Polyphenols with Anti-Proliferative Activities from Penthorum Chinense Pursh

Two new polyphenols, penthorumin C (1) and 2,6-dihydroxyacetophenone-4-O-[4ꞌ,6ꞌ-(S)-hexahydroxydiphenoyl]-β-d-glucose (2), along with four known polyphenolic acids, pinocembrin-7-O-[4ꞌꞌ,6ꞌꞌ-hexahydroxydiphenoyl]-β-d-glucose(3), pinocembrin-7-O-[3ꞌꞌ-O-galloyl- 4ꞌꞌ,6ꞌꞌ-hexahydroxydiphenoyl]-β-d-glucose (4), thonningianin A (5), and thonningianin B (6) were isolated from Penthourm chinense. All compounds were evaluated for their anti-proliferative activity in HSC-T6 cells, and 2 and 5 showed significant activity, with IC50 values of 12.7 and 19.2 μM, respectively.


Introduction
Oxidative stress due to the imbalance in oxidant and antioxidant status has been implicated in the pathogenesis of several diseases, including cancer.Several papers have reported the role of free radical mediated oxidative damage in multi-stage events of carcinogenesis.Polyphenolic acids are a complex OPEN ACCESS group of compounds that have attracted attention in the last few years because of their widespread occurrence on plants and their significant biological activities [1,2].Polyphenols may have therapeutic health effects for a variety of chronic pathological conditions including cancer, neurodegenerative diseases, diabetes, and cardiovascular diseases due to the antioxidant activity.Previous reports have showed that polyphenolic acids are the inhibition of hepatic stellate cells proliferation, considering to be associated to liver fibrosis [3].
Penthorum chinense Pursh, (Saxifragaceae), is widely distributed in eastern Asia, (China, Japan, Korea, and eastern Russia) [4].In China, where it is both consumed as food and used in traditional Chinese medicine, whole plant products prepared from P. chinense is used to alleviate "heat"-associated disorders, detoxification, to promote circulation, and to treat liver problems, and to protect the spleen [5].P. chinense metabolites have shown anti-oxidant and antitumor activities [6][7][8][9].Flavonoids, triterpenoids, polyphenols, and lignans have been isolated from P. chinense [10].Two new polyphenols (compounds 1 and 2), along with four polyphenols (compounds 3-6) were isolated from the aerial parts of P. chinense (Figure 1).Herein, we describe the separation and structural characterization of these compounds, as well as their anti-proliferative activity in HSC-T6 cells induced by PDGF.

Structure Elucidation
An 80% ethanolic extract of dried P. chinense whole plant was suspended in distilled water and extract with petroleum ether, EtOAc and n-BuOH.The EtOAc soluble fraction was concentrated under reduced pressure to produce a residue that was subjected multiple chromatography, two new compounds 1 and 2 and four known compounds were isolated and identified.
Compound 1 was obtained as white powder.The molecular formula C 26 H 24 O 17 was deduced from the HERSIMS ion peak at m/z 609.1076 [M+Na] + (calcd m/z 631.0901), exhibiting fifteen degrees of hydrogen deficiency.Its IR spectrum contained characteristic absorptions for hydroxyl (3407.6 cm −1 ), carbonyl (1724.0cm −1 ) and aromatic ring (1619.9,1454.0 cm −1 ) moieties.Interpretation of the NMR spectra (Table 1) suggested the presence of a glucose (C-1 to C-6), and two galloyl (C-1ꞌ to C-7ꞌ; C-1ꞌꞌ to C-7ꞌꞌ) groups (Ikuko et al., 2000).The glucopyranose moiety was determined to have a β-configuration at C-1 with the large coupling constant of H-1 (J = 7.8 Hz).The remaining six carbons were assigned as one carbonyl (δ C 173.6), one methylene (δ C 35.0), two methines (δ C 55.7, 75.4) including one (δ C 75.4) connected to an oxygen atom, and one doubly oxygen-substituted quaternary carbon (δ C 116.8).The HMBC spectrum (Figure 2) exhibited long-range correlations from H 2 -4ꞌꞌꞌ to C-1ꞌꞌꞌ, C-2ꞌꞌꞌ, C-3ꞌꞌꞌ, and C-5ꞌꞌꞌ, consistent with a cyclopentyl ring.The presence of a 1,4,6-tri-O-substituted-β-glucopyranose was deduced from HMBC correlations from H-4 to C-7ꞌꞌ, from H-1 to C-7ꞌ, and from H 2 -6 to C-6ꞌꞌ.The HMBC correlations from H-1ꞌꞌꞌ to C-1ꞌꞌ, C-5ꞌꞌ, C-6ꞌꞌ and from H-1ꞌꞌꞌ, H-4ꞌꞌꞌ, H-5ꞌꞌꞌ to C-6ꞌꞌꞌ indicated that the cyclopentyl ring was connected to C-6ꞌꞌ and C-6ꞌꞌꞌ via C-1ꞌꞌꞌ/C-6ꞌꞌ and C-5ꞌꞌ/C-6ꞌꞌꞌ.The glucopyranose, substituted galloyl moieties, and an additional ring account for 15 degrees of hydrogen deficiency.On the basis of the molecular formula and the chemical shift of C-2ꞌꞌꞌ (δ C 116.8), the cyclopentyl ring was also connected to C-5ꞌꞌ via oxygen linkage to form the remaining ring [11].The absolute configuration of glucose was proposed to be D by comparison of its specific rotation, [α] D −21° (c 0.16, MeOH), with those reported for related compounds [12,13].The relative configuration of 1 was confirmed by observance of NOESY correlations (Figure 3), that were observed between H-3ꞌꞌꞌ to H-4ꞌꞌꞌ α, and H-5ꞌꞌꞌ, H-1ꞌꞌꞌ to H-4ꞌꞌꞌ β.These correlations suggested that H-3ꞌꞌꞌ was α-oriented and both H-5ꞌꞌꞌ and H-1ꞌꞌꞌ were both β-orientated.To minimize the energy of 1 the OH-2ꞌꞌꞌ was assigned as β-orientation.The structure of compound 1 (penthorumin C) was thus assigned as shown in Figure 1.

