Characterisation of water-soluble proanthocyanidins of Pyracantha fortuneana fruit and their improvement in cell bioavailable antioxidant activity of quercetin

Food Chemistry 169 (2015) 484–491

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Characterisation of water-soluble proanthocyanidins of Pyracantha fortuneana fruit and their improvement in cell bioavailable antioxidant activity of quercetin Chun-Fang Zhao ⇑, Dou Jian Lei, Guang Hao Song, Hua Zhang, Hang Xu, Long-Jiang Yu ⇑ Institute of Resource Biology and Biotechnology, Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China

a r t i c l e

i n f o

Article history: Received 16 March 2014 Received in revised form 16 July 2014 Accepted 17 July 2014 Available online 4 August 2014 Keywords: Proanthocyanidins Interaction Bioavailability Cell antioxidant activities Small molecular polyphenols

a b s t r a c t Proanthocyanidins (PCs) with poor bioavailability were argued for their health benefits. In this study, water-soluble polymeric polyphenolic PCs fractions from Pyracantha fortuneana fruit were used to investigate whether the presence of PCs is correlated with the increased cell antioxidant activities (CAA) of quercetin (Q). The results indicated that the most decrement in the values of EC50, which Q inhibited peroxyl radical-induced DCFH oxidation effective in the HepG2 cells, was observed to be 2.91 (vs. control 5.97) in the present of the fraction with 15.8 of the average degree of polymerisation of PCs (ADP). Also, the order of efficacy was the same with the ADP of PCs. Further, this effect is associated with the improvement of the solubility and stability of Q after the addition of the PCs. Our current study suggests that the additive effects of PCs on small molecular polyphenols may be responsible for their antioxidant benefits in vivo. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Proanthocyanidins (PCs) are naturally occurring compounds which are the most widespread polyphenols in the human diet occurring at significant levels including fruits, vegtables, tea and red wine etc. They are a class of polyphenolic compounds that take the form of oligomers or polymers of polyhydroxy flavan-3-ol units, such as (+)-catechin (C) and (+)-epicatechin (EC), gallocatechin (GC), epigallocatechin (EGC), and their derivatives. PCs were

Abbreviations: ADP, average degree of polymerisation; ABAP, 2,20 -azobis(2amidinopropane); C, catechin; C-S, catechin-cysteaminothio; C-glu, catechin glucosides; CAA, cell antioxidant activity (ies); DCFH-DA, 20 ,70 -didhlorofluoresceine; DCFH, 20 ,70 -dichlorofluorescin; DCF, dichlorofluorescein; EC, epicatechin; EC-S, epicatechin-cysteaminothio; EGC, epigallocatechin; EGC-S, epigallocatechincysteaminothio; EC-glu, epicatechin glucosides; GC, gallocatechin; GC-S, gallocatechin-cysteaminothio; Q, quercetin; PCs, proanthocynadins; B2, procyanidin B2; PFF, Pyracantha fortuneana fruits; SMP, small molecular polyphenols; ORAC, Oxygen radical antioxidant capacity; TOSC, total oxyradical scavenging capacity; FRAP, ferric reducing antioxidant power; TEAC, Trolox equivalent antioxidant capacity; TRAP, total radical-trapping antioxidant parameter. ⇑ Corresponding authors at: Institute of Resource Biology and Biotechnology, Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China. Tel./fax: +86 27 87792265. E-mail addresses: [email protected] (C.-F. Zhao), [email protected] (L.-J. Yu). http://dx.doi.org/10.1016/j.foodchem.2014.07.091 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

found to have direct effects on the intestinal mucosa and protect it against oxidative stress or the actions of carcinogens (Nandakumar, Singh, & Katiyar, 2008). PCs are increasingly used as natural food nutrient additives, functional antioxidants, pharmacological agents (Fernandez & Labra, 2013) fromblueberry (Ma et al., 2013), cranberry (Pappas & Schaich, 2009), pine bark extracts (Jerez, Tourino, Sineiro, Torres, & Nunez, 2007), Pyracantha fortuneana fruits (PFF) (Zhao et al., 2013), and other dark-coloured fruits (Wu, Dastmalchi, Long, & Kennelly, 2012; Wu et al., 2013). However, research on PCs is limited and many questions still remain to be answered. The most query may be that PCs are not absorbed, which have been indicated by numerous feeding studies with animals and humans (Crozier, Del Rio, & Clifford, 2010). Many in vitro experiments with pure PCs have shown antitumor-related activities but few have been confirmed in vivo (Yang, Sang, Lambert, & Lee, 2008). The analytical data based on in vitro indicated that the antioxidant effectiveness in aqueous phase of PCs increases with degree of polymerisation for compounds constituted by C or EC as the monomer unit and the number of free hydroxyls (Porto, Laranjinha, & de Freitas, 2003). A combination of antioxidants was found in vivo to be superior with respect to the desired effect compared to the application of single antioxidants (Ray et al., 2006). PCs from grape skin and seed extracts in a simulated digestion demonstrated a significant activity on the

