High-performance liquid chromatographic determination of oligomeric procyanidins from dimers up to the hexamer in hawthorn

An HPLC method using UV diode array detection was developed for analysing procyanidins qualitatively and quantitatively up to the hexameric level in hawthorn samples. The analysed compounds included procyanidin dimers B-2, B-4 and B-5, procyanidin trimers C-1, epicatechin-(4beta-->8)-epicatechin-(4beta-->6)-epicatechin and epicatechin-(4beta-->6)-epicatechin-(4beta-->8)-epicatechin, a tetramer D-1 and a pentamer E-1 both consisting of (-)-epicatechin units linked through C-4beta/C-8 bonds. The concentrations of two unknown tetramers and a hexamer F were also quantified. The oligomeric procyanidins (OPs) were specifically determined due to the development of a method for isolating them from hawthorn during sample preparation. The pattern of oligomeric procyanidins in the leaves, flowers and fruits was similar, but the concentrations varied depending on the part of the plant. The concentration in leaves was 1.6%, in flowers 1.2% and in fruits 0.2% of the dry mass. The method was validated with respect to repeatability, recovery, linearity, and sensitivity. The repeatability for the quantitative analytical method of all the OPs in leaves was 7.7%, in flowers 8.8%, and in fruits 12.3%. The recovery of the main OPs ranged from 91 to 97%. The correlation coefficients of calibration curves were between 0.997 and 1.000. The limits of quantitation for different procyanidin standards were 0.05-0.12 mg/ml, when 10 microl of each standard solution was injected into the HPLC.     

Procyanidins from Myrothamnus flabellifolia

From the ethyl acetate soluble fraction of an acetone-water extract of the twig tips of Myrothamnus flabellifolia Welw. (Myrothamnaceae), a variety of flavan-3-ols (epicatechin, epigallocatechin and their 3-O-galloylated analogues) and procyanidins (DP 8)-catechin], B4 [catechin-(4alpha --> 8)-epicatechin], B6 [catechin-(4alpha --> 6)-catechin] and catechin-(4alpha --> 8)-epigallocatechin along with the galloylated analogues B4-3'-O-gallate, procyanidin B2-3'-O-gallate [epicatechin-(4beta --> 8)-epicatechin-3-O-gallate], B2-3,3'-di-O-gallate, procyanidin B5-3,3'-O-gallate [epicatechin-3-O-gallate-(4beta --> 6)-epicatechin-3-O-gallate], catechin-(4alpha --> 8)-epigallocatechin-3-O-gallate, the trimer procyanidin C1-3'-O-gallate[epicatechin-(4beta --> 8)-epicatechin-(4beta --> 8)-epicatechin-3-O-gallate] and the new epicatechin-3-O-gallate-(4beta --> 6)-epicatechin-3-O-p-hydroxybenzoate. The structures were elucidated by 1D- and 2D-NMR experiments of their peracetylated derivatives, partial acid-catalysed degradation with phloroglucinol, ESI-MS and CD spectra.     

Large-scale isolation of flavan-3-ol phloroglucinol adducts by high-speed counter-current chromatography

Flavan-3-ol phloroglucinol adducts were synthesised through acid catalysed degradation of a procyanidins-rich grape seed extract in the presence of phloroglucinol. The reaction mixture (3.3 g) was fractionated without further sample preparation using the all-liquid chromatographic technique of high-speed counter-current chromatography (HSCCC). Selected solvent systems were hexane-ethyl acetate-methanol-water (0.1:5:0.1:5, v/v/v/v) and (1.5:10:1.5:10, v/v/v/v). The fractions obtained were found to contain almost pure compounds, in some cases final purification was achieved by preparative HPLC. The so-obtained pure standards of (+)catechin-(4alpha-->2)-phloroglucinol, (-)epicatechin-(4beta-->2)-phloroglucinol, (+)catechine, (-)epicatechin-3-O-galloyl-(4beta-->2)-phloroglucinol, (-)epicatechin, and (-)epicatechin gallate are required for quantification of acid-catalysed phloroglucinol degradation products of procyanidins.     

