Catechin specification

PPO from tea reacts effectively with both 3 -4 and 3 -4 -5-hydroxylated catechins, with specificity for the o-diphenol (43,44). Studies defining the kinetics of PPO from tea in relation to substrate type are lacking as of this writing (ca 1997). Tea PPO has good functionaUty in the pH range 4.6—5.6 (43,45-48). 

The Folin-Ciocalteu assay is the most widely used method to determine the total content of food phenolics (Fleck and others 2008). Folin-Ciocalteu reagent is not specific and detects all phenolic groups found in extracts, including those found in extractable proteins. A disadvantage of this assay is the interference of reducing substances, such as ascorbic acid (Singleton and others 1999). The content of phenolics is expressed as gallic acid or catechin equivalents. 

On patients with cancer, the effects of green tea catechins, soy isoflavones and quercetin as chemoprotective/chemotherapeutic agents have also been studied. Although results have not been entirely satisfactory, a partial response has been achieved in some trials. For example, small decreases in plasma concentration of prostate-specific antigen were observed in prostate cancer patients who consumed soy isoflavones. Nevertheless, results in individuals with premalignant disease who consumed green tea polyphenols support their advancement into phase III clinical intervention trials aimed at the prevention of PIN, leukoplakia, or premalignant cervical disease (Thomasset and others 2006). 

The sugars attached to the anthocyanin molecule are in order of relative abundance glucose, rhamnose, galactose, xylose, arabinose, and glucuronic acid. The molecule may also contain one or more of the acyl acids p-coumaric, caffeic, and ferulic or the aliphatic acids malonic and acetic esterified to the sugar molecules. Extracts of anthocyanins invariably contain flavonoids, phenolic acids, catechins and polyphenols. The net result is that it is impossible to express the chemical composition accurately. Specifications usually present tinctorial power, acidity, per cent solids, per cent ash and other physical properties. 

Finally, Weinges et al. (31, 33) postulated the formation of dimers such as 27 or 28, 29 or 30 directly from catechins without involving 3,4-flavandiols (leucoanthocyanidins). This process has never been demonstrated in fruits directly and specifically not in grapes. Only the proanthocyanin dimers have been positively identified through the formation of catechins and anthocyanidins during acid hydrolysis. Dimer formation proceeds by enzymatic oxidation of two molecules of catechin... 

An alternative colorimetric method relies on the reaction with vanillin under acidic conditions. A 2-mL aliquot of a freshly prepared solution of vanillin (1 g/100 mL) in 70% sulfuric acid is added to 1 mL of aqueous plant extract. The mixture is incubated in a 20°C-waterbath and after exactly 15 min. the absorbance at 500 nm read. The concentration of proanthocyanidins is expressed as (+)-catechin equivalents (used for the standard curve). This assay is specific for flavonols. As a consequence, when using this assay to determine the concentration of condensed tannins, widely distributed monomeric flavonols, such as catechin (1.39) and epicatechin (1.90), can interfere (Hagerman and Butler, 1989). 

The major metabolic pathways for tea catechins include glucuronidation, sulfation, and methylation. There are species and tissue-specific differences in... 

Next we evaluated a series of resveratrol analogs and catechins for their ability to compete for specific [3H]-resveratrol binding in PII fraction. Interestingly, polyphenols that display neuroprotective action are the most potent ones to compete for specific [3H]-resveratrol binding with K values ranging from 25 nM (for EGCG) to 102 nM (for resveratrol), whereas molecules including EC and EGC were inactive. 

The branch pathway for anthocyanin biosynthesis starts with the enzymatic reduction of dihydrofiavonols to their corresponding flavan 3,4-diols (leucoanthocyanidins) by substrate-specific dihydroflavonol 4-reductases (DFR). Flavan 3,4-diols are the immediate precursors for the synthesis of catechins and proanthocyanidins. Catechins are formed by enzymatic reduction of the flavan 3,4-diols in the presence of NADPH to leucoanthocyanidins, which are subsequently converted to anthocyanidins by the 2-oxoglutarate-dependant dioxygenase, anthocyanidin synthase. Further glycosylation, methylation, and/or acylation of the latter lead to the formation of the more stable, colored anthocyanins (Scheme 1.1). The details of the individual steps involved in flavonoid and isoflavonoid biosynthesis, including the biochemistry and molecular biology of the enzymes involved, have recently appeared in two excellent reviews.7,8... 

