Purification and application of α-galactosidase from germinating coffee beans (<Emphasis Type="Italic">Coffea arabica</Emphasis>)

The objective of this study was to prepare and purify α-galactosidase (α-Gal) from germinating coffee beans. The molecular weight of purified α-Gal from germinating coffee beans was determined to be 38.8 kDa by SDS-PAGE. Then the purified α-Gal was applied to synthesize galactosyl β-cyclodextrin using melibiose as donor and β-cyclodextrin as acceptor. Galactosyl β-cyclodextrin was isolated by preparative high-performance liquid chromatography (HPLC) and its structure was analyzed and elucidated by HPLC, fourier transform infrared spectrometer, electro spraying ionization-mass spectrometry, and ¹³C nuclear magnetic resonance. Our results strongly demonstrate that the synthesized product is a mono-6-O-α-d-galactopyranosyl-β-cyclodextrin and the purified α-galactosidase could transfer one galactosyl residue directly to the β-cyclodextrin ring and synthesize the mono-6-O-α-d-galactosyl β-cyclodextrin.     

Effect of dietary fiber and butyric acid on lysosomal enzyme activities in streptozotocin-induced diabetic rats

The effect of dietary fiber and butyric acid on lysosomal enzyme activities in different tissues of streptozotocin-induced diabetic rats was studied by analyzing the specific activities of N-acetyl-β-d-glucosaminidase, β-d-glucuronidase, β-d-galactosidase, and acid phosphatase in various tissues. An alteration in the activities of these lysosomal enzymes in different tissues was observed in streptozotocin-induced diabetic rats. Dietary fiber and butyric acid supplementation at 500 mg/kg body weight/day was found effective in modulating the altered glycosidases activity in liver, spleen, heart, lungs, testis, and brain during diabetes. Our results also indicated that butyric acid along with dietary fiber is involved in minimizing the oxidative stress-induced tissue damage thereby regulating the altered enzyme activities in different tissues during diabetes.     

Stability of naturally occurring 2,5-dimethyl-4-hydroxy-3 [2H]-furanone derivatives

The stability, in aqueous buffer solutions at different pH values (pH 2.0–8.0, interval: 1.5 pH units), of 2,5-dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, DMHF, 1), its methoxy derivative 2,5-dimethyl-4methoxy-3[2H]-furanone (methoxyfuraneol, mesifurane, DMMF, 2 and the glycosidically bound forms DMHF β-D-glucopyranoside 3 and DMHF 6′-O-malonyl-β-Dglucopyranoside 4 was investigated over a period of 32 days at 23 °C. Only slight decomposition of 2 and 3 was observed, whereas 1 and 4 were found to be unstable at all pH values. In addition, 3 and 4 were subjected to enzymatic hydrolysis. In contrast to the rapid hydrolysis of 3, the malonylated glycoside, 4, remained unaffected by enzymatic treatment with β-glucosidase (Emulsin). Using a pectinolytic enzyme preparation (Rohapect D5L; R?hm, Darmstadt, Germany) with esterase activities, hydrolysis of 4 was achieved. Received: 25 September 1996     

Production of Cell Membrane-Bound α- and β-Glucosidase by Lactobacillus acidophilus

Hydrolytic enzymes, viz. α- and β-glucosidase, were produced from indigenous isolate, Lactobacillus acidophilus, isolated from fermented Eleusine coracana. Production of these enzymes was enhanced by optimizing media using one factor at a time followed by response surface methodology. The optimized media resulted in a 2.5- and 2.1-fold increase in α- and β-glucosidase production compared with their production in basal MRS medium. Localization studies indicated 80% of the total activity to be present in the cell membrane-bound fraction. Lack of sufficient release of these enzymes using various physical, chemical, and enzymatic methods confirmed their unique characteristic of being tightly cell membrane bound. Enzyme characterization revealed that both α- and β-glucosidase exhibited optimum catalytic activity at 50 °C and pH 6.0 and 5.0, respectively. K _m and V _max of α-glucosidase were 4.31 mM and 149 μmol min⁻¹ mL⁻¹ for p-nitrophenyl-α-d-glucopyranoside as substrate and 3.8 mM and 120 μmol min⁻¹ mL⁻¹ for β-glucosidase using p-nitrophenyl-β-d-glucopyranoside as the substrate.     

