HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 11, 2014, pp. 2619-2626
Received, 9th October, 2014, Accepted, 28th October, 2014, Published online, 30th October, 2014.
DOI: 10.3987/COM-14-13103

■ Two New Triterpene Saponins from the Tubers of Stachys sieboldii

Hyeon Kyung Cho, Chung Sub Kim, Won Se Suh, Ki Hyun Kim, and Kang Ro Lee*

Natural Products Laboratory, School of Pharmacy, Sungkyunkwan University, 300 Chunchun-dong, Jangan-ku, Suwon 440-746, Korea

Abstract

Chemical investigation of the MeOH extract of tubers of Stachys sieboldii MIQ. (Labiatae) led to two new triterpene saponins (1 and 2) named sieboldii saponin B and C, together with six known triterpenes (3-8). The structural elucidation of the new compounds was based on MS, 1H- and 13C-NMR, and 2D NMR analysis (1H-1H COSY, HMQC, HMBC, and NOESY) as well as acid hydrolysis. All the isolated compounds 1-8 were reported from this source for the first time.

Stachys sieboldii MIQ. (Labiatae) is widely distributed in North America, Asia, and Europe,1 and has been used for the treatment of various gastrointestinal problems, senile dementia, and ischemic stroke.2 Antioxidant, antitumor, and antimicrobial activities on this plant have been reported.3 However, only a few phytochemical studies on S. sieboldii have been reported. We have recently reported the isolation of triterpene saponins in the EtOAc and CHCl3-soluble layers from this plant.3 In continuing research on this source, two new triterpene saponins (1 and 2) and six known triterpene saponins (3-8) were isolated from the BuOH-soluble layer in the MeOH extract. The compounds 3-8 were reported from this source for the first time. The chemical structures of these new compounds were determined on the basis of 1D and 2D NMR spectroscopic data analysis (1H- and 13C-NMR, 1H-1H COSY, HMQC, HMBC, and NOESY), as well as chemical means.
Compound 1 was obtained as a colorless gum, and its molecular formula C42H68O15 was inferred from the positive ion HR-ESI-MS m/z 835.4457 [M + Na]+ (calcd. for 835.4456). The 1H-NMR spectrum of 1 (Table 1) showed the signals of olefinic proton at δH 5.47 (1H, br t, J = 5.9 Hz, H-12), oxygenated methine protons at δH 4.27 (1H, m, H-2), 3.73 (1H, m, H-3), and 3.53 (1H, d, J = 3.0 Hz, H-19), one methine proton at δH 2.06 (1H, d, J = 3.0 Hz, H-18), seven tertiary methyl protons at δH 1.49 (3H, s, H-27), 1.20 (3H, s, H-23), 1.15 (3H, s, H-29), 1.09 (3H, s, H-26), 0.97 (3H, s, H-25), 0.96 (3H, s, H-30), and 0.83 (3H, s, H-24), and two sugar anomeric protons at δH 6.26 (1H, d, J = 8.4 Hz, H-1′) and 5.75 (1H, d, J = 7.7 Hz, H-1′′). The 13C-NMR spectrum of 1 displayed a total of 42 carbon signals, of which 30 carbons were to be assigned to the aglycone and the remaining 12 carbons to the sugar moieties. The 13C-NMR and DEPT spectra included one carboxylic carbon at δC 177.2, seven methyl carbons at δC 29.3, 28.8, 24.6, 24.5, 22.0, 17.5, and 16.4, two olefinic carbons at δC 144.3 and 123.4, three oxygenated methine carbons at δC 80.9, 79.2, and 65.9, three methine carbons at δC 48.7, 48.0, and 44.4, eight methylene carbons at δC 42.5, 33.1, 32.5, 29.7, 28.5, 27.5, 24.0, and 18.4, six quaternary carbons at δC 46.3, 42.0, 40.2, 38.6, 38.6, and 35.3, as well as 12 remaining signals at δC 104.3, 78.9, 78.8, 75.7, 72.6, and 63.6, and at δC 93.6, 78.4, 78.1, 78.0, 70.4, and 61.7 assignable to two glucose moieties, respectively. From these data, compound 1 was presumed to be of ole-12-ene-28-oic acid glycosyl ester. The 1H and 13C NMR data of 1 were found to be very similar to those of rivaloside D4 with major differences in the downfield shift of C-2′ (δC 78.4 for 1; δC 74.0 for rivaloside D), indicating that 1 possessed a glucopyranosyl-(1→2)-glucopyranoside unit. The full NMR assignments and connectivities were determined by 1H-1H COSY, HMQC, and HMBC (Figure 2A). The sugar sequence was determined on the basis of 1D and 2D NMR spectrum. The positions of the glucoses were confirmed by the HMBC correlations of H-1′/C-28 and H-1′′/C-2′ (Figure 2A).

