b Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, China;
c Institute of Traditional Chinese Medicine & Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, China
The common figwort Scrophularia ningpoensis Hemsl. (Scro- phulariaceae) is one of over 300 known species of the genus Scrophularia,which is well known for its variety of iridoids . Its dried roots are used as anti-inflammatory agents for the treatment of fever,swelling,sore throat,and constipation [2, 3]. A series of chemical constituents including iridoids, phenylethanoid glycosides,triterpene saponins,flavones,and volatile oils have been isolated from S. ningpoensis [4, 5, 6]. Our previous study on this plant led to the isolation of monoterpene pyridine alkaloids,iridoid glycosides,phenylpropanoid glycosides and phenolic acids,some of which showed inhibitory activity against KCl induced [Ca2+]i increase in rat cardiomyocytes and cardio-protective effects against the apoptosis induced by H2O2 [7, 8]. Further investigations on this plant have resulted in the isolation of two new iridoid glycosides,named scrophularianoids A (1) and B (2) (Fig. 1). In this paper,the isolation and structural elucidation of the two new compounds and their activity in an in vitro myocardial protective bioassay are reported.2. Experimental 2.1. General
The dried roots of S. ningpoensis Hemsl. were collected from Pan’an,Zhejiang Province,China,in September 2011,and authenticated by Zeng-Xi Guo,director pharmacist of TCM (Traditional Chinese Medicine),at Zhejiang Institute for Food and Drug Control,Zhejiang Province,China. A voucher specimen was deposited at College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,China. 2.2. Extraction and isolation
The dried roots of S. ningpoensis (25 kg) were chopped into small pieces and extracted with 70% EtOH under reflux twice for 2 h each time. The combined extracts were concentrated by evaporation to yield a residue of 5.6 kg,which was chromatographed on a D101 column eluted with EtOH/H2O gradiently. The 50% EtOH eluted fraction (250.0 g) was then separated using silica gel column chromatography (CC) by gradient elution with CH2Cl2/MeOH (100:0 → 0:100) to afford 10 fractions. Fraction 8 (91.7 g) was fractionated on silica gel CC eluted with CH2Cl2/MeOH (95:5 → 0:100) to give 10 subfractions. Subfraction 5 (10.2 g) was applied to an ODS column using stepwise gradient mixtures of MeOH/H2O (10:90 → 100:0) system. Subfraction 5.5 (1.7 g) was further passed over an HW-40 column with MeOH/H2O (10:90 → 80:20) gradiently. Purification of the eluate of 10% MeOH by semi-preparative HPLC with 44% MeOH gave 1 (7.0 mg) and 2 (5.4 mg). 2.3. Scrophularianoid A
Yellowish amorphous powder; [α]D25 - 40.8 (c 0.5,MeOH). UV (MeOH) λmax (log ε): 232 (3.16),279 (4.26) nm; IR (KBr,cm-1): υmax 3366,2936,1711,1637,1577,1496; HR-ESI-MS m/z 533.1637 [M+Na]+,calcd. for C24H30O12Na: 533.1635; 1H NMR and 13C NMR data,see Table 1.