Studies on the constituents of bark of Parameria laevigata Moldenke

One new trimeric proanthocyanidin, epicatechin-(2beta-->O-->7, 4beta-->6)-epicatechin-(2beta-->O--->7, 4beta-->8)-epicatechin (5) and two new tetrameric proanthocyanidins, epicatechin-(2beta-->O-->7, 4beta-->8)-[epicatechin-(4beta-->6)]-epicatechin-(4beta-->8)-epicatechin, named as parameritannin A-1 (6), and epicatechin-(2beta-->O-->5, 4beta-->6)-[epicatechin-(2beta-->O-->7, 4beta-->8)]-epicatechin-(4beta-->8)-epicatechin, named as parameritannin A-2 (7), have been isolated from the bark of Parameria laevigata Moldenke (Apocynaceae) along with the two known dimers, proanthocyanidin A-2 (1) and proanthocyanidin A-6 (2), and two trimers, cinnamtannin B-1 (3) and aesculitannin B (4). These structures were elucidated by spectral and chemical evidence.     

Relationship between Neuroprotective Effects and Structure of Procyanidins

This study evaluated the relationship between the neuroprotective effects of procyanidins and their structural characteristics. In vitro, a rat pheochromocytoma cell line (PC12) was exposed to the grape seed-derived procyanidin monomers: catechin (C), epicatechin (EC), and epicatechin gallate (ECG); the procyanidin dimers: procyanidin B1 (B1), procyanidin B2 (B2), procyanidin B3 (B3), procyanidin B4 (B4), procyanidin B1-3-O-gallate (B1-G), and procyanidin B2-3-O-gallate (B2-G); and the procyanidin trimers: procyanidin C1 (C1) and N-acetyl-l-cysteine (NAC) for 24 h. Cells were then incubated with 200 μM H₂O₂ for 24 h. In vivo, zebrafish larvae (AB strain) 3 days post-fertilization were incubated with NAC or procyanidins (C, EC, ECG, B1, B2, B3, B4, B1-G, B2-G, C1) in 300 µM H₂O₂ for 4 days. Different grape seed procyanidins increased the survival of PC12 cells challenged with H₂O₂, improved the movement behavior disorder of zebrafish caused by H₂O₂, inhibited the increase of ROS and MDA and the decrease of GSH-Px, CAT, and SOD activities, and up-regulated the Nrf2/ARE pathway. The neuroprotective effects of the procyanidin trimer C1 treatment group were greater than the other treatment groups. These results suggest that the neuroprotective effect of procyanidins is positively correlated with their degree of polymerization.     

Separation of eight selected flavan-3-ols on cellulose thin-layer chromatographic plates

The potential of microcristaline cellulose as sorbent in the separation of eight compounds: (+)-catechin (C), (-)-epicatechin (EC), (-)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), (-)-epigallocatechin gallate (EGCg), procyanidin B1 and procyanidin B2 was studied. Cellulose HPTLC plates prewashed in water (not necessary, when water was used as developing solvent) and dried with a hair dryer, bandwise application and development in horizontal developing chamber (sandwich configuration) gave the best results. Detection was performed using vanillin-H3PO4 reagent. Four new developing solvent systems were proposed: water, 1-propanol-water (20:80, v/v), 1-propanol-water-acetic acid (4:2:1, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v), and at least two of them were needed for the differentiation between all eight compounds. Surprisingly, water enabled the separation of epimers C from EC and GC from EGC, as well as the dimers procianidin B1 and B2. Additionally, C, EGC, B1 and B2 were separated from all the other compounds. The best choice for developing solvent is given for each of the studied compounds. The best separation of the five main catechins (EC, GC, EGC, ECg, EGCg) present in green tea extract was achieved using 1-propanol-water-acetic acid (20:80:1, v/v). The chromatograms of oak bark extract developed in solvents with higher water content (1-propanol-water (1:4, v/v) and 1-propanol-water-acetic acid (20:80:1, v/v)) showed less bands than chromatograms developed in solvents with higher organic modifier content (e.g. 1-propanol-water-acetic acid (4:2:1, v/v)). It was proved that such behavior was due to the presence of procyanidins beside the main component catechin.     

