B-type Linkages Research Articles

Chemical Transformation of B- to A-type Proanthocyanidins and 3D Structural Implications.

In nature, proanthocyanidins (PACs) with A-type linkages are relatively rare, likely due to biosynthetic constraints in the formation of additional ether bonds to be introduced into the more common B-type precursors. However, A-type linkages confer greater structural rigidity on PACs than do B-type linkages. Prior investigations into the structure-activity relationships (SAR) describing how plant-derived PACs with B- and complex AB-type linkages affect their capacity for dentin biomodification indicate that a higher ratio of double linkages leads to a greater interaction with dentin type I collagen. Thus, A-type PACs emerge as particularly intriguing candidates for interventional functional biomaterials. This study employed a free-radical-mediated oxidation using DPPH to transform trimeric and tetrameric B-type PACs, 2 and 4, respectively, into their exclusively A-type linked analogues, 3 and 5, respectively. The structures and absolute configurations of the semisynthetic products, including the new all-A-type tetramer 5, were determined by comprehensive spectroscopic analysis. Additionally, molecular modeling investigated the conformational characteristics of all trimers and tetramers, 1-5. Our findings suggest that the specific interflavan linkages significantly impact the flexibility and low-energy conformations of the connected monomeric units, which conversely can affect the bioactive conformations relevant for dentin biomodification.

Affinity of Tannins to Cellulose: A Chromatographic Tool for Revealing Structure-Activity Patterns.

Food, feed and beverage processing brings tannins into contact with macromolecules, such as proteins and polysaccharides, leading to different chemical and physical interactions. The interactions of tannins with proteins are well known but less is known about the affinity of tannins to polysaccharides. We used bacterial cellulose from nata de coco as a model compound to investigate how tannins and cellulose interact by adsorption measurements using UPLC-DAD. We also explored how the structure of tannins influences these interactions. The model tannins included nine individual structurally different hydrolysable tannins (HTs) and eight well-defined proanthocyanidin (PA) fractions with different monomeric units, mean degree of polymerization and both A- and B-type linkages. Tannins were found to have both strong and weak interactions with bacterial cellulose, depending on the exact structure of the tannin. For HTs, the main structural features affecting the interactions were the structural flexibility of the HT molecule and the number of free galloyl groups. For PAs, prodelphinidins were found to have a higher affinity to cellulose than procyanidins. Similarly to HTs, the presence of free galloyl groups in galloylated PAs and the flexibility of the PA molecule led to a stronger interaction. Adsorption measurements by UPLC-DAD proved to be a sensitive and rapid tool to evaluate the affinity of tannins to cellulose.

Mechanistic Understanding of Tyrosinase Inhibition by Polymeric Proanthocyanidins from Acacia confusa Stem Bark and Their Effect on the Browning Resistance of Fresh-Cut Asparagus Lettuce.

Tyrosinase inhibitors are capable of preventing unfavorable enzymatic browning of fruits and vegetables. In this study, the capacity of Acacia confusa stem bark proanthocyanidins (ASBPs) to inhibit tyrosinase activity was evaluated. ASBPs were shown to be a high-potential inhibitor of tyrosinase with IC50 values of 92.49 ± 4.70 and 61.74 ± 8.93 μg/mL when using L-tyrosine and L-DOPA as the substrate, respectively. The structural elucidation performed with UV-vis, FT-IR spectroscopy, ESI-MS and thiolysis coupled to HPLC-ESI-MS suggested that ASBPs had structural heterogeneity in monomer units and interflavan linkages and consisted mainly of procyanidins dominant with B-type linkages. To gain insights into the inhibitory mechanisms of ASBPs against tyrosinase, different spectroscopic and molecular docking methods were further conducted. Results validated that ASBPs possessed the ability to chelate copper ions and could prevent the oxidation process of substrates by tyrosinase. The hydrogen bond formed with Lys-376 residue played a key role in the binding force of ASBPs with tyrosinase that induced a certain alteration in the microenvironment and secondary structure of tyrosinase, resulting in the enzymatic activity being ultimately restricted. It was also observed that ASBPs treatment effectively inhibited the activities of PPO and POD to retard the surface browning of fresh-cut asparagus lettuce and thus extended their shelf-life. The results provided preliminary evidence supporting the exploitation of ASBPs into potential antibrowning agents for the fresh-cut food industry.

Gut Microbiota as a Novel Tool to Dissect the Complex Structures of Black Tea Polymers.

