 1238, 1186, 1119, 1075, 1014, 988, 770, 686, 543 cm−1; 1H and 13 C NMR spectroscopic data was shown in Table 1; HR-ESI– FTMS (negative-ion mode): m/z =655.2251 [M−H]− (calcd. for C30H39O16: 655.2243). Ningposide II (3): Colorless amorphous powder; [α] 25 D : − 98.4 (c 0.10, MeOH); UV (MeOH): λmax (log ε) = 279 (2.42), 239 (1.92) nm; νmax = 3423, 2930, 1695, 1638, 1338, 1239, 1188, 1120, 1077, 1016, 990, 772, 687, 544 cm−1; 1 H and 13C NMR spectroscopic data was shown in Table 1; HR-EI–MS (negative-ion mode): m/z = 655.2248 [M−H]− (calcd. for C30H39O16: 655.2243). 2.4. Acid hydrolysis and sugar analysis Each compound (2 mg of 1–3) was refluxed with 2 mL 2 M HCl (dioxane–H2O) at 100 °C for 4 h. After the dioxane was removed, the solution was diluted with H2O and extracted with EtOAc (1 mL × 3). The aqueous layer was evaporated under vacuum, diluted repeatedly with H2O, and then evaporated under vacuum to obtain a neutral residue. Then the residue was dissolved in pyridine (300 μL) and 4 mg of L-cysteine methyl ester hydrochloride was added. The mixture was heated in an oil
J. Hua et al. / Fitoterapia 93 (2014) 67–73
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Table 1 NMR data for compounds 1–3 (MeOD, 500 MHz) (δ in ppm, J in Hz). 1 δC Aglycone 1 2 3 4 5 6 7 8 O\CH3 Feruloyl 1′ 2′ 3′ 4′ 5′ 6′ β α C_O O\CH3 Glucose 1″ 2″ 3″ 4″ 5″ 6″ 1‴ Arabinose 1‴ 2‴ 3‴ 4‴ 5‴
133.6 117.6 148.1 147.9 113.5 121.7 72.6
2 δH (HSQC)
37.1 57.1
\ 6.75 \ \ 6.82 6.69 3.75 4.04 2.82 3.88
128.2 112.4 149.9 151.3 117.1 124.8 148.2 115.6 169.1 57.0
\ 7.18 \ \ 6.81 7.08 7.65 6.38 \ 3.80
(3H, s)
104.8 75.7 76.3 72.9 75.6 69.7 105.2
4.37 3.30 3.62 4.89 3.72 3.60 3.86
(d, 7.5) (m) (t, 9.5) (t, 9.5) (m) (d, 11.5) (dd, 2, 11.5)
105.6 74.6 72.9 70.0 67.2
4.24 3.47 3.56 3.75 3.46 3.81
(d, 6.5) (m) (d, 8.5) (m) (d, 12.5) (dd, 2, 12.5)
(d, 2)
(d, 8) (dd, 2, 8) (d, 7) (dd, 2.5, 7) (2H, t, 7.5) (3H, s)
(d, 2)
(d, 8) (dd, 2, 8) (d, 16) (d, 16)
Aglycone 1 2 3 4 5 6 7 8 9 10 Cinnamoyl 1′ 2′, 6′ 3′, 5′ 4′ β α C_O Glucose 1″ 2″ 3″ 4″ 5″ 6″ Galactose 1‴ 3‴ 5‴ 6‴
bath at 60 °C for 1.5 h, and then 300 μL of HMDS–TMCS (hexamethyldisilazane–trimethylchlorosilane, 2:1) was added. Then the mixture was heated in an oil bath at 60 °C for another
3
δC
δH (HSQC)
δC
δH (HSQC)
95.1 \ 144.3 107.6 73.9 78.2 46.8
6.16 (d, 1.5)
95.3 \ 144.3 107.6 74.0 78.2 46.8
6.20 (d, 1.5)
89.2 56.1 23.2
6.40 4.94 \ 3.75 2.01 2.26 \ 2.93 1.53
(d, 6.5) (d, 1.5, 6.5)
89.2 56.1 23.2
6.41 4.95 \ 3.77 2.02 2.28 \ 2.95 1.54
136.3 129.7 130.5 132.0 146.6 120.7 169.2
\ 7.58 7.39 7.39 7.66 6.50 \
(2H, m) (2H, m) (m) (d, 16) (d, 16)
136.3 129.7 130.5 132.0 146.6 120.7 169.2
\ 7.59 7.40 7.40 7.67 6.50 \
(2H, m) (2H, m) (m) (d, 16) (d, 16)
100.4 74.8 76.6 81.3 77.3 62.5
4.65 3.28 3.56 3.59 3.48 3.90 3.99
(d, 8) (m) (m) (t, 8.5) (m) (d,12) (dd, 2.5, 12)
100.0 74.6 87.6 70.7 78.4 63.4
4.68 3.42 3.62 3.44 3.29 3.75 3.95
(d, 8) (m) (t, 8.5) (m) (m) (d, 12) (dd, 2.5, 12)
105.2 75.4 78.4 71.9 78.6 63.0
4.41 3.23 3.38 3.32 3.34 3.66 3.87
(d, 7.5) (t, 8) (m) (m) (m) (d, 12) (dd, 2, 12)
105.7 76.1 78.3 72.1 78.7 63.2
4.59 3.27 3.38 3.29 3.32 3.64 3.89
(d, 8) (t, 8) (m) (m) (m) (d, 12) (dd, 2, 12)
(d, 4) (d, 15) (dd, 4, 15) (s) (3H, s)
(d, 6) (d, 1.5,6) (d, 4) (d, 15) (dd, 4, 15) (s) (3H, s)
