Proanthocyanidin Fraction Research Articles

Preparative separation and bioactivity of oligomeric proanthocyanidins

Oligomeric proanthocyanidins are among the most widely distributed polyphenols in the plant kingdom, being endowed with antioxidant, anti-inflammatory, antimicrobial, immunomodulatory, antitumor and cardioprotective activities. Since oligomeric proanthocyanidins are found in limited amounts in plants and plant-derived products, there is a great need for efficient separation methodologies that can supply analytical quality standards or sufficient material for further bioassays. By efficiently providing a good molar mass distribution of oligomeric proanthocyanidins according to their degree of polymerization, Sephadex LH is the most used sorbent in column chromatography, together with other size exclusion polymers, such as Toyopearl, Diaion, MCI gel CHP or Amberlite. Combination of normal and reversed-phase high pressure liquid chromatography can significantly reduce the total number of chromatographic steps required to obtain high purity oligomeric proanthocyanidins, whereas hydrophilic interaction liquid chromatography on Diol phases can yield proanthocyanidins with a higher degree of polymerization. By lacking a solid support, countercurrent separations can solve issues related to irreversible adsorption, giving higher recoveries, low sample denaturation and a facile scale-up from analytical to industrial production. By taking advantage of the complementarity of all these chromatographic techniques, high-purity oligomeric proanthocyanidin fractions or individual compounds can be obtained on a large scale in a reduced number of separation steps.

Litchi (Litchi chinensis Sonn.) flower proanthocyanidin fraction exhibited protective efficacy to suppress nickel-induced expression for vascular endothelial growth factor in HepG2 cells.

The protective efficacy of litchi (Litchi chinensis Sonn.) flower proanthocyanidin fraction (LFPF) composed of (-)-epicatechin and proanthocyanidin A2 against vascular endothelial growth factor (VEGF) generation induced by nickel (Ni) in hepatocellular carcinoma (Hep G2) cells was studied. VEGF is an angiogenic inducer, which promotes tumor angiogenesis, leading to rapid tumor growth and metastasis. VEGF could be substantially induced in the Ni-mediated Hep G2 cells. Through LFPF treatment, the Ni-induced VEGF generation could be suppressed significantly. The inhibition of HIF-1α expression by blocking phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways, and the suppression of Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT 3), and Raf-1 proto-oncogene, serine/threonine kinase (RAF1)/mitogen-activated protein kinase (MEK1/2)/extracellular-signal-regulated kinase (ERK1/2) pathways are important molecular mechanisms for the LFPF action. LFPF should probably reduce the risk of liver cancer in Ni-contaminated environments by inhibiting VEGF expression. PRACTICAL APPLICATIONS: LFPF mainly contained (-)-epicatechin and proanthocyanidin A2. Our results demonstrated that LFPF considerably suppressed the Ni-induced VEGF expression through inhibition of JAK2/STAT 3 and RAF1/MEK1/2/ERK1/2 pathways and prohibited HIF-1α expression through blocking PI3K/AKT/mTOR pathway. Litchi flowers might have the potential to diminish the liver cancer risk in a Ni-contaminated environment through suitable treatment.

Extraction of Proanthocyanidins from Chinese Wild Rice (Zizania latifolia) and Analyses of Structural Composition and Potential Bioactivities of Different Fractions.

Due to the importance of proanthocyanidin bioactivity and its relationship with chemical structure, ultrasound-assisted extraction and purification schemes were proposed to evaluate the proanthocyanidin content and analyze the structural composition and potential bioactivities of different proanthocyanidin fractions from Chinese wild rice (Zizania latifolia). Following an optimized extraction procedure, the crude wild rice proanthocyanidins (WRPs) were purified using n-butanol extraction, chromatography on macroporous resins, and further fractionation on Sephadex LH-20 to yield six specific fractions (WRPs-1–WRPs-6) containing proanthocyanidin levels exceeding 524.19 ± 3.56 mg/g extract. Structurally, (+)-catechin, (−)-epicatechin, and (−)-epigallocatechin were present as both terminal and extension units, and (−)-epicatechin was the major extension unit, in each fraction. This is the first preparation of WRP fractions with a different mean degree of polymerization (mDP), ranging from 2.66 ± 0.04 to 10.30 ± 0.46. A comparison of the bioactivities of these fractions revealed that fractions WRPs-1−WRPs-5 had significant DPPH radical scavenging activities, whereas fraction WRPs-6 with a high mDP showed better α-glucosidase and pancreatic lipase inhibitory effects. These findings should help define possible applications of WRPs to functional foods or nutraceuticals.