The substrates of the polyphenol oxidase enzymes are phenolic compounds present in plant tissues, mainly flavonoids. These include catechins, anthocyanidins, leucoantho-cyanidins, flavonols, and cinnamic acid derivatives. Polyphenol oxidases from different sources show distinct differences in their activity for different substrates. Some specific examples of polyphenolase substrates are chlorogenic acid, caffeic acid, dicatechol, protocatechuic acid, tyrosine, catechol, di-hydroxyphenylalanine, pyrogallol, and catechins. 

TMS production involves one specific functional group (-OH, -COOH, =NH, -NH2, or -SH), which loses an activated hydrogen and is replaced by a trimethylsilyl group (Proestos et ah, 2006). To achieve silylation, some authors have used BSTFA (N,0-hA(trimethyl-silyl)trifluoroacetamide) and TMCS (trimethylchlorosilane) successfully in several matrices (e.g. aromatic plants, cranberry fixiit) (Zuo et ah, 2002 Proestos et ah, 2006). Using silylated derivatives is advantageous for several reasons phenols and carboxylic acids are prone to silylation, these compounds can be derivatized in the same part of the process, and the minor products do not impede analysis and are well documented (Little, 1999 Stalikas, 2008). A two-step methylation procedure was used to analyze catechins and tannins in plant extracts. The first step used trimethylsilyl diazomethane (TMS-diazomethane) to pre-methylate the sample, and the second step used thermally assisted hydrolysis and methylation (THM). The pre-methylation step with TMS-diazomethane stabilized the dimer molecule m/z 540) by minimizing isomerization and reducing reactivity. (Shadkami et ah, 2009). 

Catechins and proanthocyanidins can be detected at 280 nm using a UV detector. However, peak intensity at this wavelength is low, and many other phenolic compounds also adsorb light at 280 nm. Fluorescent detection provides better sensitivity and specificity than UV detection. The excitation and emission spectra of procyanidin dimers are shown in Figure 8.5. Excitation at 276 nm and emission at 316 nm had been used in earlier studies however, an examination of the fluorescent spectra indicated this was not the optimal condition. Excitation and emission wavelengths were set to 230 and 321 nm, respectively, in our most recent study, which caused a 5-fold increase in peak intensity (Robbins et ah, 2009). 

Kivits, G.A.A. van der Sman, F.J.P. Tijburg, L.B.M. 1997. Analysis of catechins from green and black tea in humans a specific and sensitive colorimetric assay of total catechins inbiological fluids. Int. J. Food Sci. Nutr. 48 387-392. 

Nevertheless, the substrate specificity of peroxidases is so wide that secretory plant peroxidases are capable of accepting a plethora of natural compounds as substrates, such as indoles [43], porphyrins [44,45] including chlorophylls [46,47], terpenoids such as lutein [48], unsaturated lipid acids such as linoleic acid [49], alkaloids [50-52] including betacyanins [53], phenolics such as benzoic acids [54,55], DOPA [56], coumarins [38,57], stylbenes [58], catechins [36,59], chalcones [60], flavonols [61,62], isoflavones [63,64], cinnamyl alcohols and cinnamic acids [65,66], anthocyanins [67,68] and ascorbic acid [69,70],... 

The flavonols and their glycosides contribute to specific taste characteristics such as bitterness and astringency in berry fruits and their products (Shahidi and Naczk, 1995). The molecular structure of flavonols lacks the conjugated double bonds of the anthocyanins, and they are thereby colorless. They may, however, contribute to discoloration of berry fruits, as they are readily oxidized by O-phenoloxidase in the presence of catechin and chlorogenic acid. Discoloration may also occur as a consequence of complex formation with metallic ions. On the other hand, the flavonol glycoside rutin is known to form complexes with anthocyanins, thus stabilizing the color of these compounds. 

SPE on a CLX cartridge was applied to separate acidic phenols such as chlorogenic acid (95) from neutral phenols such as (—)-epicatechin (2), (+)-catechin (3), phloridzin (96) and quercitrin (100). The neutral phenols were determined in apple juice by capillary LC with UVD at 280 nm, as an alternative to conventional HPLC. LOD were from 9 pg for 96 to 97 pg for 3. HPLC analysis with MS and DA-UVD showed that apple pomace is a good potential source for phenoUcs. The usefulness of arbutin (9) as specific marker for pear products was placed in doubt (see Section II.A °). 

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