The Relationship between Procyanidin Structure and Their Protective Effect in a Parkinson’s Disease Model

This study evaluated the effect of grape seed-derived monomer, dimeric, and trimeric procyanidins on rat pheochromocytoma cell line (PC12) cells and in a zebrafish Parkinson’s disease (PD) model. PC12 cells were cultured with grape seed-derived procyanidins or deprenyl for 24 h and then exposed to 1.5 mm 1-methyl-4-phenylpyridinium (MPP⁺) for 24 h. Zebrafish larvae (AB strain) 3 days post-fertilization were incubated with deprenyl or grape seed-derived procyanidins in 400 µM 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 4 days. The results showed that the procyanidin dimers procyanidin B1 (B1), procyanidin B2 (B2), procyanidin B3 (B3), procyanidin B4 (B4), procyanidin B1-3-O-gallate (B1-G), procyanidin B2-3-O-gallate (B2-G), and the procyanidin trimer procyanidin C1 (C1) had a protective effect on PC12 cells, decreasing the damaged dopaminergic neurons and motor impairment in zebrafish. In PC12 cells and the zebrafish PD model, procyanidin (B1, B2, B3, B4, B1-G, B2-G, C1) treatment decreased the content of reactive oxygen species (ROS) and malondialdehyde (MDA), increased the activity of antioxidant enzymes glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD), and upregulated the expression of nuclear factor-erythroid 2-related factor (Nrf2), NAD(P)H: quinone oxidoreductase 1 (NQO1), and heme oxygenase-1 (HO-1). These results suggest that in PC12 cells and the zebrafish PD model, the neuroprotective effects of the procyanidins were positively correlated with their degree of polymerization.     

Excess molar volume measurements of ternary mixtures [2-propanol+ethyl acetate+n-hexane] and their binary constituents at 298.15, 308.15 and 313.15 K

The excess molar volume of the ternary mixture [2-propanol?+?ethyl acetate?+?n-hexane], and its binary constituents; [2-propanol?+?ethyl acetate], [2-propanol?+?n-hexane] and [ethyl acetate?+?n-hexane] were evaluated by the mixtures density measurements over the whole concentration range at three temperatures 298.15, 308.15 and 313.15?K. The excess molar volumes data were fitted to the Redlich–Kister (RK) type equation and the parameters of this equation have been calculated and presented for the studied mixtures.     

Separation of caffeoylquinic acids and flavonoids from Asteris souliei by high‐performance counter‐current chromatography and their anti‐inflammatory activity in vitro

Eleven compounds were successfully separated from Asteris souliei by using a two‐step high‐performance counter‐current chromatography method. The first step involved a reversed phase isocratic counter‐current chromatography separation using hexane/ethyl acetate/methanol/water (1:0.8:1:1 v/v/v/v), which produced three fractions, the first two of which were mixtures. The second step used step‐gradient reversed‐phase counter‐current chromatography with hexane/butanol/ethyl acetate/methanol/water (1:0.5:3.5:1:4 v/v/v/v/v) initially followed by hexane/ethyl acetate/methanol/water (1:2:1:2 v/v/v/v) to separate Fraction 1 into seven compounds; and hexane/ethyl acetate/methanol/water (1:1:1:1.2 v/v/v/v) to separate Fraction 2 into three further compounds. The chemical structures of the separated compounds were identified by ESI‐MS and NMR spectroscopy (¹H and ¹³C). Baicalin ( 5 ), eriodictyol ( 7 ), apigenin‐7‐glycoside ( 8 ), quercetin ( 9 ), luteolin ( 10 ), and apigenin ( 11 ) showed obvious inhibitory effects on lipopolysaccharide‐induced nitric oxide production in RAW264.7 cells at a concentration of 10 μg/mL.     

Preparative isolation of huperzines A and B from Huperzia serrata by displacement centrifugal partition chromatography

The pH-zone refining centrifugal partition chromatography technique was used to separate the two acetylcholinesterase inhibitors huperzines A and B from a crude alkaloid extract of the club moss Huperzia serrata. Complete co-elution of huperzines A and B was initially observed with the well-known methyl tert-butyl ether-acetonitrile-water (4:1:5, v/v/v) solvent system with triethylamine (8mM) as the displacer and methane sulfonic acid (6mM) as the retainer. An efficient biphasic system was designed on the basis of solvent association that provided selectivity in the elution mode: n-heptane/ethyl acetate/n-propanol/water (5:15:35:45, v/v/v/v). Lowering the bridge solvent content (n-propanol) of this system increased the polarity difference between the two phases thus adapting it to the pH-zone refining mode. Thus, the purification of these compounds was achieved using the biphasic system n-heptane/ethyl acetate/n-propanol/water (10:30:15:45, v/v/v/v) with triethylamine (8mM) as the displacer and methane sulfonic acid (6mM) as the retainer.     

