Identification and characterization of oligomeric proanthocyanidins with significant anti-cancer activity in adzuki beans (Vigna angularis)

The aim of the present study was to characterize and evaluate the anti-cancer activity of proanthocyanidin-enriched fractions from adzuki beans. For this purpose, we concentrated proanthocyanidins from adzuki beans (Vigna angularis) into five fractions using Amberlite XAD-1180N, Toyopearl HW40F, and Sepacore C-18 reverse-phase flash column chromatography. Proanthocyanidin-enriched fractions were characterized as (epi)catechin hexamer, heptamer, and octamer, epigallocatechin-(epi)catechin pentamer, and epigallocatechin-(epi)catechin hexamer using electrospray ionization time-of-flight mass spectrometry and thiolytic degradation. These fractions showed significant anti-cancer activity against the human PC-3 prostate cancer cell line. They also significantly suppressed the expression of the fatty acid-binding protein 5 gene, which plays critical roles in cell growth and metastasis in prostate cancer.


Introduction
Adzuki beans (Vigna angularis) are an important legume that are used in traditional foods such as confectionaries (e.g., Wagashi, Manju, and Yokan) in Japan. They are cultivated in Japan and other East Asian countries. Red adzuki beans have strong antioxidant activity, and have been used in traditional Chinese medicines for various purposes [1]. The main pigments in adzuki beans are proanthocyanidins [1,2]. Proanthocyanidins are a class of oligomeric or polymeric phenol compounds that contain flavan-3-ol units. They show a wide variety of biological activities such as suppression of endothelin 1 synthesis [3], anti-inflammatory activity [4], and inhibition of cancer cell proliferation [5,6,7,8,9,10]. Recently, we reported that epicatechin pentamer and hexamer synthesized in our laboratory inhibited cancer metastasis and invasion [11]. The structure of proanthocyanidin consists of a catechin and/or epicatechin polymer linked through 4-8 or 4-6 interflavan bonds. Sometimes these compounds contain epigallocatechin and/or gallate. These compounds showed significant cytotoxic activity against the human PC-3 prostate cancer cell line. Moreover, they suppressed the expression of the cancer-promoting gene, fatty acid-binding protein 5 (FABP5) [12,13].
Proanthocyanidin-enriched fractions from with significant anticancer activity in adzuki bean have not been so far concentrated and characterized. Therefore, the aim of this work was to characterize and evaluate the anti-cancer activity of proanthocyanidin-enriched fractions from adzuki beans. For these purposes, we concentrated proanthocyanidins using gel filtration chromatography and analyses by highperformance liquid chromatography (HPLC).

Materials
Amberlite XAD-1180N was purchased from Organo Corporation (Tokyo, Japan), and Toyopearl HW 40F from Tosoh Corporation (Tokyo, Japan). Adzuki beans were purchased from Imuraya Co. Ltd, Japan in 2015. Standard samples of catechin tetramer and pentamer, and epicatechin dimer to hexamer were synthesized in our laboratory [11].

Instruments
1 H and 13 C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX 500 spectrometer at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR. Chemical shifts (δ ppm) in the 1 H NMR spectra are expressed relative to the signal of tetramethylsilane adjusted to 0.00 ppm, and coupling constants are expressed in hertz. Liquid chromatography-mass spectrometry (LC-MS) was performed using a Waters Xevo QTOF (quadrupole time-of-flight) mass spectrometer and an ACQUITY UPLC (ultra-performance liquid chromatography) system.

HPLC method for separation of polyphenolic compounds
The crude sample was diluted in acetonitrile (10 mg/mL) and filtered through a 0.45-μm PTFE membrane filter before injection. Twenty microliters of sample were injected into the HPLC column (InertSustain C18; 250 mm Â 4.6 mm i.d., 5 μm particle size) and eluted with mobile phase A (0.2% acetic acid) and phase B (acetonitrile). The flow rate was 0.2 mL/min and the column temperature was 30 C. A gradient elution at 10 min intervals was performed as follows: 0-10 min, 100% A; 10-30 min, 94% A and 6% B; 30-45 min, 80% A and 20% B; and 45-60 min, 100% B.