Ripening and ethylene biosynthesis in controlled atmosphere stored apricots

 Ethylene synthesis and metabolism were studied in apricot fruits (Prunus armeniaca L. cv. Búlida, under controlled atmosphere (CA) conditions (20% CO₂ and 20% O₂; 20% CO₂ and 1% O₂; 0.03% CO₂ and 1% O₂) and at a temperature of 2  °C. In control apricots stored outdoors in air containing 20% O₂ and 0.03% CO₂, the evolution of the physical and chemical parameters characteristic of ripening (loss of texture, decrease in acidity and increase in soluble solids) was slow during the storage period. However, for apricots stored in CAs containing 1% O₂ and 20% or 0.03% CO₂, these parameters did not show significant changes during the storage period. Ethylene production was completely inhibited in apricots stored in the three CAs, while control fruits showed a maximum ethylene level after 2 weeks of storage. This inhibition was accompanied by lower levels of 1-aminocyclopropane-1-carboxylic acid, free and total, which were more pronounced in apricots stored in the CAs containing 20% CO₂. In apricots stored in the CAs containing 1% O₂, the ethanol content increased after 1 week of storage, and this caused a smell and taste that made the fruits unsuitable for consumption. Received: 16 October 1998     

Analytical assessment of destoned and organic-destoned extra-virgin olive oil

Outcomes related with the concentrations of bioactive compounds in new functional virgin olive oil kinds (destoned or organic-destoned) are referred to in this research paper. We paid attention to their nutraceutical metabolome and noticeably to their concentration in biophenolics and vitaminic antioxidants (tocopherols, tocotrienols and β-carotene) and to their sensory and hedonistic traits. There was evidence that the innovative destoning technique, owing to its soft action during olive processing, meaningfully affected the profile of the non-glyceryl-containing components and noticeably the levels of phytochemicals more related to human health and nutrition. We observed that the profile of the major lipidome (triacylglycerols and ω₉, ω₆ and ω₃ fatty acids) of the new natural produces did not differ significantly from that of the reference ones. But they were richer in biophenols (oleuropein and ligstroside aglycons, lignans, flavonoids, phenol acids and phenol alcohols) as well as in α- and γ-tocopherol and α- and γ-tocotrienol. Moreover, they stood out for their harmonic flavour and aroma, higher levels of green volatiles and more marked fragrance. They also had excellent hedonistic properties, higher stability/endurance to oxidation, and longer shelf-life. Their bionutritional traits were further enhanced when organic olives were submitted to destoning during processing. They can thus contribute to reduce risks concerning several current human pathologies (including mainly cardiovascular diseases and cancer types).     

Changes in ethylene and CO2 during the ripening of apples

The internal concentrations and production of both ethylene and CO₂ were measured during the maturation and ripening of Cox's Orange Pippin apples. Within 12 hours of the onset of the respiration climacteric there was no measurable increase in the production of ethylene and the data indicate that the increase in ethylene production is synchronous with the increased CO₂ production that marks the start of ripening. It therefore appears that a factor other than a change in the rate of ethylene production determines the time at which apples commence ripening.     

Carbohydrate changes in ripening Capsicum annuum in relation to textural degradation

 Within 8 days of harvest, fruits of Capsium annum showed striking changes in texture when ripened at room temperature. Physiologically, they showed a significant weight loss and organoleptically, a change in textural firmness which was evident microscopically, as extensive cellular damage. The associated biochemical changes during ripening were: (1) an increase in free-sugar levels, (2) an increase in in situ hydrolysis of some hemicellulose fractions (Hem a, b and c) and (3) a general increase in the activity of cellulase, α-mannosidase, laminarinase, polygalacturanase, galactanase, mannanase, β-galactosidase and hemicellulase (HCe) activity on Hem b and c. In contrast the activity of xylanase, pectin methyl esterase and HCe on Hem a decreased during ripening. Cold storage (8°C) held textural changes in check, resulting in a longer shelf-life for cold-stored fruits compared to fruits stored at room temperature. Received: 14 May 1997     

Multiple α-galactosidases from <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> ZU-G1: purification,characterization and application in soybean milk hydrolysis