C.-F. Zhao et al. / Food Chemistry 169 (2015) 484–491

inhibition of the angiotensin I-converting enzyme by the extracts, while the purified extracts lost their ability to inhibit the enzyme (Fernandez & Labra, 2013). Our previous research also indicated that the recovery rate of total antioxidant activity was only 65.9% when the optimised extract was fractionated into four individual fractions (Zhao et al., 2013). These studies suggest that the health beneficial effects of PCs may result from the interaction (s) with other components in their sources. In fact, there are about 5–25% small molecular polyphenols (SMP) such as quercetin (Q) and its glucosides, monomers or other ployphenols in the PCs-rich foods (Manach, Williamson, Morand, Scalbert, & Remesy, 2005). While SMP could be selectively absorbed in the gut, and have been well reported on their pharmacokinetics, although the peak in plasma for almost all SMP are not high enough to execute their physicological function (Pappas & Schaich, 2009). These evidences have led to increased interest in the improvement of bioavailability of SMP and the underline mechanism of interactions between SMP and PCs, especially with polymeric PCs. The cellular antioxidant activity (CAA) assays have been developed to measure the antioxidant activity of antioxidants in foods using cultured human HepG2 liver cancer cells (Wolfe & Liu, 2007). It allows us to determine the potential biological activities f the samples in response to a need for a more biologically representative method than the common chemistry antioxidant activity assays. In this assay prototype, 20 ,70 -dichlorofluorescin diacetate (DCFH-DA) was utilised as a probe in cultured HepG2 cells, in which, nonpolar DCFH-DA is uptaken by the cells and deacetylated by cellular esterases to form polar 20 ,70 -dichlorofluorescin (DCFH). The reduced form (DCFH) can trap the peroxyl radicals within the cells generated from 2,20 -azobis(2-amidinopropane) (ABAP) lead to the formation of fluorescent compound dichlorofluorescein (DCF). Since level of fluorescence formed within the cells is proportional to the level of oxidation, the decrease in cellular fluorescence compared to the control cells indicates the antioxidant capacity of the antioxidants existing within the cells (Song et al., 2010). Thus, the CAA assay reflects not only the antioxidant activity but also bioavailable potential. Polyphenols, particularly to PCs, could interact with other macromolecules in foods through non-covalent and covalent associations, which affected strongly the quality of polyphenol-rich food products, as well as on biological systems. These interactions have been well documented in publications (Le Bourvellec & Renard, 2012). For example, the hydrophobic interactions between polyphenols and proteins could involve aromatics rings of polyphenols and hydrophobic sites of proteins, such as pyrrolidine rings of prolyl residues, while hydrogen bonding occurs between H-acceptor sites of the proteins and the hydroxyl groups of the polyphenols (Carvalho, Povoas, Mateus, & de Freitas, 2006). The interaction between quercetin and catechin was observed in a mimical matrix to be positive effects on the concentrations and antiradical activity of both compounds in vitro (Turan, Gulsen, Makris, & Kefalas, 2007). Procyanidin B2 and hyperoside were found to improve the solubilisation and bioavailability of hypericin, which resulted in a significant accumulation of hypericin in rat plasma (Butterweck, Lieflander-Wulf, Winterhoff, & Nahrstedt, 2003). However, there have been so far no reports on the interactions between PCs and SMP in the literature. The present study was designed to investigate whether the presence of PCs is correlated with the increased CAA of Q, in which polymeric PCs, as main polyphenols in our diets, act as mediators on the antioxidant actions in vivo. Water-soluble high polymeric PCs of PPF, which were further fractionated from the previous optimal extracts (Zhao et al., 2013), and pure compound Q were chosen as the typical PCs and SMP to evaluate the influence on CAA, solubility and stability of Q.

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2. Materials and methods 2.1. Reagents Catechin (C), epichatechin (EC), quercetin (Q), procyanidin B2, were purchased from Sigma–Aldrich, Inc.(St. Louis, MO). Gallic acid, cysteamine hydrochloride, DCFH-DA, ABAP, fetal bovine serum (FBS, GIBCO, USA), DMEM medium (GIBCO, USA), and antibiotics (penicillin/streptomycin, Hyclone, USA) were obtained from Sanyi Biomedicals, Inc. (Wuhan, Hubei). Methanol, acetone, acetonitrile were obtained from Tedia Biomedicals, Inc. (Wuhan, Hubei). The HepG2 cells were obtained from the China Center for Type Culture Collection, CCTCC.