Preparative purification of plasmin activity stimulating phenolic derivatives from Gastrodia elata using centrifugal partition chromatography

Gastrodia rhizome, a dried and steamed tuber of Gastrodia elata Blume (Orchidaceae), has been traditionally used in Korea, China and Japan for the treatment of neurological and nervous disorders such as headaches, dizziness, vertigo and convulsive illnesses. The ethyl acetate and water extracts of G. elata stimulated plasmin activity. The active ethyl acetate fraction was subjected to centrifugal partition chromatography (CPC) with a two‐phase solvent system, composed of n‐hexane–ethyl acetate–methanol–water (3:7:4:6, v/v) followed by semi‐preparative HPLC purification to separate active compounds and the water fraction was purified by Diaion HP‐20 resin and semi‐preparative HPLC. In ethyl acetate extract, 4‐hydroxybenzyl alcohol (1), 4‐hydroxybenzoic acid (2), 4‐hydroxybenzaldehyde (3), 4‐ethoxymethylphenol (4), 4,4′‐oxybis(methylene)diphenol (5) and 4,4′‐methylenediphenol (6) were obtained with high purities. Parishin (7) and parishin B (8) were isolated from water extract. Among isolated compounds, 4‐hydroxybenzyl alcohol (1), 4‐hydroxybenzaldehyde (3) and 4‐ethoxymethylphenol (4) significantly stimulated plasmin activity. Copyright © 2015 John Wiley & Sons, Ltd.     

Preparative separation of five flavones from flowers of Polygonum cuspidatum by high‐speed countercurrent chromatography

A preparative high‐speed countercurrent chromatography method was successfully used for the isolation of five minor flavones from Polygonum cuspidatum flowers. Among them, three compounds were obtained from P. cuspidatum for the first time. A twin two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (1:6:3:6, v/v/v/v) and petroleum ether/ethyl acetate/methanol/water (2:4:3:3, v/v/v/v) was developed. Compounds were obtained from the fraction B and fraction C prepurified by silica gel column chromatography. Five minor compositions, 6.8 mg of hesperidin, 11.2 mg of phloridzin, 4.9 mg of luteolin, 5.3 mg of hyperin, and 3.7 mg of luteoloside were obtained from 140 mg of the fraction B and 110 mg of fraction C with a purity of 95.3, 96.4, 98.0, 96.8, and 95.3%, respectively, as determined by high‐performance liquid chromatography. The structures of these compounds were identified by ¹H and ¹³C NMR spectroscopy.     

Kinetic and thermodynamic barriers to carbon and oxygen alkylation of phenol and phenoxide ion by the 1-(4-methoxyphenyl)ethyl carbocation

Rate constant ratios for addition of the three nucleophilic sites of phenol to the 1-(4-methoxyphenyl)ethyl carbocation (1+) in 50/50 (v/v) trifluoroethanol/water were determined from the relative yields of the three phenol adducts, and absolute rate constants were determined from product rate constant ratios for addition of phenol and azide ion to 1+ using k(az) = 5 x 10(9) M(-1) s(-1) for the diffusion-limited reaction of azide ion. A selectivity of 230:20:1 was determined for alkylation of phenol at oxygen, C-4 and C-2 to form 1-OPh and biphenyls 1-(4-C6H4OH) and 1-(2-C6H4OH), respectively, and of 2:2:1 for alkylation of the corresponding nucleophilic sites of phenoxide ion in diffusion-limited reactions. The Mayr nucleophilicity parameter for C-4 of phenol is N = 2.0. Encounter-limited addition of phenoxide ion to 1+ to form 1-OPh is faster than encounter-limited addition of oxygen anions that are either more or less basic than phenoxide ion. Only the products of solvolysis are observed from acid-catalyzed cleavage of 1-OPh in 50/50 (v/v) trifluoroethanol/water, but a 50% yield of biphenyls 1-(4-C6H4OH) and 1-(2-C6H4OH) are observed from spontaneous cleavage of 1-OPh, where the leaving group is phenoxide ion, because of the very low kinetic barriers to collapse of the ion pair intermediate 1+.PhO-. The 230-fold larger rate constant for O-compared to C-2-alkylation of phenol is due primarily to the larger thermodynamic driving force for oxygen addition. There are similar Marcus intrinsic barriers for these two reactions.     

