Two New Oleanane-Type Triterpenoids from Platycodi Radix and Anti-proliferative Activity in HSC-T6 Cells

Two new oleanane-type triterpenoids, named platycodonoids A and B (1, 2), together with five known saponins, including platycodin D (3), deapioplatycodin D (4), 3-O-β-d-glucopyranosyl polygalacic acid (5), 3-O-β-d-glucopyranosyl platycodigenin (6) and polygalacin D (7), were isolated from the roots of Platycodon grandiflorum. On the basis of spectral data and chemical evidence, the structures of the new compounds were elucidated as 2β,3β,23,24-tetrahydroxy-28-nor-olean-12-en-16-one (1) and 2β,3β,23,24-tetrahydroxy-28-nor-olean-12-en-16-one-3-O-β-d-glucopyranoside (2). Compounds 1–7 were evaluated for their in vitro anti-proliferative activity against the HSC-T6 cell line.


OPEN ACCESS
antiobesity and glucose metabolism regulation [13][14][15][16][17], anti-atherosclerotic [18], and anti-hyperlipidemic [19] activities. Chemical investigation of Platycodi Radix revealed that triterpenoid saponins were the main chemical components, and more than 55 triterpenoid saponins have been isolated from Platycodi Radix to date [20,21]. Based on the structures of the aglycones, the triterpenoid saponins are classified into three types: the platycodigenin type, platycogenic acid A lactone type and polygalacic acid type [22]. It has been previously demonstrated that Platycodi Radix showed protective effects against acute ethanol, acetaminophen-, carbon tetrachloride-, thioacetamide and cholestasis-induced hepatotoxicity or hepatic injury in mice and inhibited the progress of hepatic fibrosis in rats [23][24][25][26][27][28][29][30]. In our preliminary pharmacological study, the 70% EtOH extract of Platycodi Radix was also found to exhibit significant protective activities against liver fibrosis in rats. In a continued effort to search for possible hepatoprotective component from this herb, an investigation of Platycodi Radix was undertaken, and this has led to the isolation of two new triterpenoids [an aglycone and its saponin, named platycodonoids A (1) and B (2) (Figure 1)], which possess a rare 28-nor-oleanane-type with a C-16 keto group in the aglycone structure. Besides, five known saponins were isolated and identified as platycodin D (3) [31], deapio-platycodin D (4) [21], 3-O-β-D-glucopyranosyl polygalacic acid (5) [32], 3-O-β-D-glucopyranosyl platycodigenin (6) [33] and polygalacin D (7) [32,34], by comparison of their IR, NMR and MS data with literature values. To the best of our knowledge, it was first time triterpenoids such a platycodonoids A and B having a 28-nor-oleanane-type skeleton are reported from the genus Platycodon.

Results and Discussion
The air-dried roots of P. Grandiflorum were extracted three times with 70% EtOH under reflux. The combined extract was chromatographed over a macroporous adsorbing resin column and partitioned as described in the Experimental section. After repeated column chromatography, two new compounds 1 and 2 and five known saponins 3-7 were isolated and identified.

Scheme 1. The plausible biogenetic origin of compounds 1 and 2.
Compounds 1-7 were evaluated for their anti-proliferative activities against the Hepatic Stellate Cell (HSC)-T6 line using the 3-(4,5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT) assay [36]. Colchicine was used as positive control in this study (IC 50 value < 10 μM). Among the tested compounds, compounds 1, 3, 4 and 7 were the most potent, showing IC 50 values of 5.27, 1.77, 8.24 and 1.04 μM, respectively. The IC 50 values for the remaing compounds, 2, 5 and 6, were 69.63, 8150.23, and 13.36 μM, respectively. It was reported that saponins from Platycodi Radix prevented the increase in the serum levels of hepatic enzyme markers (alanine aminotransferase and aspartate aminotransferase) and reduced oxidative stress, such as glutathione content and lipid peroxidation, in the liver in a dose-dependent manner [23][24][25][26][27][28][29][30]. The reason why compounds 1, 3, 4 and 7 delayed the formation of liver fibrosis need to be further studied. In the structure-activity relationship of these oleanane-type triterpenoids, the presence of a free carboxyl functional group at C-28 seemed not to be related to the hepatoprotective activity (compounds 5 and 6). When forming C-28 glycosides, the presence of the glycosides affected the activity, and the number of monosaccharides in the sugar moiety increased the activity (compounds 3, 4 and 7). In contrast, when came to the 28-nor-oleanane-type triterpenoids 1 and 2, the aglycone was more active than its corresponding glycoside.

Plant Material
Platycodi Radix was collected from Taihe

Acid Hydrolysis of Compound 2
Compound 2 (3.0 mg) was refluxed with 1M HCl (dioxane-H 2 O, 1:1, 2 mL) at 90 °C for 3 h in a water bath. After dioxane was removed, the solution was extracted with EtOAc (2 mL × 3 times). After evaporating to dryness, the monosaccharide portion was analyzed by gas chromatography after conversion of the hydrolysates into corresponding alditol acetates. Only D-glucose was detected. The EtOAc portion was washed with H 2 O and evaporated to yield the aglycone. The aglycone was identified by TLC together with compound 1.

In Vitro Inhibitory Activity on Cell Proliferation
Tested compounds 17 were dissolved in DMSO (final concertration, 0.1%). Inhibitory activity of compounds 17 against HSC-T6 cell line was evaluated by the MTT assay [36]. Briefly, cells at the exponential growth phase were harvested and seeded into a flatbottom 96-well plate. A total of 90 μL containing 5 × 10 4 cells was added to each well of the plate and incubated for 24 h in a 5% humidified CO 2 at 37 C. HSC-T6 cells were treated with vehicle or compounds at concerntration of 0.01, 0.1, 1, 10, 100 and 1000 μg/mL. After 48 h of incubation at 37 C, 20 μL/well, MTT was then added and the plate was again incubated at 37 C for 4 h. Reduction of MTT to formazan was measured in an ELISA plate reader at 570 nm. Inhibitory activity of compounds 17 on cell proliferation (% of control) was calculated as 100 × (absorbance of treated compound-absorbance of background light)/(absorbance of control-absorbance of background light). Data were expressed as the mean of the three independent experiment. Colchicin was used as a positive control.