Oligomerization Mechanisms of Tea Catechins Involved in the Production of Black Tea Thearubigins.

Thearubigins (TRs) are chemically ill-defined black tea pigments composed of numerous catechin oxidation products. TRs contain oligomeric components; however, the oligomerization mechanisms are poorly understood. The comparison of the 13C nuclear magnetic resonance (NMR) spectra of TRs with different molecular sizes suggested the participation of A-ring methine carbons in the oligomerization. Crushing fresh tea leaves with phloroglucinol, a mimic of the catechin A-rings, yielded the phloroglucinol adducts of the B-ring quinones of pyrogallol-type catechins and dehydrotheasinensins, indicating that intermolecular oxidative couplings between pyrogallol-type B-rings and A-rings are involved in the oligomerization. This is supported by the comparison of the 13C NMR spectra of the oligomers generated from the dehydrotheasinensins and epicatechin. Furthermore, the presence of the quinones or related structures in the catechin oligomers is shown by condensation with 1,2-phenylenediamine. The pyrogallol-type catechins account for approximately 70% of tea catechins; therefore, the B-A ring couplings of the pyrogallol-type catechins are important in the catechin oligomerization involved in TR production.

New degradation mechanism of black tea pigment theaflavin involving condensation with epigallocatechin-3-O-gallate.

Theanaphthoquinone (TNQ) is the initial and main oxidation product of theaflavin, a representative black tea pigment. Nevertheless, TNQ is virtually undetected in the high-performance liquid chromatography analysis of black tea leaves using photodiode array detection. To elucidate the degradation mechanism of theaflavin in the black tea production process, this study investigated the reaction of TNQ with epigallocatechin-3-O-gallate (EGCg), which is the most abundant polyphenol in tea leaves. In citrate-phosphate buffer solution at pH 6 and room temperature, TNQ reacted nonenzymatically with EGCg to afford three products, whose structures were determined on the basis of spectroscopic data. The results indicated that the double bond of the ortho-naphthoquinone moiety in TNQ reacted with the autoxidation product of EGCg. This study demonstrates novel reactions occurring in the process of theaflavin degradation, which might be involved in the formation of thearubigins, the major black tea pigments composing oligomeric catechin oxidation products.

Ellagitannin Digestion in Moth Larvae and a New Dimeric Ellagitannin from the Leaves of Platycarya strobilacea.

Ellagitannins (ETs) are plant polyphenols with various health benefits. Recent studies have indicated that the biological activities of ETs are attributable to their degradation products, including ellagic acid and its gut microflora metabolites, such as urolithins. Insect tea produced in the Guangxi region, China, is made from the frass of moth larvae that feed on the ET-rich leaves of Platycarya strobilacea. Chromatographic separation of the Guangxi insect tea showed that the major phenolic constituents are ellagic acid, brevifolin carboxylic acid, gallic acid, brevifolin, and polymeric polyphenols. Chemical investigation of the feed of the larvae, the fresh leaves of P. strobilacea, showed that the major polyphenols are ETs including pedunculagin, casuarictin, strictinin, and a new ET named platycaryanin E. The new ET was confirmed as a dimer of strictinin having a tergalloyl group. The insect tea and the leaves of P. strobilacea contained polymeric polyphenols, both of which were shown to be composed of ETs and proanthocyanidins by acid hydrolysis and thiol degradation. This study clarified that Guangxi insect tea contains ET metabolites produced in the digestive tract of moth larvae, and the metabolites probably have higher bioavailabilities than the original large-molecular ETs of the leaves of P. strobilacea.

Production Mechanisms of Black Tea Polyphenols.

Black tea accounts for 70-80% of world tea production, and the polyphenols therein are produced by enzymatic oxidation of four tea catechins during tea fermentation. However, only limited groups of dimeric oxidation products, such as theaflavins, theasinensins, and theacitrins, have been isolated from black tea and chemically characterized. This is largely because of the complexity and heterogeneity of the oxidation products. To determine structures and production mechanisms of uncharacterized black tea polyphenols, in vitro model fermentation experiments using pure catechins and polyphenol oxidase have been applied, and basic oxidation mechanisms have been established. Contemporary methods, such as LC-MS, are also effective to identify catechin oxidation products in black tea. Despite ongoing efforts, almost 60% of the solids in black tea infusion remain uncharacterized. These compounds include the so-called thearubigins, which are a heterogeneous mixture of uncharacterized catechin oxidation products with oligomeric structures. This review summarizes the current knowledge of the production mechanisms of representative black tea polyphenols and presents recent progress in characterization of thearubigins.