Efficacy of Proanthocyanidins from Pelargonium sidoides Root Extract in Reducing P. gingivalis Viability While Preserving Oral Commensal S. salivarius.

Bacterial resistance to antibiotics and the disruption of beneficial microbiota are key problems in contemporary medicine and make the search for new, more efficient infection treatment strategies among the most important tasks in medicine. Multicomponent plant-derived preparations with mild antibacterial activity created by many simultaneous mechanisms together with anti-inflammatory, innate immune and regenerative capacity-stimulating properties are good candidates for this therapy, and proanthocyanidins are among the most promising compounds of this sort. In this study, we have isolated proanthocyanidins from Pelargonium sidoides DC root extract and characterized and compared the composition, antioxidant properties and antibacterial activity of the proanthocyanidin fraction with those of the whole extract. The results revealed that proanthocyanidins had significantly stronger antioxidant capacity compared to the root extract and exhibited a unique antibacterial action profile that selectively targets Gram-negative keystone periodontal and peri-implant pathogenic strains, such as Porphyromonas gingivalis, while preserving the viability of beneficial oral commensal Streptococcus salivarius. The finding suggests that proanthocyanidins from Pelargonium sidoides root extract are good candidates for the prolonged and harmless treatment of infectious diseases.

Comparison of phytochemical profiles and antiproliferative activities of different proanthocyanidins fractions from Choerospondias axillaris fruit peels

Epidemiological studies had indicated that the incidence of colorectal cancer is inversely correlated to the consumption of proanthocyanidins (PAs). In this study, five PAs fractions isolated from Choerospondias axillaris fruit peels with different mean degree of polymerization (mDP) were characterized and their antiproliferative effects on Caco-2 cells were investigated. MALDI-TOF-MS results indicated that PAs fractions were composed of procyanidins with DP up to 13-mers. All PAs fractions induced dose and time-dependent reductions of Caco-2 cell viability. The IC50 values of PAs fractions with increasing mDP were 240 ± 16.0, 143 ± 9.53, 87.0 ± 3.55, 44.3 ± 4.89, and 42.8 ± 2.20 μg/mL after 24 h incubation, respectively. The observed reduction in Caco-2 cell viability was due to apoptosis via the activation of caspase-9, caspase-3, caspase-8 and the elevation of intracellular ROS generation. Moreover, the extent of the reduction in cell viability after exposure to PAs fractions was positively correlated with their mDP and galloylation. These results indicate that Choerospondias axillaris peel is a potential source of natural chemopreventive agents for the treatment of colorectal cancer.

Controlled water deficit modifies the phenolic composition and sensory properties in Cabernet Sauvignon wines

Abstract This study investigates the chemical composition and sensory properties of wines from Cabernet Sauvignon vines grown under controlled water deficit in two consecutive seasons. The wines were made from fruit of grapevines that were maintained under three water status levels (i.e. T1, Ψstem = −0.8 MPa; T2, Ψstem = −0.9 MPa; T3, Ψstem = −1.0 MPa) from veraison until harvest. Our results suggest that wine phenolic composition was affected by controlled water deficit, where T3 wines exhibited a higher concentration of total phenols, total anthocyanins and chroma (C*) in both seasons. These results coincide with the principal component analysis that indicated a substantial separation between years and deficit irrigation. We found that irrigation treatments only produce differences in concentration, but not in anthocyanin composition in both years. Separation of proanthocyanidins fractions by solid phase extraction using Sep-Pak Plus tC18 cartridges showed only a change in the concentration of the monomeric fraction in 2014 season, but not in the proportion of the different proanthocyanidins fractions in both seasons. Finally, the sensory composition of wines showed differences that depend on the season and resulted in more red fruits, more fullness perception in mouth and more color intensity in wines from less irrigated treatments.