High‐performance countercurrent chromatography separation of Peucedanum cervaria fruit extract for the isolation of rare coumarin derivatives

For the first time, rare major and minor compounds from fruits of Peucedanum cervaria were isolated. High‐performance countercurrent chromatography with two different solvent systems, heptane/ethyl acetate/methanol/water (3:2:3:2 and 2:1:2:1, v/v), was successfully used in the reversed‐phase mode. A scale‐up process from analytical to semipreparative in a very short time was developed. The structures of isolated compounds were evaluated by high‐performance liquid chromatography with diode array detection and electrospray ionization mass spectrometry, gas chromatography with mass spectrometry, and one‐ and two‐dimensional NMR spectroscopy. (8S,9R)‐9‐(3‐Methylbutenoyloxy)‐O‐acetyl‐8,9‐dihydrooroselol (compound B), (8S,9R)‐9‐(2‐methyl‐Z‐butenoyloxy)‐O‐acetyl‐8,9‐dihydrooroselol (edultin, compound C), and (8S,9R)‐9‐acetoxy‐O‐(2α‐methylbutyryl)‐8,9‐dihydrooroselol (compound D) were obtained using heptane/ethyl acetate/methanol/water (2:1:2:1, v/v) in <40 min. The method yielded 4.6 mg of a mixture of compounds B and C (11:89) and 3.7 mg of compound D. These amounts were obtained from the crude extract (0.5 g) in a single run. Although the compounds are known, their isolation by countercurrent chromatography and the analysis of their relative stereochemistry by two‐dimensional NMR spectroscopy have been performed for the first time. Additionally, heptane/ethyl acetate/methanol/water (3:2:3:2, v/v) led to the isolation of oxypeucedanin (1.2 mg; compound A). This is the first time that angular dihydrofuranocoumarin was isolated from plant extract by countercurrent chromatography.     

Separation of ternary systems of hydrophilic ionic liquid with miscible organic compounds by RPLC with refractive index detection

A rapid and simple analytical method was developed for the simultaneous and quantitative determination and separation of hydrophilic imidazolium ionic liquids (ILs) (1-butyl-3-methylimidazolium chloride, [C(4)mim]Cl; 1-hexyl-3-methylimidazolium chloride, [C(6)mim]Cl; 1-octyl-3-methylimidazolium chloride, [C(8)mim]Cl; 1-allyl-3-methylimidazolium chloride, [Amim]Cl; or 1-allyl-3-methylimidazolium bromide, [Amim]Br) with miscible ethyl acetate and EtOH and their mixtures using reverse phase liquid chromatography coupled with refractive index detection (RPLC-RI). The influence of 60 to 100% (volume percentage) methanol in the mobile phase on the IL systems ([C(4)mim]Cl, [C(6)mim]Cl, [C(8)mim]Cl, [Amim]Br, or [Amim]Cl)-ethyl acetate-EtOH was investigated. The optimum mobile phase for the system [C(8)mim]Cl-ethyl acetate-EtOH, [C(4)mim]Cl-ethyl acetate-EtOH, [Amim]Br-ethyl acetate-EtOH and [Amim]Cl-ethyl acetate-EtOH was methanol/water (60:40, v/v), and methanol/water (70:30, v/v) for [C(6)mim]Cl-ethyl acetate-EtOH. Under optimum mobile phase conditions for each system, the RSD of the retention time ranged from 0.02 to 0.04%, and the RSDs of the peak area percent ranged from 0.23 to 1.85%, which showed good reproducibility of the RPLC-RI method. The RPLC-RI method can determine IL, ethyl acetate, and EtOH simultaneously in 5 min, and the analytes, especially IL, can be eluted completely. The results show that the RPLC-RI method can be used to separate and determine ILs in mixtures with organic compounds simultaneously and quantitatively.     

