Proanthocyanidin assay

The proanthocyanidin assay is carried out in a solution of butanol-concentrated hydrochloric acid, where proanthocyanidins (condensed tannins) are converted to antho-cyanidins (products of autoxidation of carbocations formed by cleavage of interfla-vanoid bonds) (Matus-Cadiz and others 2008). 

The following functional group assays are used proanthocyanidin assay, vanillin assay and dimethylaminocinnamaldehyde (DMACA) assay. Details to the experimental designs are listed in Tab. (3.1) to (3.3). 

Proanthocyanidin Assay (Synonym Acid Butanol Assay, formarly Leucoanthocyanin method) ... 

Tables (4.1) and (4.2) show a comparison of absorbance data obtained with the proanthocyanidin assay. Values for purified procyanidins as well as for extracted and unextracted plant tissues containing only procyanidins are listed. The data clearly demonstrate that calibration factors not only depend on experimental variables but also on the matrix. The use of calibration factors does therefore hardly produce true estimates for contents in plant extracts.

The only correct quantitation of results from the proanthocyanidin assay is to use purified procyanidin fractions from the matrix under analysis. Calibrations should be prepared in the extracts themselves to correct for matrix effects as much as possible. A similar approach has been used by Li et al. [67] who produced a calibration curve with purified... 

Table 4.1. Absorbance Data of Cyanidin and Procyanidins Using the Proanthocyanidin Assay...

Table 4.2. Absorbance Data in the Proanthocyanidin Assay for Extracts and Unextracted Plant Tissues Containing Exclusively Procyanidins...

The condensation reaction may take place at position 6 or 8. Position 6 of the flavan skeleton is favored because it is usually less sterically hindered than position 8. In contrast to the proanthocyanidin assay the condensation reaction runs without depolymerization of the proanthocyanidins [135]. Consequently monomers also react [116] and a multitude of different dyes are produced depending on the complexity of the sample. The maxima of the chromophores of the resulting dyes are not influenced by the B-ring hydroxylation pattern (i.e. procyanidin-prodelphinidin ratio) like in the proanthocyanidin assay [116]. Absorbances are however influenced by the substitution pattern on the ring at which condensation takes place. Condensation products of phloroglucinol nucleis show absorbance maxima at about 500 nm, whereas resorcinol and pyrogallol nucleis exhibit absorbance maxima around 520 nm [77]. 

A major drawback of all functional group assays is that a satisfactory standard does not exist. For a given sample the most appropriate standard is a purified procyanidin fraction prepared from the same matrix. The isolation and characterization of such purified fractions are laborious. Added to that procyanidins undergo oxidation, complexation and self-polymerization very easily, rendering such purified fractions only reproducible to a limited degree. At least in the proanthocyanidin assay the color reaction depends not only on the polyphenols themselves, but also on the matrix. The use of specified proanthocyanidins as a standard in a suitable blank matrix is an attempt to correct for such effects [67],... 

Sun BS, Ricardo-da-Silva JM and Spranger MI. 1998. Critical factors of vanillin assay for catechins and proanthocyanidins. J Agric Food Chem 46 A261-A21A. 

Butler LG, Price ML, Brotherton JE (1982) VaniUin assay for proanthocyanidins (condensed tannins) modification of the solvent for estimation of the degree of polymerization. J Agric Food Chem 30 1087-1089... 

Moved] Cranberry fruit of Early Black cultivar was fractionated chromatographically and fractions were analyzed for flavonoid content. The effects of the flavonoid fractions and ursolic acid, an abundant triterpenoid in cranberry peel, were assessed in two models of colon cancer and one model of breast cancer. Clonogenic soft agar assays were used to determine the effect of these compounds on tumor colony formation in HCT-116, HT-29 and MCF-7 cells. MTT and trypan blue assays were performed to assess their ability to inhibit tumor cell proliferation. TUNEL assays were performed to assess apop-totic response to the cranberry compounds. The proanthocyanidins inhibited tumor colony formation in HCT-116 and HT-29 cells in a dose-dependent manner, with greater effect on the HCT-116 cell line. Ursolic acid strongly inhibited tumor colony formation in both colon cell lines. These compounds also decreased proliferation in all three tumor cell lines with the HCT-116 cell line most strongly affected. (150 words)... 

