A new 3,4-seco-lupane triterpenene glycosyl ester from the leaves of Eleutherococcus sessiliflorus

Abstract A new minor 3,4-seco-lupane triterpenene glycosyl ester, named sessiloside-A1 (1), along with three known 3,4-seco-lupane triterpenenes were isolated from the which alcohol extract of the leaves of Eleutherococcus sessiliflorus (Rupr. & Maxim.) S.Y. Hu by silica gel column chromatography, and their structures were determined by spectroscopic methods (UV, IR, NMR and HRMS). Compound 1 was elucidated to be β-D-glucopyranosyl ester of chiisanogenin. At the same time, a new efficient two-step enzymatic hydrolysis method was established to transform chiisanoside (2) → divaroside (3) → 1. Graphical Abstract


Introduction
Eleutherococcus species were widely distributed in Northeast China, Japan and the Korean peninsula. Traditionally, they have been used as treatment for several diseases such as diabetes, tumors, and rheumatoid arthritis (Bian et al. 2017). Especially the stems, leaves and fruits of E. sessiliflorus (Rupr. & Maxim.) S.Y. Hu that is a synonym of Acanthopanax sessiliflorus (Rupr. & Maxim.) Seem are safe and non-tox, which were approved as a new food raw material by the Chinese Ministry of health in 2008. E. sessiliflorus is rich in secondary metabolites of 3,4-seco-lupane triterpenenes, of which the most studied is chiisanoside. As the main active ingredient, chiisanoside exhibits a series of significant pharmacological effects, such as antidepressant, against acute liver injury, anti-cancer and so on (Bian et al 2018).
However, the content of 1 in the leaves of E. sessiliflorus is approximately 0.07-0.11%. Previous studies have found that enzymatic hydrolysis can efficiently convert glycosides into secondary glycosides and their aglycones, and has been widely used in the extraction and separation of ginsenosides (Upadhyaya et al. 2016;Fu et al. 2017). Therefore, we further studied the enzymatic hydrolysis of 2 to get the minor glycoside 1.

Results and discussion
Repeated column chromatography of the ethanol extract of the leaves of E. sessiliflorus led to the isolation of compound 1-4. Comparison of the NMR data with reported values led to identification of known compounds as chiisanoside (2), divaroside (3), chiisanogenin (4). The structure of new compound 1 was elucidated by UV, IR, NMR and HRMS.
Compound 1 was obtained as white needles (ethanol  absorption spectrum (KBr, max ) of compound 1 shows an absorption at 3412 cm À1 indicates the existence of hydroxyl (-OH), the signals at 2956-2884 cm À1 reveal the saturated carbon-hydrogen stretching vibrations, 1749 and 1713 cm À1 indicate carbonyl stretching vibrations, 1641 cm À1 demonstrates terminal olefin carbon-carbon double bond stretching vibration, 1377 cm À1 demonstrates symmetric deformational vibration of methyl, and 1068 cm À1 reveals carbon-oxygen stretching vibration. The 1 H and 13 C NMR spectra of 1 (Table S1) also revealed the presence of one glucosyl unit, and were similar to those of 3, with the exception of proton and carbon resonances for the lack of a sugar moiety. The carbon signal for C-6' (d C 62.20 ppm) shifted 6. 8 ppm downfield compared with 3, suggesting that 1 was lack of a glycosyl compared with 3 at C-6'. As for the glucosyl unit, an anomeric proton signal at d H 6.44 ppm (d, J ¼ 8.1 Hz) and a carbon signal at d C 95.55 ppm (C-1') as well as the oxygenated methine and methylene carbon signals at d C 79.56 (C-3'), 78.95 (C-5'), 74.36 (C-2'), 71. 11 (C-4'), and 62.20 ppm (C-6'), indicating the presence of a b-D-glucopyranosyl group. The connectivity between the glucopyranosyl unit (C-1') and the C-28 of the aglycone was verified by the presence of a cross-peak between d H 6.44 (C-1') and d C 175.00 (C-28) in the HMBC spectrum as well as the chemical shift of the anomeric carbon signal d C 95.55 (C-1'). Therefore, the structure of 1 was elucidated to be 1a,11a-dihydroxy-3, 4-seco-lupa-4(23),20(30)-diene-3,28-dioic acid 3,11-lactone 28-O-b-D-glucopyranoside. All carbon signals and hydrogen signals were assigned (Table S1), and the correctness of structure analysis was further verified according to the HMQC and HMBC spectra. At the same time, a new efficient two-step enzymatic hydrolysis method was established to transform 2 ! 3 ! 1. Using 50% ethanol aqueous solution (pH 4.3) as solvent, 2 was hydrolysed into 3 by rhamnosidase at 25 C and the conversion rate within 1 week was about 52%. Using 50% ethanol aqueous solution (pH 4.3) as solvent, 3 was hydrolysed into 1 by b-glucosidase at 40 C, and the conversion rate within 3 h was about 98%.

Conclusion
A new minor 3,4-seco-lupane triterpenene glycosyl ester, named sessiloside-A1 (1), alongwith three known 3,4-seco-lupane triterpenenes were isolated from the leaves of E. sessiliflorus, and their structures were determined by using spectroscopic methods. At the same time, a new efficient two-step enzymatic hydrolysis method was established to transform 2 ! 3 ! 1.