Yellowish amorphous powder; [α]D25- 50.0 (c 0.5,MeOH). UV (MeOH) λmax (log ε): 232 (3.41),279 (4.26) nm; IR (KBr,cm-1): υmax 3370,2926,1691,1635,1578,1514; HR-ESI-MS m/z 565.1904 [M+Na]+,calcd. for C25H34O13Na: 565.1897; 1H NMR and 13C NMR data,see Table 1. 2.5. Sugar analysis
The absolute configuration of the sugar moiety was determined by the method reported by Tanaka et al. . Compounds 1 and 2 (2 mg,each) were heated in 2 mol/L aqueous HCl for 2 h at 90 ℃. The mixture was evaporated to dryness in vacuo,and then the residue was dissolved in H2O and extracted with CHCl3. The aqueous layer was collected. After drying in vacuo,the residue was dissolved in pyridine (1 mL) containing L-cysteine methyl ester (1 mg) (Sigma,USA) and the mixture was heated at 60 ℃ for 1 h. Then,o-tolyl isothiocyanate (5 μL) (Alfa Aesar,UK) was added to the mixture,which was heated at 60 ℃ for 1 h. The reaction mixture was directly analyzed by HPLC. Analytical HPLC was performed on a reversed-phase C18 column (250 mm Χ .6 mm i.d.,5 μm,Phenomenex,Gemini) at 35 ℃ with isocratic elution of 25% CH3CN containing 0.1% formic acid for 40 min at a flow rate 0.8 mL/min. Peaks were detected by a UV detector at 250 nm. The derivatives of 1 and 2 both gave one peak at tR 21.6 min. The derivatives of D-glucose and L-glucose (Sigma,USA) were subjected to the same method. The peaks were recorded at tR 20.2 (L-glucose) and 21.6 (D-glucose) min,respectively. 3. Results and discussion Compound 1 was isolated as a yellowish amorphous powder, [α]D25 - 40.8 (c 0.5,MeOH). The molecular formula C24H30O12 was determined on the basis of HR-ESI-MS (m/z 533.1637 [M+Na]+). Its UV spectrum exhibited the characteristic cinnamoyl chromophore absorption at 279 nm. Its IR spectrum displayed absorption bands at 1711 cm-1 for conjugated ester carbonyl group(s),1637,1577, and 1496 cm-1 for aromatic ring(s),and 3366 cm-1 for OH group(s). The 1H NMR spectrum of 1 showed resonances for a cinnamoyl group at δ 7.62-7.65 (m,2H,H-2′′ ,6′′),7.40-7.42 (m,3H, H-3′′ ,4′′ ,5′′),7.75 (d,1H,J = 16.0 Hz,H-7′′),and 6.59 (d,1H, J = 16.0 Hz,H-8′′),and a sugar moiety including an anomeric proton signal at δ 4.58 (d,1H,J = 8.0 Hz,H-1′) together with five proton signals at δ 3.22 (m,1H,H-2′),3.38 (m,1H,H-3′),3.30 (m, 2H,H-4′ ,5′),3.86 (br d,1H,J = 11.6 Hz,H-6a′) and 3.66 (m,1H,H- 6b′). Remaining resonances indicated a characteristic iridoid skeleton at δ 5.95 (d,1H,J = 0.8 Hz,H-1),6.37 (d,1H,J = 6.4 Hz, H-3),5.00 (dd,1H,J = 6.4,1.2 Hz,H-4),3.80 (t,1H,J = 4.0 Hz,H-6), 2.07 (dd,1H,J = 14.0,4.8 Hz,H-7a),1.84 (dd,1H,J = 14.0,3.2 Hz,H- 7b),and 2.73 (br s,1H,H-9),and two methylene protons at δ 4.22 (dd,2H,J = 11.6,6.8 Hz,). The 13C NMR spectrum of 1 in combination with the DEPT and HSQC spectra indicated the presence of 24 carbons including a cinnamoyl group at δ 168.6, 146.9,135.9,131.7,130.2 (Χ2),129.5 (Χ2),and 118.8,a sugar moiety at δ 99.8,78.4,77.7,74.7,71.8,and 62.9,and an iridoid skeleton at δ 143.1,108.5,92.7,79.3,78.0,72.9,69.5,59.2,and 43.2. After acid hydrolysis and derivatization of 1 using the reported method ,an HPLC analysis of the derivatives revealed the presence of D-glucose. Additionally,the β-configuration was established based on the coupling constant (8.0 Hz) of the anomeric proton.
According to the 1H NMR and 13C NMR data,an iridoid glycoside similar to harpagoside was proposed ,except for the absence of a methyl group and the presence of an oxygenated methylene (δ 69.5) at C-8. HMBC correlation from δ 2.73 (H-9) to δ 69.5 further confirmed the above deduction. The partial structures of the iridoid,cinnamoyl,and glucose moiety were assigned by detailed analyses of 1H-1H COSY,HSQC and HMBC spectra. The glucose moiety was attached at C-1 of iridoid,based on the HMBC correlations observed at H-1/C-1′ and H-1′/C-1. Furthermore,the linkage of the cinnamoyl group to iridoid was established at C-8 by the HMBC long-range correlation at H-10/C-9′′ .