UPLC-ESI-TOFMS analysis
LC-MS was performed using a Waters Xevo QTOF mass spectrometer equipped with an ACQUITY UPLC system. The heated capillary and spray voltage were maintained at 300 C and 3.0 kV, respectively. Nitrogen was used at 80 psi for the sheath gas and 20 psi for the auxiliary gas. Full scan mass spectra in the range m/z 100-5000 were acquired in positive ion mode with a scan speed of one scan per second. The tandem mass spectrometry collision gas was helium with a collision energy of 30% of the 5 V end-cap maximum ticking voltage.
2.6. Thiolytic degradation of Fr. 3 in Sepacore C18 reverse-phase column chromatography and LC-MS analysis Constitutional flavan-3-ol units of Fr. 3 were analyzed using a HPLC-DAD-ESI-MS (HPLC-diode-array-detection (DAD)-electrospray ionization (ESI)-mass spectrometry (MS)) system after thiolytic degradation, which was performed according to the method of Hamauzu et al. [14]. In a screw-cap test tube, an aliquot (100 μL) of a methanolic solution of Fr. 3 was mixed with 100 μL of methanol containing 0.30 mol/L HCl and 10% (w/v) 2-mercaptoethylamine hydrochloride (cysteamine hydrochloride). The mixture was incubated for 30 min at 75 C. To assess the occurrence of isomerization, reaction at 50 C for 90 min was also performed. The reaction mixture was added to two times its volume of water, filtered using a 0.45-μm membrane filter, and analyzed using an ACQUITY UPLC separation module coupled with a Waters Micromass Q-micro MS(/MS) system that was controlled with MassLynx v4.1 software (Waters, Milford, MA). The column was a Phenomenex Luna C 18 (150 Â 4.6 mm, 5 μm particle size) with a security guard cartridge (4.0 Â 3.0 mm). The solvents used were: (A) 0.5% (volume fraction) formic acid in 5% (volume fraction) acetonitrile, and (B) 0.5% (volume fraction) formic acid and 20% (volume fraction) 2-propanol in acetonitrile. The gradient program started with 0% solvent B (0-1 min), and was slowly increased to 5% solvent B at 10 min, 5% B at 15 min, 40% B at 40 min, and 90% B at 50 min. The flow rate was 0.5 mL/min. The MS was performed in negative ion mode to analyze the thiolytically degraded mixture with a capillary voltage of À3.0 kV and nebulizer gas (N 2 ) temperature of 350 C (gas flow rate of 600 L/h). The cone voltage was set at 25 V. Mass spectra were scanned over the m/z range 100-1000. Analysis in positive ion mode was also conducted for confirmation. Thiolysis of procyanidin B2, a dimer of (À)-epicatechin was conducted to confirm the retention time of (À)-epicatechin cysteamine thioether. The peak area obtained at 280 nm was used to calculate the mean degree of polymerization (mDP) because addition of cysteamine does not affect the absorbance.

Cell lines, cell culture, and reagents
Human prostate cancer cell line PC-3 was purchased from the Health Science Research Resources Bank (Osaka, Japan). The cells were maintained in a monolayer culture at 37 C and 5% CO 2 in RPMI-1640 (R8755, Sigma-Aldrich) supplemented with 10% charcoal-stripped fetal bovine serum (No. 04-201-1, Biological Industries) and 1% antibioticantimycotic mixed stock solution (No. 09366-44, Nacalai Tesque). The cells were treated with various concentrations of grape stem extract for 48 h.

Viable cell count assay
The degree of cell proliferation was evaluated with Cell Count Reagent SF (Nacalai Tesque, No. 07553-15) according to the manufacturer's instructions. The cells were plated in 96-well plates and treated with the indicated concentrations of test compounds derived from adzuki beans extract for 48 h. The absorbance at 450 nm was measured using a microplate reader after the addition of the Cell Count Reagent.