Thearubigins, polymers of tea catechins, account for more than 20% of the black tea polyphenols and have been reported to be the active components in black tea. However, the chemical structures and underlying mechanisms regarding how the thearubigins, being poorly bioavailable, generate in vivo health benefits are still largely unknown. Using germ-free and specific pathogen-free husbandry conditions combined with LC/MS-based nontargeted and targeted metabolomic analyses, we investigated the role of intestinal bacteria in thearubigin metabolism. Theaflavins and theasinensins were identified as the major microbial metabolites of thearubigins, suggesting that these molecules are the building units for the complex thearubigins. To further confirm this, thearubigin depolymerization was done using menthofuran in an acidic condition. Menthofuran-conjugated theaflavins, theasinensins, and catechins as well as their free forms were detected as the major degradation products of thearubigins. This indicated that theaflavins and theasinensins could be further polymerized through B-type proanthocyanidin linkages. Furthermore, four microbial degradation products were able to be detected in urine samples, suggesting that they can be absorbed into the circulatory system. Using the combination of microbial degradation, metabolomics, and chemical degradation, our results demonstrate that thearubigins are the complex polymers of theaflavins, theasinensins, and catechins and can be metabolized by gut microbiota to their corresponding bioactive and bioavailable smaller molecular metabolites.

Isolation, Characterization, Antioxidant Activity, Metal-Chelating Activity, and Protein-Precipitating Capacity of Condensed Tannins from Plum (Prunus salicina) Fruit.

The type of polymeric condensed tannins from plum fruit (Prunus salicina) (PCT), the degree of polymerization and the distribution of polymers were characterized by MALDI-TOF MS and NMR spectroscopy. The metal-binding capacity of PCT with five metal ions (Cu2+, Zn2+, Al3+, Fe2+, and Fe3+) was characterized by a fluorescence quenching method. The results demonstrated the following: epicatechin was the basic unit occurring in PCT, and A-type and B-type linkages were the most common between the structural units of the polymers. The PCT have a strong antioxidant activity, which is comparable with that of the synthetic antioxidant BHA. The quenching mechanism of the PCT’s fluorescence intensity by Zn2+, Cu2+, and Al3+ was different from that of Fe3+ and Fe2+. Fe3+, Al3+ and Fe2+ had much higher affinities for the PCT than Zn2+ and Cu2+. A simple UV-Vis spectra method was developed to determine the protein-precipitating capacity of tannins. Bovine serum albumin (BSA) was effectively precipitated by tannins isolated from plum fruits, Chinese gallnut, sorghum grain, and Platycarya strobilacea at pH values between 4.5 and 5.0. A statistically significant linear relationship (p < 0.0001 or p < 0.0003) existed between the amount of tannin–protein complex formed and the amount of tannins added to the reaction mixture. The slopes of these lines indicated the protein-precipitating capacity of tannins.

Structure, degree of polymerization, and starch hydrolase inhibition activities of bird cherry (Prunus padus) proanthocyanidins

The structure of proanthocyanidins extracted from bird cherry fruits was characterized by HPLC-ESI/MS2 and MALDI-TOF/MS analyses, and their subunits and mean degree of polymerization (mDP) were investigated by thiolysis reaction, and the inhibition activity against starch hydrolases measured using the high-throughput turbidity assay. This is the first mass spectrometric analysis to thoroughly investigate the structure and mDP of proanthocyanidins in bird cherry fruits. Bird cherry proanthocyanidins were categorized as oligomeric proanthocyanidins (mDP = 5.6), which constituted of (epi)gallocatechins and (epi)catechins. The proanthocyanidins increased from a (epi)gallocatechin-[(epi)catechin]3 tetramer to a (epi)gallocatechin-[(epi)catechin]11 dodecamer through the addition of one (epi)catechin with both A-type and B-type linkages. The proanthocyanidins had potent α-amylase and α-glucosidase inhibition activities with IC50 values of 0.19 ± 0.01 µg/mL and 0.18 ± 0.006 µg/mL, comparing favorably to commercial drug acarbose. Bird cherry oligomeric proanthocyanidins are a promising starch hydrolase inhibitor for the application of potential functional food components.