2.2. Extraction and fractionation of extracts The water-soluble PCs of P. fortuneana fruit (PFF) in this study were started from the water-soluble fractions (FA), and the whole procedure was showed in Scheme 1. The FA was prepared according our previous descriptions with a little modification (Zhao et al., 2013). In briefly, the optimised extract was obtained under the extraction conditions of 71% ethanol, 51 °C, and pH 3.2. Then, 5.0 g of the extract were suspended in water (100 ml) and separated by successive liquid–liquid extraction using hexane (100 ml), ethyl acetate (100 ml). This process yielded three fractions, only one of the aqueous phase was further used as fractionation. The aqueous fraction (FA) was vacuum lyophilized at 10 °C and kept at 4 °C till it was used. About 0.91 g FA solid weight were obtained. The experiments were conducted in triplicate and then, obtained FA solids were combined into one. PCs fractionations of the FA were conducted with minor modifications to the procedure, as described in some studies (Jerez et al., 2007). A sample of FA (400 mg/4 ml of 50% methanol) was applied to a 50  2.5 cm i.d. Sephadex LH-20 column. After manually packed by elution with 50% methanol in water, the column was eluted with 50% methanol in water, the column was eluted with 60% methanol in water (MeOH), 75% MeOH, 90% MeOH, 80% methanol with 10%Acetone and 10% water, 65% methanol with 20%

Scheme 1. Preparations of PCs fractions from PFF (here AON represents as acetone).

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acetone and 15% water, 40% methanol with 30% acetone and 30% water, and 70% acetone with 30% water, respectively, from F1 to F8, at a flow rate of 3 ml/min. The elution of PCs were collected, and monitored using UV-spectrophotometric analysis at 280 nm (with a Mapada UV-1600PC spectrophotometer (Shanghai, China), or online UV spectrophotometer (Dalian, China), for the samples of F1–F5, at 400 nm for F6–F8 in order to avoid the distraction of the acetone in the mobile phases. The samples were evaporated under vacuum to remove organic solvents, then were vacuum lyophilized. The remnants were dissolved in 50% methanol into a concentration of 5 mg/ml solid weight.

for fluorescence versus time was integrated to calculate the CAA value at each concentration point of Q:

R where SA is the integrated area under the sample fluorescence R versus time curve and CA is the integrated area under the control group curve. The CAA unit of Q was expressed as lg/ml per 100 lg/ ml Q concentration. The Y value was defined here as the calculation according formula 2:

2.3. Characterisation of PCs fractions

Y¼

The fractions F1–F8 were diluted to 1 mg/ml PCs (solid) concentration, then they were employed to assay Structural composition of PCs with thiolysis tests, respectively. The thiolysis procedures were conducted a modified method (Jerez et al., 2007). In brief, the terminal flavan-3-ols units were released by acid cleavage in the presence of cysteamine whereas the extension moieties were released as the cysteamine derivatives. An aliquot (200 ll) of the fraction was treated with 200 ll of 2 N HCl in methanol of the thiolysis mixture, which consisted of cysteamine hydrochloride (500 mg). The mixture (400 ll) was kept at 65 °C in a water bath for 30 min. The reaction mixture was filtered through 0.45 l PTFE filter and analysed in HPLC–MS. The characterisation of fractions gave values of average degree of polymerisation (ADP), which was calculated as the ratio of total units (nmol) to terminal units (nmol), and procyanidin–prodeliphidin ratio was estimated as molar ratio of catehin derivatives to gallocatehin derivatives (Arimboor & Arumughan, 2012). The proportions of C, EC, GC or EGC were used as terminal units, while the proportions of their cysteamine derivatives, such as C-S, EC-S, ECG, and EGC-S, were used as the extended units.

The median effective concentration (EC50) was calculated from the median effect plot of log Y versus log C). The EC50 values were stated as mean (SD for triplicate sets of data obtained from the same experiment. EC50 values of PCs were converted to CAA values, which are expressed as lg/ml of quercetin equivalents using the mean EC50 value for quercetin from eight separate experiments EC50.

 CAA ¼

R  SA  100 1R CA

CAA 100  CAA

ð1Þ

ð2Þ

2.6. Measurement of solubility of quercetin in PCs fractions Q solid was chosen at a concentration of 0.17 mM (50 lg/ml) in FASSIF matrix (Li et al., 2013) with or without PCs in a proportion of 0.17 mM solid weight F1–F8. These mixtures were incubated at 37 °C for 30 min. After centrifugation, the quantification of Q in the supernatant was conducted by HPLC. FASSIF (fasted state simulated intestinal fluid) was prepared according the reference (Dressman, Amidon, Reppas, & Shah, 1998) as following: NaTC 3 mM, Lecithin 0.75 mM, KH2PO4 3.9 g, KCl 7.7 g, NaOH ad pH 6.5, water ad 1 L. Treatments were carried out in triplicate. The solubilities of Q in matrix were expressed as mean (lg/ml) ± SD and the portion to control (absent in PCs) (%) for triplicate data.