Quantitative real-time PCR (qPCR)
Cells were plated in six-well plates and grown to 50% confluence. The cells were treated with the indicated concentrations of test compounds for 48 h. Total RNA was extracted from these cells 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 quantitative real-time PCR (qPCR) RT Master Mix (Toyobo No. FSQ-301). qPCR analyses were performed with the StepOne Real-Time PCR system (Applied Biosystems/Thermo Fisher Scientific) using THUNDERBIRD SYBR qPCR Mix (Toyobo No. QPS-201).

Measurement of apoptosis by assay for caspase-3 activity
Assays for caspase-3 activity were carried out using BD Cytofix/ Cytoperm™ Kit (BD Biosciences, No. 554714), according to the manufacture's protocol. Purified rabbit anti-active caspase-3 (BD Phar-mingen™, No. 559565) was used as the first antibody (1 st Ab) and FITC-conjugate anti-rabbit Ig G (Jackson ImmunoResearch, No. 711-096-152) was used for the second antibody (2 nd Ab). Briefly, after treatment of cells with 1% w/v ABE or 500 nmol/L of carnitine palmitoyltransferase (CPT) for 48 h, the cells were collected and prepared by the same method as described in cell cycle analysis. The cells were diluted in PBS and fixed with BD Cytofix/Cytoperm Fixation and Permilization Solution on ice in the dark for 20 min. The cells were washed with the washing buffer and then reacted with 1 st Ab at room temperature. Next, the cells were washed with the washing buffer and then reacted with 2 nd Ab at room temperature. After the reaction, the cells were diluted in PBS, and flow cytometry was performed with FACSCalibur flow cytometer (Becton Dickinson, Japan), and the data obtained were analyzed utilizing Cell Quest Software. For each sample, 1 Â 10 4 cells were recorded.

Cell cycle analysis
Effects of test compounds on the phase distribution of cell cycle were assessed by flow cytometry. Briefly, after treatment of cells with 1% w/v ABE or the control (vehicle) for 48 h, floated cells were discarded by aspiration and the attached cells were trypsinized and thereafter washed twice with cold PBS, and centrifuged. The pellets were used for sample preparation for flow cytometry using BD Cycletest Plus DNA Reagent Kit (BD Biosciences, No.340242), according to the manufacturer's protocol. Flow cytometry was performed with FACSCalibur flow cytometer (Becton Dickinson, Japan), and the data were analyzed utilizing Cell Quest software. For each sample, 1 Â 10 4 cells were recorded.

Statistical analysis
GraphPad Prism 7 was used for statistical analysis. Data are expressed as means AE standard deviations. Comparisons between two groups were performed with two-tailed Student's t-tests. For comparisons of more than two groups, one-way analysis of variance (ANOVA) with Dunnett's post-hoc test or two-way ANOVA with Sidak's post-hoc test was used. Statistical significance is indicated by *P < 0.05 or **P < 0.01.

Concentration of proanthocyanidins using gel filtration chromatography
The main aims of this research were characterization and evaluation of the antitumor activity of proanthocyanidin-enriched fractions from the adzuki beans. To the best of our knowledge, this is the first study on characterization of proanthocyanidin-enriched fractions from adzuki beans, which have structures ranging from epicatechin trimer to  nonamer and can contain the pyrogallol moiety. The purification scheme for the water extracts at room temperature using gel filtration chromatography is shown in Fig. 1. The use of Amberlite XAD-1180N for the first stage and Toyopearl HW 40F before HPLC purification allowed for purification of higher oligomerized proanthocyanidins. The HPLC results for the ethyl acetate-eluted fraction from the Amberlite XAD-1180N resin are shown in Fig. 2. We identified catechin glucopyranoside, (epi)catechin dimer, procyanidin B4, arecatannin A1, arecatannin A2, (epi)catechin nonamer, piceid, and dehydroquercetin rhamnoside. These compounds were identified using ESI-TOFMS, NMR, and by comparison with spectra of known compounds or synthesized procyanidin B2, B4 [15], arecatannin A1, and arecatannin A2 [11,16]. This fraction did not show significant cytotoxic activity or suppression of the expression of FABP5.
Next, we evaluated the methanol eluted fraction from the Amberlite XAD-1180N resin because this fraction showed significant activity (Fig. 3).
We further purified this fraction using Toyopearl HW40F to afford three fractions. Fraction 3 (60% acetone-eluted fraction from Toyopearl HW 40F, called as ABE) (Fig. 3) showed significant cytotoxic activity, and we further purified this fraction using flash chromatography with a Sepacore C18 reverse-phase column to afford five fractions (Fig. 4).