The relative stereochemistry of the aglycone was assumed to be similar with that of 2α,3α,19α-trihydroxy-olean-12-en-28-oic acid by comparing 13C-NMR5 and corroborated by NOESY cross-peaks of H-2/H-3 and H-25, H-3/H-24, H-5/H-9, H-9/H-27, H-24/H-25, H-25/H-26, H-18/H-19 and H-29, and H-19/H-29 (Figure 2B). Acid hydrolysis of 1 with 1 N HCl yielded 2α,3α,19α-trihydroxy-olean-12-en-28-oic acid and D-glucose {[α]+49.4° (c 0.04 in H2O)}. The anomeric configurations for two glucoses were defined as β for glucose from the coupling constant of 8.4 and 7.7 Hz.6 These data indicated the structure of 1 to be 28-O-[β-D-glucopyranosyl-(1→2)-β-D- glucopyranosyl]-2α,3α,19α-trihydroxy-olean-12-en-28-oic acid, named sieboldii saponin B.

Compound 2 was obtained as a colorless gum, and its molecular formula C42H66O15 was inferred from the positive ion HR-FAB-MS m/z 811.4480 [M + H]+ (calcd. for 811.4480). The 1H-NMR spectrum of 2 (Table 1) showed the signals of olefinic proton at δH 5.43 (1H, brt, J = 3.5 Hz, H-12), exomethylene protons at δH 4.76 (1H, br s, H-30a) and 4.71 (1H, br s, H-30b), oxygenated methine protons at δH 4.60 (1H, m, H-3) and 4.41 (1H, m, H-2), oxymethylene protons at δH 4.08 (1H, d, J = 10.9 Hz, H-24a) and 3.71 (1H, d, J = 10.9 Hz, H-24b), four tertiary methyl protons at δH 1.63 (3H, s, H-23), 1.06 (3H, s, H-27), 1.05 (3H, s, H-26), and 0.96 (3H, s, H-25), one secondary methyl proton at δH 1.02 (3H, d, J = 6.4 Hz, H-29), as well as two sugar anomeric protons at δH 6.15 (1H, d, J = 8.2 Hz, H-1′) and 5.63 (1H, d, J = 7.7 Hz, H-1′′). The 13C-NMR spectrum of 2 displayed a total of 42 carbon signals, 30 of which were assigned to the aglycone and the remaining 12 carbons to the sugar moieties. The 13C-NMR and DEPT spectra included one carboxylic carbon at δC 176.9, five methyl carbons at δC 25.1, 24.9, 18.7, 18.7, and 17.7, four olefinic carbons at δC 154.7, 139.6, 127.5, and 106.3, two oxygenated methine carbons at δC 75.5 and 67.5, one oxygenated methylene carbon at δC 66.4, four methine carbons at δC 56.8, 50.8, 49.5, and 38.8, eight methylene carbons at δC 44.6, 39.9, 33.8, 35.3, 30.5, 25.7, 25.3, and 20.1, five quaternary carbons at δC 49.8, 46.5, 43.8, 41.7, and 39.8, together with 12 remaining signals at δC 106.3, 80.6, 80.4, 77.4, 73.9, and 64.9, and at δC 95.0, 80.1, 79.6, 79.5, 72.1, and 63.5 assignable to two glucose moieties. The NMR data of 2 was similar to those of urs-12-en-28-oic acid,7,8 except for the presence of oxymethylene group and additional two glucose groups terminal double bond.