2.4. Cell culture and treatment 2.7. Measurement of stability of quercetin in procyanidin B2 mixtures HepG2 cells were grown in Complete Medium (WME supplemented with 10% FBS, 10 mM Hepes, 2 mM L-glutamine, 5 lg/mL insulin, 0.05 lg/mL hydrocortisone, 50 units/mL penicillin, 50 lg/ mL streptomycin, and 100 lg/mL gentamycin) and were maintained at 37 °C and 5% CO2 as described previously (Song et al., 2010). The cellular antioxidant activities (CAA) of Q assisted by PFF PCs were determined using the protocol described in publications (Song et al., 2010; Wolfe & Liu, 2007). HepG2 cells were seeded at a density of 6  105 per well on a 96-well microplate in 100 lL of Complete Medium per well. After 24 h culturing, the growth medium was removed, and the wells were washed with 100 lL of PBS. Wells were treated with 200 lL of treatment medium containing solvent control (blank control), control test, or tested groups including in a proportion of 60 lmol/L of PCs in F1–F8 within and without 10, 20, 30, 40, 50 lmol/L of Q, added by DCFH-DA in a final concentration of 25 lM for 1 h. Then, the supernatants were removed and washed twice with 100 lL of PBS. Then 600 lM ABAP was applied to the cells of control test and tested group in 200 lL of oxidant treatment medium, and the 96-well microplate was placed into a multilabel Reader Envision (Perkin Elmer, Waltham, MA, USA) at 37 °C. Emission at 538 nm was measured after excitation at 485 nm every 5 min for 1 h. 2.5. Analysis of cell antioxidant activity (CAA) The analysis of CAA was conducted using the previous method (Song et al., 2010; Wolfe & Liu, 2007). In briefly, after blank subtraction of the initial fluorescence values, the area under the curve

In order to find out complete effect on solubility of Q in the present of PCs, a mixture containing 5 mM pyruvic acid, 20 lg/ml FeCl3 and in ethanol/acetate buffer (1/1, v/v, pH 3.5) was chosen for the measurement of stability of Q in matrix, which could mimic real pH environment in polyphenol-rich food systems, and avoid a potential microbial spoilage (Turan et al., 2007). The model matrix was designed as consisting of the amounts of 0.33 mM (100 lg/ml) Q and 0.33 mM procyanidin B2 (200 lg/ml) in a final volume of 5 ml. The solution was placed in a dark glass vial, and incubated in a water bath at 55 °C. Samples were withdrawn on at day 0, 2, 4, 6, 8, 10, 15, 20, respectively, basis over a period of 20 days. After filtration of 0.45 l PTFE filter, the quantification of Q and its possible oxidant products in the solutions were analysed by HPLC and LC–MS. Treatments were carried out in duplicate. The stability of Q in matrix was compared by its remaining rate (%) of the concentration to pure Q (control) expressed as mean (%) ± SD for duplicate data. 2.8. HPLC and LC–MS analysis Q and catechin(C), epi-catechin (EC), gallocatechin (GC), epigallocatechin (EGC) and their thiolytic products C-CYT, EC-CYT, ECG-CYT and GC-CYT were determined using HPLC and LC–MS. Chromatographic column was used with an Agilent ZORBOX SBphenyl column (5 lm, 250 mm  4.6 mm, PN: 880975-912). The flow rate was 1.0 mL/min, and the injection volume was 20 lL. The column temperature was kept at 25 °C. For the Q analysis, 0.5% of formic acid, 39.5% water and 60% acetonitrile were

