Isolation and characterization of a novel oligomeric proanthocyanidin with significant anti-cancer activities from grape stems (Vitis vinifera)

Novel proanthocyanidin fractions from grape stem extracts were purified using Amberlite XAD-1180N, Sephadex-LH-20, Toyopearl HW40F and reverse phase high-performance liquid chromatography. Two key compounds were estimated as epigallocatechin-(epicatechin)7 gallate using electron-spray ionization time-of-flight mass spectrometry. Epigallocatechin-(epicatechin)7 gallate (compound 1) showed significant anti-cancer activity in PC-3 prostate cancer cells. In particular, compound 1 suppressed the gene expression of fatty acid-binding protein 5 (FABP5), which is involved in promoting cell proliferation and metastasis in prostate cancer cells.


Table of contents
The most active fraction, 60% acetone-eluted fraction (fraction 3) in Toyopearl HW40 chromatography was evaluated for bioactivity assessed by qPCR. Fraction 3 was further purified by reverse phase HPLC to get compound 1 and compound 2.             HPLC condition for supplementary Figure 11 and 12.
The crude sample of fraction 3 was diluted in MeCN (10 mg/ mL) and filtered through a 0.45 µm PTFE membrane filter prior to injection. 20 µL of sample was injected into an InertSustain C18 column (3 µm, 250 mm x 4.6 mm i,d.) and eluted with mobile phase A (0.1% formic acid) and B (0.1% formic acid in MeCN). The flow rate was 0.5 mL/min.
The crude sample of fraction 3 was diluted in MeCN (10 mg/ mL) and filtered through a 0.45 µm PTFE membrane filter prior to injection. 20 µL of sample was injected into an InertSustain C18 column (3 µm, 250 mm x 4.6 mm i,d.) and eluted with mobile phase A (0.1% formic acid) and B (0.1% formic acid in MeCN). The flow rate was 0.5 mL/min.

ESI-LC-TOFMS/MS
Liquid chromatography/mass spectra (LCMS) were acquired using a Waters Xevo QTOF mass spectrometer equipped with an Acquty UPLC HPLC system. The heated capillary and spray voltage were maintained at 250 o C and 4.5 kV, respectively. Nitrogen was operated at 80 psi for sheath gas flow rate and 20 psi for auxiliary gas flow rate. The full scan mass spectra from m/z 50-5000 were acquired in the positive ion mode with a scan speed of one scan per second. The MS/MS collision gas was helium with collision energy of 30% of the 5 V end-cap maximum ticking voltage. The purity of compound 1 was assumed to be about 90% from the eluates of the reverse phase HPLC at the final step of purification. And used directly for bioassays in the present study. each well, they were agitated by pipetting. The number of cells was measured with the hemocytometer. After treatment of PC-3 cells with test compound such as EGCG or compound 1 for 48 h, the cells were observed under the microscope and the number of cells was counted.

qPCR (quantitative real-time PCR).
Experimental procedure for bioassay by qPCR was carried out as described in the previous study 1 . Cells were plated in 6-well plates and allowed to reach 50% confluent growth.
The cells were treated with the indicated concentrations of test compounds such as compound 1 (30 µmol/L) for 48 h. Total RNA of these cells was extracted using the Plant RNA Purification Reagent (Invitrogen No. 12322-012), and 1 µg of total RNA was reverse transcribed into cDNA using the ReverTra Ace qPCR . qPCR analyses were performed with the StepOne Real-Time PCR system (Applied Biosystems) using THUNDERBIRD® SYBR® qPCR .
The sequences of the FABP5, GAPDH and 18S rRNA RPL27 primers for qPCR are as follows 1 :