α-Galactosidase had applied in food and feed industries for hydrolyzing raffinose series oligosaccharides (RO) that are the factors primarily responsible for flatulence upon ingestion of soybean-derived products. The objective of the current work was to purify the α-galactosidase of Aspergillus foetidus ZU-G1 and compared the biochemical and hydrolytic properties of three major α-galactosidase forms (α-gal I, α-gal II and α-gal III). The molecular mass of the purified enzyme as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 106.3, 49.7 and 109.9 kDa, respectively. Its optimum reaction temperature was 60 °C and stable below 50 °C. The optimum pH of α-gal I and α-gal III was 5.0 and α-gal II was 4.0. Under 28 °C conditions for 24 h, α-gal I was stable at pH 4.0, α-gal II was stable at pH 6.0, and α-gal III was pH 5.0. α-Galactosidase was completely inhibited by Ag⁺. CuSO₄·5H₂O and SDS were powerful inhibitors of α-gal I and α-gal III but had little effect to α-gal II. EDTA did not strongly affect α-gal I and α-gal III, while strongly affect α-gal II. CaCl₂·2H₂O, MgSO₄·7H₂O and MnSO₄·7H₂O were activation for α-gal I, α-gal II and α-gal III. No significant inhibition of enzymes activity was observed in the presence of raffinose, lactose as well as other sugars tested. Synthetic substrate p-nitrophenyl-α-d-galactopyranoside was not preferentially hydrolyzed than natural substrates, such as melibiose, stachyose and raffinose. Under 40 and 50 °C incubation for 1–5 h, the stachyose of soybean milk was degraded by α-gal I, α-gal II and α-gal III and strongly hydrolyzed by α-gal II, and the raffinose of soybean milk was completely hydrolyzed by α-gal II and weakly hydrolyzed by α-gal I and α-gal III. The distinct hydrolytic and biochemical properties of α-gal I, α-gal II and α-gal III further signify the α-galactosidase of A. foetidus ZU-G1 was propitious to soybean milk and related food industry.     

Storage of olives (Olea europaea) under CO2 atmosphere: Effect on anthocyanins,phenolics, sensory attributes and in vitro antioxidant properties

Green, unripe olives were subjected to post-harvest treatment under a CO₂ atmosphere for a period of 12 days. The total polyphenol (TP), total flavonoid (TFd) and total anthocyanin (TA) contents, along with the antioxidant and sensory characteristics, were continuously monitored during the treatment on a 24 h-interval basis, in order to identify possible changes in the quality of olives related mainly to changes in the polyphenolic contents. The storage of olives under CO₂ atmosphere resulted in pronounced increases in TP and TF contents, mainly within the first 3-5 days, but TA exhibited a different pattern of evolution. Furthermore, storage under CO₂ contributed to flavour appearance with the development of fruity/floral notes, and reduced bitterness. The in vitro antioxidant properties of the CO₂-treated sample showed notable increases compared with the sample stored under regular atmospheric conditions. It was concluded that storage of olives under a CO₂ atmosphere resulted in the appearance of desired sensory attributes, by decreasing bitterness and developing aroma and colour, and the functional (antioxidant) properties were improved. This approach may be used as an alternative, chemical-free means of table olive debittering.     

Synthesis of oligosaccharides with lactose and N-acetylglucosamine as substrates by using β-d-galactosidase from Bacillus circulans