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employed as mobile phase. The detection was performed at 370 nm. For the analysis of flavanol unit and their cysteamine derivatives, 0.1% trifluoroacetic acid (TFA) aqueous solution and 0.08% TFA in acetonitrile were employed as mobile phase A and mobile phase B, respectively. The mobile phase in the gradient procedure was 8–23% B during 0–10 min, 23–60% B during 10–30 min, 60– 80% B during 30–40 min, 80–8% B during 40–45 min. The detection was performed at 280 nm. The qualifications of C, EC and Q were conducted by comparison of available standard substances. The others were identified based on LC–MS analysis. Standard calibration graphs of C, EC and Q were prepared in the concentrations of 5–200 lg/mL, and was recorded at 280 nm. The calibration curves showed good linearity between 15 lg/mL and 180 lg/mL for EC and C, 1.6–95 lg/mL for Q. The regression equations for EC, Q, C were Y = 7.04X  16.54 (R2 = 0.9878), Y = 42.4X  306.5 (R2 = 0.9998), Y = 10.4X + 2.4 (R2 = 0.9918), respectively, where Y is the area of peak, X represent concentration of compound (lg/mL). Limits of detection (LOD) and limits of quantification (LOQ) for individual compounds in standard solutions were also calculated as S/N = 3 and S/N = 10, respectively, where S/N is the signal-to-noise ratio. The values of LOD for EC, Q, C were 2, 0.5, 1 lg/mL, respectively, and LOQ were in turn 5, 2.5, 3 lg/mL, Samples were analysed twice and mean values were used for calculation. The mass spectrometer used was an ion trap electrospray mass spectrometer equipped with an ESI source and an ion trap mass analyzer controlled via Windows XP based on ChemStation software (version B.04.01 SP1, Agilent, USA). The mass parameters for the oxidative products of Q were as follows: capillary voltage, 4.9 kV; fragmentation voltage, 45 V; drying gas temperature, 450 °C; gas flow (N2), and 9 L/min. The ESI mass spectra were scanned from m/z 100 to 1000 at a scan rate of 1.5 s/cycle in positive mode. For the analysis of flavanol unit and their cysteamine derivatives, the mass parameters were conducted as following: capillary voltage, 2.5 kV; fragmentation voltage, 45 V; drying gas temperature, 350 °C; gas flow (N2), and 7 L/min. The ESI mass spectra were scanned from m/z 100 to 700 at a scan rate of 1.5 s/ cycle in both positive and negative mode. 2.9. Calculation and statistics The results were processed using Origin v 8.0. The data were subjected to an ANOVA. P values <0.05 were considered as statistically significant. 3. Results 3.1. Characterisation of water-soluble proanthocyanidins (PCs) of P. fortuneana fruit The starting-material of this work was crude fraction, obtained in previous works from the water-soluble fractions of P. fortuneana fruit, named FA in this paper. In our previous investigation (Zhao et al., 2013), both extracts and crude fractions, including the water-soluble fractions, were analysed by HPLC–MS to be rich in polyphenols. The chromatographic profiling has shown that there are more than 20 small molecular polyphenols the fruit such rutin, quercitin and its hexoside, epi-catechin-3-glucosides. The high molecular weight components such PCs, however, could not be clearly established from the HPLC–MS analyses. Table 1 showed that FA was complex, around eight fractions with different recovery and degrees of polymerisation, which were named by F1–F8, respectively, could be obtained by the further fractionation on a Sephadex LH-20 column. Under the fractionating

Table 1 The solvents of preparation and results of analysis of PCs fractions from PFF crude aqueous fraction.

F1 F2 F3 F4 F5 F6 F7 F8

PCs recovery (%)

mDP

Procyanidin (%)

85.6 ± 2.3 9.0 ± 0.8 1.5 ± 0.2 0.8 ± 0.2 1.5 ± 0.3 1.7 ± 0.5 1.8 ± 0.4 4.2 ± 0.9

6.2 ± 0.4 5.0 ± 0.2 8.3 ± 0.3 8.9 ± 0.4 9.5 ± 0.3 10.7 ± 0.3 13.9 ± 0.3 15.8 ± 0.4

92.6 ± 2.3 88.3 ± 2.1 84.2 ± 1.8 96.1 ± 2.6 97.8 ± 2.7 94.5 ± 2.4 98.0 ± 2.8 99.0 ± 2.9

and monitoring conditions, a major fraction was eluted as F1, followed by F2 and F8, yielded 85.6%, 9.0%, and 4.2% of FA, respectively. Other fractions, such as F7, F6, F5, F3 and F4 had less than 2% yield. The elution orders of the fractions were accorded with their averages of polymeric degree (APD), varying from 6 to 15.8 for the fractions F2 to F8, which indicated that smaller molecular compounds were eluted first and bulkier PCs were recovered last. The APD and procyanidin ratio of the individual fraction were estimated from the compositions of their depolymerisation products with cysteamine. The analysis and identification of PCs in the typical fractions are shown in Fig. 1. The chromatographic peaks of C and EC were determined by comparing their retention times with the corresponding referent substances. The LC–MS experiments of standard substance C and EC in positive ion mode showed the presence of the pseudo molecular ion peak at m/z 291 and m/z 332, corresponding to the intense [M+H]+ and [M+ACN+H]+. Several obvious new peaks appeared in the chromatograms of the thiolysized fractions, which were simpler than their untreated samples (Fig. 1), such as the peaks of the retention time at 9.3 min, 17.4 min and 25.2 min. The peaks at 9.3 min showed the ion peak with m/z 291.1 and 368 in the MS results. The fragment ion peak of m/z 368 was calculated as [M+SCH2CH2NH2+H]+ ([M+77]+), which might correspond to the reaction products C-S of C or EC-S of EC. For the chromatographic peak at 17.4 min, the MS results showed the ion peak at m/z 453.2 and 531. The MS data of m/z 531 had a obvious exact ion peak in total ion chromatography, suggesting the peak may be the molecular ion peak. The MS data of m/ z 531 was further calculated to be [453.2+SCH2CH2NH2+ H]+([453.2 + 77]+), which suggested the presence of catechin glucosides (C-glu) or epi-catechin glucosides (EC-glu). MS/MS analysis of ion at m/z 453 resulted in daughter ions at m/z 436 ([MH2O+H]+) and m/z 323 ([MH2OTFA+H]+). This further supported the assigned structure of C-glu or EC-glu. Thus, the chromatographic peak at 17.4 min was assigned as C(EC)-glu-S. For the assignment of the peak at retention time 25.2 min, the MS data showed a mixed with the ion peaks at m/z 581, m/z 404 and m/z 368. MS/MS analysis of ion at m/z 404 indicated the presence of the daughter ions at m/z 311.9 and m/z 291 and m/z 404 resulted from ions at m/z 581, indicating the presence of C or EC or their reaction products. Being considered the chromatographic peak at 9.3 min, which also appeared in the molecular ion peak at m/z 368, the peak with retention time of 17.4 min was therefore assigned as EC-S. The assignments of GC, EGC and their reaction products GC-S and EGC-S in the chromatogram were conducted by using the software that comes with the instrument inquiry. For example, when the parameter of 307 ([M+1]+ was set, there was a extract ion peak at 24.0 min in total ion chromatogram (Fig. 1). Although we could not confirmed whether the peak represents GC or EGC, here it does not affect the calculation of the characteristic masses and structures of PCs. According to the MS adduct observations above, GC(EGC)-S might have main ion peak at m/z 383 ([M+77]+, while

