Proanthocyanidins determination

Similar to catechins, several studies have reported that proanthocyanidins exhibit a more or less pronoimced antibacterial activity. Chimg et al. [76] reported that proanthocyanidins determine the growth inhibition of strains of Aeromonas spp.. Bacillus spp., Clostridium botulinum, Clostridium per-fringens, Enterobacter spp., Klebsiella spp., Proteus spp.. Pseudomonas spp.. Shigella spp., S. aureus. Streptococcus spp., and Vibrio spp. 

Proanthocyanidins are an important group of di- to oligomeric flavonoids in plants. Four proanthocyanidins (procyanidin B3, prodelphinidin B4, ECG-(4 8)-ECG and GC-(4 8)-EGCG) were determined quantitatively in tea. The amounts in fresh tea leaves were between 1 and 2 g/kg per compound (Nakabayashi, 1991). The occurrence of proanthocyanidins may serve as a criterion for the differentiation between fermented and non-fermented teas (Kiehne et al, 1997). 

Flavan-3,4-diols FIavan-3,4-diols, also known as leucoanthocyanidins, are not particularly prevalent in the plant kingdom, instead being themselves precursors of flavan-3-ols (catechins), anthocyanidins, and condensed tannins (proanthocyanidins) (see Fig. 5.4). Flavan-3,4-diols are synthesized from dihydroflavonol precursors by the enzyme dihydroflavonol 4-reductase (DFR), through an NADPH-dependent reaction (Anderson and Markham 2006). The substrate binding affinity of DFR is paramount in determining which types of downstream anthocyanins are synthesized, with many fruits and flowers unable to synthesize pelargonidin type anthocyanins, because their particular DFR enzymes cannot accept dihydrokaempferol as a substrate (Anderson and Markham 2006). 

The R locus determines the presence (R) or absence (r) of anthocyanins in the seed coat. R is required (with i and T) to produce black seed [10]. However the identity of the gene product encoded by this locus has not been reported. Todd and Vodkin [25] have demonstrated that brown seed coats (r) contain proanthocyanidin (PAs) and black seed coats (R) contain anthocyanins in addition to PAs and suggested that R acts subsequent to the formation of leucoanthocyanidin but previous to the formation of anthocyanins. UDP-glucose flavonoid 3-0-glucosyltransferase (UF3GT) should be considered a candidate gene of the R locus but its identiflcation has not yet been reported. 

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)... 

Nesi, N. et al.. The Arahidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed. Plant Cell, 13, 2099, 2001. 

As discussed above, the development of mild MS techniques has led to further progress in the determination of proanthocyanidin size distribution. In particular, ESI-MS studies have demonstrated that prodelphinidin and procyanidin units coexist within the polymers, where they seem distributed at random. A list of mass signals attributed to proanthocyanidins detected in grape or wine extracts is given in Table 5.2. 

Based on the currently available data, grape anthocyanin and flavonol profiles are determined by genetic characteristics whereas their content varies with the vine growing conditions. Available information still appears too limited and contradictory to draw any conclusion on the impact of genetic and environmental factors on grape proanthocyanidin composition. 

Finally, reactions of flavonoid and nonflavonoid precursors are affected by other parameters like pH, temperature, presence of metal catalysts, etc. In particular, pH values determine the relative nucleophilic and electrophilic characters of both anthocyanins and flavanols. Studies performed in model solutions showed that acetaldehyde-mediated condensation is faster at pH 2.2 than at pH 4 and limited by the rate of aldehyde protonation. The formation of flavanol-anthocyanin adducts was also limited by the rate of proanthocyanidin cleavage, which was shown to take place at pH 3.2, but not at pH 3.8. Nucleophilic addition of anthocyanins was faster at pH 3.4 than at pH 1.7, but still took place at pH values much lower than those encountered in wine, as evidenced by the formation of anthocyanin-caffeoyltartaric acid adducts, methylmethine anthocyanin-flavanol adducts,and flavanol-anthocyanin adducts. The formation of pyranoanthocyanins requiring the flavylium cation was faster under more acidic conditions, as expected, but took place in the whole wine pH range. Thus, the availability of either the flavylium or the hemiketal form does not seem to limit any of the anthocyanin reactions. 

The various MS methods to determine the molecular composition of the constituent monomeric units in proanthocyanidins oligomers are summarized in Ref. 257. Contributions focusing on proanthocyanidin analysis via the HPLC-MS protocol included a wide range of plant-derived foods and beverages, and are summarized in Refs. 12, 258-261. In addition, references to additional significant contributions in this area are readily available via several of the excellent electronic search engines that are at our disposal. 

Primary antibodies, see Antibodies Proanthocyanidins. see also Polyphenolics Probes, in immunoblotting avidin-biotin-coupled antibodies, 209-210 directly conjugated antibodies, 207-209 Probe spectrofluorometry, determining protein hydrophobicity, 301 -304 ProBlot membranes, electroblot and elution of proteins, 189-190, 193-197 Processed solid foods, drip loss... 

Proanthocyanidins Extraction, Purification, and Determination of Subunit Composition by HPLC... 

Proanthocyanidins are polymeric flavonoid compounds composed of flavan-3-ol subunits (unitii.3), and are responsible for bitterness and astringency in some foods and beverages. This unit describes methods for extracting and purifying proanthocyanidins, and for determining their subunit composition by HPLC. Based upon HPLC results, the average degree of polymerization and the conversion yield for purified proanthocyanidins can be determined. 

Because proanthocyanidins are susceptible to oxidation, the amount of time the flask is left on the rotary evaporator should be limited to the time necessary to remove the acetone. This is most easily determined by observing the rotary evaporator condenser. Because water has a higher surface tension than acetone, condensation of water is observed as fogging of the condenser portion of the rotary evaporator. 

The potential impurities will vary according to the plant tissue extracted, and therefore the exact washing volume will vary. It is important to determine the impurities present and their retention properties on the column to minimize impurities in the final proanthocyanidin and maximize proanthocyanidin recovery. For this step, the use of a spectrophotometer is helpful in monitoring the eluate. Some typical impurities and monitoring wavelengths include organic acids (215 nm), flavan-3-ol monomers (280 nm), hydroxycinnamic acids (320 nm), andflavonols (365 nm). Anthocyanins are observable in the visible spectrum. 

In this protocol, the subunit composition and the conversion yield of purified proanthocyanidins is determined by reversed-phase HPLC. The instrument has been selected because of its ability to run a gradient and because it can acquire UV/Vis spectra, which can be useful for compound identification. 

Subunits that react with phloroglucinol are derived from proanthocyanidin extension subunits. Subunits that have not reacted with phloroglucinol are derived from proanthocyanidin terminal subunits or were present as monomeric flavan-3-ols. For quantitation, the sample peak areas of the individual subunits are compared with a(+)-catechin standard, and individual quantities are determined using the relative response factors shown in Table 11.4.1. 

Determine the conversion yield by summing the mass of the subunits (not including the phloroglucinol moiety) and dividing by the starting mass of proanthocyanidins ... 

The extraction of proanthocyanidins is the first step in determining their subunit composition. A number of extraction systems have been investigated in different plant tissues. The most common solvent systems are acetone and methanol with various amounts of water and with or without acid. In general, it has been found that an aqueous acetone system gives the best results in terms of total amount extracted. 

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