Photochemistry synthesis. Part 2: Enantiomerically pure polyhydroxy-1,1,3-triarylpropan-2-ols

A new method to open the heterocyclic ring of flavan-3-ols via photolytic cleavage of the ether bond, with stereoselective trapping of the intermediates with phloroglucinol to obtain phloroglucinol grafted derivatives of flavan-3-ols, was developed. Photolysis of catechin and epicatechin, respectively, in the presence of phloroglucinol yielded the enantiomeric (1S,2S)- and (1R,2R)-1,3-di(2,4,6-trihydroxyphenyl)-1-(3,4-dihydroxyphenyl)propan-2-ols, respectively. The absolute configuration at C-1 and C-2 was determined by electronic circular dichroism (experimental and calculated) and these results confirmed that the trapping mechanism is controlled by the C-3 configuration of the flavan-3-ol.     

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.     

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.     

Characterization of monomeric and oligomeric flavan-3-ols from barley and malt by liquid chromatography-ultraviolet detection-electrospray ionization mass spectrometry

The pattern of the monomeric and oligomeric flavan-3-ols for 10 barley varieties and the corresponding malts were identified and quantified using high-performance liquid chromatography-ultraviolet detection-electrospray ion trap mass spectrometry. The Folin-Ciocalteau and the vanillin spectrophotometric assays were used for the assessment of the total polyphenol and total flavan-3-ol content, respectively, and the antioxidant activity was determined as the 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity and the ferricyanide reducing power. Catechin and prodelphinidin B3 were respectively the major monomeric and dimeric flavan-3-ols. Moreover, prodelphinidin B3 was shown to be the main contributor for the radical scavenging activity both for barley and malt.     

Changes of flavan-3-ols with different degrees of polymerization in seeds of 'Shiraz', 'Cabernet Sauvignon' and 'Marselan' grapes after veraison

Flavan-3-ols consist of flavan-3-ol monomers and polymers with different degrees of polymerization (DP). In this study, flavan-3-ol extracts from grape seeds were well separated into three fractions including monomers, oligomers (2 < DP < 10) and polymers (DP > 10), by means of normal-phase HPLC-MS. The different patterns of these three fractions were analyzed in three Vitis vinifera cultivars ('Shiraz', 'Cabernet Sauvignon' and 'Marselan') seeds from veraison to harvest. The results showed: (1) polymers were the main form of flavan-3-ols in grape seeds and monomers accounted for only a small proportion; (2) the contents of flavan-3-ol monomers in the seeds of three grape cultivars all exhibited a gradually decreasing trend with a little fluctuation, whereas the patterns of the change of contents of oligomers and polymers were extremely different among grape cultivars; the contents of flavan-3-ol oligomers were enhanced in the seeds of 'Cabernet Sauvignon', but were reduced in the other two cultivars; (3) with regard to the proportion of flavan-3-ols with a certain DP to total flavan-3-ols, both flavan-3-ol monomers and flavan-3-ols with low DP fell in proportion, while the flavan-3-ols with high DP increased correspondingly. These findings indicate that flavan-3-ol polymerization in developing seeds is variety-dependent and may be genetically regulated.     

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.     

Retention behavior of oligomeric proanthocyanidins in hydrophilic interaction chromatography

A novel method was developed for the separation of proanthocyanidins (PAs; oligomeric flavan-3-ols) by hydrophilic interaction chromatography (HILIC) using an amide-silica column eluting with an aqueous acetonitrile mobile phase. The best separation was achieved with a linear gradient elution of acetonitrile-water at ratios of 9:1 to 5:5 (v/v) for 60 min at a flow rate of 1.0 ml/min. Under these HPLC conditions, a mixture of natural oligomeric PAs (from apple) was separated according to degree of polymerization (DP) up to decamers. The DP of each separated oligomer was confirmed by LC/electrospray ionization MS. In further HILIC separation studies of 15 different flavan-3-ol and oligomeric PA (up to pentamer) standards with an isocratic elution of acetonitrile-water (84:16), a high correlation was observed between the logarithm of retention factors (log k) and the number of hydroxyl groups in their structures. The coefficient of this correlation (r2=0.9501) was larger than the coefficient (r2=0.7949) obtained from the correlation between log k and log P(o/w) values. These data reveal that two effects, i.e. hydrogen bonding between the carbamoyl terminal on the column and the hydroxyl group of solute oligomer and hydrophilicity based on the high-order structure of oligomeric PAs, corporately contribute to the separation, but the hydrogen bonding effect is predominant in our HILIC separation mode.     