Thiolytic degradation products
Thiolysis of Fr. 3 in the Sepacore C18 reverse-phase column revealed that the main degradation product was (À)-epicatechin cysteamine thioether (peak 2 in Fig. 1), which generated a molecular ion at m/z 364 ([M -H] À ) and gave a fragment ion at m/z 287 in negative mode. This characteristic fragmentation of (À)-epicatechin cysteamine thioether has been reported by Selga et al. [19]. (þ)-Catechin cysteamine thioether (peak 1, [M -H] À at m/z 364) was also detected as a product corresponding to the extension unit, although it was present at a lower level than epicatechin cysteamine thioether. (þ)-Catechin and (À)-epicatechin (peaks 3 and 4, respectively, [M-H] À at m/z 289) were detected as thiolysis products without cysteamine conjugation, indicating that these are terminal units of proanthocyanidins in Fr. 3. Although (þ)-catechin can be produced from (À)-epicatechin by epimerization during thiolysis at 75 C, reaction at 50 C could not eliminate the (þ)-catechin peak. The mDP calculated using the peak area of 280 nm was seven and this was consistent with the result obtained by TOF-MS analysis, which showed that the heptamer was the main component of Fr. 3. Thus, the results suggest that Fr. 3 contains procyanidins with a mDP of around seven and constituent units composed of (À)-epicatechin as the major extension units and (þ)-catechin as the major terminal units (see Fig. 5).

ESI-TOF-MS analysis of Fr. 3 and 4
The MS results for these fractions showed pseudo-molecular ion peaks with a difference of 288 atomic mass units (amu), which corresponded to an oligomer of (epi)catechin. For example, the peaks at m/z 2307, 2019, and 1731 corresponded to the masses for octamer, heptamer, and hexamer (epi)catechin units. In addition, the peaks at m/z 2324 and 2036 corresponded to the masses for (epi)gallocatechin containing octamer and heptamer (epi)catechin units. The data from thiol analysis suggested that it is highly possible that the terminal unit is catechin. Thus, the main component of Fr. 3 is (epicatechin) 6 -catechin. Similarly, the main components of Fr. 4 are (epicatechin) 7 -catechin and its (epi)gallocatechin containing compound.

Effects of various concentrations of test fractions on PC-3 prostate cancer cell proliferation
The anti-cancer activities against the PC-3 prostate cancer cell line for fractions obtained using ABE or Sepacore C18 reverse phase column chromatography are shown in Fig. 7. The Sepacore C18-purified Frs. 1-5, and especially Fr. 3 and Fr. 4, showed significant cell growth inhibitory activities. The results obtained using Fr. 3 and Fr. 4 are shown in Fig. 7. The inhibitory activities of test fractions against cell proliferation has also been observed in colorectal cancer cells (HCT116) (data not shown).

Effects of test fractions on cell cycle distribution
Cell growth and proliferation are related to cell cycle progression. In this study with FACS analysis, treatment of PC-3 prostate cancer cells with ABE for 48 h induced an increase in the G2/M phase population from 28.8 % to 40.5 % and a G1/G0 phase decrease from 62.7 % to 48.3 %. ABE blocked the PC-3 prostate cancer cell cycle at the G2 phase within 48 h (Fig. 8).