30 min. After centrifugation, the supernatant was analyzed by GC under the following conditions: capillary column, CP-SIL 5CB (0.32 mm × 30 mm); detection, FID; detector temperature,
Fig. 1. The chemical structure of the three new compounds.
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a)
b)
Fig. 2. Selected HMBC correlations for 1–3. a. Selected HMBC correlations for 1. b. Selected HMBC correlations for 2 and 3.
280 °C; injection temperature, 250 °C; initial temperature, 140 °C, and then raised to 280 °C at 10 °C/min; carrier, N2 gas. The standard monosaccharides were subjected to the same
reaction and GC–MS analysis. Under these conditions, the derivatives of L-arabinose, D-glucose and D-galactose were detected at 18.22, 19.75 and 19.91 min, respectively.
Fig. 3. The structure of the isolated phenylpropanoid glycosides.
J. Hua et al. / Fitoterapia 93 (2014) 67–73
2.5. Assay procedure for α-Glucosidase inhibitory activity A microplate-based screening method was adopted and modified with reference to previous literature [10,11]. a total of 100 μL of reaction mixture contained 25 μL of 0.1 mol/L phosphate buffer (pH 6.8), 25 μL of substrate solution (2.5 mmol/L pNPG in 0.1 mol/L phosphate buffer), 25 μL varying concentration of experimental drugs, and 25 μL of α-Glucosidase solution (0.2 U/mL α-Glucosidase in 0.1 mol/L phosphate buffer). After incubation of the plates at 37 °C for 15 min, 25 μL of Na2CO3 (0.2 mol/L) was added to each well to stop the reaction. The absorption was measured at 405 nm using Multiskan plate reader (Thermo Labsystems, UK). The inhibitory rate (%) was calculated according to the formula: [[1 − (ODtest − ODblank)] / (control ODtest − control ODblank)] × 100%. 3. Results and discussion Compound 1 was obtained as amorphous white powder. A molecular formula of C30H38O15 is assigned for 1 based on
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the deprotonated molecular ion determined at [M−H]− m/z 637.2104 (calcd. for C30H37O15: 637.2137) in the HR-ESI–MS. The 1 H NMR signals of 1 at δH 7.18 (d, J = 2 Hz), δH 6.81 (d, J = 8 Hz), δH 7.08 (dd, J = 2, 8 Hz) and δH 6.75 (d, J =2 Hz), δH 6.82 (d, J = 8 Hz), δH 6.69 (dd, J = 2, 8 Hz) suggested the presence of two 1,3,4-trisubstituted phenyl groups. In addition, the signals at δH 7.65 (d, J = 16 Hz) and δH 6.38 (d, J = 16 Hz) of two trans olefinic protons indicated that one of these two might be assigned to a feruloyl group. And the signals of a broad triplet signal at δH 2.82 (2H, t, J =7.5 Hz) and two non-equivalent protons at δH 3.75 (d, J =7 Hz) and δH 4.04 (dd, J = 2.5, 7 Hz) suggested that another phenyl group was assigned to a 1,3,4-trisubstituted phenylethanol moiety. Besides, the signals for two anomeric protons at δH 4.37 (d, J = 7.5 Hz) and δH 4.24 (d, J = 6.5 Hz) suggesting that 1 was a phenylpropanoid diglycoside. Compound 1 was then hydrolyzed with hydrochloric acid to yield a D-glucose and a L-arabinose, which was consistent with the above conclusion. The NMR data of 1 were similar to that of the known phenylpropanoid glycoside, 2-(3-hydroxy-4-methoxyphenyl) ethyl-6-O-α-L-arabinopyranosyl-β-D-Glucopyranoside (6) [3]
Fig. 4. The structure of the isolated iridoids.