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Fig. 1. The LC–MS analysis and identification of thiolysis reaction products of PCs in the typical the fraction.

there was no obvious peak in the case of m/z 383, which suggested that different molecular structure may result in different adduct ions. When 343 ([MGC-S+HCl]+) was entered, two extract ion peaks present at 20.0 min and 33.8 min, respectively. 3.2. The effects of PCs on the CAA of Q The CAA of Q in the presence of different fractions was examined using the CAA assay. The EC50 values for the pure Q compound and the samples are shown in Table 2 and typical time course of fluorescein reaction is present in Fig. 2. In the presence of PCs, Q had lower EC50 values than Q in the case of pure Q compound system, which was designed as absence of the fractions. The addition of F8 was the most effective at Q inhibiting peroxyl radical-induced DCFH oxidation (P > 0.01), followed by F7, F3, F2, and F1 (vs. Q, P > 0.05). The order of efficacy was the same with the ADP of PCs in the fractions. Theoretically, polymeric polyphenols cannot be absorbed by cells since they fail to pass membrane (Yang et al., 2008). In this study, time course of fluorescein reaction did not show obvious difference when comparing the samples only containing PCs without Q with the control (absence of both PCs and Q) (unlisted results). These indicate that the improved CAA of Q resulted from PCs additions, and the addictive effects increase with the risings of mDAP of PCs (Tables 1 and 2). 3.3. PCs effect on stability of Q In order to study potential influence of PCs on the improved bioavailability of Q and the antioxidant characteristics, we selected pure compounds of procyanidin B2 and Q as material in a ready-established model matrix system solution (Jerez et al.,

2007). Incubation of the matrix at 55 °C for 20 days resulted in an almost complete degradation of Q in the absence of procyanidin B2, while it only showed a smaller progressive decline in the presence of procyanidin B2, Q was degraded by 50% (Fig. 3A). The variations were not only in quantification of Q, but also in qualitative aspect. Fig. 4 showed that there was only one peak at retention time 7.4 min in the spectrum of HPLC at the beginning of the experiments, while several obvious new peaks rose in, especially in the peak at retention time 6.6 min in the end of the experiments (Fig. 4B). Further, the areas of peak had an increasing tendency with the time course (Fig. 3A). In this study, the oxidation products formed during the incubation were identified by LC–MS in positive ion mode. The MS data of the stability experiment showed that it is a mixed picture either at 6.8 min or 7.5 min. But the pseudo molecular ion peak at m/z 303 ([M+H]+) and m/z 325 ([M+Na]+) for Q at 7.5 min, m/z 317 ([M+H]+) for the one of oxidation product of Q at 6.8 min. Interestingly, the increasing tendency of oxidation product is negatively related to that of Q concentration, indicated that the degradation of Q underwent mainly oxidation during the incubation, and the presence of procyanidin B2 could curb effectively the oxidation. These results suggest PCs additive effects to Q, both in chemical stability and bioavailability. 3.4. PCs effect on solubilisation of Q In order to study PCs possible influence on Q improved bioavailability whether is correlated to increased water-solubility of Q, we determined solubility of Q in the presence of 60 lmol/L F1–F8 in a simulated intestinal fluid medium and the results are displayed in Fig. 3B. The data demonstrate that the solubilisation of Q is significantly increased by 6.7–97.4% in presence of PCs in the fractions of

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Fig. 2. Time course of fluorescein reaction (A) and the calculation of EC50 (B) with ABAP in the blank, Control, 10, 20, 30, 40, 50 lmol/L Q, and Q in the presence of F1, F2, F3, F7, F8, results are the mean.