Production of Ellagitannin Hexahydroxydiphenoyl Ester by Spontaneous Reduction of Dehydrohexa-hydroxydiphenoyl Ester.

Amariin is an ellagitannin with two dehydrohexahydroxydiphenoyl (DHHDP) moieties connecting glucose 2,4- and 3,6-hydroxy groups. This tannin is predominant in the young leaves of Triadica sebifera and Carpinus japonica. However, as the leaves grow, the 3,6-DHHDP is converted to its reduced form, the hexahydroxydiphenoyl (HHDP) group, to generate geraniin, a predominant ellagitannin of the matured leaves. The purified amariin is unstable in aqueous solution, and the 3,6-(R)-DHHDP is spontaneously degraded to give HHDP, whereas 2,4-(R)-DHHDP is stable. The driving force of the selective reduction of the 3,6-DHHDP of amariin is shown to be the conformational change of glucose from O,3B to 1C4. Heating geraniin with pyridine affords 2,4-(R)-DHHDP reduction products. Furthermore, the acid hydrolysis of geraniin yields two equivalents of ellagic acid. Although the reaction mechanism is still ambiguous, these results propose an alternative biosynthetic route of the ellagitannin HHDP groups.

Oligomerization mechanism of tea catechins during tea roasting.

Roasting of green tea causes oligomerization of tea catechins, which decreases the astringency. The aim of this study was to elucidate the oligomerization mechanism. The 13C NMR spectrum of the oligomer fraction showed signals arising from catechin and sugar residues. Heating of epigallocatechin-3-O-gallate with 13C-labeled glucose (150 °C for 2 h) suggested that condensation of sugars with catechin A-rings caused the oligomerization. The dimeric product obtained by heating for a shorter period (30 min) suggested cross-linking occurred between sugars and catechin A-rings. Furthermore, heating of phloroglucinol, a catechin A-ring mimic, with glucose, methylglyoxal, and dihydroxyacetone, confirmed that the basic mechanism included reaction of the catechin A-ring methine carbons with carbonyl carbons of glucose and their pyrolysis products.

Solubility of Tannins and Preparation of Oil-Soluble Derivatives.

Tannins are plant defense substances that exhibit a strong astringent taste and precipitate proteins, leading to the inhibition of protein functions; however, owing to their relatively low toxicity, tannins must be accumulated in high concentrations in cell vacuoles. Therefore, the solubility of tannins is crucial for their functions. In this review, the structure and reactions of tannins related to solubility; insolubilization of persimmon proanthocyanidins on fruit ripening; pigment formation from cinnamon procyanidins by reaction with cinnamaldehyde in case of wounding; and insolubilization of ellagitannins in chestnut wood is discussed. In addition, the development of functional polyphenols including oil-soluble tea catechins is introduced.

Utilization of Flavonoid Compounds from Bark and Wood. III. Application in Health Foods.

Dietary supplements ACAPOLIA® and ACAPOLIA PLUS have been sold in Japan under the classification "Foods in General" for a number of years. In April 2015, the classification of "Foods with Function Claims" was introduced in Japan to make more products available to the public that were clearly labeled with functional claims based on scientific evidence. In order to obtain recognition of ACAPOLIA PLUS under this new classification, the following information needed to be established. The safety of the bark extract of Acacia mearnsii was shown from the history of the long-term safe consumption of the extract as a health supplement, together with several additional clinical safety tests. Robinetinidol-(4α,8)-catechin was detected by high performance liquid chromatography (HPLC) in the supplement and was suitable for use as the basis of the quantitative analysis. In clinical tests, the amount of change in the plasma glucose concentration in the initial 60 min after rice consumption by a test group who had been given the Acadia extract was significantly lower than the glucose concentration in the group that was given a placebo. The blood glucose incremental areas under the curve (IAUC) in the first 60 min after rice consumption were also significantly lower in the Acacia group. The functional mechanisms were explained in terms of the inhibition of the absorption of glucose in the small intestine and the reduction in the activity of the digestive enzymes caused by proanthocyanidins derived from A. mearnsii bark. As a result, ACAPOLIA PLUS was accepted as a "Food with Function Claims" in August 2016. ACAPOLIA PLUS is now sold under this new classification. The growth of a typical intestinal bacterium is inhibited by an extract containing flavonoid compounds from A. mearnsii bark; thus, one of the future directions of study must be a comprehensive investigation of the effect that flavonoid compounds, proanthocyanidins, have on intestinal bacteria.

