A New Furostanol Glycoside from Tribulus terrestris

Besides two known glycosides, a new furostanol glycoside was isolated from the Fruits of Tribulus terrestris L. The structure of the new furostanol glycoside was established as 26-O-β-D-glucopyranosyl-(25S)-5α-furostane-20(22)-en-12-one-3β, 26-diol-3-O-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-β-D-galactopyranoside (1) on the basis of 1D and 2D-NMR techniques, including COSY, HMBC, and HMQC correlations.


Introduction
Tribulus terrestris L. is an annual plant found around the world. Its fruits have been used in traditional Chinese medicine for treatment of eye problems, edema, abdominal distention, emission, morbid leucorrhea, sexual dysfunction and veiling. It also has been used as a medicine in India, South Africa and Japan. Some steroidal saponins have previously been isolated from this plant. Many pharmaceutical preparations and food supplements with these saponins as the active compound have OPEN ACCESS been commercially available. Examples of these are "Tribestane" and "Vitanone", which have been used to treat impotency, as well as "tribusaponins" and "Xin-nao-shu-tong", which have been used for the treatment of cardiovascular disease [1,2]. Recently, more than fifty steroidal saponins have been isolated from this plant [3][4][5][6][7][8][9][10][11][12][13]. In a preceding paper, we had reported the isolation and structural elucidation of three steroidal glycosides obtained from the fruits of this plant [14,15]. As a continuation to this study, we now describe the isolation and structural elucidation of a new furostanol glycoside obtained from the crude saponins of the fruits of T. terrestris.
Our investigation on the constituents in the ethanol extract of the plant led to the isolation of a new furostanol glycoside 1, along with two known constituents: chloromaloside A (2) [16] and 25(R, S)-5α- [17]. Herein, we describe the isolation and structure elucidation of the new compound 1.

Results and Discussion
Compound 1, isolated as a white powder, was deduced to possess a furostanol structure by the Ehrlich test.

General
The optical rotations were measured on a WZZ-15 autopolarimeter. The HRESIMS was recorded on IonSpec HiResESI FT-ICR and the ESIMS was recorded on LCQ-1700 ESIMS instrument. The NMR spectra were obtained on a Bruker AM-500 instrument, using TMS as internal standard. HPLC was carried out using a Waters 600 HPLC system (pump: waters 600 E power, a DDL-31 light scattering Detector detected at 128 C and a waters 486 UV Detector detected at 203nm) equipped with a Waters Novapak C 18 column (i.d. 25 × 100 mm and 8 × 100 mm) with a mobile phase flow rate of 10 mL min -1 for prep HPLC and 1 mL min -1 mobile phase for analysis, respectively. Column chromatography was carried out on silica gel (200-300 mesh Qingdao Marine Chemical Inc., P. R. China), and macroporous resin D 101 made by Tianjin Gel Co. (Tianjin, China). Reversed phase column chromatographic separations were carried out using ODS (200-300 mesh) provided by Fuji Chem. TLC was performed on silica gel plates (Kieselgel 60 F254, Merck) and RP C 18 silica gel plates (Merck). The spots on TLC were visualized by UV light (254/366 nm) and sprayed with 10% H 2 SO4, followed by heating.

Plant Material
The fruits of Tribulus terrestris L. were purchased from Jilin Medicinal Material Corporation, Changchun, China and identified by Professor Minglu Deng, Changchun College of Traditional Chinese Medicine. A voucher specimen (No.06091) has been deposited in the Herbarium of Academy of Traditional Chinese Medicine and Material Medica of Jilin Province.

Acid Hydrolysis of 1
Compound 1 (10 mg) were dissolved in 1 mol/L HCl in MeOH-H 2 O (1:1) and refluxed for 2 h. The reaction mixture was neutralized with NaHCO 3 . The water phase was chromatographed on the silica gel HPTLC with the system of n-BuOH-i-PrOH-H 2 O (10:5:4, homogenous), then the brown coloured spots were visualized by spraying with phenylamine-ortho-benzenedicarboxylic acid reagent followed by heating. The glucose, galactose and rhamnose were detected by comparison with the authentic samples.   (3), were isolated from the Fruits of T. terrestris. Many pharmaceutical preparations with these saponins as the active compound have been commercially available. Examples of these are "tribusaponins" and "Xin-nao-shu-tong", which have been used for the treatment of cardiovascular disease. This finding may provide a hint in the search for new and bioactive components from this plant.