Isomerisation of benzophenones and direct observation of intermediates in the aromatic acylation of phloroglucinol derivatives with trifluoroacetic anhydride

Acylation of methyl and benzylethers of orcinol with the same ethers of phloroglucinol carboxylic acid using trifluoroacetic anhydride (TFAA) in chloroform or dichloro methane afforded both unsymmetrical and symmetrical benzophenones. Magnetic resonance measurements were performed on the reactions between various deuterium labelled methylethers of phloroglucinol and phloroglucinol carboxylic acid. The reaction was found to proceed via several equilibria resulting in a total scrambling of the aromatic rings around the carbonyl function. The occurrence of an intermediate in the reaction was observed and is thought to consist of an ion pair between a protonated benzophenone and trifluoroacetate ion which rapidly catalyses an exchange between TFAA and TFA.     

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.     

Antidiarrhoeic effect and dereplication of the aqueous extract of Annona crassiflora (Annonaceae)

We investigated the antidiarrhoeic effect of the aqueous extract of Annona crassiflora leaves (AEAC). The AEAC decreased the diarrhoeic stools and enteropooling induced by castor oil, without altering total faecal output; moreover, the distance travelled by charcoal meal in the intestine was increased. Twenty-eight compounds were identified by LC-DAD-MS in the AEAC, including flavonoids, alkaloids and proanthocyanidins. In addition, two oligomeric series of condensed tannins of up to nine flavan-3-ol units were characterised by MALDI-MS. These data suggest that the antidiarrhoeic effect of the AEAC is related to its ability to inhibit intestinal secretion and/or to increase intestinal absorption. Moreover, the prokinetic effect of AEAC, together with its inhibitory effect on enteropooling induced by castor oil, explains why this extract decreased diarrhoeic faeces without altering the total faecal output. All these effects are in agreement with the pharmacological activity reported in the literature for many of the secondary metabolites identified.     

Improving the Phloroglucinolysis Protocol and Characterization of Sagrantino Wines Proanthocyanidins

Proanthocyanidins are key metabolites that explain wine sensorial character (bitterness and astringency) and red wine color changes during aging. Therefore, a fast and accurate method to evaluate the degree of polymerization and the structural composition of the polymeric proanthocyanidins is a crucial analytical tool. Phloroglucinolysis is the most used method for this analysis but, unfortunately, the phloroglucinol adducts of the monomeric flavan-3-ols are not commercially available, making the results less accurate. The aim of this work was the isolation by semi-preparative high performance liquid chromatography (HPLC) of these non-commercial compounds and their use for the development of an accurate UHPLC-MS/MS protocol. The purity of each adduct was established via quantitative ¹H-nuclear magnetic resonance (NMR) measurements with 3-trimethylsilyl-propionic-d4 acid sodium salt as the calibration standard. The developed method was applied to evaluate the proanthocyanidins profile of Sagrantino di Montefalco wines in comparison to other well-known tannic wines. Commercial, 6–8 years old Sagrantino wines were demonstrated to be very rich in epicatechin type B procyanidins, to have low galloylation %, and to have a high mean degree of polymerization of the proanthocyanidins with respect to the other analyzed wines.     