Phenolic composition and sensory characteristics of Cabernet Sauvignon wines: effect of water stress and harvest date

SummaryThe effect of three water status level and two harvest date 55 and 64 days after veraison (DAV) on phenolic and sensory composition of Cabernet Sauvignon wines were investigated. The later harvest date led to wine with a higher alcohol content. Total phenols varied from 1439.66 to 1643.08 mg L−1 with higher values at 64 DAV. No differences were observed between irrigation treatments. For total tannins and anthocyanins, no differences were found between harvest date. Separation of proanthocyanidins by Sep‐Pak Plus tC18 cartridges showed only differences in concentration but not in the proportion of proanthocyanidin fractions. The wines from the most restricted treatment had a better colour and the same aroma of red fruits, persistence, astringency, fullness and bitterness, as the wine from the treatment with highest irrigation. Under the assay conditions, it was possible to obtain wines with a similar chemical and sensory composition earlier and using less irrigation water.

Three new flavonoids, proanthocyanidin, and accompanying phenolic constituents from Feijoa sellowiana.

Our investigation of phenolic constituents of fruits, flower buds, and leaves of Feijoa sellowiana led to the isolation of twenty-one phenolics including three new gossypetin glycosides 1-3, and also the purification of a proanthocyanidin fraction. A high-performance liquid chromatography method for simultaneous analysis of phenolic constituents was established and then used to investigate the phenolic profiles of the parts of the plant species, to show the presence of characteristic flavonoids and ellagic acid derivatives or ellagitannins in the extracts from fruits, flower buds, and leaves. The branch extract profile also suggested the presence of alkylated ellagic acids as characteristic constituents. Inhibitory effects of feijoa flavonoids on mushroom tyrosinase were seen, although in some cases this may have resulted from direct interaction with the enzyme. Cytotoxic effect of the proanthocyanidin fraction was also shown.

Different Inhibitory Potencies of Oseltamivir Carboxylate, Zanamivir, and Several Tannins on Bacterial and Viral Neuraminidases as Assessed in a Cell-Free Fluorescence-Based Enzyme Inhibition Assay.

Neuraminidase is a key enzyme in the life cycle of influenza viruses and is present in some bacterial pathogens. We here assess the inhibitory potency of plant tannins versus clinically used inhibitors on both a viral and a bacterial model neuraminidase by applying the 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA)-based activity assay. A range of flavan-3-ols, ellagitannins and chemically defined proanthocyanidin fractions was evaluated in comparison to oseltamivir carboxylate and zanamivir for their inhibitory activities against viral influenza A (H1N1) and bacterial Vibrio cholerae neuraminidase (VCNA). Compared to the positive controls, all tested polyphenols displayed a weak inhibition of the viral enzyme but similar or even higher potency on the bacterial neuraminidase. Structure–activity relationship analyses revealed the presence of galloyl groups and the hydroxylation pattern of the flavan skeleton to be crucial for inhibitory activity. The combination of zanamivir and EPs® 7630 (root extract of Pelargonium sidoides) showed synergistic inhibitory effects on the bacterial neuraminidase. Co-crystal structures of VCNA with oseltamivir carboxylate and zanamivir provided insight into bacterial versus viral enzyme-inhibitor interactions. The current data clearly indicate that inhibitor potency strongly depends on the biological origin of the enzyme and that results are not readily transferable. The therapeutic relevance of our findings is briefly discussed.

Investigation of the flavan-3-ol patterns in willow species during one growing-season

Flavonoids, proanthocyanidins (PAs) and salicylic alcohol derivatives are the main groups of ingredients in Salix needed as defensive tools and signal molecules, but have also pharmaceutical importance. The present study investigated total PA content, complete PA pattern, the oligomeric/total PAs quotient and the contents of catechin and epicatechin during one growing-season for the leaves and this year's sprouts in ten willows (Salix pentandra L. ♂, S. alba L. ♂, S.fragilis L. ♀, S.caprea L. ♂ & ♀, S.cinerea L. ♂, S.caprea x cinerea ♂, S. daphnoidesVill. ♂ & ♀ and S.purpurea L. ♀; all Salicaceae). Comparison of the different species revealed distinct seasonal fluctuations of the oligomeric and polymeric PA fractions, but the contents of both groups always developed in the same direction. All willows prefer the synthesis of PAs with DP-2 - DP-4 within the oligomeric fraction (DP-2 - DP-10) and species with rather low PA contents like S.purpurea (0.1-2.6mg/g) as well as species with rather high PA contents like S.alba (3.8-14.7mg/g) were found. Only slight gender specific differences could be observed for both sexes of S.daphnoides and S.caprea. The PA pattern of the hybrid S.caprea x cinerea seems to be influenced by both parents. Thus, the accumulation of the oligomeric PAs accorded to S.caprea and the polymeric PAs matched S.cinerea resulting in an overall depression of PAs in the sprouts and a varying seasonal trend in the leaves. In contrast, the content of catechin remained high and seemed to be not influenced in the hybrid. Although only one individual of each Salix species could be considered in this screening study, the present results demonstrate the variability of the flavan-3-ol pattern within the genus Salix but also some preliminary correlations could be observed. Future studies with more Salix species will provide more insights into chemotaxonomic correlations.