Unveiling structure–activity relationships of proanthocyanidins with dentin collagen

ObjectiveTo elucidate the structure–activity relationships (SARs) of proanthocyanidins (PACs) with type I collagen using sixteen chemically defined PACs with degree of polymerization (DP) 2–6. MethodsUnder a dentin model, the biomimicry of PACs with type I collagen was investigated by dynamic mechanical analysis (DMA) and infrared spectroscopy. The dentin matrix was modified with PACs from Pinus massoniana [monomers (Mon-1 and Mon-2), dimers (Dim-1-Dim-4), trimers (Tri-1-Tri-4), tetramers (Tet-1-Tet-5), and hexamer (Hex-1)]. A strain sweep method in a 3-point bending submersion clamp was used to assess the viscoelastic properties [storage (E’), loss (E”), and complex moduli (E*) and tan δ] of the dentin matrix before and after biomodification. Biochemical analysis of the dentin matrix was assessed with FTIR spectroscopy. Data were statistically analyzed using one-way ANOVA and post-hoc tests (α = 0.05). ResultsDP had a significant effect on modified dentin moduli (tetramers ≈ trimers > hexamers ≈ dimers > monomers ≈ control, p < 0.001). Trimers and tetramers yielded 6- to 8-fold increase in the mechanical properties of modified dentin and induced conformational changes to the secondary structure of collagen. Modifications to the tertiary structure of collagen was shown in all PAC modified-dentin matrices. SignificanceFindings establish three key SARs: (i) increasing DP generally enhances biomimicry potential of PACs in modulating the mechanical and chemical properties of dentin (ii) the secondary structure of dentin collagen is affected by the position of B-type inter-flavanyl linkages (4β → 6 and 4β → 8); and (iii) the terminal monomeric flavan-3-ol unit plays a modulatory role in the viscoelasticity of dentin.

Characterization of bioactive cranberry fractions by mass spectrometry

Increasing evidence indicates that fruits contain functional bio-active compounds that have several preventive and therapeutic health benefits. Our group has recently conducted studies to assess the potential effects of cranberry polyphenolic fractions on intestinal Caco-2/15 epithelial cells and a substantial reduction in oxidative stress and inflammation was observed. The aim of the present work was to determine the polyphenolic species most likely responsible for the observed biological activity. Low, medium, and high molecular weight cranberry fractions were generated with a Sephadex LH-20 column by elution with 60% MeOH, 100% MeOH, and 70% acetone, respectively. The total phenolic content in these fractions was determined by the Folin–Ciocalteu method. A combination of LC–MS and MALDI-TOF methods were used to characterize the nature of polyphenolic compounds in the cranberry extracts. High resolution mass spectrometry was used to generate empirical formulae for the detected species. The low molecular weight fraction was essentially constituted of small phenolic acids (hydroxycinnamic and hydroxybenzoic acids). The medium fraction was mostly composed of anthocyanin, flavonols (quercetin, myricetin, isorhamnetin, kaempferol), procyanidins monomers (epicatechin, catechin), dimers, and few small oligomers. The heavy fraction was devoid of phenolic acids and anthocyanins, and it contained for the most part oligomers and polymers of procyanidins. Proanthocyanidins oligomers up to n = 22 were detected, which is, to our knowledge, the largest individual polymers reported to date. With this approach, it was also possible to distinguish between the A-type and B-type linkages.

Comparison of Protein Precipitation Ability of Structurally Diverse Procyanidin-Rich Condensed Tannins in Two Buffer Systems.

The protein precipitation (PP) of bovine serum albumin (BSA), lysozyme (LYS), and alfalfa leaf protein (ALF) by four procyanidin-rich condensed tannin (CT) samples in both 2-[N-morpholino]ethanesulfonic acid (MES) and a modified Goering-Van Soest (GVS) buffer is described. Purified CT samples examined included Vitis vinifera seed (mean degree of polymerization [mDP] 4.1, 16.5% galloylated), Tilia sp. flowers (B-type linkages, mDP 5.9), Vaccinium macrocarpon berries (mDP 8.7, 31.7% A-type linkages). and Trifolium pratense flowers (B-type linkages, mDP 12.3) and were characterized by 2D NMR (>90% purity). In general, CTs precipitated ALF > LYS ≥ BSA. PP in GVS buffer was 1 to 2.25 times greater than that in MES buffer (25 °C). The GVS buffer system better reflects the results/conclusions from the literature on the impacts mDP, galloylation, and A-type linkages have on PP. Determinations of PP using the MES buffer at 37 °C indicated that some of these differences may be attributed to the temperature at which GVS buffer determinations are conducted. In vitro PP studies using the GVS buffer may offer better guidance when selecting CT-containing forages and amendments for ruminant feeding studies.

