Notes
Cyprotuoside C and Cyprotuoside D, Two New Cycloartane Glycosides from the Rhizomes of Cyperus rotundus
2018 Volume 66 Issue 1 Pages 96-100
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2018 Volume 66 Issue 1 Pages 96-100
Cyprotuoside C (1) and cyprotuoside D (2), two new cycloartane glycosides were isolated from the ethanol extract of the rhizomes of Cyperus rotundus. Their structures were identified as 24R-9,10-seco-cycloartan-1(10),9(11)-dien-3β,7β,24,25-tetraol 3-O-β-D-xylopyranosyl-(1→4)-[α-L-arabinopyranosyl-(1→6)]-β-D-glucopyranosyl-25-O-β-D-glucuronide (1) and 9,10-seco-cycloartan-1(10),9(11),23(24)-trien-3β,7β,25-triol 3-O-β-D-xylopyranosyl-(1→4)-{α-L-arabinopyranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)]}-β-D-glucopyranosyl-25-O-β-D-glucuronide (2) by spectroscopic methods.
Cyperus rotundus (C. rotundus), a perennial herb, is widespread in the tropical and subtropical regions all over the world. The rhizomes of C. rotundus is a kind of traditional Chinese medicine (TCM) named “Xiangfuzi,” which is broadly used in folk medicine as an anti-inflammatory, antidepressant, analgesic, and antiemetic remedy for dysentery and women’s diseases.1–3) Previous investigations confirmed that the phytochemical constituents of C. rotundus contained terpenoids, steroids and flavonoids.4–6) Among them, the terpenoids are the characteristic secondary metabolites.1) In recent years, we have put our efforts to the discovery of secondary metabolites with various skeletons, in particular triterpene glycosides, from TCM.7–10) In our ongoing research on the triterpene glycosides of this plant, we isolated two new cycloartane glycosides, cyprotuoside C (1) and cyprotuoside D (2) (Fig. 1). This paper reports the isolation and structure elucidation of the two new triterpene glycosides.
Cyprotuoside C (1) was isolated as a white amorphous powder. Its molecular formula, C52H84O23, was determined on the basis of its [M+Na]+ peak (m/z 1099.5303) in the high resolution electrospray ionization (HR-ESI)-MS and 13C-NMR data. IR spectrum showed the absorptions for the hydroxyl (3346 cm−1) and olefin (1650 cm−1) functionalities. A distortionless enhancement by polarization transfer (DEPT) experiment showed that the 52 carbons present in the 13C-NMR spectrum consisted of 7 methyl, 11 methylene, 27 methine, and 7 quaternary carbons. The 1H-NMR spectrum displayed six methyl singlets at δ 0.76, 0.93, 0.99, 1.02, 1.53, 1.58, one methyl doublet at δ 0.94 (3H, d, J=6.4 Hz), two olefinic protons at δ 5.31 (1H, dd, J=4.5, 2.4 Hz) and 5.41 (1H, br d, J=5.5 Hz), and a pair of diagnostic methylene protons at δ 2.74 (1H, d, J=14.4 Hz) and 3.03 (1H, d, J=14.4 Hz). Correspondingly, seven methyl carbons (δ 15.7, 16.1, 19.2, 24.2, 24.8, 21.7 and 19.1), four olefinic carbons (δ 120.2, 139.8, 119.3 and 136.9) and an isolated methylene carbon (δ 45.5) were exhibited in the13C-NMR spectrum (Table 1), respectively. The above information implied that compound 1 might possess a 9,10-seco-cycloartane