In the present study, β-d-galactosidase from Bacillus circulans was proved to be a suitable biocatalyst for the production of N-acetyl-oligosaccharides with lactose and N-acetylglucosamine (GlcNAc) as biocatalyst. During the hydrolysis of lactose, apart from no ultraviolet absorption oligosaccharides such as β-d-Galp-(1 → 6)-d-Glcp (6′-allolactose) and β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-Glcp (4′-galactosyl-lactose), the formation of four N-acetyl-oligosaccharides was followed by high-performance liquid chromatography with a diode-array detector. The four N-acetyl-oligosaccharides were isolated from the reaction mixture and identified to be as β-d-Galp-(1 → 4)-d-GlcpNAc (LacNAc, I), β-d-Galp-(1 → 6)-d-GlcpNAc (allo-LacNAc, II), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (III), β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-β-d-Galp-(1 → 4)-d-GlcpNAc (IV) by authentic standards and the spike technique or high-resolution mass spectrometry with an electrospray ionization source and nuclear magnetic resonance spectroscopy. Furthermore, the effects of synthetic conditions including reaction temperature, concentration of substrate, molar ratio of donor/acceptor and enzyme concentration on the formation of N-acetyl-oligosaccharides were examined. We found that the optimal synthetic conditions were different for production of oligosaccharides with β-(1 → 4) linkages and β-(1 → 6) linkage. The optimal reaction conditions for I, III and IV were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 1.0 U/mL of enzyme. Under such conditions, the N-acetyl-oligosaccharides formed were composed of 28.75% of I, 2.29% of II, 9.47% of III and 5.67% of IV. On the other hand, suitable reaction conditions found for II were 40 °C, 0.50 M lactose and 0.50 M GlcNAc and 2.0 U/mL of enzyme.     

Flavonoid glycosides from cowpea seeds (Vigna sinensis K.) inhibit LDL oxidation

Six flavonoid glycosides were isolated from the n-butanol fraction of cowpea seeds (Vigna sinensis K.) through silica gel (SiO₂) and octadecyl silica gel (ODS) column chromatographies. Based on their chemical structures determined via interpretation of spectroscopic data including NMR, MS, and IR, the compounds were identified as kaempferol 3-O-β-d-sophoroside (1), quercetin 3-O-β-d-sophoroside (2), isoquercitrin (3), hyperin (4), catechin 7-O-β-d-glucopyranoside (5), and quercetin 3-O-β-Dglucopyranosyl( 1→6)-O-β-d-galactopyranoside (6). This is the first report of the isolation of these flavonoids from this plant. Among these flavonoids, compound 2, 5, and 6 significantly inhibited LDL oxidation exhibiting 96.0±0.1 (IC₅₀: 3.9 μM), 96.8±1.7 (IC₅₀: 2.9 μM), and 97.4±0.1% (IC₅₀: 3.5 μM) inhibition, respectively, at a concentration of 40 μM.     

On the non-enzymatic liberation of limonin and C17-epilimonin from limonin-17-β-d-glucopyranoside in orange juice

Limonin is well-known to induce the so-called “delayed” bitterness which is developing during standing and/or processing of orange juice. The objective of this study was to investigate the hydrolytic liberation of limonin from its precursor limonin-17-β-d-glucopyranoside in aqueous model solutions and orange juice samples. Quantitative model studies on the degradation of limonin-17-β-d-glucopyranoside under acidic conditions as well as during storage of orange juice revealed the formation of limonin besides a second hydrolysis product of yet unknown structure. After isolation, the structure of that compound was unequivocally determined for the first time as the C₁₇-epimer of limonin, named C₁₇-epilimonin. In order to investigate the generation of these triterpenoid lactones during storage as well as heat-treatment of orange juice, freshly squeezed orange juice was stored for up to 4 weeks at 4 and 20 °C or was heated at 70 and 100 °C and, then, both limonoids were quantitatively determined by means of high performance liquid chromatography-mass spectrometry/mass spectrometry (multiple reaction monitoring). The data obtained showed first evidence for the use of the C₁₇-epilimonin/limonin ratio as a suitable marker for the analytical determination of the thermal input applied during processing orange juice products.     

Effect of dressings "(aliños)" on olive texture: cellulase, polygalacturonase and glycosidase activities of garlic and lemon present in brines

 Texture is a basic characteristic of plant products for human consumption, being an essential factor in acceptance or rejection by the consumer. Dressed olives are highly valued for their excellent organoleptic characteristics, but bear the drawback of low stability. They are mainly sold loose, without any preservatives, and only a small proportion is packed after pasteurization. This work investigates the enzymatic activities of the endogenous enzymes present in products used as dressings such as garlic and lemon, and their capacity to be solubilized in brine and remain active throughout storage. In preparations with lemon and garlic, a progressive increase in the cellulase and polygalacturonase activity was observed with storage time. Of all the glycosidases assayed in the brine, only α-galactosidase activity was detected in garlic preparations. Environmental factors such as the presence of salt and/or the pH dependence of the enzymes were also studied. Received: 14 June 2000 / Revised version: 24 August 2000     