The relative stereochemistry of 2 was presumed to be similar with that of 2α,3α,24-trihydroxy-ursa-12,20(30)-dien-28-oic acid by comparing 13C-NMR,5 and was reconfirmed by NOESY cross-peaks of H-2/H-3 and H-25, H-3/H-24, H-5/H-9, H-9/H-27, H-24/H-25, H-25/H-26 and H-18/H-29 (Figure 2B). The anomeric configurations for two glucoses of 2 were confirmed by the same method as 1. And the positions of the glucoses were confirmed by the HMBC correlations of H-1′/C-28 and H-1′′/C-2′ (Figure 2B). Thus, the structure of 2 was established as 28-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl]-2α,3α,24-trihydroxy-ursa-12,20(30)-dien-28-oic acid,5 and named sieboldii saponin C.
The known compounds were identified as methyl 3-epimaslinate (3),9 2α,3α,24-trihydroxyolean-12- ene-28-oic acid (4),9 28-O-[β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl]-2α,3α,24-trihydroxy-olean- 12-ene-28-oic acid (5),5,10 28-O-β-D-glucopyranosyl-2α,3α,19α,24-tetrahydroxy-olean-12-en-28-oic acid (6),11 kaji-ichigoside F1 (7),12,13 and 2α,3α-dihydroxyursan-12-en-28-oic acid (8)9,14 by comparing their spectroscopic data with those in the literatures. The compounds 3-8 were reported from this source for the first time.

EXPERIMENTAL
General. HR-ESI-MS and HR-FAB-MS data were obtained on a JEOL JMS700 mass spectrometer. Preparative HPLC was conducted using a Gilson 306 pump with a Shodex refractive index detector and Econosil RP-C18 10 μ column (250×10 mm). RP-C18 silica gel (YMG GEL ODS-A, 12 nm, S-75 μm) and silica gel 60 (Merck, 70-230 mesh and 230-400 mesh) were used for column chromatography. TLC was performed using Merck precoated Silica gel F254 plates and RP-18 F254s plates. NMR spectra were recorded on a Varian UNITY INOVA 700 NMR spectrometer operating at 700 MHz (1H) and 175 MHz (13C) with chemical shifts given in ppm (δ). A Hewlett-Packard (HP) GC system 6890 Series equipped with a 5973 Mass Selective Detector (MSD) system. The system was controlled by the Enhanced ChemStation Version B.01.00 program. The capillary column used for GC was an Agilent J&W HP-5MS UI (30.0 m×0.25 mm i.d., 0.25 μm film thickness coated 5% diphenyl 95% dimethylpolysiloxane).

Plant material. The tubers of S. sieboldii were collected at Yecheon, Gyeongsangbuk-Do, Korea, in June 2012, and identified by one of the authors (K. R. Lee). A voucher specimen (SKKU-NPL 1211) was deposited in the herbarium of the School of Pharmacy, Sungkyunkwan University, Suwon, Korea.