Ion mobility-mass spectrometry for the separation and analysis of procyanidins.

Procyanidins are polymeric flavan-3-ones occurring in many plants with antioxidant and other beneficial bioactivities. They are composed of catechin and epicatechin monomeric units connected by single carbon-carbon B-type linkages or A-type linkages containing both carbon-carbon and carbon-oxygen-carbon bonds. Their polymeric structure makes analysis of procyanidin mixtures always difficult. Evaluation of procyanidins according to degree of polymerization (DP) using high-performance liquid chromatography (HPLC) is time-consuming and at best has resolved polymeric families up to DP-17. To expedite studies of procyanidins, the utility of positive ion electrospray ion mobility-mass spectrometry (IM-MS) was investigated for the rapid separation and characterization of procyanidins in mixtures. Applying IM-MS to analyse structurally defined standards containing up to five subunits, procyanidins could be resolved in less than 6ms not only by degree of polymerization but also by linkage type. A-type procyanidins could be resolved from B-type and both could be at least partially resolved from mixed-type procyanidins of the same DP. IM-MS separated higher order procyanidins with DP of at least 24 from extracts of cranberry. As DP increased, the abundances of multiply-charged procyanidins also increased. During IM-MS of ions of similar m/z, the ion drift times decreased inversely with increasing charge state. Therefore, IM-MS was shown to separate mixtures of procyanidins containing at least 24 interconnected subunits in less than 16ms, not only according to DP, but also according to linkage type between subunits and charge state.

Crown Procyanidin Tetramer: A Procyanidin with an Unusual Cyclic Skeleton with a Potent Protective Effect against Amyloid-β-Induced Toxicity.

The structure of a new procyanidin tetramer, which we call a crown procyanidin tetramer, with an unprecedented macrocyclic structure has been characterized for the first time. Its comprehensive spectroscopic analysis revealed that it is a symmetric procyanidin tetramer composed of four (−)-epicatechin sub-units linked alternatively via 4β→8 or 4β→6 B-type interflavanyl linkages to form the macrocyclic structure. This NMR-characterized carbon skeleton has never been reported before for procyanidins in grape or in wine, neither in the plant kingdom. Surprisingly, the crown procyanidin tetramer appeared to be specifically localized in grape skin, contrasting with the oligomeric and polymeric procyanidins present in seed, skin, and bunch stem. Moreover, this crown procyanidin tetramer showed promising protective effects against amyloid-β induced toxicity.

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.

Browplasminin, a condensed tannin with anti-plasmin activity isolated from an aqueous extract of Brownea grandiceps Jacq. flowers

Ethnopharmacological relevanceFollowing Venezuelan traditional medicine, females with heavy menstrual blood loss (menorrhagia) drink Brownea grandiceps Jacq. flowers (BG) decoctions to reduce the bleeding. In a previous study, we demonstrated that BG aqueous extract (E) possesses a potent anti-fibrinolytic activity capable of inhibiting plasmin, the main serine-protease that degrades fibrin. It is widely known that plasmin inhibitors are often used as anti-fibrinolytics to reduce bleeding during surgeries with high risk of blood loss such as cardiac, liver, vascular, tooth extraction and large orthopedic procedures, as well as for menorrhagia treatments. The aim of this work was to isolate and characterize from BGE the compound responsible for the reported anti-fibrinolytic activity. Materials and methodsA decoction of BG was prepared; then it was homogenized, centrifuged and lyophilized to obtain BGE. Subsequently the extract was fractionated by gel filtration and reverse phase using HPLC and the active compound was characterized by MALDI-ToF MS. The kinetic parameters of anti-plasmin activity were evaluated by an amidolytic assay using a chromogenic substrate; also the anti-plasmin activity was estimated by fibrin plate method. Data were analyzed by nonparametric statistics. ResultsThe active compound was a condensed tannin denominated Browplasminin, which is capable of inhibiting the plasmin activity in a dose-dependent manner when measured in fibrin plates or by the amidolytic activity method; it also has a minor effect on the FXa activity. However, it does not affect the activity of other serine-proteases such as trypsin, t-PA or u-PA. Browplasminin consists predominately of heteroflavan-3-ols of catechin with B-type linkages, and extents up to heptadecamers (~ 5000Da), with hexose residues attached to the polymer that presents a high degree of galloylation. Its IC50 for plasmin was 47.80μg/mL and for FXa was 237.08μg/mL, while the Ki were 0.76 and 61.61μg/mL for plasmin and FXa, respectively. ConclusionsThe overall outcome of this study suggests that Browplasminin could be responsible for reducing heavy menstrual bleeding in women because its kinetic parameters showed that is a good plasmin inhibitor.

