Bioactive Protopanaxatriol Type Saponins Isolated from the Roots of Panax Notoginseng (Burk.) F. H. Chen

Seven new protopanaxatriol type saponins, 20S-sanchirhinosides A1 (1), A2 (2), A3 (3), A4 (4), A5 (5), and A6 (6), and sanchirhinoside B (7) were obtained as minor constituents from the root extract of Panax notoginseng (Burkill, F. H. Chen), which showed protection effects against antimycin A induced mitochondrial oxidative stress. Their structures were elucidated by chemical and spectroscopic methods (IR, HRESI-TOF-MS, 1D and 2D NMR). Among them, compounds 4, 6 and 7 showed significant protective effects against antimycin A-induced L6 cell injury.


Introdution
Reactive oxygen species (ROS) cause protein and DNA injuries and further induce pathological changes, such as heart failure [1], neuronal injury [2] and ischemia reperfusion [3]. A lot of natural products show potential ROS scavenging effects and are used as antioxidant agents.
Panax notoginseng (Burkill, F. H. Chen), have been cultivated in China for more than 400 years. As a traditional Chinese medicine, whose root components have several medicinal properties and are used for stenching the blood, dispersion of gore and reduction of the pain caused by blood diseases, etc. The main components in this plant were identified to be saponins, flavonoids, dencichine and polysaccharides [4]. During the course of our characterization studies on the bioactive constituents from the roots of P. notoginseng, the 70% EtOH extract showed significant protective effects against antimycin A-induced L6 cell injuries. Seven new protopanaxatriol type saponins: 20S-sanchirhinosides A 1 (1), A 2 (2), A 3 (3), A 4 (4), A 5 (5), and A 6 (6) and sanchirhinoside B (7) were obtained as minor constituents from it. In this paper, we report the protect effects of P. notoginseng 70% EtOH extract and new compounds 1-7 against antimycin A-induced mitochondrial oxidative stress.

Plant Material
The dried roots of P. notoginseng (Burkill, F. H. Chen) were collected from Wenshan, Guangxi province, China and identified by Dr. Li Tianxiang. The voucher specimen was deposited at the Academy of Traditional Chinese Medicine of Tianjin University of TCM (No. 20120505).

Mitochondrial Oxidative Stress Protect Effects Assay
Antimycin A was used to induce mitochondrial oxidative stress [11]. Briefly, L6 cells (Cell Resource Center, IBMS, CAMS/PUMC, Beijing, China) were plated at a density of 5 × 10 4 cells/well in Dulbecco's modified Eagle's medium (DMEM, Thermo Scientific, UT, USA) supplemented with 10% calf serum (Thermo Scientific) in a 96-well plate and were incubated at 37 °C for 24 h. Cells were treated with or without 10 μmol/L sample DMSO solution (final DMSO concentration was 0.5%). One hour later, medium was removed and 100 μg/mL antimycin A (Sigma Co. Ltd, MO, USA) in 200 μL DMEM was added to each well, The MTT assay was performed 24 h later to detect the cell survival rate. Probucol was used as positive control.

Statistical Analysis
Values are expressed as mean ± S.D. All the grouped data were statistically performed with SPSS 11.0. Significant differences between means were evaluated by one-way analysis of variance (ANOVA) and Tukey's Studentized range test was used for post hoc evaluations. p < 0.05 was considered to indicate statistical significance.

Conclusions
Antimycin A is known to cause the leakage of superoxide radicals from cell mitochondria by inhibiting mitochondrial electron transport [12]. Compared with normal group, 100 µg/mL antimycin A induced significant L6 cell injury, while 10 µM probucol showed increased cell survival rate effects compared with the antimycin treated group. From the bioactive 70% EtOH extract of P. notoginseng roots, seven new protopanaxatriol type saponins, 20S-sanchirhinosides A 1 -A 6 (1-6), and sanchirhinoside B (7) were obtained. Among the new compounds, 4, 6 and 7 showed significant protective effects against antimycin A-induced L6 cell injury. This research will benefit investigation of trace bioactive chemical constituents of P. notoginseng root. On the basis of the activity screening results, further studies of the antioxidant mechanisms of compounds 1-7 are necessary.