The relative configuration of 1 was determined by comparison with reported literature values. The chemical shift of C-1 at δ 92.7 indicated a cis-fused iridoid by comparing with the chemical shift of C-1 (δ 100-105) in trans-fused iridoids . The C-1 shift (δ 92.7) together with the difference between the C-3 and C-4 shifts (Δδ 34.6) demonstrated a 6β-O-substituted iridoid compared to the C-1 shift (δ > 99) and the difference between the C-3 and C-4 shifts (Δδ > 47) for 6a-O-substituted compounds . On the basis of the above evidence,1 is believed to be a new iridoid glycoside,designated as scrophularianoid A.
Compound 2 was obtained as a yellowish amorphous powder, [α]D25-50.0 (c 0.5,MeOH). HR-ESI-MS gave a quasimolecular ion peak at m/z 565.1904 [M+Na]+,corresponding to the molecular formula C25H34O13. Its UV absorption λmax at 279 nm was indicative of a cinnamoyl chromophore. Its IR spectrum showed absorption bands of OH groups (3370 cm-1),conjugated ester carbonyl group(s) (1691 cm-1) and aromatic ring(s) (1635,1578, 1514 cm-1). The 1H NMR and 13C NMR spectra of 2 were also similar to those of harpagoside . Proton signals at δ 7.58-7.61 (m,2H,H-2′′ ,6′′),7.39-7.41 (m,3H,H-3′′ ,4′′ ,5′′),7.66 (d,1H, J = 16.0 Hz,H-7′′),and 6.49 (1H,d,J = 16.0 Hz,H-8′′),together with carbon signals at δ 168.7,146.3,136.0,131.7,130.2 (×2),129.4 (×2),and 120.2,suggested the presence of a cinnamoyl moiety. Proton signals at δ 4.68 (d,1H,J = 7.6 Hz,H-1′),3.28 (m,1H,H-2′), 3.41 (d,1H,J = 8.8 Hz,H-3′),3.33 (m,1H,H-4′),3.35 (m,1H,H-5′), 3.93 (dd,1H,J = 12.0,2.0 Hz,H-6a′) and 3.70 (m,1H,H-6b′), together with carbon signals at δ 100.3,78.2,78.1,74.8,72.2,and 63.4,belonged to a sugar moiety. Acid hydrolysis followed by an HPLC analysis of the derivatives using an authentic sample as reference  confirmed the presence of D-glucose. Additionally,the configuration of the anomeric proton was deduced to be β based on the coupling constant (7.6 Hz) of the anomeric proton.
The remaining 1H NMR and 13C NMR signals of 2 were similar to those of harpagoside,except for the absence of an olefinic bond at C-3 and C-4,and the presence of two oxygenated methines (δ 98.2, 71.2) and an additional methoxy group (δ 57.5). HMBC correlation from δ 3.54 to δ 98.2 (C-3) indicated the attachment of the methoxy group at C-3.
The relative configuration of 2 was determined by comparison with 1 and further supported by NOESY analysis. The correlations between H-1 and H-10,H-10 and H-4,and H-4 and H-6 indicated that C4-OH was in β-orientation. The α-configuration of C3-OCH3 was established based on the coupling constant (J3,4 = 8.0 Hz). Thus,2 is a new iridoid derivative and is named scrophularianoid B.
HR-ESI-MS,UV,IR,1H NMR,13C NMR,DEPT,1H-1H COSY,HSQC, HMBC and NOESY spectra of compounds 1 and 2 are supplied in Supporting information.
In our preliminary in vitro myocardial protective bioassay, compounds 1 and 2 were evaluated by the MTT assay. However, both of them showed little effect against H2O2-induced apoptosis in cardiomyocytes. 4. Conclusion
In conclusion,the 70% EtOH extract of the roots of S. ningpoensis gave two new iridoid glycosides,named scrophularianoids A (1) and B (2). The myocardial protective bioassay indicated that they both had little cardioprotective effect against the apoptosis induced by H2O2. Acknowledgments
The authors thank Ming Zhu and Zeng-Xi Guo,director pharmacists of TCM,at Zhejiang Institute for Food and Drug Control,Zhejiang Province,China for the collection and identification of the plant materials. We are grateful to Institute of Traditional Chinese Medicine & Natural Products,College of Pharmacy,Ji’nan University for collecting the optical rotation data and HR-ESI-MS spectra. Appendix A. Supplementary data
Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2014.05.007.
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