Structural characterization and bioactivity of proanthocyanidins from indigenous cinnamon (Cinnamomum osmophloeum).

Barks and twigs of common species of cinnamon with abundant proanthocyanidins are used as a spice, fold medicine or supplement. Cinnamomum osmophloeum is an endemic species in Taiwan and coumarin was not detected in the oil of the C. osmophloeum twig. The present study aimed to evaluate the relationship between the bioactivities and proanthocyanidins of C. osmophloeum twig extracts (CoTE). The n-butanol soluble fraction from CoTE was divided into 10 subfractions (F1-F10) by Sephadex LH-20 gel chromatography. The antihyperglycemic activities were examined by α-glucosidase, α-amylase and protein tyrosine phosphatase 1B inhibitory assays. Total antioxidant activities were examined by 2,2-diphenyl-1-picrylhydrazyl free radical scavenging and ferrous ion-chelating assays. The results revealed that subfractions F6-F10, with high proanthocyanidin contents, showed excellent antihyperglycemic and antioxidant activities. Subfractions F6-F10 were analyzed further by matrix-assisted laser desorption/ionization-time of flight/mass spectrometry and thiolysis-reversed-phase high-performance liquid chromatography/tandem mass spectrometry methods. The results showed that the mean degrees of polymerization of proanthocyanidins in subfractions F6-F10 ranged from 3.5 to 5.1, with the highest degrees of polymerization of proanthocyanidins reaching 8 in subfractions F8-F10. Two compounds in F6 were identified as cinnamtannin B1 and parameritannin A1. These proanthocyanidins contained at least one A-type and major B-type linkages. These results demonstrate that proanthocyanidins are associated with their antihyperglycemic and antioxidant activities in CoTE. © 2016 Society of Chemical Industry.

Galloylated proanthocyanidins from shea (Vitellaria paradoxa) meal have potent anthelmintic activity against Ascaris suum

Proanthocyanidins (PA) from shea (Vitellaria paradoxa) meal were investigated by thiolytic degradation with benzyl mercaptan and the reaction products were analysed by high performance liquid chromatography–mass spectrometry. These PA were galloylated (≈40%), contained only B-type linkages and had a high proportion of prodelphinidins (>70%). The mean degree of polymerisation was 8 (i.e. average molecular size was 2384Da) and epigallocatechin gallate (EGCg) was the major flavan-3-ol subunit in PA. Shea meal also proved to be a potentially valuable source for extracting free flavan-3-ol-O-gallates, especially EGCg (575mg/kg meal), which is known for its health and anti-parasitic benefits. Proanthocyanidins were isolated and tested for bioactivity against Ascaris suum, which is an important parasite of pigs. Migration and motility tests revealed that these PA have potent activity against this parasitic nematode.

Potent anthelmintic activity of galloylated proanthocyanidins from shea meal (Vitellaria paradoxa)

Proanthocyanindins (PAs) from shea meal (SM), a by-product obtained after lipid extraction of the nuts, contained B-type linkages, had a high ratio of prodelphinidins (73%) and were galloylated (42%). The average polymer size was 8 flavan-3-ol subunits (≈2384 Daltons) and epigallocatechin gallate was the major subunit. Purified PA fractions from SM were tested in vitro for anthelmintic properties against gastrointestinal nematodes from ruminants (H. contortus and T. colubriformis) [1] by the larval exsheathment inhibition assay and from pigs (A. suum) by the larval migration inhibition assay. Results showed that PAs from SM have a potent anthelmintic activity against those parasites similar to white clover (Trifolium repens) flowers (WCF) [1, 2] (EC50 µg/mL; SM: 55.1, 16.5, 75.9; WCF: 37.4, 14.5, 110.1 for A. suum, H. contortus and T. colubriformis respectively). WCF PAs are constituted almost exclusively of prodelphinidin (PD) compared to SM (98% vs. 73%) but do not contained galloylated PAs. Studies [1, 2] have shown that anthelmintic activity of PAs was mainly associated with their PD ratio but our current results suggest that galloylation can be a major factor to anthelmintic activity and SM as a potential nutraceutical anthelmintic feed for controlling parasitic nematodes.