triterpenoid skeleton.11–13) Acid hydrolysis of 1 produced a triterpenoid aglycone (1a), and D-glucose, D-xylose, L-arabinose and D-glucuronic acid as sugar residues which were identified by comparison with the respective authentic samples by GC analysis. The 1H- and 13C-NMR spectra of 1a confirmed that the structure of the aglycone moiety (1a) was the same as secomacrogenin B13) (Table 1). Thus, the structure of the aglycone of 1 was determined as 24R-9,10-seco-cycloartan-1(10),9(11)-dien-3β,7β,24,25-tetraol. Anomeric region in the 1H- and 13C-NMR spectra of 1 revealed signals for four anomeric protons at δ 4.91 (1H, d, J=8.0 Hz), 5.03 (1H, d, J=7.6 Hz), 5.06 (1H, d, J=7.6 Hz) and 5.56 (1H, d, J=7.8 Hz) with their corresponding anomeric carbons at δ 102.6, 105.3, 105.5 and 105.0, respectively (Table 2). The relative stereochemistry of each monosaccharide was determined as β-D-glucose, β-D-xylose, α-L-arabinose and β-D-glucuronic acid based on the coupling constants of anomeric protons and 13C-NMR data of the sugar moieties. The linkage of the sugar residues was deduced from the heteronuclear multiple bond correlation (HMBC) spectrum. HMBC spectrum showed cross peaks between the signals at δH 4.91 and δC 78.9 (C-3 of the aglycon), δH 5.56 and δC 79.8 (C-4 of the inner glucose), δH 5.06 and δC 68.1 (C-6 of the inner glucose) and δH 5.03 and δC 81.2 (C-25 of the aglycon) (Fig. 2). Therefore, the Xyl-(1→4)-[Ara-(1→6)]-Glc structure of the trisaccharide moiety was concluded to be linked to C-3 of the aglycone, and the glucuronic acid moiety to the C-25 of the aglycone, respectively. The combined application of 1H–1H correlation spectroscopy (1H–1H COSY), total correlation spectroscopy (TOCSY), heteronuclear single quantum correlation (HSQC) and HMBC experiments allowed the sequential assignments of all resonances for each monosaccharide. The rotating frame Overhauser enhancement spectroscopy (ROESY) correlations among H-7, H-5, H-29, H-28 and H-21, and H-3, H-30, H-8 and H-18 established the stereochemistry of H-7α and H-3β (Fig. 3). Based on all above, the structure of 1 was formulated as 24R-9,10-seco-cycloartan-1(10),9(11)-dien-3β,7β,24,25-tetraol 3-O-β-D-xylopyranosyl-(1→4)-[α-L-arabinopyranosyl-(1→6)]-β-D-glucopyranosyl-25-O-β-D-glucuronide, named cyprotuoside C.
| No. | 1a) | 1ab) | 2a) | |||
|---|---|---|---|---|---|---|
| δH | δC | δH | δC | δH | δC | |
| 1 | 5.31 (dd, 4.5, 2.4) | 120.2 | 5.31 (dd, 4.8, 2.4) | 117.3 | 5.33 (dd, 4.4, 2.4) | 120.4 |
| 2 | 1.87 (m), 2.15 (m) | 30.8 | 1.91 (m), 2.33 (m) | 32.4 | 1.85 (m), 2.19 (m) | 31.1 |
| 3 | 3.97 (dd, 9.4, 6.0) | 78.9 | 3.55 (dd, 9.6, 5.8) | 75.0 | 3.99 (dd, 9.6, 6.2) | 79.2 |
| 4 | — | 37.1 | — | 38.21 | — | 37.6 |
| 5 | 1.72 (dd, 12.4, 5.2) | 55.5 | 1.71 (dd, 12.4, 5.0) | 45.3 | 1.71 (dd, 12.4, 5.0) | 55.3 |
| 6 | 1.86 (m), 1.98 (m) | 35.2 | 1.82 (m), 1.91 (m) | 36.1 | 1.84 (m), 1.98 (m) | 35.2 |
| 7 | 3.93 (ddd, 12.0, 7.2, 4.6) | 74.4 | 3.91 (ddd, 11.8, 7.2, 4.4) | 74.6 | 3.92 (ddd, 12.0, 7.2, 4.4) | 74.4 |