Stereoisomeric flavour compounds LXXVI: direct enantioseparation, structure elucidation and structure-function relationship of 4-tert-butyl-α-methyldihydrocinnamaldehyde

 Using enantioselective gas chromatography and heptakis(2,3-di-O-acetyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin (DIAC-TBDMS-β-CD) as the chiral stationary phase, the direct enantioseparation of 4-tert-butyl-α-methyldihydrocinnamaldehyde was achieved. The threshold values and odour characteristics of the enantiomers were investigated by enantioselective gas chromatography/olfactometry. In order to elucidate stereochemical features, the carbonyl-function was oxidized to the corresponding acid and diastereomeric amides were generated with (S)-2-amino-2-phenyl-ethanol [L(+)-α-phenylglycinol] as the enantiopure reagent. After separation and isolation by high-performance liquid chromatography, absolute configurations were deduced from X-ray structure elucidation of a pure stereoisomer. Amide cleavage, reduction and selective oxidation yielded the enantiomers of 4-tert-butyl-α-methyldihydrocinnamaldehyde. Received: 7 October 1996 / Revised version: 18 November 1996     

Determination of the carotenoid content in selected vegetables and fruit by HPLC and photodiode array detection

 Epidemiological studies have shown inverse correlations between the consumption of vegetables and fruit rich in carotenoids and the incidence of cancer and cardiovascular diseases. A total of 22 species of vegetables (including potatoes) and 28 of fruit (including rhubarb) were analysed for their contents of carotenoids by reversed-phase high-performance liquid chromatography (RP-HPLC) and photodiode array detection. A total of 27 carotenoids (among them β-carotene, lutein and violaxanthin also as cis isomers) were identified and quantified. Lutein, β-carotene (trans and cis forms) and violaxanthin were the predominant carotenoids in all green vegetables. Yellow and yellow-red vegetables and fruit contained β-carotene, α-carotene, β-cryptoxanthin and α-cryptoxanthin. Antheraxanthin and neoxanthin were found in nearly all produce. Lycopene was the predominant carotene in tomatoes, papayas and grapefruit. Vegetables with more than 10 mg of total carotenoids per 100-g edible portion were kale (34.8), red paprika (30.4), parsley (25.7), spinach (17.3), lamb’s lettuce (16.0), carrots (15.9) and tomatoes (12.7). In the case of fruit, grapefruit (3.5), papayas (3.4) and nectarines (2.4) were pre-eminent with more than 2 mg of total carotenoids (except for phytoene, phytofluene and ζ-carotene) per 100 g. Received: 27 March 1996     

Characteristics of olive fruits and extra‐virgin olive oils obtained from olive trees growing in Appellation of Controlled Origin ‘Sierra Mágina’

Olives and olive oils from Appellation of Controlled Origin (ACO) ‘Sierra Mágina’ have been analysed during the 1997/98 harvest. The territory of this ACO has been subdivided into four zones of different characteristics. The olives were harvested on two occasions (November 1997 and January 1998). Various parameters were analysed, such as the ripening index, the average volume, the average weight of 100 olives and 100 stones, the pulp/stone ratio, the industrial yield, etc. From these olives, olive oils were extracted using an Abencor system, and the free acidity, the peroxide index, the coefficients K₂₇₀ and K₂₃₂ and the fatty acid composition were determined. The analysis of the results obtained, as well as principal component analysis, demonstrates a marked variability in the fruits, but the corresponding olive oils show fairly homogeneous compositions that are difficult to distinguish by sensory analysis. The olive oils from this ACO are extra‐virgin olive oils characterised by low acidity, a high content of oleic acid and a high ratio of unsaturated to saturated fatty acids. The most important sensory attributes are fruity, woody‐fig, green, bitter and pungent. Copyright © 2003 Society of Chemical Industry     