Characterization of Proanthocyanidin Oligomers of Ephedra sinica.

Ephedra sinica, an important plant in Chinese traditional medicine, contains a complex mixture of proanthocyanidin oligomers as major constituents; however, only the minor components have been chemically characterized. In this study, oligomers with relatively large molecular weights, which form the main body of the proanthocyanidin fractions, were separated by adsorption and size-exclusion chromatography. Acid-catalyzed degradation in the presence of mercaptoethanol or phloroglucinol led to the isolation of 18 fragments, the structures of which were elucidated from their experimental and TDDFT-calculated ECD spectra. The results indicated that (−)-epigallocatechin was the main extension unit, while catechin, the A-type epigallocatechin–gallocatechin dimer, and the A-type epigallocatechin homodimer, were identified as the terminal units. Among the degradation products, thioethers of gallocatechin with 3,4-cis configurations, a B-type prodelphinidin dimer, a prodelphinidin trimer with both A- and B-type linkages, and a prodelphinidin dimer with an α-substituted A-type linkage were new compounds. In addition, a phloroglucinol adduct of an A-type prodelphinidin dimer, a doubly-linked phloroglucinol adduct of epigallocatechin, and a unique product with a flavan-3-ol skeleton generated by the rearrangement of the aromatic rings were also isolated.

Cross-modal interactions and effects of the level of expertise on the perception of bitterness and astringency of red wines

The present study investigates the role of sensory interactions and the level of expertise of consumers on the perception of bitterness and astringency in a red wine complex matrix.In the first experiment, a commercial red wine was spiked with quinine sulphate to produce two levels of bitterness. Seven samples with high and seven with low bitterness were further spiked with one of the following aroma vectors: vegetal, herbal, spicy, roasted, animal, dried fruit or with tartaric acid to modulate sourness. In the second experiment, the wine was spiked with an astringent proanthocyanidin fraction to produce two levels of astringency and with one of the seven vectors used in the first experiment or with quinine sulphate to modulate bitterness. Fourteen and 16 spiked samples of experiments 1 and 2, respectively, were sensory evaluated together with control samples to assess the intensity of the six aroma vectors, sourness, bitterness and astringency. Evaluation was performed using a descriptive analysis technique based on intensity rating by three groups of participants with different levels of expertise. The effect of the addition of one aroma vector, sourness or bitterness on bitter or astringent intensity was evaluated by ANOVA.No significant effect of aroma, sourness or bitterness was observed for astringent intensity, confirming that in these red wines, cross-modal interactions are not relevant in astringency. Bitter-aroma interactions were demonstrated for animal aroma just for less-experienced consumers (novices) attributed to a hedonic synergism. Significant decreases in bitterness due to sour vector were observed regardless consumer expertise. This could be explained in terms of interactions at both cognitive and receptor level.

Separation and purification of polyphenols from red wine extracts using high speed counter current chromatography.