Optimization of a high-performance liquid chromatography method for the analysis of complex polyphenol mixtures and application for sainfoin extracts (Onobrychis viciifolia)

A pentafluorophenylpropyl (PFP) stationary phase was tested for the simultaneous determination of several classes of phenolic compounds. The chromatographic results were compared with those obtained by using a bifunctional phase constituted of octadecyl and phenylpropyl bonded silica and three conventional C18 columns. The elution gradient was optimized with 5% formic acid and sodium acetate in combination with acetic acid as additives and methanol as solvents. For these evaluations, a complex phenolic extract of Onobrychis viciifolia (sainfoin) and test mixtures containing 54 standard substances including 2 simple phenolic compounds, 1 amino acid, 4 hydroxybenzoic acids (HBA), 6 hydroxycinnamic acids (HCA), 3 flavan-3-ols, 9 anthocyanins, 2 dihydroflavonols, 1 chalcone, 4 flavones, 1 isoflavone and 21 flavonols have been assayed. The perfluorinated column showed good resolution for the studied phenolic compounds which have the following elution order: HBA, HCA, flavan-3-ols, anthocyanins, dihydroflavonols, flavones, flavonols and isoflavones. Compared with other columns, it provides longer elution ranges for HBA, HCA and flavan-3-ols and increased retention times for all compound classes except anthocyanins which were similarly retained on a C18 column. Its selectivity is different from C18 and bifunctional phases. A high-performance liquid chromatography (HPLC) method with diode array detection (DAD) and post-column derivatization with p-dimethyl-aminocinnamic aldehyde (DMACA) has been validated for the analysis of individual phenolic compounds from a sainfoin plant extract (O. viciifolia).     

“水包水”型阳离子聚丙烯酰胺乳液的合成

The emulsion of cationic polyacrylamide was prepared by a soap-free polymerization,using water as media,the oligomer that prepared by myself as emulsifier and stabilizer,(NH_4)_2S_2O_8 as initiator.The effect of the amount of isopropanol,oligomer and the ratio of DMC/AM on the molecular weight of cationic polyacrylamide were all studied.The best amount of isopropanol,oligomer are 25%,20% of the total monomer and the ratio of DMC/AM is 3/7.     

Examining the Performance of Two Extraction Solvent Systems on Phenolic Constituents from U.S. Southeastern Blackberries

Two common extraction solvent systems, namely acidified aqueous methanol and acidified aqueous acetone, were used to extract blackberry phenolics, and the antioxidant properties of the recovered extracts were compared. The crude extracts were fractionated into low- and high-molecular-weight phenolics by Sephadex LH-20 column chromatography. The hydrophilic-oxygen radical absorbance capacity (H-ORAC_FL), ferric reducing antioxidant power (FRAP), and the cellular antioxidant activity (CAA) assays were employed as indices to assess antioxidant capacity of the extracts and their respective fractions. The methanolic solvent system displayed a greater efficiency at extracting anthocyanin and flavonol constituents from the blackberries, while the acetonic solvent system was better at extracting flavan-3-ols and tannins. Anthocyanins were the dominant phenolic class found in the blackberries with 138.7 ± 9.8 mg C3G eq./100 g f.w. when using methanol as the extractant and 114.6 ± 3.4 mg C3G eq./100 g f.w. when using acetone. In terms of overall antioxidant capacity of blackberry phenolics, the acetonic solvent system was superior. Though present only as a small percentage of the total phenolics in each crude extract, the flavan-3-ols (42.37 ± 2.44 and 51.44 ± 3.15 mg/100 g f.w. in MLF and ALF, respectively) and ellagitannins (5.15 ± 0.78 and 9.31 ± 0.63 mg/100 g f.w. in MHF and AHF, respectively) appear to account for the differences in the observed antioxidant activity between the two solvent systems.     

Rotation planar extraction and rotation planar chromatography of oak (Quercus robur L.) bark

The versatile novel instrument for rotation planar extraction and rotation planar chromatography was exploited for the investigation of oak bark (Quercus robur L.). The same instrument enabled extraction of the bark, analytical proof of (+)-catechin directly in the crude extract and also its fractionation. Additionally, epimeric flavan-3-ols, (+)-catechin and (-)-epicatechin were separated by analytical ultra-micro rotation planar chromatography on cellulose plates with pure water as developing solvent. A comparison of the extraction of oak bark with 80% aqueous methanol by rotation planar extraction and medium pressure solid-liquid extraction was carried out and both techniques were shown to be suitable for the efficient extraction of oak bark. The raw extracts and fractions on thin-layer chromatography showed many compounds that possessed antioxidant activity after spraying with 1,1-diphenyl-2-picrylhydrazyl. Rotation planar fractionation of 840 mg of crude oak bark extract on silica gel gave 6.7 mg of pure (+)-catechin in one run.     