PFF (P < 0.05). The most increasement in Q solubility was found in the presence of F3 and F2, followed by F1, F4, F5, F6, F8, and F7, respectively. The adductive effects of different fractions on Q solubility showed differences. The values of solubility of Q increased from 17.2 lg/ml (Q in absence of fractions) to 34, 33.5, 32.2, 30, 23, 20.4, 18.9 and 18.4 lg/ml, respectively, by 97.4%, 94.9%, 87.4%, 79.1%, 36.5%, 18.6%, 9.9% and 6.7% compared to control (pure Q, absence of PCs), for F3, F2, F1, F4, F5, F6, F8, and F7, respectively. While these variations were not significant between F1, F2, F3 and F4, as well as F6, F8, and F7 (P > 0.05). 4. Discussions PCs, which represent a significant percentage of polyphenol intaken in our dietary vegetables and fruits, have been reported to possess substantial antimutagenic activities and in the prevention or reduce of the risk of coronary heart disease (Nandakumar et al., 2008) because their antioxidant activities greater than

Fig. 3. Examination of the stability and the solution of Q in the presence and absence of PCs. (A) The stability of Q in the presence and absence of procyanidin B2 determined by HPLC during the incubation (day) and (B) the solubility of Q in the presence and absence of PCs in the fractions of PFF.

standard antioxidants like trolox and ascorbic acid. The analytical data based on in vitro indicated that the antioxidant effectiveness in aqueous phase of PCs increases with degree of polymerisation for compounds constituted by C or EC as the monomer unit and the number of free hydroxyls (Porto et al., 2003). PCs are increasingly used as natural food nutrient additives or pharmacological agents such as grape seed extracts (Fernandez & Labra, 2013), Cranberry products (Pappas & Schaich, 2009), pine bark extracts (Jerez et al., 2007). However, PCs exhibit poor bioavailability, especially to high molecular weight polymers, which are not absorbed at all in most animal models (Pappas & Schaich, 2009). On the contrary, SMP such as Q and C, or smaller oligomeric PCs could be absorbed in the gut, have been well reported on their pharmacokinetics. Q has been studied extensively and well understood in vitro and in vivo (Lotito, Zhang, Yang, Crozier, & Frei, 2011). In vitro, Q and its hydrolysed glyosides can be absorbed by intestine enterocyte, in which Q is mostly metabolized to glucuonides or sulphate conjugates and exported into the circulation. After consumption of large quantities of Q to rats, the concentration of Q in plasma was found to be 7.70 lM (Lotito et al., 2011), although the

Table 2 The results of CAA (EC50) from pure Q and the addition of the fractions.

EC50 (lM) a Fitted equation R2 a

Q

Q + F1

Q + F2

Q + F3

Q + F7

Q + F8

5.97 ± 0.12 1.03x  0.80 0.9882

5.24 ± 0.15 0.93x  0.67 0.9749

4.55 ± 0.23 0.81x  0.53 0.9414

3.64 ± 0.20 0.52x  0.29 0.9556

3.42 ± 0.22 0.36x  0.19 0.9484

2.91 ± 0.11 0.74x  0.34 0.9824

x Represent log C (concentration of Q lmol/L).

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Fig. 4. Putative structure proposed for the oxidation of Q on the basis of LC–MS data. (A) MS data of Q at tR 7.5 min; (B) MS data of putative oxidation of Q (O-Q); (C) HPLC on day 0 and (D) HPLC on day 15.

concentration peak in plasma is not high enough to execute their physicological function (van Dorsten et al., 2010). Therefore, there is interesting to improve the bioavailability of Q. CAA assay may provide a better prediction of antioxidant behaviour in biological systems, because it mimics some of the cellular processes that occur in vivo, being superior to the traditional chemistry based antioxidant activity analysis such as Oxygen radical antioxidant capacity (ORAC), total oxyradical scavenging capacity (TOSC), ferric reducing antioxidant power (FRAP), Trolox equivalent antioxidant capacity (TEAC), total radical-trapping antioxidant parameter (TRAP) (Wolfe & Liu, 2007). Thus, the CAA assay reflects on not only the antioxidant activity but also some aspects of bioavailability, is a cell available antioxidant activity. The polyphenols with CAA must first have the capability of cell uptake. In the current work, we hypothesize that the bioeffects of dietary PCs are conducted by associating with the improvement of Q bioavailability. Our study was designed to investigate whether the presence of PCs is correlated to increased CAA of Q. Interestingly, the fractions in this study increased significantly the CAA of Q (Table 2 & Fig. 2). Moreover, the improvement of CAA for Q increased with the mDP of PCs in the fractions. While fluorescein reaction of inhibiting peroxyl radical-induced DCFH oxidation was found to unchanged in the absence of Q but in presence of the serial of fraction (the results unlist). Our results in CAA experiments indicate that PCs promoted the CAA of Q through the improvement of bioavailability of Q, and this is supported further by the data obtained from the experiments of stability and solubilisation of Q (Fig. 3A, B; Fig. 4 Table 2). In our limit knowledge, this may be first direct evidence which indicate PCs can have an additive effect on Q bioavailable antioxidant activity. The biological evaluation of Q in various test models is bounded by its poor water solubility. In the former studies, it has shown that the bioavailability of quercetin glycoside was promoted by the