Polyphenols are important compounds of red wine owing to their contribution to sensory properties and antioxidant activities. In this study, high-speed counter-current chromatography (HSCCC) coupled with semi-preparative HPLC was used for large-scale separation and purification of polyphenols from red wine extracts. With the solvent system of hexane-ethyl acetate-water (1-50-50), various oligomeric procyanidins including monomer catechin, epicatechin, dimers B1, B2; phenolic acids including coutaric acid, caftaric acid and other type of polyphenols were largely separated within 370min and most of these compounds presented high yields (0.97mg to 13.79mg) with high purity (90.34% to 98.91%) after the semi-preparative HPLC isolation. Using the solvent system of Methyl tert-Butyl Ether (MTBE) - n-butyl alcohol- acetonitrile-water (1-40-1-50, acidified with 0.01% trifluoroacetic acid (TFA)) by one-step HSCCC of 100mg of the red wine extracts, the major anthocyanins, i.e., malvidin-3-O-glucoside, delphinidin-3-O-glucoside and peonidin-3-O-glucoside, as well as two polymeric proanthocyanidin fractions were successfully separated one another within 320min. The yields of malvidin-3-O-glucoside, delphinidin-3-O-glucoside and peonidin-3-O-glucoside were 12.12mg, 1.78mg and 11.57mg with the purity of 92.74%, 91.03% and 91.21%, respectively. Thiolysis-UPLC analysis indicated that the two polymeric proanthocyanidin fractions presented high purity, with mean degree of polymerization of 7.66±0.12 and 6.20±0.09, respectively. The further experiments on the antioxidant activities by DPPH radical test, FRAP assay and ABTS method showed that all of the isolated procyandins and anthocyanins and the two polymeric proanthocyanidin fractions, with exception of phenolic acids possessed much greater antioxidant activities compared to standard Trolox andl-ascorbic acid (2-14 times).

Supercritical fluid extraction of polyphenols from grape seed (Vitis vinifera): Study on process variables and kinetics

Polyphenol extraction from white grape seeds using supercritical carbon dioxide (SC-CO2) containing different percentages of ethanol-water mixture (57% v/v) as co-solvent was carried out at 40°C. Box–Behnken experimental design was used to optimize the process variables: pressure (80, 100, 120bar), CO2 flow rate (2, 4 and 6kg/h) and co-solvent percentages (10, 15, and 20% (w/w)). Total polyphenols (TPC) and fractionation of proanthocyanidins were determined.The statistically generated optimum extraction conditions to obtain the highest total polyphenol concentration were a pressure of 80bar, CO2 flow rate of 6kg/h and 20% (w/w) co-solvent. The value achieved in validation experimental was 7132mg GAE/100g DM. Under these operating conditions a good extraction of FI (>1000mg catechin/100g DM) and FII (>800mg catechin/100g DM) are obtained.

Effect of the proanthocyanidin fraction from Medemia argun on the in vitro growth and activity of selected soil microorganisms

Effect of the proanthocyanidin fraction from Medemia argun on the in vitro growth and activity of selected soil microorganisms

Relationship Between Antioxidant Capacity, Proanthocyanidin and Anthocyanin Content During Grape Maturation of Touriga Nacional and Tinta Roriz Grape Varieties

To investigate antioxidant capacity in seeds and skins during grape maturation and its relationship with anthocyanin and proanthocyanidin content, two Portuguese red grape varieties, Touriga Nacional and Tinta Roriz (Vitis vinifera L.) were studied. Two analytical methods were used for antioxidant capacity analysis: the DPPH and ABTS methods. Proanthocyanidins from seeds and skins were separated into monomers, oligomers and polymers, while 13 individual anthocyanins from the skins were also evaluated by HPLC. For both grape varieties studied, antioxidant capacity from the skins and seeds was characterised during grape maturation by a general decrease, mainly in the first weeks after veraison, followed by stabilisation and a slight increase in the values in the last three weeks of ripening. A similar tendency was observed for the amount of all the different proanthocyanidin fractions quantified. Our results also showed that seeds are an important source of proanthocyanidins with respect to the grape berry skins. Seeds were also the grape berry fraction with the highest antioxidant capacity when compared to the antioxidant capacity content of the skins. For the 13 individual monomeric anthocyanins quantified during grape maturation, evolution was generally characterised by a continuous increase in the values. However, for some of the individual anthocyanins, the continuous increase was followed by stabilisation or a decrease in the values in the last weeks of ripening. Finally, there was a positive relationship between the different proanthocyanidin fractions and antioxidant capacity of both grape varieties studied; while a negative relationship during grape maturation was obtained for individual anthocyanins.

Supercritical antisolvent precipitation of polyphenols from grape marc extract

Supercritical anti-solvent (SAS) conditions to obtain the maximum enrichment of total polyphenols from grape marc extract was studied by 23 factorial design. Operating at 12MPa, 45°C and 0.99 CO2 molar fraction, a relative enrichment of 350% of total polyphenols as well as a relative enrichment of proanthocyanidins fractions between 300 and 450% was achieved. Under these process conditions, microparticles in the order of 5μm size were produced. The content of the main phenolic acid (gallic acid) and flavonoids (catechin, epicatechin, quercetin, malvidin and procyanidin B2) for the different precipitates was determined using high performance liquid chromatography. Starting from 100g of grape marc, the amount of total polyphenols obtained by SAS ranged from 60 to 300mg GAE(GallicAcidEquivalent)/100g DGM(DriedGrapeMarc).

