A new pregnane hexaglycoside from Adonis multiflora

Abstract

A new pregnane hexaglycoside, named amurensioside L (1), was isolated from the whole plant of Adonis multiflora. Amurensioside L (1) emerged to be new pregnane hexaglycoside which we fully spectroscopically characterized. The chemical structure of compound 1 was established as 14β,20R-epoxy-3β-hydroxypregna-4,6-dien-18-oic acid-20-lactone 3-O-β-d-glucopyranosyl-(1 → 4)-O-β-d-diginopyranosyl-(1 → 4)-O-β-d-cymaropyranosyl-(1 → 4)-O-β-d-cymaropyranosyl-(1 → 4)-O-β-d-oleandropyranosyl-(1 → 4)-O-β-d-oleandropyranoside on the basis of spectroscopic analyses including nuclear magnetic resonance spectrometry, high-resolution fast atom bombardment mass spectrometry, and infrared spectroscopy.

Introduction

Adonis is a genus of perennial flowering herbs of the crowfoot family, Ranunculaceae, native to Europe and Asia. Korean Adonis comprised three species, A. multiflora Nishikawa & Koki Ito, A. amurensis var. uniflora Makino, and A. pseudoamurensis Wang (Suh et al. 2002).

A. multiflora is native to Korea, Japan, and Manchuria, and grows only on Jeju Island in Korea. A. multiflora has been mainly cultivated for an ornamental purpose, so far. It is a spring ephemeral herb growing in temperate deciduous forests, and can reach 20–25 cm tall at flowering with up to four yellow flowers per stem. The leaves are smooth, and sepals are pale yellow, oval, oblong, cuneate, or elliptic (Lee et al. 2003). However, with exception of a report on its inhibitory effect on elastase and tyrosinase activity (Moon et al. 2010), there have been no phytochemical and biological report on A. multiflora. We previously used thin-layer chromatography (TLC) to confirm the presence of pregnane glycosides in ethanolic whole plant extracts of A. multiflora based on UV absorption patterns and characteristic dark blue colors produced by spraying plates with a 10 % H₂SO₄ solution followed by heating. In this study, we isolated and identified new pregnane glycosides present in whole plants of A. multiflora.

Materials and methods

Isolation of pregnane glycoside

Whole plants of A. multiflora were collected on the mountains of Jeju Island Korea. Dried whole plants of A. multiflora (1.5 kg) were extracted with 70 % aqueous EtOH at room temperature for 24 h. The concentrated EtOH extracts (106 g) were poured into H₂O (3 L) and successively extracted with CH₂Cl₂ (3 L × 3), AcOEt (3 L × 3), and n-BuOH (2.5 L × 3) to afford four fractions, namely, CH₂Cl₂ (AAC, 2.6 g), AcOEt (AAE, 0.7 g), n-BuOH (AAB, 12 g), and H₂O (AAW, 89.2 g). The AAC (2.6 g) was applied to column chromatography (c.c.) [silica gel (SiO₂), ϕ 4 × 11 cm, CH₂Cl₂–MeOH = 18:1 → 15:1 → 7:1, 1.6 l of each] yielding 16 fractions, AAC-1–AAC-16. Fr. AAC-7 [194 mg, elution volume/total volume (V _e/V _t) 0.17–0.12] was applied to octadecyl silica gel (ODS) c.c. (3 × 5 cm) and eluted with MeOH–H₂O (3:1, 1.2 l) to afford nine fractions along with a purified compound 1 [AAC-7-5, 78 mg, yield 0.0052 %, V _e/V _t 0.78–0.82, TLC (ODS) Rf 0.47, MeOH–H₂O = 7:1 (SiO₂) Rf 0.42, CHCl₃–MeOH = 10:1].

Compound: 1 white amorphous powder; [α]$_{D}^{25}$ + 2.1° (c 0.82, MeOH); infrared spectroscopy (IR, KBr, ν): 3420, 1730, and 1622 cm⁻¹; negative high-resolution fast atom bombardment mass spectrometry (HR/FAB/MS m/z 1223.6206 [M–H]⁻ (calcd. for C₆₂H₉₅O₂₄ 1223.6213)); ¹H and ¹³C nuclear magnetic resonance spectrometry (NMR) (see Table 1).

Table 1 ¹H- (400 MHz) and ¹³C-NMR (100 MHz) data of amurensioside L (1) and its acetate (1a) (CDCl₃)

Acetylation of amurensioside L.