| 8 | 2.18 (d, 12.0) | 55.6 | 2.06 (d, 12.0) | 55.7 | 2.18 (d, 12.0) | 55.4 |
| 9 | — | 136.9 | — | 135.6 | — | 136.7 |
| 10 | — | 139.8 | — | 139.4 | — | 139.8 |
| 11 | 5.41 (br d, 5.5) | 119.3 | 5.41 (d, 6.0) | 126.2 | 5.39 (br d, 5.4) | 119.0 |
| 12 | 2.12 (m), 1.95 (m) | 34.3 | 1.97 (m), 2.08 (m) | 37.7 | 2.09 (m), 1.91 (m) | 34.1 |
| 13 | — | 45.8 | — | 46.0 | — | 45.6 |
| 14 | — | 49.5 | — | 48.3 | — | 49.0 |
| 15 | 1.51 (m), 1.59 (m) | 33.6 | 1.50 (m), 1.66 (m) | 35.6 | 1.49 (m), 1.57 (m) | 33.8 |
| 16 | 1.17 (m), 1.88 (m) | 29.4 | 1.41 (m), 1.98 (m) | 28.8 | 1.08 (m), 1.89 (m) | 28.4 |
| 17 | 1.60 (m) | 52.2 | 1.61 (m) | 51.0 | 1.66 (m) | 53.7 |
| 18 | 0.93 (s) | 16.1 | 0.72 (s) | 15.3 | 0.94 (s) | 16.3 |
| 19a | 3.03 (d, 14.4) | 45.5 | 3.03 (d, 14.4) | 45.3 | 3.01 (d, 14.4) | 45.2 |
| 19b | 2.74 (d, 14.4) | 2.77 (d, 14.4) | 2.72 (d, 14.4) | |||
| 20 | 1.43 (m) | 37.5 | 1.41 (m) | 36.6 | 1.41 (m) | 37.3 |
| 21 | 0.94 (d, 6.4) | 19.1 | 0.90 (d, 6.0) | 18.8 | 0.96 (d, 6.0) | 19.8 |
| 22 | 2.11 (m), 1.13 (m) | 34.7 | 1.51 (m), 1.26 (m) | 33.6 | 2.27 (m), 1.89 (m) | 39.6 |
| 23 | 1.95 (m), 1.32 (m) | 28.6 | 1.53 (m), 1.37 (m) | 28.8 | 5.64 (m) | 129.8 |
| 24 | 3.81 (dd, 6.6, 5.4) | 78.8 | 3.35 (dd, 6.6, 5.7) | 79.1 | 5.49 (d, 16.0) | 138.5 |
| 25 | — | 81.2 | — | 73.5 | — | 78.4 |
| 26 | 1.53 (s) | 24.8 | 1.18 (s) | 23.9 | 1.43 (s) | 27.6 |
| 27 | 1.58 (s) | 21.7 | 1.23 (s) | 26.9 | 1.43 (s) | 27.6 |
| 28 | 0.99 (s) | 19.2 | 0.94 (s) | 19.3 | 1.01 (s) | 18.9 |
| 29 | 1.02 (s) | 24.2 | 1.04 (s) | 24.7 | 1.06 | 24.7 |
| 30 | 0.76 (s) | 15.7 | 0.73 (s) | 13.9 | 0.79 | 16.1 |
a) Compound 1a was tested in C5D5N. b) Compounds 1 and 2 was tested in CDCl3.
| Position | 1 | 2 | ||
|---|---|---|---|---|
| δH | δC | δH | δC | |
| 1′ | 4.91 (d, 8.0) | 102.6 | 4.87 (d, 8.0) | 101.1 |
| 2′ | 3.87 (m) | 75.4 | 4.53 (m) | 79.4 |
| 3′ | 4.20 (m) | 76.7 | 4.10 (m) | 75.5 |
| 4′ | 4.55 (m) | 79.8 | 4.56 (m) | 79.9 |
| 5′ | 4.02 (m) | 74.6 | 4.11 (m) | 75.8 |
| 6′ | 4.73 (dd, 2.5, 11.8) | 68.1 | 4.76 (dd, 2.0, 11) | 68.2 |
| 4.81 (dd, 5.4, 11.8) | 4.82 (dd, 5.2, 11) | |||
| 1″ | 5.56 (d, 7.8) | 105.0 | 5.54 (d, 7.8) | 105.3 |
| 2″ | 4.01 (m) | 74.9 | 4.03 (m) | 74.8 |
| 3″ | 4.32 (m) | 78.6 | 4.29 (m) | 78.4 |
| 4″ | 4.24 (m) | 71.1 | 4.23 (m) | 70.9 |
| 5″ | 3.94 (m), 4.25 (m) | 67.2 | 3.96 (m), 4.27 (m) | 67.5 |
| 1‴ | 5.06 (d, 7.6) | 105.5 | 5.07 (d, 7.6) | 105.7 |
| 2‴ | 4.49 (m) | 72.6 | 4.48 (m) | 72.6 |
| 3‴ | 4.01 (m) | 74.5 | 4.02 (m) | 74.4 |
| 4‴ | 4.26 (m) | 70.2 | 4.24 (m) | 69.9 |
| 5‴ | 3.69 (m), 4.27 (m) | 67.7 | 3.70 (m), 4.28 (m) | 67.6 |
| 1‴′ | 5.03 (d, 7.6) | 105.3 | 6.13 (br s) | 101.4 |
| 2‴′ | 4.13 (m) | 75.1 | 4.68 (m) | 72.2 |
| 3‴′ | 4.35 (m) | 78.1 | 4.36 (m) | 72.4 |
| 4‴′ | 4.61 (m) | 73.6 | 4.38 (m) | 74.1 |
| 5‴′ | 4.67 (d, 10) | 78.1 | 4.80 (m) | 69.5 |
| 6‴′ | 173.8 | 1.81 (d, 6.8) | 18.7 | |
| 1‴″ | 5.05 (d, 7.6) | 105.4 | ||
| 2‴″ | 4.11 (m) | 75.0 | ||
| 3‴″ | 4.34 (m) | 78.1 | ||
| 4‴″ | 4.61 (m) | 73.8 | ||
| 5‴″ | 4.66 (d, 10) | 78.0 | ||
| 6‴″ | 173.8 | |||