Extraction and isolation. The tubers of S. sieboldii (5 kg) were extracted with 80% MeOH three times at room temperature to yield 1 kg of material. The resultant MeOH extracts were suspended in distilled water (800 mL x 3) and then successively partitioned with hexane, CHCl3, EtOAc, and n-BuOH, yielding residues weighing 7 g, 20 g, 12 g, and 24 g, respectively. The n-BuOH-soluble layer (24 g) was chromatographed on a Diaion HP-20 column eluting with a gradient solvent system consisting of 20% MeOH, 40% MeOH, 60% MeOH, 80% MeOH, and 100% MeOH to yield five subfractions (B1-B5). Fraction B3 (2.3 g) was purified using a silica gel (230-400 mesh, 50 g) column eluted with CHCl3-MeOH (5:1) to yield four fractions (B31-B34). Fraction B31 (47 mg) and B34 (60 mg) was purified by reversed-phase preparative HPLC with MeCN-H2O (3:7) and MeCN-H2O (33:67), respectively to obtain compounds 1 (7 mg, tR = 16.0 min) and 2 (3 mg, tR = 10.9 min). The CHCl3-soluble layer (20 g) was separated on RP-C18 silica gel open column (230-400 mesh, 550 g) eluting with a gradient solvent system of MeOH-H2O (1:1 and 1:0), yielding nine fractions (C1-C9). Fraction C4 (8 g) was separated on a RP-C18 silica gel with 80% MeOH and further separated by silica gel column using n-hexane-EtOAc (1:1) to give six fractions (C41-C46). Fraction C45 (280 mg) was purified by reversed-phase preparative HPLC using 75% MeCN to yield compound 3 (9 mg, tR = 20.0 min). Fraction C7 (273 mg) was chromatographed on RP-C18 silica gel open column with 80% MeOH and further separated by reversed-phase preparative HPLC using 60% MeCN to yield compound 4 (7 mg, tR = 15.3 min). The EtOAc-soluble layer (12 g) was loaded on RP-C18 silica gel open column and eluted with a gradient of MeOH-H2O (1:1 and 1:0) to yield eight fractions (E1-E8). Fraction E3 (1 g) was subjected to Lobar-A column using CHCl3-MeOH (20:1) as the eluant and then purified by reversed-phase preparative HPLC with MeOH-H2O (3:2) to give compound 6 (3 mg, tR = 15.8 min). Fraction E6 (75 mg) was separated on silica gel column with CHCl3-MeOH (20:1) and then purified by reversed-phase preparative HPLC using 40% MeCN to yield compound 5 (3 mg, tR = 18.0 min) and 7 (3 mg, tR = 18.8 min). Compound 8 (3 mg, tR = 12.4 min) was obtained from fraction E7 (70 mg) by using reversed-phase preparative HPLC (85% MeOH).
Sieboldii saponin B (1): colorless gum; [α]+11.2 (c 0.05, MeOH); IR (KBr) νmax 3420, 2938, 1738, 1378, 1209, 1056 cm−1; 1H (C5D5N, 700 MHz) and 13C-NMR (C5D5N, 175 MHz) see Table 1; HR-ESI-MS m/z 835.4457 [M + Na]+ (calcd. for 835.4456).

Sieboldii saponin C (2): colorless gum; [α]+8.7 (c 0.05, MeOH); IR (KBr) νmax 3420, 2936, 1740, 1368, 1216, 1055 cm−1; 1H (C5D5N, 700 MHz) and 13C-NMR (C5D5N, 175 MHz) see Table 1; HR-FAB-MS m/z 811.4480 [M + H]+ (calcd. for 811.4480).

Acid Hydrolysis of Compounds 1 and 2. Compound 1 (1 mg) was shaken with 2 mL of 1 N HCl for 1.5 h at 80 °C. After cooling, the hydrolysate was extracted with CHCl3 and the CHCl3 extract was evaporated in vacuo to yield 2α,3α,19α-trihydroxy-olean-12-en-28-oic acid5 as a colorless gum. The sugar in water layer was identified as D-glucose by co-TLC (EtOAc-MeOH-H2O=9:3:1, Rf value: 0.2) with D-glucose standard (Aldrich Co., U.S.A.). Compound 2 (1 mg) was treated in the same method to give 2α,3α,24-trihydroxy-ursa-12,20(30)-dien-28-oic acid5 and sugar.

Determination of the Sugar. The sugar obtained from the hydrolysis of compounds 1 and 2 was dissolved in anhydrous pyridine (0.1 mL) and L-cysteine methyl ester hydrochloride (2 mg) was added. The mixture was stirred at 60 °C for 1.5 h. After the reaction mixture was dried in vacuo, the residue was trimethylsilylated with 1-trimethylsilylimidazole (0.1 mL) for 2 h. The mixture was partitioned between n-hexane and H2O (0.3 mL each), and the organic layer (1 μL) was analyzed by GC-MS. The identification of D-glucose was detected by co-injection of the hydrolysate with standard silylated samples, giving single peaks at 16.414 min. Retention time of authentic sample treated in the same way with 1-trimethylsilylimidazole in pyridine was 16.328 min.

ACKNOWLEDGEMENTS
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2012R1A5A2A28671860). We are thankful to the Korea Basic Science Institute (KBSI) for the measurements of NMR and MS spectra.

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