Compound 1 (10 mg) was dissolved in pyridine (2 mL), added by acetic anhydride (2 mL) drop by drop in ice bath and stirred at room temperature for 8 h. The reaction mixture was poured in H₂O (30 mL) and extracted with AcOEt (30 mL × 3). The organic phase was successively washed with 5 % HCl solution (60 mL), a saturated NaHCO₃ (60 mL), and a saline water (60 mL). And the solution was dehydrated by pouring anhydrous MgSO₄, filtered, and concentrated in vacuo. The concentrates were purified by SiO₂ c.c. (Pasteur pipette, 1 × 7 cm, CHCl₃–MeOH = 18:1, 40 mL) to give an acetylated compound 1a [9.8 mg, TLC (SiO₂) Rf 0.85, CHCl₃–MeOH = 10:1].

Compound 1a: white amorphous powder; IR (KBr, ν): 3010, 1735, and 1625 cm⁻¹; ¹H and ¹³C NMR, see Table 1.

Results and discussion

EtOH extracts of the whole plants of A. multiflora were partitioned into CH₂Cl₂, AcOEt, n-BuOH, and H₂O fractions. The TLC analysis indicated that the pregnane glycosides were mainly included in CH₂Cl₂ fraction. The SiO₂ and ODS column chromatography for the CH₂Cl₂ resulted in the isolation of a new pregnane glycoside (1) with the isolation yield 0.0052 % from dry plant. The chemical structure was determined based on the spectroscopic data such as NMR, HR/FAB/MS, and IR spectra.

