New technology for large preparation of a series of bioactive polyphenols from by-products of vinification

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Introduction
Proanthocyanidins (PAs) are oligomers and polymers of flavan-3-ol units presented essentially in epidermal tissue of plants, leaf, bark, solid parts of fruits, so some agricultural residues or agro-industrial by-products, such as fruit pomaces, are often rich sources of these compounds [1].According to the hydroxylation pattern of flavan-3-ols, PAs can be classified into various sub-groups, including essentially procyanidins, prodelphinidins, propelargonidins, profisetinidins, and prorobinetinidins.
PAs, as the fifth class of plant biopolymers, was still not familiar thirty years ago, but during the last two decades, some spectacular advances in scientific research on these compounds have been achieved.The explosion of research activities on PAs is mainly due to the discovery of various biological activities of their low-molecular compounds, i.e., flavan-3-ol monomers (commonly catechins) and oligomers (OPCs), which are likely the most powerful antioxidants known: they are 50 times more powerful than vitamin E and 20 times than vitamin C [2].Today various OPCs have been available but very expensive.This is mainly because over 90% PAs in natural plants are present in the polymeric form (PPCs), which are structurally-complex and non-nutritive [3][4].This fact would be the main reason why the majority of published works in the literature were only those related to catechins and OPCs.However, from enological point of view, both OPCs and PPCs are important phenolic compounds responsible for sensory properties of red wine, so during the last twenty years, one important topic of our research works has been the study on grape and red wine OPCs and PPCs, including their fractionation [5][6], isolation and purification [6][7][8][9], identification [7][8][9][10][11][12][13], quantification [14][15], condensation reactions [10][11][12], and biological activities [15][16][17][18][19].Among these, one of the interesting works was the acidic degradation of grape and wine PPCs in the presence of nucleophiles, releasing flavan-3-ols (catechins) and flavan-3-ol-nucleophile derivatives [5,20].This is a unique method allowing to determine the structural composition of highly polymerized proanthocyanidins, which can not be achieved by commonly-used methods such as HPLC, ESI-MS and NMR.
This communication will present our recent work about large preparation of a series of bioactive polyphenols and their derivatives from grape seeds and grape skinsthe major components of grape pomaces, firstly by degradation of PPCs in the presence of nucleophiles, followed by preparative separation of the degraded products using HSCCC.
Acidic degradation in the presence of the nucleophiles has been often used for structural analysis of plant polymeric proanthocyanidins.Numerous nucleophiles used include thiolactic acid, benzene-p-sulphinic acid, benzenethiol, benzyl mercaptan, 2-mercaptoethanol and phloroglucinol [23].All these nucleophilic reagents allow degrade polymeric proanthocyanidins into their constitutive units or derivatives, but with different degradation efficiencies [23].
A French research group firstly characterized the structure of grape polymeric proanthocyanidins by thiolysed degradation using benzyl mercaptan as nucleophile followed by HPLC analysis [24][25][26]; Using the same degradation approach, red wine polymeric proanthocyanidins [5] and apple highly-polymerized procyanidins [27] were also structurally characterized.
Matthews et al. [23] compared the degradation methods for estimation of proanthocyanidins in the presence of the two most widely used nucleophiles, benzyl mercaptan and phloroglucinol.These authors recommended the use of benzyl mercaptan rather than phloroglucinol due to the higher yields of depolymerization.However, Kennedy and Jones [28] reported that the degradation method for proanthocyanidins under the proposed analytical conditions using phloroglucinol was more effective and favourable to that using benzyl mercaptan.In fact, phloroglucinol was preferred is also due to its odorless while benzyl mercaptan is very smelly and toxic and probably for this reason, phloroglucinol has been mostly used as nucleophile for analysis of structural composition of proanthocyanidins in various plant tissues.More recently, other non-toxic and bioactive nucleophiles, i.e., captopril, L-cysteine, and tiopronin were also tested for degradation of grape and wine PPCs in our laboratory.

HSCCC technology for separation of polyphenols
High-speed countercurrent chromatography (HSCCC) is based on continuous liquid-liquid partitioning, which enables one to eliminate irreversible adsorption on solid supports.As a relative new separation technology for natural plants, HSCCC is especially suited to large and efficient separations of polar compounds like polyphenols and their derivatives [7-9, 20-22, 29].Compared to conventional liquid-solid separation methods, HSCCC offers several advantages, as it does not suffer from irreversible analyte adsorption and features better loading capacity, higher sample recovery, lower sample denaturation risk, minimised tailing, and decreased solvent consumption [29][30][31].
Several factors can affect the separation efficiency of HSCCC, including solvent system, elution mode, revolution speed and column temperature.Among these factors, the most important one is the suitable selection of a two-phase solvent system, which provides the ideal partition coefficient (K) for the target compounds.Therefore, the partition coefficient (K) of one compound is critical for its successful isolation and separation by HSCCC.Another important parameter is the separation factor (α), which indicates the peak resolution between the target compounds.Both partition coefficient (K) value and separation factor (α) were used as evaluation parameters for selection of the two-phase solvent system [32].The partition coefficients (K) were determined by HPLC.The K values of target components were calculated according to the equation K = AU/AL, where AU and AL are the peak areas of target compounds in the upper phase and lower phase respectively and the separation factor (α) was defined as the ratio of partition coefficients between two compounds (α = K1/K2, K1 ≥ K2).

