Dammarane-type Triterpene Saponins from the Flowers of Panax notoginseng

Four new dammarane-type triterpene saponins named floranotoginsenosides A (1), B (2), C (3) and D (4), together with five known triterpene saponins, were isolated from the flowers of Panax notoginseng. Their structures were elucidated on the basis of spectral and chemical evidence.


Introduction
Root of Panax notoginseng (Burk) F. H. Chen (Araliaceae), commonly referred to notoginseng and "San-Qi", is a well-known medicinal herb, used historically in traditional Chinese medicine as a hemostatic agent that both invigorates and builds blood. The flowers of P. notoginseng have been used for treating hypertension, vertigo, tinnitus and laryngopharyngitis [1]. The saponin fraction of the notoginseng flowers has been proven to have hepatoprotective effect [2]. More than 60 dammaranetype saponins named ginsenosides, notoginsenosides and gypenosides have been hitherto isolated from this plant [3,4], among which about 20 saponins were obtained from its flower buds [5,6].
We herein report the isolation and structural elucidation of four new dammarane-type triterpene saponins 1-4, together with five known saponins 5-9 from the flowers of Panax notoginseng.

Results and Discussion
The water layer of methanolic extracts of notoginseng flowers was separated by MCI gel CHP 20P and ODS column chromatographies, and purified by preparative HPLC (ODS) to afford compounds 1-9. Compounds 5-9 were identified as gypenoside LXIX (5) [7], 3,12,20S-trihydroxy-25-hydroperoxydammar-23-ene-3-O-[-D-glucopyranosyl(1→2)--D-glucopyranosyl]-20-O-[-D-xylopyranosyl(1→6)]--D-glucopyranoside (6) [8], notoginsenoside FP 2 (7) [4], floraginsenoside O (8) [9] and gypenoside LXXI (9) [10], respectively, by comparison of their spectral data with those described in the literature. Compounds 7 and 8 were previously isolated from Panax notoginseng and P. ginseng, respectively, while compounds 5, 6 and 9 were isolated from Gynostemma pentaphyllum. In the 13 C-NMR spectrum of 1, 30 carbon signals due to the aglycone moiety were in good agreement with those of 5, especially the olefinic carbon signals ( C 123.8 and 141.9), and the oxygenated carbon signal ( C 71.5) of the side chain, suggesting that the aglycone of compound 1 is the same as that of 5, in which a double bond located at C-23, 24 and a hydroxyl group of notoginsenoside-C [12], except for the presence of signals for a -D-xylopyranosyl moiety and absence of signals for a-D-glucopyranosyl moiety. This suggested that 2 has the same aglycone and similar glycosylation manner with that of notoginsenoside-C with the only difference in the -Dxylopyranosyl moiety as the terminal sugar of C-20 in 2. Reduction of 2 by triphenylphosphine afforded gypenoside LXXI (9), which strongly supports the proposed structure. Thus the structure of floranotoginsenoside B was characterized as shown for 2 ( Figure 1).   13 C-NMR spectra closely resembled those of 3, especially the signals arising from the sugar moiety and the rings A-D, indicating that 4 is a also dammaranes bidemoside. In comparison of the 13 C-NMR spectra of 4 with that of 3, the upfield shift of C-24 (Δδ C -13.7) and C-26 (Δδ C -2.9), as well as the downfield shift of C-25 (Δδ C +3.4) were observed, indicating that a hydroxyl group is located at C-24 position in 4. This was supported by chemical shift comparison of H-24 ( H 4.42) and C-24 ( C 76.4) with that of 9 and related compounds with the same side chain structure [13]. When 3 was treated by triphenylphosphine, 4 was obtained. Therefore, the structure of floranotoginsenoside D was determined to be the formula 4 ( Figure 1).

General
Optical rotations were measured with a JASCO DIP-370 digital polarimeter. 1 H-and 13 C-NMR spectra were recorded on Varian Unity plus 500 and Varian Gemini 300 spectrometers. Coupling constants (J) are expressed in Hz, and chemical shifts are given on a  (ppm) scale with tetramethylsilane as an internal standard. MS were recorded on a JEOL JMS DX-303 spectrometer, and glycerol was used as a matrix for FAB-MS measurement. HR-ESI-MS was preformed on a Q-TOF mass spectrometer (Bruker Daltonics, MA, USA). Column chromatographies were performed with Kieselgel 60 (70 -230 mesh, Merck), MCI-gel CHP 20P (75 -150 mm, Mitsubishi Chemical Co. Ltd., Japan), Chromatorex ODS (100 -200 mesh, Fuji Silysia Chemical). Preparative HPLC (Shimadzu ODS) was performed on a Tosoh apparatus equipped with a CCPM solvent delivery system. Thin layer chromatography (TLC) was performed on precoated Kieselgel 60 F 254 plates (0.2 mm thick, Merck), and spots were detected by ultraviolet (UV) illumination and by spraying 10% sulfuric acid reagent.

Extraction and Isolation
Air-dried flowers of Panax notoginseng (800 g) purchased from a herbs market in Kunming, Yunnan Province, P.R. China in August, 1997 were extracted three times with methanol at room temperature. After removal of the solvent by evaporation in vacuo, the extract was suspended in water and extracted successively with Et 2 O, EtOAc, n-BuOH. The H 2 O layer (40.8 g of 168.5 g) was subjected to column chromatography over MCI-gel CHP 20P eluted from 0 % to 100 % MeOH to afford four fractions. Fraction-2 (3.15 g) eluted by (50-60 % MeOH) was chromatographed on MCIgel CHP 20P (eluted with 50 % to 80 % MeOH) and Chromatorex ODS (eluted from and 50 to 100 % MeOH) to afford a saponin fraction (0.89 g). This fraction were purified by preparative HPLC to give compounds 6 (37.7 mg), 2 (25.  Table 1.    Table 1. Reduction of 2 and 3: Triphenylphosphine (10 mg) was added to a solution of 2 (10 mg) in MeOH (5 ml). After stirring at room temperature for 4 hr, the solution was evaporated to dryness under reduced pressure, then subjected to silica gel chromatography (CHCl 3 -MeOH-Water: 8:2:0.2) to afford 9 (6.1 mg) whose 1 H-and 13 C-NMR spectral data are completely identical with those of gypenoside LXXI. Reduction of 3 (10 mg) in a manner similar to that described for 2 yielded floranotoginsenoside D (4, 5.6 mg).

Conclusions
Among four new darmmarane-type triterpene saponins isolated from notoginseng flowers, compounds 2 and 3 have a hydroperoxyl group. Although hydroperoxydammarane triterpene saponins have been previously isolated from the roots of Panax notoginseng [12], this was the first report of the isolation of this kind of saponins from the notoginseng flowers. These results may contribute to better understanding on the chemical characteristics of this medicinal herb.