Senna alata leaves are a good source of propelargonidins

Proanthocyanidins (PA) in Senna alata leaves were investigated by thiolysis with benzyl mercaptan, LC–MS and NMR and consisted of almost pure propelargonidins with <6% procyanidins, had B-type linkages and a mean degree of polymerisation of three. Epiafzelechin was the major flavan-3-ol subunit (>94%) and epicatechin a minor constituent (6.4%) in residual PA and mainly detected as an extension unit.

Structure, antioxidant and α-amylase inhibitory activities of longan pericarp proanthocyanidins

Longan pericarp proanthocyanidins (LPPs) were extracted and examined by structural and bioactive analyses. By 1H and 13C NMR spectra, LPPs were identified for the first time containing dominant catechin/epicatechin units with a mean degree of polymerisation of 3.3. Consistent with this result, electron spray ionisation (ESI) mass spectra revealed that the predominant LPPs were poly(epi)catechin with abundant A-type and B-type linkages. Matrix-assisted laser desorption ionisation time of flight (MALDI-TOF) mass spectra of LPPs showed dominant [M + Na]+ peaks that corresponded to procyanidin trimers–octamers. The presence of galloyl group in monomeric units of LPPs was confirmed by FT-IR, NMR, ESI and MALDI-TOF mass spectrometry (MS). Moreover, LPPs had a strong antioxidant activity of 1.23 × 104 µmol TE/g as measured by oxygen radical scavenging capacity (ORAC), and α-amylase inhibitory activity with IC50 for uncompetitive α-amylase inhibition of 0.075 mg/mL. These results indicate LPPs are promising antioxidant which could be applied as potential functional food components.

Developmental profile of anthocyanin, flavonol, and proanthocyanidin type, content, and localization in saskatoon fruits (Amelanchier alnifolia Nutt.).

Saskatoons (Amelanchier alnifolia Nutt.) are small fruits that contain substantial quantities of flavonoids. To further characterize and understand the role of these flavonoids during fruit development, anthocyanins, flavonols, and proanthocyanidins were identified, quantified, and localized over development in cultivars that produce blue-purple or white fruit at maturity. Anthocyanin content was low in young fruit and then dramatically increased as the fruit transitioned into ripening only in the pigmented-fruit (blue-purple) cultivars. Proanthocyanidins with both A-type and B-type linkages were detected in fruit, with (-)-epicatechin as the most abundant proanthocyanidin subunit. Flavonol and proanthocyanidin content was high in, and localized throughout, the tissues of young fruit and in the developing seed coats, with levels decreasing as the fruit expanded. Our data show that flavonoid type, content, and tissue localization vary throughout development in saskatoon fruit. These data can be used to target specific fruit developmental stages and flavonoid classes for optimization of health-beneficial flavonoid content.

Antioxidant Activity and Proanthocyanidin Profile of Selliguea feei Rhizomes

Proanthocyanidins from the rhizomes of Selliguea feei (PSFs) were solvent-extracted and fractionated by Sephadex LH-20 column chromatography to give a 2.42% isolated yield (dry matter basis). 1H-NMR spectroscopy revealed the mean degree of polymerization (mDP) to be 2.6. 13C-NMR analysis showed typical signals for afzelechin/epiafzelechin units. Clear peaks at 76 ppm and 84 ppm indicated that both stereoisomers (afzelechin/epiafzelechin) are present. In agreement with the NMR spectra, the ESI-MS spectrum indicated that PSFs are mainly monomers to trimers consisting of afzelechin/epiafzelechin units with A-type and B-type interflavanyl linkages. A trimer was purified and identified as demethylated selligueain B. Thiolysis confirmed the structure and the thiolytic products, methyl 2-[(2R, 3R, 4S)-3,5,7-trihydroxy-2-(4-hydroxyphenyl)chroman-4-yl]acetate (1) and 4β-(carboxymethyl)sulphanylepiafzelechin-(2β→O→7,4β→8)-epiafzelechin methyl ester (2), were purified and characterized. Selligueain A, demethylated selligueain B, compounds 1 and 2 possess high antioxidant capacity at 1.18 × 104, 1.16 × 104, 0.95 × 104 and 1.29 × 104 µmol TE/g, respectively.