Effects of test fractions on caspase-3 activity assessed by FACS
Next, we examined whether ABE could induce apoptosis in PC-3 cells. We evaluated the caspase-3 activity to elucidate that cells exposed to ABE were actually undergoing apoptosis (Fig. 9).

Effects of test fractions on the expression of the cancer-promoting gene FABP5
Next, we searched for potential anti-cancer agents by assessing inhibitory activity for gene expression of the cancer-promoting gene FABP5. The FABP5 gene is epigenetically regulated during human prostate carcinogenesis and is involved in growth promotion and metastasis in prostate cancer cells [12,13,17,18] High-expression of FABP5 is responsible for promotion of cell growth and invasion in various cancer cells [17,18], suggesting that it plays a critical role in tumorigenesis of various cancer cells. The fractions derived from ABE, which suppress the expression of the FABP5 gene, might be promising chemo-preventive agents against prostate cancer metastasis (Fig. 6). ABE, Fr.3, and Fr. 4 significantly suppressed the expression of FABP5 at mRNA levels (Fig. 6).
We have previously reported on the synthesis of epicatechin oligomers and suppression of the FABP5 gene expression by them, and found that epicatechin pentamer significantly suppressed the expression of FABP5 at mRNA and protein levels [11]. No suppression was observed for the units less than tetramers. Epicatechin oligomers longer than pentamers showed significant activity. Fractions 3 and 4 contained (epicatechin) 6 -catechin and (epicatechin) 7 -catechin (Fig. 2), which might be involved in suppression of expression of the FABP5 gene (Fig. 10).

Conclusions
In conclusion, we concentrated proanthocyanidins from adzuki bean extracts by newly developed methods and characterized them. The Values are expressed as the mean AE standard deviation of three independent experiments. **P < 0.01 and ns ¼ not significant from two-way ANOVA followed by Sidak's multiple comparison test. Fig. 8. Effects of ABE on the cell cycle in PC-3 prostate cancer cells. Cells treated with the test fraction (ABE) for 48 h were collected and stained with propidium iodide using a BD Cycletest™ Plus DNA Reagent Kit (Becton Dickinson and Company BD Biosciences) obtained from Phoenix Flow Systems. Following FACS analysis, cell cycle distributions were further analyzed by Cell Quest software. *P < 0.05 and ns ¼ not significant from two-way ANOVA followed by Sidak's multiple comparison test. Fig. 9. Effects of ABE on apoptosis-inducible activity in PC-3 prostate cancer cells. Five μmol/L CPT (carnitine palmitoyltransferase) or 1% w/v ABE was used for the assay. The graph presents the mean values of the FITC (fluorescein isothiocyanate) fluorescence activities. A shift of the mean FITC fluorescence activities of mock cells to the right side was taken as an increase in apoptosis. The values are presented the rate of induction of apoptosis compared with that of the control (vehicle). Data were analyzed using Student's t-test (**P < 0.01). significantly suppressed the expression of the FABP5 gene in PC-3 cells. Cells treated with these compounds for 48 h were collected and the FABP5 mRNA level was evaluated by qPCR. Relative FABP5 expression levels were quantified by normalized levels. The data are shown as the mean AE standard deviation of three independent experiments. **P < 0.01 from one-way ANOVA followed by Dunnett's multiple comparison test. bioactive fractions were (epicatechin) 6 -catechin and (epicatechin) 7catechin and their derivatives containing (epi) gallocatechins. These fractions had significant anti-cancer activity and suppressed the expression of the cancer-promoting gene FABP5. Our results suggest that proanthocyanidin-enriched fractions from adzuki bean extracts might be potentially chemopreventive agents for various cancers, including prostate cancer. Further in vivo and mechanistic studies are needed to confirm these anti-cancer activities.