Olive glutaraldehyde-like compounds against plant pathogenic bacteria and fungi

Aqueous solutions from table olives constitute a major environmental problem because of their high mineral and organic contamination but they also contain compounds with antimicrobial properties. In this study, the bactericidal and fungicidal activities of salt-free table olive solutions against several phytopathogenic micro-organisms were examined. The storage solutions of black ripe olives showed noticeable bactericidal activity against species of Erwinia, Clavibacter, Agrobacterium and Pseudomonas, although the washing waters from Spanish-style green olives were less effective. In fact, black olive storage solutions diluted to 20–30% were able to reduce the inoculated population of Erwinia amylovora and Pseudomonas syringae up to 6–7 log. This bactericidal activity was correlated with the presence of olive glutaraldehyde-like compounds such as the dialdehydic form of decarboxymethyl elenolic acid in these solutions, either free or linked to hydroxytyrosol. Experiments in vitro demonstrated that the bactericidal activity of these substances could explain most of the activity of the black olive storage solutions. Moreover, these salt-free table olive solutions also showed fungicidal activity against species of Phytophthora, Colletotrichum, Alternaria, Botrytis and Pestalotiopsis. The black olive storage solutions were more fungicidal than the washing solutions from Spanish-style green olives, with mycelial growth inhibition of up to 100% being attained, in some instances. These findings open the possibility of using table olive wastewaters in agriculture for pest management.     

Antioxidant activity of lignans from fringe tree (Chionanthus virginicus L.)

Fringe tree (Chionanthus virginicus L.), a shrub of the eastern part of America, used as a raw material by pharmaceutical industries for cholagoque, diuretic, tonic and the preparation of homeopathic tinctures. The identification of lignans as major antioxidant components and determination of their antioxidant activities are of considerable interest, because of the role they play in pharmacological actions. The potential antioxidant activity of the lignans such as phillyrin, pinoresinol-β-d-glucoside (PDG) and pinoresinol di-β-d-glucoside (PDDG) from root bark of fringe tree (C. virginicus L.) were examined by different antioxidant tests including; 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH^•) scavenging, superoxide anion radical (O₂ ^•-) scavenging, total antioxidant activity by ferric thiocyanate method, reducing activity by Fe³⁺–Fe²⁺ transformation, hydrogen peroxide (H₂O₂) scavenging and ferrous metal (Fe⁺²) chelating activities. Phillyrin, PDG and PDDG, as antioxidants, neutralized the activities of radicals and inhibited the peroxidation reactions of linoleic acid emulsion. The total antioxidant activity was determined according to the ferric thiocyanate method. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol and trolox, which is a water-soluble analogue of tocopherol, were used as the reference antioxidant compounds. Phillyrin, PDG and PDDG showed 67.6, 77.3 and 64.2% inhibition on lipid peroxidation of linoleic acid emulsion, respectively, at the concentration of 20 μg/mL. On the other hand, BHA, BHT, α-tocopherol and trolox exhibited 74.4, 71.2, 54.7 and 20.6% inhibition on peroxidation of linoleic acid emulsion, respectively, at the above mentioned concentration. In addition, phillyrin, PDG and PDDG were effective on DPPH^•, ABTS^•+ and O₂ ^•- scavenging, H₂O₂ scavenging, total reducing power and metal chelating effect on ferrous ions activities. Also, BHA, BHT, α-tocopherol and trolox were used as references antioxidants for these various antioxidant activities.     

Phenolic composition of rhubarb

Extracts of different parts (leaves, petioles and rhizomes) of domestic garden rhubarb (hybrids of Rheum rhabarbarum L. and Rheum rhaponticum L.) were investigated for their content of phenolic ingredients. Two stilbenes (trans-rhapontigenin, trans-desoxyrhapontigenin), five stilbene glycosides (trans-rhaponticin, cis- and trans-desoxyrhaponticin, trans-resveratrol-4′-O-β-d-glucopyranoside, trans-piceatannol-3′-O-β-d-glucopyranoside) and seven flavonoids [rutin, quercetin-3-O-glucuronide, isovitexin, 6,8-di-C-β-d-glucosylapigenin, 6-C-β-d-glucosyl-8-C-β-d-arabinosylapigenin (schaftoside), 6-C-β-d-arabinosyl-8-C-β-d-glycosylapigenin (isoschaftoside), (+)-catechin] were unequivocally established. Separation was done in two steps. Multilayer countercurrent chromatography was applied to separate different extracts of plant material. Preparative HPLC was then used to obtain pure substances. The purity and identity of isolated compounds was confirmed by different NMR experiments, HR-MS and HPLC-DAD analysis.