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Table 2 α-Glucosidase inhibition assay results of compounds 1–15. Compd IC50, mM
Acarbose 0.37 ± 0.01
1 2 3 4 ND ND ND 5.51 ± 1.12
5 1.62 ± 0.29
6 7 ND 12.01 ± 0.633
with the exception of the signals of an additional feruloyl group. In the HMBC spectra, the correlation between δH 4.37 (Glc, H-1″) and δC 72.6 (C-7), and the δH 4.89 (Glc, H-4″) and δC 169.1 (C_O), which indicated that the feruloyl group and the phenylethanol moiety were linked to C-1″ and C-4″ of the glucosyl, respectively. Moreover, the clear correlation of δH 4.24 (ara, H-1‴) to δC 69.7 (C-6″) indicated that the arabinose should be attached to C-6″ of glucosyl. Therefore, the structure of 1 was elucidated as 2-(3-hydroxy-4-methoxyphenyl)ethyl-4O-feruloyl-6-O-α-L-arabinopyranosyl-β-D-Glucopyranoside. It is a new phenylpropanoid glycoside, and therefore a trivial name, Scrophuside, was designated for it. Compound 2 exhibited a deprotonated molecular ion [M− H]− at m/z 655.2251 (calcd. for C30H39O16: 655.2243) in HR-ESI–MS, suggesting a molecular formula of C30H40O16. The 1H NMR data of 2 at δH 6.16 (d, J = 1.5 Hz), δH 6.40 (d, J = 6.5 Hz), δH 4.94 (d, J = 1.5, 6.5 Hz), δH 2.26 (dd, J = 4, 15 Hz), δH 2.01 (d, J = 15 Hz) and δH 1.53 (3H, s) were the easily distinguishable signals of iridoid aglycone. And the signals of five aromatic protons [δH 7.58 (2H, m), 7.39 (3H, m)], together with two trans olefinic protons [δH 7.66 (d, J = 16 Hz), 6.50 (d, J = 16 Hz)] indicated the presence of the trans-cinnamoyl moiety. Two methine proton signals at δH 4.65 (d, J = 8 Hz) and δH 4.41 (d, J = 7.5 Hz) indicated that 2 should be an iridoid diglycoside. Moreover, compound 2 was hydrolyzed with hydrochloric acid to yield a D-glucose and a D-galactose, which is consistent with the deduction. The NMR spectral data of 2 were similar to those reported for harpagoside (15) [5], except for the signals of an additional hexose group and a different chemical shift at C-4″ position [δC 81.3 (about + 9.3 ppm)] [9]. In the HMBC spectrum, the long range correlations between δH 4.65 (Glc, H-1″) and δC 95.1 (C-1), δH 4.41 (d, J = 7.5 Hz) and δC 81.3 (Glc C-4″) revealed that the glucose was attached to C-1 of iridoid aglycone and the galactose was linked at C-4″ of glucose. Therefore, the structure of 2 was elucidated as 4″-O-β-D-galactopyranosylharpagoside, and named it as Ningposide I (Figs. 1 and 2, Table 1). Compound 3 was obtained as a white amorphous powder with the molecular formula C30H40O16 based on a deprotonated molecular ion [M−H]− at m/z 655.2248 (calcd. for C30H39O16: 655.2243), which indicated that 2 and 3 are isomers. The spectroscopic data of 3 were highly similar to 2, except for some NMR spectral data of the glucose group. Acid hydrolysis of compound 3 produced a D-glucose and a D-galactose same as 2 indicating that the different NMR data may be caused by different substituted locations of the galactose group. In the 13 C NMR spectra, the C-3″ (δC 87.6) of 3 displayed about 6.3 ppm downfield from that of 2 due to the known effects of O-glycosylation, which suggested that the galactose was linked at the C-3″ of glucose in 3 [9]. The HMBC correlation between δH 4.59 (d, J = 8 Hz) and δC 87.6 (Glc C-3″) further