Scalbert et al. (1989) described several methods to determine the concentration of hydrolysable and condensed tannins (proanthocyanidins see Chapter 1, section 3.13.1) in wood extracts. Tannins are complex and heterogeneous In addition to the distinction between the flavonoid-based condensed tannins and the gallic acid-based hydrolysable tannins, each group can display a large degree of chemical variability that can affect the efficacy of the different assays. Interference of chemically related nontannin compounds, such as flavonoids, can in certain cases bias the results. 

A colorimetric assay involves the oxidative cleavage of proanthocyanidins with ferrous sulfate. To 0.5 mL of aqueous plant extract is added a 5-mL portion of an acidic solution of ferrous sulfate (77 mg of FeS04.7H20 dissolved in 500 mL of 2 3 HCl/ -butanol). The tubes are loosely covered and placed in a water bath at 95°C for 15 min. The absorbance is read at 530 nm. The concentration of proanthocyanidins is expressed as cyanidin equivalents (used for the standard curve). The molecular extinction coefficient emoi that can be used to convert the absorbance values to a concentration is equal to 34700 L mol cm 1. 

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). 

In a typical assay using toluene-a-thiol as a nucleophile, a proanthocyanidin solution (50 pL, in 70% acetone) was placed in a 250-pL polypropylene microtube. The solvent was evaporated at 25 °C under vacuum. Methanol (50 pL) was added to dissolve the residue. Then 50 pL of methanol, acidified with concentrated HCl (3.3%, v/v), and 100 pL of toluene-a-thiol (5% v/v in methanol) were added into this microtube. The microtube was placed into a 1.5-mL vial and sealed with... 

There are several caveats associated with this assay that may affect accuracy and precision. (+)-Catechin is the natural form in proanthocyanidins. Part of (+)-catechin epimerizes at the C2 position to form (+)-epicatechin during depolymerization. Similarly, part of (—)-epicatechin epimerizes to form (—)-catechin as an artifact. (+)-Catechin and (+)-epicatechin are an epimer pair in solution (similar to a- and 3-glucose in solution), i.e. they are chiral isomers that cannot be separated on a common reversed-phase HPLC column. The degree of epimerization increases with reaction temperature and time. Depolymerization at room temperature for 10 h caused less than 10% of flavan-3-ols to undergo epimerization. Toluene-a-thiol also causes the heterocyclic ring fission of flavan-3-ols to form adducts that... 

MALDI-TOF-MS assay, purified proanthocyanidins in acetone were mixed with a matrix solution ( ra 5-3-indoleacrylic acid, 5 mg/100 pL in 80% aqueous acetone). The mixture (0.2 pL) was applied on a stainless steel target and dried at room temperature. Dried mixtures were subject to MALDI-TOF-MS using anN2 laser as the ionization and reflection mode for mass separation. Proanthocyanidins trimers to nonomers were detected (Krueger et ah, 2003). 

Bagchi et al. (1997) studied the oxygen free-radical scavenger ability of grape seed proanthocyanidins using a chemiluminescence assay and cytochrome c reduction. The results revealed that on a weight basis (100 mg/L), the grape seed proantho-cyanidin extract was a better inhibitor of both superoxide anion and hydroxyl radical than vitamin C and vitamin E succinate. 

The proanthocyanidins are widely distributed in nature and often represent the active compounds of medicinal plants or plants showing human health beneficial effects. Reports of several in vitro assays demonstrate potentially significant antiviral, antibacterial, molluscicidal, enzyme-inhibiting, antioxidant, and radical-scavenging properties. " Their potential to interact with biological systems rests, at least in part, on the ability to form complexes with other biomolecules, thus emphasizing the necessity to accurately define... 

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