Cyprotuoside D (2) was obtained as a white amorphous powder. Its HR-ESI-MS showed a quasi-molecular ion peak at m/z 1227.5779 (calculated value 1227.5775) [M+Na]+, which indicated a molecular formula of C58H92O26. The 1H- and 13C-NMR spectroscopic data of 2 were similar to those of 1, with the exception of a double bond [δH 5.49 (1H, d, J=16 Hz) and 5.64 (1H, m); δC 138.5, 129.8] located at C-23 and C-24 in 2, instead of a hydroxyl group linked to C-24 in 1. This was revealed by the HMBC correlations from H-23 to C-20, C-22, C-24 and C-25, H-24 to C-22, C-23, C-25, C-26 and C-27 as well as the 1H–1H correlations of the spin system H-22/H-23/H-24 (Fig. 2). Furthermore, comparison of 1H- and 13C-NMR spectra of 2 with those of 1, indicated that 2 had one more rhamnosyl group [δH 6.13 (1H, br s) and 1.81 (3H, d, J=6.8 Hz); δC 101.4, 72.2, 72.4, 74.1, 69.5, 18.7] attached at C-2′ of the inner glucose. The suggestion was in accord with the observation of the downfield shift of C-2′ signal from δC 75.4 in 1 to δC 79.4 in 2. This was further established by the HMBC correlation from H-1‴′ [δH 6.13 (1H, br s)] to C-2′ (δC 79.4) (Fig. 2). Acid hydrolysis of 2 was performed in the same manner as that of 1 and D-glucose, D-xylose, L-arabinose, L-rhamnose and D-glucuronic acid as sugar residues were identified by GC analysis from 2. The coupling constants of anomeric protons and 13C-NMR data of the sugar moieties demonstrated that the glucose, xylose and glucuronic acid have a β-configuration, while the arabinose and rhamnose have an α-configuration. Consequently, the structure of 2 was established as 9,10-seco-cycloartan-1(10),9(11),23(24)-trien-3β,7β,25-triol 3-O-β-D-xylopyranosyl-(1→4)-{α-L-arabinopyranosyl-(1→6)-[α-L-rhamnopyranosyl-(1→2)-]}-β-D-glucopyranosyl-25-O-β-D-glucuronide, named cyprotuoside D.
Optical rotations were measured using a JASCO P-1010 digital polarimeter (Horiba, Tokyo, Japan) and IR spectra were obtained from a PerkinElmer, Inc. Spectrum One FT-IR spectrometer (PerkinElmer, Inc., Waltham, U.S.A.). MS data were detected on a Finnigan LCQ Advantage Spectrometer (Thermo Scientific, Massachusetts, U.S.A.) and a Shimadzu GC-MS model QP2010 Plus spectrophotometer (Shimadzu, Kyoto, Japan), respectively. NMR spectra were recorded on 400 MHz FT-NMR spectrometer (Varian Inova AS 400, Varian, U.S.A.). GC analysis was performed on an Agilent Technologies HP6890 gas chromatograph equipped with an H2 flame ionization detector (Agilent Technologies Inc., California, U.S.A.). The column was HP-5 quartz capillary column (30 m×0.32 mm×0.25 µm) with the following conditions: column temperature 180–260°C; carrier gas, N2 (2 mL/min); injector and detector temperature, 270°C. Column chromatography separations were carried out on silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd., China), octadecyl silane (ODS) (50 mesh, AA12S50, YMC), and Diaion HP-20 (Pharmacia, U.S.A.). All other chemicals and reagents of analytical grade were obtained from Sigma-Aldrich, unless indicated otherwise.