Compound 1 was isolated as a white amorphous powder, which showed UV absorption, and appeared as a dark blue spot on the TLC plates upon spraying with 10 % sulfuric acid followed by heating. The IR spectrum showed absorbance bands corresponding to hydroxyl (3420 cm⁻¹), ester (1730 cm⁻¹), and olefin (1622 cm⁻¹) groups. The molecular weight was determined to be 1224 from the molecular ion peak m/z 1223 [M–H]⁻ in the negative FAB-MS spectrum, and a molecular formula of C₆₂H₉₆O₂₄ was determined from the highly resolved molecular ion peak ([M–H]⁻, m/z 1223.6206, calc. for C₆₂H₉₅O₂₄, 1223.6213) in the negative HR-FAB-MS. The ¹H and ¹³C NMR spectra of compound 1 were very similar to those of a pregnane hexaglycoside previously isolated from A. amurensis, amurensioside F (Kuroda et al. 2010), with the exception of the presence of two double bonds instead of one. We also noted signals representative of one aldohexose and five 2,6-deoxy-3-methyl aldohexoses that appeared as six hemiacetals [δ_C 104.8, 101.6, 100.0, 98.5, 98.3, 98.2; δ_H 4.95 (br.d, J = 9.5 Hz), 4.79 (br.d, J = 9.6 Hz), 4.77 (d, J = 7.8 Hz), 4.64 (dd, J = 9.6, 1.6 Hz), 4.55 (dd, J = 9.6, 1.6 Hz), 4.38 (dd, J = 9.6, 1.6 Hz)], five methoxies [δ_C 57.9, 56.5, 56.5, 56.4, 56.2; δ_H 3.46 (s), 3.45 (s), 3.38 (s), 3.35 (s), 3.32 (s)], and five doublet methyls [δ_C 18.3, 18.2, 18.1, 18.1, 17.1; δ_H 1.27 (d, J = 6.0 Hz), 1.26 (d, J = 6.0 Hz), 1.21 (d, J = 0.0 Hz), 1.20 (d, J = 6.0 Hz), 1.17 (d, J = 6.0 Hz)]. An aglycone was observed, consisting of one ester carbon of a five-membered lactone ring (δ_C 175.6), one ketal carbon (δ_C 113.3), one olefin quaternary carbon (δ_C 143.8), three olefin methines [δ_C 129.4, 126.0, 124.6; δ_H 5.94 (dd, J = 10.0, 2.8 Hz), 5.67 (br.d. J = 10.0 Hz), 5.40 (br.s)], one oxygenated quaternary carbon (δ_C 89.9), one oxygenated methine (δ_C 74.5; δ_H 4.25, br.dd, J = 7.0, 2.0 Hz), and two singlet methyls [δ_C 17.4, 15.5; δ_H 1.59 (s), 0.94 (s)]. The complicated overlapping of NMR signals in the oxygenated and upfield regions prevented full identification of all signals, and thus acetylation of compound 1 was performed. The acetate 1a showed one single spot with higher Rf value than compound 1 on the SiO₂ TLC. In addition, no hydroxyl group absorption bands were observed in the IR spectrum. The NMR data of 1a such as six hemiacetals [δ_C 101.6, 100.4, 100.2, 98.6, 98.4, 98.4; δ_H 4.92 (br.d, J = 10.0 Hz), 4.74 (br.d, J = 8.8 Hz), 4.61 (br.d, J = 9.6 Hz), 4.52 (br.d, J = 9.6 Hz), 4.37 (d, J = 7.8 Hz), 4.36 (br.d, J = 9.5 Hz)], five methoxies [δ_C 59.1, 59.1, 56.6, 56.5, 56.0; δ_H 3.37 (s), 3.35 (s), 3.34 (s), 3.33 (s), 3.32 (s)], five methyls [δ_C 18.4, 18.4, 18.2, 18.1, 17.1; δ_H 1.25 (d, J = 6.4 Hz), 1.24 (d, J = 6.4 Hz), 1.23 (d, J = 7.8 Hz), 1.18 (d, J = 6.4 Hz), 1.14 (d, J = 6.4 Hz)], four acetyls [δ_C 169.1, 170.1, 169.2, 170.2, 20.7, 20.62, 20.60. 20.60; δ_H 2.04 (s), 1.99 (s), 1.969 (s), 1.965 (s)], and numerous methylenes and oxygenated methines due to the sugar moiety confirmed the sugar of 1a as 2,3,4,6-tetraacetyl-β-d-glucopyranosyl-(1 → 4)-O-β-d-diginopyranosyl-(1 → 4)-O-β-d-cymaropyranosyl-(1 → 4)-O-β-d-cymaropyranosyl-(1 → 4)-O-β-d-oleandropyranosyl-(1 → 4)-O-β-d-oleandropyranose. The key signals of the aglycone moiety were identified as one ester carbon (δ_C 175.9), one ketal carbon (δ_C 113.4), one olefin quaternary carbon (δ_C 143.8), three olefin methines [δ_C 124.7, 129.5, 126.1; δ_H 5.91 (dd, J = 10.0, 2.4 Hz), 5.62 (br.d, J = 10.8 Hz), 5.37 (br.s)], one oxygenated quaternary carbon (δ_C 90.0), one oxygenated methine [δ_C 74.6; δ_H 4.22 (br.dd, J = 7.1, 2.1 Hz)], and two singlet methyls [δ_C 17.5, 15.6; δ_H 1.55 (s), 0.90 (s)], indicating that compound 1a has one more double bond compared to the aglycone of amurensioside F, adonilide. The positions of the sugars and key functional groups were determined from the HMBC experiment. The correlations of H-1′/C-3; H-4/C-2, C-6, and C-10; H-6/C-8; H-7/C-5, C-8, and C-14; H-17/C-14; H-16 and H-21/C-20; and H-12b/C-18 in the HMBC spectrum of 1a confirmed the positions of the sugar, β-d-oleandropyranose, the double bonds, the ketal, and the ester of the lactone ring, as shown in Fig. 1. Therefore, 1a was identified as 2″″″, 3″″″, 4″″″, 6″″″-tetraacetyl-14β,20R-epoxy-3β-hydroxypregna-4,6-dien-18-oic acid-20-lactone 3-O-β-d-glucopyranosyl-(1 → 4)-O-β-d-diginopyranosyl-(1 → 4)-O-β-d-cymaropyranosyl-(1 → 4)-O-β-d-cymaropyranosyl-(1 → 4)-O-β-d-oleandropyranosyl-(1 → 4)-O-β-d-oleandropyranoside. All remaining carbon and major proton signals of compounds 1 and 1a were identified by analysis of the DEPT, ¹H–¹H COSY, NOESY, HSQC, and HMBC spectra (Table 1), as well as by comparison of the data with those of amurensioside F (Kuroda et al. 2010). Finally, the chemical structure of compound 1 was determined to be 14β,20R-epoxy-3β-hydroxypregna-4,6-dien-18-oic acid-20-lactone 3-O-β-d-glucopyranosyl-(1 → 4)-O-β-d-diginopyranosyl-(1 → 4)-O-β-d-cymaropyranosyl-(1 → 4)-O-β-d-cymaropyranosyl-(1 → 4)-O-β-d-oleandropyranosyl-(1 → 4)-O-β-d-oleandropyranoside, a new pregnane glycosides which we named amurensioside L.

Fig. 1

Chemical structure of amurensioside L (1) and its acetate (1a)

Pregnane glycosides have been reported to show a variety of biological activities such as antimicrobial activity, antioxidant (Reddy et al. 2013), and cytotoxic activity against HSC-2 (Kuroda et al. 2010).