Preparation of bioactive polyphenols from grape seeds
Degradation methods for polymeric proanthocyanidins in the presence of nucleophiles have been used only for their structural analysis.In our laboratory, the degradation methods were, for the first time, employed for the purpose of producing bioactive polyphenols.Thus, by selection of suitable nucleophile, the abundant non-nutritive polymeric proanthocyanidins, from grape and wine or other plant tissues, were effectively transformed into low-molecularweight bioactive molecules.
Grape seed, one of the richest sources in procyanidins, is the important component of grape pomaces from vinification.Zhang et al. [20] used phloroglucinol as nucleophile for acidic degradation of polymeric proanthocyanidins from a grape seed into monomer catechins and their nucleophilic derivatives.Each of the phloroglucinolysis products was successfully separated and isolated in large amount by semi-preparative HSCCC technique under the optimized conditions based on a selection of suitable solvent system.The optimized solvent system consisted of n-hexane-ethyl acetate-water (1:80:80, v/v/v) with a combination of head-tail and tailhead elution modes.By only one-step HSCCC separation, the purity of each obtained phloroglucinolysis product, including monomer catechins and their nucleophile derivatives was above 76%, verified by UPLC. Figure 1 presents the HSCCC chromatogram on the separation of phloroglucinolysis products using head to tail (H-T) and tail to head (T-H) elution modes.
In addition to phloroglucinol, tiopronin was lately experimented, in our laboratory, as a novel thiolcontaining nucleophile for depolymerizing grape seed polymeric proanthocyanidins, with the purpose of producing low-molecular-weight bioactive compounds [21].Tiopronin is a well-known diffusible antioxidant, an antidote to heavy metal poisoning and a radioprotective agent and has powerful protective effects on carbon tetrachloride-induced hepatotoxicity [33].By using this nucleophile, grape seed polymeric proanthocyanidins could be converted into catechins and flavan-3ol−tiopronin derivatives, each of which could be further isolated by HSCCC, with or without the aid of semipreparative HPLC.Direct HSCCC could successfully isolate epicatechin gallate, epicatechin-tiopronin conjugate and epicatechin gallate-tiopronin conjugate, with the yields of 6.5, 29.8, and 10.3 mg and purity of 90.3, 93.7, and 89.5%, by each run.Higher purity of these compounds could be obtained by further semipreparative HPLC.Furthermore, all these degradation products were verified to possess potent antimicrobial activity, with epicatechin gallate the strongest.
Very recently, other non-toxic and bioactive nucleophiles, i.e., captopril and L-cysteine were also tested for degradation of grape seed PPCs in our laboratory [22].L-cysteine is a nature amino acid inhibiting insulin release from the pancreatic β-cell [34]; captopril is orally active antihypertensive agent [35][36].By using these new bioactive nucleophiles in degradation of grape seed PPCs, followed by HSCCC and semi-preparative under the optimized separation conditions, a series of new antioxidants including free catechins and their conjugates were effectively separated, each of them with high purity and high yield, as shown in Tables 1-2 [7][8].
Table 1.Purity and yield of L-cysteine degradation products by HSCCC separation and semi-preparative HPLC (adopted from Tian et al. [22]).

Compounds
Purity (%) Yield (mg) C 93.2±2.Furthermore, all these degradation products presented high antioxidant activities of which the most potent are those galloylated due to presence of a gallate group, leading that the activities of hydroxies were more reactive than others [34].
In a word, grape seed procyanidins are presented essentially in the polymeric form, which are non-nutritive.
The proposed methods permitted transform efficiently such polymers into low-molecular-weight bioactive polyphenols and their derivatives.

Preparation of bioactive polyphenols from grape skins
Quantitatively, grape skins are the most important part of grape pomaces, particularly white grape pomaces from vinification.Polyphenols in grape skins are more complex and diverse than in grape seeds.Grape skins contain not only procyanidins (as grape seeds), but also other phenolic compounds such as flavonols, flavanonols, flavones, derivatives of cinnamic acids and tartaric acid [37]; for grape skins from red varieties, they contain also large amounts of anthocyanins [38].The mean polymerization degree of proanthocyanidins in grape skins may reach 80 [25].As compared with grape seeds, this value is between 25-30 [5,24].Because the disposal of grape skins may also cause environmental problems during wine-making process, the recovery of phenolic compounds from these waste materials is of undoubtedly economic and social significance.
As shown in Figure 2, grape skin polyphenolic extract could be successfully separated, under the optimized HSCCC conditions, into 8 fractions during less than 400 min of elution.For each run, 300 mg of sample loading yielded total of 260.6 mg of different phenolic fractions, with recovery over 86%.
From each of these fractions, a preparative-HPLC separation was applied to isolate individual polyphenols.As shown in Figures. 3 and 4, total of fifteen individual compounds were obtained, most of which presented high yields and purity (all over 90%).
It should be mentioned that the last three fractions from HSCCC (Fig. 2) were those composed of polymeric proanthocyanidins.From this point of view, it is suggested that these fractions can also be degraded using suitable nucleophiles, followed by re-chromatography on HSCCC and/or with preparative HPLC, for the purpose of producing low-molecular-weight bioactive polyphenols.

Conclusion
It is for the first time that, in our laboratory, the different non-toxic and bioactive nucleophiles were applied in degradation of grape seed polymeric procyanidins for the purpose of producing low-molecular-weight bioactive polyphenols.The proposed technology appeared feasible and cost-efficient which can also be applicable for grape skin polymeric proanthocyanidins.The new bioactive compounds obtained are expected to have the possibility for their use in functional food, pharmaceutical and/or cosmetic sectors.

Figure 2 .
Figure 2. HSCCC chromatogram on the separation of grape skin extract using tail to head (0-300 min) and head to tail (300 min-end)