Characterization of the XIAP-Inhibiting Proanthocyanidin Fraction of the Aerial Parts of Ephedra sinica.

The X-linked inhibitor of apoptosis protein is a cellular protein that inhibits the activity of mammalian caspases and promotes resistance to apoptosis. The ethanol extract of the aerial parts of Ephedra sinica has been identified to possess inhibitory activity of the X-linked inhibitor of apoptosis protein by an in vitro fluorescence polarization assay using the BIR3 domain of the X-linked inhibitor of apoptosis protein. Bioactivity-guided fractionation identified proanthocyanidin-enriched fractions as the active principles. The most active fraction showed an IC50 value of 27.3 µg/mL (CI95: 25.9-28.9 µg/mL) corresponding to 9.6 µM (CI95: 9.1-10.1 µM) calculated by the use of the determined average molecular weight of 2853.5. Samples were analyzed by a thiolytic degradation/HPLC-MS assay, UHPLC-HRMS, and 1D NMR.The thiolytic degradation/HPLC-MS assay revealed a mean degree of polymerization of 9.5 ± 0.2 units (calculated average MW 2853.5) for the active fraction and 11.4 ± 0.6 units (calculated average MW 3437.0) for the most related inactive fraction. Chemical characterization identified (epi)gallocatechin (76.6 ± 1.0 % active; 80.7 ± 2.7 % inactive sample) and (epi)catechin units as building blocks. Interestingly, the investigated proanthocyanidins turned out to be a complex mixture of double linked A-type (binding 2-O-7″, 4-6″) and single linked B-type units.This study identified oligomeric proanthocyanidins as active principles of E. sinica in vitro by a fluorescence polarization assay and via protein fragment complementation analysis.

Two new proanthocyanidin trimers isolated from Cistus incanus L. demonstrate potent anti-inflammatory activity and selectivity to cyclooxygenase isoenzymes inhibition

Two new proanthocyanidin trimers have been isolated from Cistus incanus herb; gallocatechin-(4α→6)-gallocatechin-(4α→8)-gallocatechin (compound 1) and epigallocatechin-3-O-gallate-(4ß→8)-epigallocatechin-3-O-gallate-(4ß→8)-gallocatechin (compound 2). The structures were determined on the basis of 1D- and 2D-NMR (HSQC, HMBC) of their peracetylated derivatives, MALDI-TOF-MS and by acid-catalysed degradation with phloroglucinol. A more abundant proanthocyanidin oligomer was also isolated, purified and its chemical constitution studied by 13C-NMR and phloroglucinol degradation. The mean molecular weight of the polymer was estimated to be about 7 to 8 flavan-3-ol-units with a ratio of procyanidin : prodelphinidin units at 1:5, some of which are derivatised by gallic acid. Water extract and higher oligomeric proanthocyanidin fractions of C. incanus significantly inhibited TPA-induced oedema when applied topically at doses of 0.5 and 1 mg/ear in mice. Furthermore, the extracts and the pure compounds inhibited COX-1 and COX-2 activities. In addition, compound 2 exhibited an IC50 of 4.5 μM against COX-2 indicating its high selectivity towards COX-2.

Isolation of a Tetrameric A-type Proanthocyanidin Containing Fraction from Fresh Tea (Camellia Sinensis) Leaves Using High-Speed Counter-Current Chromatography

A proanthocyanidin (PA) fraction containing tetrameric A- and B-type PAs was obtained after Sephadex LH-20 chromatography and high-speed counter-current chromatography (HSCCC) of a PA extract from fresh tea (Camellia sinensis) leaves. The biphasic solvent system hexane–ethyl acetate–methanol–water system (1:5:1:5), with the aqueous phase as the mobile phase, was selected on the basis of partition coefficient (KD1.02), settling time (18 s), and stationary phase retention (60%). Electrospray ionization mass spectrometry (ESI-MS) indicated the presence of A-type linkages in the tetrameric PAs in one of the HSCCC fractions.