Separation of nine compounds from Salvia plebeia R.Br. using two‐step high‐speed counter‐current chromatography with different elution modes

Nine compounds were successfully separated from Salvia plebeia R.Br. using two‐step high‐speed counter‐current chromatography with three elution modes. Elution–extrusion counter‐current chromatography was applied in the first step, while classical counter‐current chromatography and recycling counter‐current chromatography were used in the second step. Three solvent systems, n‐hexane/ethyl acetate/ethanol/water (4:6.5:3:7, v/v), methyl tert‐butyl ether/ethyl acetate/n‐butanol/methanol/water (6:4:1:2:8, v/v) and n‐hexane/ethyl acetate/methanol/water (5:5.5:5:5, v/v) were screened and optimized for the two‐step separation. The separation yielded nine compounds, including caffeic acid ( 1 ), 6‐hydroxyluteuolin‐7‐glucoside ( 2 ), 5,7,3′,4′‐tetrahydroxy‐6‐methoxyflavanone‐7‐glucoside ( 3 ), nepitrin ( 4 ), rosmarinic acid ( 5 ), homoplantaginin ( 6 ), nepetin ( 7 ), hispidulin ( 8 ), and 5,6,7,4′‐tertrahydroxyflavone ( 9 ). To the best of our knowledge, 5,7,3′,4′‐tetrahydroxy‐6‐methoxyflavanone‐7‐glucoside and 5,6,7,4′‐tertrahydroxyflavone have been separated from Salvia plebeia R.Br. for the first time. The purities and structures of these compounds were identified by high‐performance liquid chromatography, electrospray ionization mass spectrometry, ¹H and ¹³C NMR spectroscopy. This study demonstrates that high‐speed counter‐current chromatography is a useful and flexible tool for the separation of components from a complex sample.     

粘委陵菜中的鞣质成分的研究

从枯委陵菜(potenjtilla viscosa J.Don)根中分离出七个鞣质成分, 经波谱分析, 化学降解和衍生物制备等手段确定了它们的化学结构, 分别为(+)-儿茶素(1), (+)-儿茶素3-O-β-D葡萄糖苷(2), afzelechin-(4α-8)-儿茶素(3), 原花色苷元B-3(4), 原花色苷元B-33'-O-β-D-葡萄吡喃糖苷(5), 原花色苷元C-2(6)和枯委陵菜素(potentillanin)(7), 其中5和7是首次发现的天然化合物,化合物1及2具有保肝作用。     

Separation of flavonoids from Millettia griffithii with high‐performance counter‐current chromatography guided by anti‐inflammatory activity

Millettia griffithii is a unique Chinese plant located in the southern part of Yunnan Province. Up to now, there is no report about its phytochemical or related bioactivity research. In our previous study, the n‐hexane crude extract of Millettia griffithii revealed significant anti‐inflammatory activity at 100 μg/mL, inspiring us to explore the anti‐inflammatory constituents. Four fractions (I, II, III, and A) were fractionated from n‐hexane crude extract by high‐performance counter‐current chromatography with solvent system composed of n‐hexane/ethyl acetate/methanol/water (8:9:8:9, v/v) and then were investigated for the potent anti‐inflammatory activity. Fraction A, with the most potent inhibitory activity was further separated to give another four fractions (IV, V, VI, and B) with solvent system composed of n‐hexane/ethyl acetate/methanol/water (8:4:8:4, v/v). Compound V and fraction B exhibited remarkable anti‐inflammatory activity with nitric oxide inhibitory rate of 80 and 65%, which was worth further fractionation. Then, three fractions (VII, VIII, and IX) were separated from fraction B with a solvent system composed of n‐hexane/ethyl acetate/methanol/water (8:1:8:1, v/v), with compound VIII demonstrating the most potent inhibitory activity (80%). Finally, the IC₅₀ values of compound V and VIII were tested as 38.2 and 14.9 μM. The structures were identified by electrospray ionization mass spectrometry and¹H and ¹³C NMR spectroscopy.     

Fractionation of apple procyanidins according to their degree of polymerization by normal-phase high-performance liquid chromatography

A new method was developed for the fractionation of procyanidin oligomers according to their degree of polymerization. Monomeric flavan-3-ols and low molecular mass procyanidins were selectively extracted from the lyophilized powder of apple condensed tannins (ACTs) by methyl acetate extraction. Sequentially, the separation of each oligomer from dimer to pentamer in this extract was carried out by normal-phase high-performance liquid chromatography using a silica-beads packed column. The best separation was achieved with a mobile phase system containing hexane; (1) hexane–methanol–ethyl acetate, (2) hexane–acetone. These sequential treatments can be easily adapted to large-scale fractionation.