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.

Study of the Stereochemistry and Oxidation Mechanism of Plant Polyphenols, Assisted by Computational Chemistry.

In recent years, plant polyphenols have attracted great attention due to their wide range of biological activities. Certain kinds of polyphenols have complex structures; therefore, it is difficult to elucidate their total structure, including stereochemistry. In this study, we reinvestigated the stereostructures of two major C-glycosidic ellagitannins contained in Quercus plants, vescalagin and castalagin, and revised their stereostructures based on theoretical calculations of spectroscopic data. We also determined the structures of quercusnins A and B, isolated from the sapwood of Quercus crispula, based on theoretical calculations of NMR data. The oxidation mechanism of polyphenols has not been entirely elucidated. Therefore, we have also studied the oxidation mechanism of tea catechins during black tea production. Our investigation of the oxidation mechanism of black tea pigment theaflavins revealed that the difference in the position of the galloyl ester affords different oxidation products of theaflavins. In addition, oxidation products of pyrogallol-type catechins could be classified into three types-dehydrotheasinensins, theacitrins, and proepitheaflagallins; their detailed production and degradation mechanisms were also examined.

Ferulic-Acid Esters of Oligo-glucose from A-ilium macrostemon.

Two new ferulic acid esters of oligo-glucose, 1-Ο-(E)-feruloyl-ß--D-gentiobioside (1) and 1-Ο-(E)feruloyl-{ß-D-glucopyranosy (1->6)-[ß-D-glucopyranosyl (I--2)]}-ß-D-glucopyranoside.(allimacronoid D, 2) were isolated together with 1-Ο-(E)-feruloyl-ß-D-glucopyranoside (3) and trans-ferulic acid (4) from the leaves of Allium macrostemon Bunge. The chemical structures were elucidated based on the analyses of the spectroscopic and chemical data.

Theagalloflavic Acid, a New Pigment Derived from Hexahydroxydiphenoyl Group, and Lignan Oxidation Products Produced by Aerobic Microbial Fermentation of Green Tea.

Chinese ripe pu-erh tea is produced by aerobic microbial fermentation of green tea. To clarify the microbial degradation of tea polyphenols, Japanese commercial green tea was mixed with Chinese ripe pu-erh tea, which retains microorganisms, and fermented for 5 d. Chromatographic separation yielded a novel water-soluble yellow pigment termed theagalloflavic acid. Spectroscopic and chemical evidence suggested that this pigment was produced by oxidative ring cleavage of hexahydroxydiphenoyl esters. In addition, two new oxygenated lignin metabolites, (+)-5,5'-dihydroxypinoresinol and 5-hydroxydihydrodehydrodiconiferyl alcohol, were also isolated together with known degradation products of quercetin and tea catechins.

Triterpene Galloyl Esters from Edible Acorn of Castanopsis cuspidata.

Polyphenols of edible acorns of Castanopsis cuspidata were examined and two triterpene galloyl esters were isolated. Based on two-dimensional NMR spectroscopic evidence the structures were determined to be 3,24-di-O-galloyl-2α,3ß-23,24-tetrahydroxyolean-12-en-28-oic acid (1) and 3,24-di-O-galloyl- 2α,3ß-23,24-tetrahydroxyurs-12-en-28-oic acid (2). The triterpene hexahydroxydiphenoyl esters, which had been found in the leaves, were not detected in the acorns.

Three New Oxidation Products Produced from Epigallocatechin-3- O-gallate and Epicatechin-3-O-gallate.

During chemical studies on uncharacterized black tea polyphenols, the enzymatic oxidation of a mixture of epigallocatechin-3-O-gallate (1) and epicatechin-3-O-gallate (2) was examined, and three new dimeric products together with six known catechin dimers were isolated. Two of the new compounds have tricyclo[5.2.2.0²,6]undecane and tricyclo[5.2.1.0²,6]decane carboxyl structures generated by oxidative coupling between two pyrogallol-B-rings of 1. Another new product was a dimer connected by a C-C bond between the B-ring of 1 and a galloyl group of 2.

Stereochemistry of the Black Tea Pigments Theacitrins A and C.

Theacitrins A-C are yellow pigments of black tea that are produced by oxidative coupling of gallocatechins, i.e., flavan-3-ols with pyrogallol-type B-rings. However, their stereostructures have not yet been determined. In this study, DFT calculations of NMR chemical shifts of theacitrin C (1) and TDDFT calculations of the ECD spectra of theacitrinin A (5), a degradation product of theacitrin C (1), were used to determine the stereostructure of the theacitrins. Furthermore, the preparation of theacitrins A (4) and C (1) by enzymatic oxidation of an epigallocatechin (7) and epigallocatechin-3-O-gallate (2) mixture confirmed their structural relationship.