Cytotoxicity of Penthorum Chinense Extract
Penthorum.chinense extracts were evaluated on the HSC-T6 cells for culture periods of 24 h.P. chinense extract and four fractions (Pe, Et, n-BuOH, and W), showed little toxic effect under 80 μg/mL (Figure 4).The cytotoxicity of n-BuOH fraction was stronger than other fractions (P.chinense extract, Pe, Et, and W), and half of the cells were dead when the added concentration was up to 160 μg/mL.

Effect on PDGF Induced Proliferation
Based on the cytotoxic effect on HSC-T6 cells, we evaluated the anti-proliferative activity at 80 μg/mL.In order to figure out the active constituents of P. chinense, P. chinense extract was suspended in water and partitioned successively with petroleum ether (Pe), ethyl acetate (Et), n-BuOH(n-BuOH), and water (W), and these four fractions (Pe, Et, n-BuOH, and W) were tested their inhibitory activity, together with P. chinense extract (Figure 4).In comparison with other parts of P. chinense, Et fraction showed remarkable inhibitory activity, 78.6% at 80 μg/mL.The result suggested that the active ingredients of anti-proliferation may exhibit in Et part, and some isolation and purification methods had been further applied in Et part, which led to isolation of six polyphenols 1-6, including two new isolates 1 and 2. The anti-proliferative activities of 1-6 were evaluated in parallel experiments by measuring their inhibitory activities in PDGF induced HSC-T6.Compounds 2 and 5 displayed moderate inhibitory activity, with IC 50 values of 12.7, 19.2 μM, respectively vs. 6.36 μM for Colchicine, whereas the other compounds exhibited weak activity, with IC 50 values greater than 200 μM.

Cell Culture
An immortalized rat hepatic stellate cell line, HSC-T6 (obtained from the cell bank of the Chinese Academy of Science, Shanghai, China) was were batch cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), 100 IU mL −1 and streptomycin at 37 °C in a 5% CO 2 incubator.

Cell Viability
Compounds and P. chinense extract were dissolved in dimethylsulfoxiode (DMSO).Our preliminary study showed that DMSO at a final concentration of 0.1% in media did not affect the cell viability.HSC-T6 cells seeded for 24 h in 96-well plates were exposed to different concentrations of compounds and P. chinense extract.Different concentrations of compounds were carried out in the plate for 5 consecutive wells (final volume 100 μL) and incubated for 24 h.Cell viability was calculated as 100 × (absorbance of treated compound − absorbance of background light)/(absorbance of control − absorbance of background light).

Activation of HSC-T6 Cells Induced by PDGF
HSC-T6 cells were plated in a 96-well plate.Initially, cells were cultured with DMEM containing 10% FBS for 6 h.The medium was then replaced with DMEM without FBS to starve the cells for 12 h.The cells were then cultured with DMEM that contained 5.0 ng/mL PDGF (without FBS) for 24 h.

Anti-Proliferative Activity Assay
Activated HSC, which was induced by some mediators (TGF-β and PDGF, etc.), has been long considered to be associated with liver fibrosis, and inhibition for HSC growth has been proposed as a method for treating liver fibrosis [17,18].The anti-proliferative activity in PDGF induced HSC-T6 cell was assessed by the MTT assay [19].Inhibitory activity on cell proliferation was calculated as 100 × (absorbance of treated compound − absorbance of background light)/(absorbance of model − absorbance of background light).Data were expressed as the mean of the three independent experiments.Colchicine was used as a positive control.

Figure 4 .
Figure 4.The inhibitory activity of Penthorum chinense extracts and four fractional extractions on HSC-T6 cells.Values are mean ± SD, n = 6.