ingestion of a-1,6-glucosaccharides, this was because the supplementation of a-1,6-glucosaccharides could enhance the content of Q in the water-phase fraction (Shinoki et al., 2013). Another previous study indicated that it could influence on the plasma kinetics of hypericin in the presence of procyanidin B2 and hyperoside, which contains six phenolic hydroxyls at its molecular skeleton. As a result the accumulation of hypericin in rat plasma had a significant increase. (Butterweck et al., 2003). Our current study has demonstrated a prominent increase in Q solubility based in the water-phase FASSIF matrix after supplementation with the fractions (Fig. 3B). The solubilities of Q were improved by from 97.4% to 6.7% for the group of the addition of PCs, when it is compared with pure compounds of Q (control), indicating that the increased CAA of Q is responsible for the improvement of its solubilisation influenced by PCs. One possible explanation for the improved solubilisation in Q is attributed to the interactions between the molecules of PCs and Q such as hydrogen bonds. Another reason for the increase in content of Q in the presence of PCs in the mimical matrix may lie in its improvement on chemical stability. Procyanidin B2 dramatically enhanced the preservation of Q in the model solution during 20 days (Fig. 4), and inhibited the formation of its oxidation products. This result is similar to the observation of the interaction between Q and C (Turan et al., 2007), in which Q encountered a complete disappearance while only 75% decrease for catechin duo to the formation of a co-pigment within the same time course as we use. But different from their study, no co-pigment like compound between procyanidin B2 and Q was found in our study. This may be attributed to the detective condition, in which procyanidin B2 selected in our study did not occur in the HPLC graphs. The more possible reason than the formation of co-pigment compounds we speculate is that the effect of PCs on the chemical stability of Q could be associated with improving reducing ambient of solvent systems.

C.-F. Zhao et al. / Food Chemistry 169 (2015) 484–491

As there are evidence numerously indicating that PCs have antioxidant effects in vitro, although PCs, particular to the polymeric PCs, did not be absorbed at all by neither cell nor intestine. However, the consumption of PCs-rich foods has been shown to increase the plasma antioxidant capacity also have multilateral positive effects on human health (Nandakumar et al., 2008). In addition, there are among the most abundant polyphenols in our diet and about 5–25% monomers or other ployphenols in these PCs rich sources (Manach et al., 2005), The combinations of antioxidants were found in vivo to be superior with respect to the desired effect compared to the application of single antioxidants (Ray et al., 2006). PCs from grape skin and seed extracts in a simulated digestion demonstrated that over 80% of the angiotensin I-converting enzyme inhibition by the extracts was preserved, while the purified extracts lost their ability to inhibit the enzyme (Fernandez & Labra, 2013). These results suggest that the percentage of lowmolecular-weight compounds in extracts is a key factor determining the level of bioactivity but PCs may have effects on other SMP through interactions, especially in additive effects. In a complex regulatory systems like the production and extinction of ROS in vivo, it raises the assumptions whether unspecific stimuli are at least to some extent involved in ROS as well as antioxidants. The maintenance of the redox-homeostasis may be more important than a single antioxidant action. Therefore, the modulation of the redox-state may require complex rather than single interventions (Ulrich-Merzenich, Zeitler, Vetter, & Kraft, 2009). Our results are compatible with the hypothesis that PCs may not enter cells or in plasma in where they execute elimination of oxidative stress, but involve in stabilizing the chemical property of other bioavailable polyphenols through improving reducing ambient of solvent systems, and this may be crucial to elucidate the healthy benefits of PCs. 5. Conclusion PCs promote cellular antioxidant activity of Q in the mode cells of HepG2, and enhance the bioavailability of Q. Modulation of the solubility and stability of Q derived from by PCs may be determinant of the improved bioavailability of Q. The additive effects of PCs on Q are responsible for their antioxidant benefits in vivo. Funding This work was supported by Natural Science Foundation of Hubei Province, China, Grant 2012FFB02606. Acknowledgements We would like to thank Prof. Li. Su of Key Laboratory of Molecular Biophysics Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, China, for her critical review. Also, the authors thank Dr. Gu Xiao-man of Analytical and Testing Center in Huazhong University of Science and Technology for HPLC–DAD–MS analysis. References Arimboor, R., & Arumughan, C. (2012). Effect of polymerization on antioxidant and xanthine oxidase inhibitory potential of sea buckthorn (H. rhamnoides) proanthocyanidins. Journal of Food Science, 77(10), C1036–C1041. Butterweck, V., Lieflander-Wulf, U., Winterhoff, H., & Nahrstedt, A. (2003). Plasma levels of hypericin in presence of procyanidin B2 and hyperoside: A pharmacokinetic study in rats. Planta Medica, 69(3), 189–192.

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