8 9 10 11 ND ND ND 2.16 ± 0.13
12 3.02 ± 0.16
13 3.09 ± 0.16
14 1.54 ± 0.31
15 ND
confirmed the deduction. Consequently, the structure of 3 was determined to be 3″-O-β-D-galactopyranosylharpagoside, and named it as Ningposide II (Figs. 1 and 2, Table 1). The structures of the known compounds were identified as darendoside B (4), acteoside (5), 2-(3-hydroxy4-methoxyphenyl)ethyl-6-O-α- L -arabinopyranosyl-β-DGlucopyranoside (6), 2-(3-hydroxy-4-methoxyphenyl) ethyl-O-α-L -arabinopyranosyl-(1 → 6)-O-[6-deoxy-α-Lmannopyranosyl-(1 → 3)]-β-D-Glucopyranoside (7), angoroside C (8), cistanoside D (9), trans-cistanoside D (10), 6-O-αD-galactopyranmosylharpagoside (11), 8-O-feruloylharpagide (12), 8-O-(coumaroyl)harpagide (13), ninpogenin (14), and harpagoside (15), by comparing the NMR data with that reported in the literature [3–6,12–16]. In which, compound 10 was first found in this plant (Figs. 3 and 4). Compounds 1–15 were screened for α-Glucosidase inhibitory activity according to methods described by Li Ting et al. [10,11]. Acarbose (from Bayer Schering Pharma) was used as positive control with IC50 values of 0.37 ± 0.01 mM (n = 3). Compounds 4, 5, 7, 11, 12, 13, and 14 exhibited moderate α-Glucosidase inhibitory activity, with the IC50 values of the 5.51 ± 1.12, 1.62 ± 0.29, 12.01 ± 0.63, 2.16 ± 0.13, 3.02 ± 0.16, 3.09 ± 0.16, and 1.54 ± 0.31 mM (n = 3) respectively (Table 2). The result of compound 5 is in accordance with the previous report [17]. Our findings could provide more scientific support for the medicinal use of this species. Conflict of interest The authors declare no conflict of interest. Acknowledgments This research was financially funded by the National Natural Science Foundation of China (No. 30901956), the 2011' Program for Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education and the Priority Academic Program Development of Jiangsu Higher Education Institutions. References [1] Sciences Flora of China Editorial Committee of Chinese Academy of. Flora of China. 1997;67:46–83. [2] Miyazawa M, Okuno Y. Volatile components from the roots of Scrophularia ningpoensis Hemsl. Flavour Frag J 2003;18:398–400. [3] Kajimoto T, Hidaka M, Shoyama K, Nohara T. Iridoids from Scrophularia ningpoensis. Phytochemistry 1989;28:2701–4. [4] Li YM, Jiang SH, Gao WY, Zhu DY. Phenylpropanoid glycosides from Scrophularia ningpoensis. Phytochemistry 2000;54:923–5. [5] Li YM, Jiang SH, Gao WY, Zhu DY. Iridoid glycosides from Scrophularia ningpoensis. Phytochemistry 1999;50:101–4. [6] Qian J, Hunkler D, Rimpler H. Iridoid-related aglycone and its glycosides from Scrophularia ningpoensis. Phytochemistry 1992;31:905–11. [7] Qi J, Song XC, Yu BY. New medical applications of harpagoside and its derivatives. Chinese Patent No. ZL 201110104810.8. 18/09/2012.
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