Plant MaterialsThe rhizomes of Cyperus rotundus were collected in Zhanjiang, Guangdong Province of China in September 2009, and were identified by Professor Wenqing Yin (School of Chemistry & Chemical Engineering of Guangxi Normal University, Ministry of Education Key Laboratory of Chemistry and Molecular Engineering of Medicinal Resource, Guilin). A voucher specimen (No. 20090903) has been deposited in the authors’ laboratory.
Extraction and IsolationThe dry rhizomes of C. rotundus (10 kg) were extracted three times under reflux with 95% aqueous ethanol (150 L×2 h). After removing the solvent under reduced pressure, the residue was suspended in water and then sequentially extracted with petroleum ether, CH2Cl2, EtOAc and n-BuOH. The n-BuOH extract (152 g) was submitted through a column chromatography (9×90 cm) of Diaion HP-20, eluting with H2O and CH3OH. The methanol fraction (98 g) was repeatedly chromatographed over normal phase silica gel and the eluting solutions were monitored by TLC to produce four fractions (Frs.1–4). Fraction 4 (11.3 g) was subjected to ODS column chromatography (8×30 cm) eluting with CH3OH–H2O (0 : 1–1 : 0) to obtain 5 fractions (Frs. A–E). Fraction D (6.6 g) was subjected to repeated silica gel column chromatography with CHCl3–MeOH–H2O (7 : 3 : 0.3) and then subjected to repeated ODS column chromatography with a MeOH–H2O (40 : 60–90 : 10) to yield 1 (21 mg) and 2 (18 mg).
Cyprotuoside C (1): white amorphous powder; [α]D25+71.2 (c 1.0, MeOH); IR vmax (KBr): 3346, 2929, 2354, 1721, 1650, 1451, 1384, and 1036 cm−1; HR-ESI-MS m/z: 1099.5303 [M+Na]+ (Calcd for C52H84O23Na, 1099.5301). 1H-NMR (400 MHz, C5D5N) and 13C-NMR (100 MHz, C5D5N) spectral data see Tables 1 and 2.
Cyprotuoside D (2): white amorphous powder; [α]D25+57.3 (c 1.0, MeOH); IR vmax (KBr): 3349, 2931, 2351, 1722, 1649, 1453, 1381, 1033 cm−1; HR-ESI-MS m/z: 1227.5779 [M+Na]+ (Calcd. for C58H92O26Na, 1227.5775). 1H-NMR (400 MHz, C5D5N) and 13C-NMR (100 MHz, C5D5N) spectral data see Tables 1 and 2.
Acid Hydrolysis of Compounds 1 and 2A solution (18 mg) of 1 in 1 M HCl (8 mL) was heated at 100°C for 3 h under an N2 atmosphere. After cooling, the reaction mixture was neutralized with Na2CO3, and then extracted with CHCl3 (3 ×10 mL). The CHCl3 phase was chromatographed on silica gel column eluting with CHCl3–CH3OH (9 : 1) to give compound 1a (9 mg). A solution of 2 (2 mg) in 1 M HCl (2 mL) was heated at 100°C for 3 h under an N2 atmosphere. The reaction mixture was diluted with H2O and extracted with CHCl3 (3×10 mL). The aqueous layer was concentrated to dryness to give a residue and dissolved in pyridine (1 mL), and then L-cysteine methyl ester hydrochloride (5 mg) was added to the solution. The mixture was heated at 60°C for 3 h, and trimethylchlorosilane (0.5 mL) was added, followed by heating at 60°C for 3 h. Finally, the solution was concentrated to dryness and taken up in water, followed by extraction with n-hexane (3×5 mL). The n-hexane fraction was analyzed by GC, and the retention times of the D-glucose, L-arabinose, D-xylose, D-glucuronic acid, and L-rhamnose standards were 8.96, 4.84, 6.08, 9.05, 7.31 min, respectively.
This study was supported by the National Natural Science Foundation of China (31400295), the China Spark Program (YQ2015109), Science and Technology Planning Project of Guangdong Province (2014A020221057), Distinguished Young Teachers in Higher Education of Guangdong (YQ2015109), Foundation of Education Bureau of Guangdong Province (2015KTSCX087, 2016KQNCX099), and Foundation for Youths of Lingnan Normal University (QL1401).
The authors declare no conflict of interest.