Eudesmane Sesquiterpenoids from the Wood of Platycarya strobilacea.

The wood of Platycarya strobilacea Sieb. et Zucc. (Juglandaceae) was used as incense. In this study, three new sesquiterpenes (2, 4, 5) and one nor- sesquiterpene (3) were isolated and their structures determined as 7-hydroperoxy-11-hydroxy-2,4-cycloeudesman-8-one (2), 8-hydroxy-13-nor-2,4- cycloeudesman-7-en-I 1-one (3), 2,4-cycloeudesma-7(11), 8-dien-(12,8)-olide (4), and 8-hydroxy-8,12-peroxy-2,4-cycloeudesm-7(l I)-ene (5). In addition, the absolute configuration of a previously reported sesquiterpene was revised based on the calculation of its ECD spectrum.

Characterization of the α-Amylase Inhibitory Activity of Oligomeric Proanthocyanidins from Acacia mearnsii Bark Extract.

Acacia mearnsii (Fabaceae) contains acacia polyphenols, which are a complex mixture of proanthocyanidins that are mainly composed of 5-deoxycatechin units. In this study, an aqueous extract of A. mearnsii bark was fractionated and the α-amylase inhibitory activity of each fraction was evaluated. The (13)C NMR and MS data and the pyrolysis products obtained from the active and inactive fractions were compared. The spectroscopic results clearly indicated that fractions with strong inhibitory activity contained proanthocyanidin oligomers with catechol-type B-rings rather than pyrogallol-type B-rings HPLC analysis of the pyrolysis products showed peaks for pyrocatechol were only observed in the mixtures obtained from the fractions with high inhibitory activities. In addition,(+)-pinitol was isolated as a major polyol of the extract at a level comparable with that of sucrose.

A New Flavonoid from Camellia sinensis Fermented Tea.

A new flavonoid, quercetin 3-0-α-L-rhamnopyranosyl-(l-6)-(2-E-p-coumaroyl)-D-D-glucopyranoside-4'-O-α-L-rhamnopyranoside, was isolated from the fermented tea of Camellia sinensis L. (Pu-erh tea) from China. The chemical structure was elucidated based on analyses of the spectroscopic data.

Chalcane-stilbene conjugates and oligomeric flavonoids from Chinese Dragon's Blood produced from Dracaena cochinchinensis.

A detailed chemical investigation of Chinese Dragon's Blood, which is a traditional medicine produced form the red resin of Dracaena cochinchinensis, yielded two chalcane-stilbene conjugates, named cochinchinenenes G and H, together with 25 known compounds. The structures of these compounds were determined by spectroscopic examination. HPLC analysis of the resin indicated that the major constituents were a complex mixture of oligomeric polyphenols, which were detected as a broad hump on the base line of a HPLC chromatogram. (13)C NMR analysis indicated that the oligomers were mainly composed of oxygenated chalcane units. This suggestion was supported by the results of a thiol degradation experiment with mercaptoethanol, which yielded a thioether of 4-[(4-hydroxyphenyl)propyl]-3-methoxyphenol. Furthermore, methylation followed by electrospray ionization mass spectroscopic analysis of the resulting fractions established the presence of at least one heptamer of chalcane units.

Diastereomeric Ellagitannin Isomers from Penthorum chinense.

From the dried stem of Penthorum chinense (Penthoraceae), 1-O-galloyl-4,6-(R)-hexahydroxydiphenoyl (HHDP)-ß-D-glucose and 2',4',6'-trihydroxyacetophenone 4'-O-[4,6-(R)-HHDP]-ß-D-glucoside were isolated together with their (S)-HHDP isomers. Ellagitannins with a 4,6-(S)-HHDP-glucose moiety are widely distributed in the plant kingdom; however, 4,6-(R)-HHDP glucoses are extremely rare. Lowest-energy conformers of 1-O-galloyl-(S)- and (R)-HHDP-glucopyranoses were derived by density functional theory calculations, and the calculated (1)H and (13)C NMR chemical shifts and the (1)H-(1)H coupling constants were in agreement with the experimental values. The results revealed a conformational difference of the diastereomeric macrocyclic ester rings. In addition, a new compound, 1',3',5'-trihydroxybenzene 1'-O-[4,6-(S)-HHDP]-ß-D-glucoside, was also isolated.