Extraction of flavan-3-ols from grape seed and skin into wine using simulated maceration

Simulated maceration assays were carried out in wine model systems using increasing ethanol percentages from 0 to 12.5%, in order to study the extraction of flavan-3-ol monomers and oligomers from grape skins and seeds. The amount of flavan-3-ols transferred to the solutions improves with the increase in the alcohol percentage, although flavanols were much more readily released from grape skins than from seeds. Maximum release of flavanols from the skins is reached after 24 h of maceration in 12.5% ethanol, whereas long maceration and higher ethanol percentages were required for extraction from the seeds, from which maximum flavanol extraction was observed after 2-3 weeks of maceration. Thus, the length of the maceration not only had an influence in the total levels of flavanols reached in the wines, but also in their qualitative composition. The contribution of the seeds to the flavanol composition of the model wines was found to range from about 40% at 24 h of maceration in 12.5% ethanol to as high as 90% after 3 weeks. Gallocatechin was the major flavanol contributed by the skins and catechin by the seeds of the grape variety used (Vitis vinifera cv. Viura). Flavanol extraction was also followed during winemaking of commercial red wines, reaching similar conclusions to that obtained from the model assays.     

Detection of phenolic oxidation products in cider apple juice by high-performance liquid chromatography electrospray ionisation ion trap mass spectrometry

Juice was prepared from cider apples of the cultivar "Kermerrien" under oxidative conditions. After isolation by solid-phase extraction, the phenolic fraction was subjected to high-performance liquid chromatography/electrospray ionisation mass spectrometry. SIM scans were performed at m/z values obtained in model solutions. The oxidation products, resulting from coupling between a molecule of caffeoylquinic acid and caffeoylquinic acid, catechin or dimeric flavan-3-ol, were detected.     

Chemical constituents of Nepalese propolis (II)

A novel flavanonol (1), three new isoflavones (2-4) and a new flavan-3-ol (5) were isolated along with ten other known flavonoids (6-15) from the methanolic extract of propolis collected from Chitwan, Nepal. Their structures were determined on the basis of spectral analysis.     

Amplexicine, an antioxidant flavan-3-ol from Polygonum amplexicaule

Bioassay guided fractionation of an ethanolic extract of Polygonum amplexicaule D. Don led to the isolation of amplexicine, a new flavan-3-ol (1), along with khellactone (2). The structure of the isolates was established by UV, IR, HRESI/MS and NMR, including 1D and 2D experiments. Compound 1 exhibited considerable antioxidant activity in a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay.     

High-performance liquid chromatography/electrospray ionization mass spectrometric characterization of new products formed by the reaction between flavanols and malvidin 3-glucoside in the presence of acetaldehyde

Monomer and polymer flavan-3-ols and anthocyanins are the two major groups of phenolic compounds in red wine and one of the most important reactions during red wine ageing is the condensation reaction, either direct or indirect, between these two classes of compounds. In this work, the reaction of (+)-catechin, (-)-epicatechin and dimer procyanidin B2 with malvidin 3-glucoside, in the presence of acetaldehyde, was carried out in wine model solutions at pH 1.7 and 3.2. Identification of the reaction products was carried out by high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC/ESI-MS) and MS(n) analysis. Various reaction products were detected. The structures of three condensation products - malvidin-3-glucoside-ethyl-(epi)catechin-1'-hydroxyethyl, malvidin-3-glucoside-ethyl-dimer-vinyl and malvidin-3-glucoside-ethyl-dimer-1'-hydroxyethyl - were identified in a model solution for the first time. Moreover, in the reaction solution containing dimer B2 at pH 3.2, monomer epicatechin, trimer and tetramer procyanidins were also detected indicating that, under these conditions, dimer procyanidins can either be hydrolyzed to monomers or polymerized to higher oligomer or polymers.