5,6-Didehydroginsenosides from the Roots of Panax notoginseng

Two minor novel dammarane-type saponins—5,6-didehydroginsenoside Rd (1) and 5,6-didehydroginsenoside Rb1 (2)—were isolated from the dried roots of Panax notoginseng along with sixteen known saponins. The structures of the new compounds were elucidated on the basis of spectroscopic and chemical methods.


Introduction
Root of Panax notoginseng (Burk.) F. H. Chen (Araliaceae), also called Sanqi, is a well-known herb commonly used as a medicine and dietary supplement. It has been cultivated commercially in the southwest regions of China, especially in the Wenshan region, Yunnan Province. P. notoginseng has been used in China as a drug for the treatment of haemoptysis, haemostatic and haematoma for more than 400 years [1]. Current pharmacological studies revealed that P. notoginseng and its ingredients OPEN ACCESS possess anticarcinogenic [2,3], immunoregulatory [4], anti-inflammatory [5], anti-arrhythmic [6], hepatoprotective [7] properties, as well as protective effects on cardiovascular and cerebrovascular systems [1,[8][9]. Drammarane type saponins are considered as the major bioactive constitutes in P. notoginseng [1,[10][11][12].
During our studies on the screening of bioactive ingredients from medicinal herbs with activity against vascular inflammation, the glycosidic fraction from the roots of P. notoginseng was observed to reduce atherosclerotic lesions in apoE deficient mice, an effect which may be responsible for its inhibitory action on vascular inflammation [13]. During further study on this fraction, two novel triterpene saponins, 5,6-didehydroginsenoside Rd (1) and 5,6-didehydroginsenoside Rb1 (2) ( Figure  1), were isolated from the methanolic extract of the roots together with sixteen known dammaranetype saponins. We report herein the structure elucidation of these components.

General
ESI mass spectra were recorded on an LC-MSD trap VL mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). NMR spectra were recorded on a Bruker AV-400 spectrometer (C 5 D 5 N used as solvent and TMS as an internal standard). Column chromatography was performed with D-101 macroporous absorption resin (Haiguang Chemical Industrial Company, Tianjin, China) and silica gel (200-300 mesh, Qingdao Marine Chemical Group Co., Qingdao, China). Medium Pressure Liquid Chromatography (MPLC, Büchi, Switzerland) and Agilent 1100 Series prep-HPLC apparatus (Palo Alto, CA, USA) were also used for further isolation. For detection, HPLC was performed on an Agilent 1100 series HPLC apparatus. A Zorbax SB-C18 column (250 mm × 4.6 mm, I.D., 5 μm) and a Zorbax SB-C18 guard column (12.5 mm × 4.6 mm I.D., 5 µm) were used at a 25 ºC. The mobile phase consisted of water (A) and acetonitrile (B), the detection wavelength was set at 203 nm. D-Glucose and pyridine (Reagent Plus, ≥99%) were purchased from Sigma (St. Louis, MO, USA). Trifluoroacetic acid (TFA, 99%) was purchased from Riedel-de Haën (Seelze, Germany). GC-MS was performed on an Agilent 6890 gas chromatography instrument coupled with an Agilent 5973 mass spectrometer (Agilent Technologies, Palo Alto, CA, USA).

Plant Material
The roots of P. notoginseng were collected in Wenshan region, Yunnan province, China. The botanical origin of material was identified by Dr. Xiu-ming Cui, Wenshan Prefecture Sanqi Research Institute, Yunnan Province. The voucher specimen was deposited at the Institute of Chinese Medical Sciences, University of Macau, Macao, China.

Acidic Hydrolysis of Compounds 1 and 2
Each sample (1 mg) was hydrolyzed with 2 mol·L -1 TFA (1 mL) at 100 ºC for 2 h in a sealed glass tube with a screw cap which was filled with pure nitrogen gas. The hydrolyzed solution was evaporated to dryness under 45 ºC and then methanol (1 mL) was added for further evaporation and complete removal of TFA. The hydrolysate was treated with 1 mL hydroxylamine hydrochloridepyridine solution (20 mg·mL -1 ) at 90 ºC for 30 min in a sealed glass tube fitted with a screw cap. After cooling to room temperature, acetic anhydride (1 mL) was added and heating continued for another 30 min in the resealed tube. The cooled solution was evaporated to dryness under reduced pressure at 45 ºC. The residue was dissolved in dry chloroform (2 mL). The solution was filtered through a 0.45 μm syringe filter (Agilent Technologies) prior to injection into GC-MS system. GC-MS was carried out on a HP-5MS capillary column (30 m × 0.25 mm, i.d.) coated with 0.25 μm film 5% phenyl methyl siloxane. The column temperature was set at 175 ºC and held for 7 min, then programmed at 5 ºC·min -1 to 185 ºC and held for 5 min, then at 4 ºC·min -1 to 230 ºC. Split injection (2 μL) with a split ratio of 1:50 was applied. High purity helium was used as carrier gas with flow rate of 1.0 mL·min -1 . The mass spectrometer was operated in electron-impact (EI) mode, the scan range was 40-550 amu, the ionization energy was 70 eV and the scan rate was 2.89 s per scan. The inlet, ionization source temperature were 250 and 280 ºC, respectively. The same reaction and analysis were applied for standard sugar (D-glucose). The D-glucose derivative showed a peak at t R 12.5 min. As a result, Dglucose was detected from both 1 and 2.

Conclusions
In this study, the glycosidic fraction from the roots of P. notoginseng was investigated. Two novel saponins, 5,6-didehydroginsenoside Rd (1) and 5,6-didehydroginsenoside Rb1 (2), were isolated from the roots of P. notoginseng, along with sixteen known saponins. This result will be helpful to better understand the chemical components of P. notoginseng.