Two new dammarane-type triterpenoid saponins from ginseng medicinal fungal substance

Abstract Two new triterpenoid saponins, namely ginsenoside Re8 (1) and notoginsenoside ST-8 (2), were isolated from ginseng medicinal fungal substance. Their structures were elucidated by spectroscopic data and chemical analysis.


Introduction
Panax ginseng C.A. Meyer is a distinguished tonic medicinal herb consumed worldwide. Undoubtedly, the saponin constituents, also known as ginsenosides, are the main bioactive ingredients responsible for the pharmacological efficacy of ginseng, and more than 120 saponins have been isolated and detected from ginseng (Yang et al. 2014;Xu, Yuan et al. 2015;Han et al. 2016;Zheng et al. 2016). However, rare ginsenosides present in low concentration or absent in P. ginseng often have better performances in bioactivity and bioavailability than others. Biotransformation was considered to be a highly efficient and regiospecific method to preparation of rare saponins . In our previous study, the ginseng medicinal fungal substance (GMFS) was obtained by the biotransformation of ginseng with microorganisms, which exhibited stronger antitumor activity than ginseng. In the course of exploring biologically active components from GMFS, triterpenoid saponins had been obtained . As a continuous study of this project, two new triterpenoid saponins, namely ginsenoside Re 8 (1) and notoginsenoside   (Figure 1), were isolated. Here, we report the isolation and structural elucidation of two new triterpenoid saponins 1 and 2.

General experimental procedure
HR-eSI-MS was recorded on a Q eXACTIVe (Thermo, America). 1D and 2D NMR spectra were taken on a Bruker AV 500 spectrometer (Bruker, Fllanden, Switzerland) with tetramethylsilane as the internal standard. GC analysis was performed on an Agilent GC-7820 A (FID detector) with a HP-5 column (0.32 mm × 30 m, 0.25 μm, Agilent, America). High-performance liquid chromatography (HPLC) was performed on Hitachi instrument (pump LC-2130, UV detector LC-2030) equipped with a YMC-Pack ODS-AM column (10 mm × 300 mm, 5 μm) with a flow rate 1.2 mL/min. Column chromatography was performed on silica gel (100-200 mesh, Qingdao Marine Chemical CO, Ltd, Qingdao, China). Thin layer chromatography (TLC) was performed on silica gel plates (GF 254 , Qingdao Marine Chemical CO, Ltd, Qingdao, China) and RP-18 F 254 (Merck). Fractions were monitored by TLC, and spots were visualized by spraying with 10% H 2 SO 4 in etOH, followed by heating at 105 °C.

Plant material
Ginseng was purchased from Ji Lin An Pharmaceutical Co, Ltd (Jilin province, China) on May 2012, and they were identified by professor Li-Li Weng of Changchun University of Chinese Medicine. A voucher specimen (No. RS05212012) was deposited at College of Pharmacy, Changchun University of Chinese Medicine. The GMFS was obtained by biotransformation of the ginseng with microorganisms.

Determination of absolute configuration of the sugar moieties in compounds 1 and 2
A solution of each saponin (4.0 mg each) in 1.0 M HCl (dioxane/H 2 O 1:1; 1 mL) was heated at 100 °C for 10 h in a H 2 O bath. After removal of dioxane under vacuum, the solution was extracted with etOAc (3 × 1 mL). The etOAc-soluble part of compound 2 was treated to determine the absolute configuration of C-24 in 2. The H 2 O-soluble part was concentrated on dryness, and subsequently the residue was dissolved in pyridine (each 2 mL). l-Cysteine methyl ester hydrochloride (2.0 mg each) was added to the pyridine solution. The mixture was kept at 60 °C for 1.5 h, dried under vacuum and trimethylsilylated with 1-(trimethylsilyl)-1H-imidazole (0.1 mL) at 60 °C for 1.0 h. The mixture was suspended in H 2 O (1 mL), then extracted with hexane (3 × 1 mL). The supernatant was analyzed by GC under the following conditions: capacity column, HP-5 (0.32 mm × 30 m, 0.25 μm); column temperature, 160 (4 °C/min), 200 °C (keep 5 min, 10 °C/min), 240 °C (keep 10 min); carrier gas, He at 1.0 mL/ min, split ratio 1/10; injection temperature, 270 °C; detection temperature, 280 °C; injection volume, 1.0 μL. Compounds 1 and 2 gave d-Glu and l-Rha at t R 18.42 and 15.41 min (identical to authentic standards), respectively.

Conclusions
Ginsenoside Re 8 (1) and notoginsenoside ST-8 (2), the two new triterpenoid saponins, were isolated from GMFS. Their structures were elucidated by a combination of 1D and 2D NMR, MS and chemical analysis. Biotransformation mechanism is under study.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the National Science and Technology Supporting Plan of China [grant number 2012BAI29B05].