Proanthocyanidins detection

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. 

Knowledge of the identity of phenolic compounds in food facilitates the analysis and discussion of potential antioxidant effects. Thus studies of phenolic compounds as antioxidants in food should usually by accompanied by the identification and quantification of the phenols. Reversed-phase HPLC combined with UV-VIS or electrochemical detection is the most common method for quantification of individual flavonoids and phenolic acids in foods (Merken and Beecher, 2000 Mattila and Kumpulainen, 2002), whereas HPLC combined with mass spectrometry has been used for identification of phenolic compounds (Justesen et al, 1998). Normal-phase HPLC combined with mass spectrometry has been used to identify monomeric and dimeric proanthocyanidins (Lazarus et al, 1999). Flavonoids are usually quantified as aglycones by HPLC, and samples containing flavonoid glycosides are therefore hydrolysed before analysis (Nuutila et al, 2002). 

Proanthocyanidins (PAs), also known as condensed tannins, are oligomeric and polymeric flavan-3-ols. Procyanidins are the main PAs in foods however, prodelphinidins and propelargonidins have also been identified (Gu and others 2004). The main food sources of total PAs are cinnamon, 8084 mg/100 g FW, and sorghum, 3937 mg/100 g FW. Other important sources of PAs are beans, red wine, nuts, and chocolate, their content ranging between 180 and 300 mg/100 g FW. In fruits, berries and plums are the major sources, with 213.6 and 199.9 mg/100 g FW, respectively. Apples and grapes are intermediate sources of PAs (60 to 90 mg/100 g FW), and the content of PAs in other fruits is less than 40 mg/100 g FW. In the majority of vegetables PAs are not detected, but they can be found in small concentrations in Indian squash (14.8 mg/ 100 g FW) (Gu and others, 2004 US Department of Agriculture, 2004). 

Treutter D (1989) Chemical reaction detection of catechins and proanthocyanidins with 4-dimethylaminocinnamaldehyde. J Chromatogr 467 185-193... 

Peel GJ, Dixon RA (2007) Detection and quantification of engineered proanthocyanidins in transgenic plants. Nat Prod Commun 2 1009-1014... 

ESI-MS in the positive ion mode enabled to detect [M + H] ions up to the galloylated pentamer. Analysis of wine proanthocyanidins showed the presence of additional series of species with 16 mass unit differences to signals given by grape seed procyanidins. These were attributed to the presence of (epi)gallocatechin units in mixed procyanidin prodelphinidin... 

Proanthocyanidin Signals Detected in Grape and Wine Extracts by ESI-MS in the Negative Ion Mode... 

Sano, A., Yamakoshi, J., Tokutake, S., Tobe, K., Kubota, Y., and Kikuchi, M., Procyanidin B1 is detected in human serum after intake of proanthocyanidin-rich grape seed extract, Biosci. Biotechnol. Biochem., 67, 1140, 2003. 

D Madigan, I McMurrough, MR Smyth. Determination of proanthocyanidins and catechins in beer and barley by high-performance liquid chromatography with dual-electrode electrochemical detection. Analyst 119 863-868, 1994. 

Catechin and the proanthocyanidin prodelphinidin B3 are, respectively, the major monomeric and dimeric flavan-3-ols found in barley and malt where prodelphinidin B3 is the main contributor for the radical scavenging activity [Dvorakova et al., 2007], Proanthocyanidins have also been detected in nuts. Hazelnuts (Corylus avellana) and pecans (Carya illinoensis) are particularly rich in proanthocyanidins containing ca. 5 g kg, whereas almonds (Prunus dulcis) and pistachios (Pistachio vera) contain 1.8-2.4 mg kg 1, walnuts (Juglans spp.) ca. 0.67 g kg, roasted peanuts (Arachis hypgaea) 0.16 g kg, and cashews (Anarcardium occidentale) 0.09 g kg 1 [Crozier et al., 2006c]. Dark chocolate derived from the roasted seeds of cocoa (Theobroma cacao) is also a rich source of procyanidins [Gu et al., 2004], Monomeric flavan-3-ols and the proanthocyanidin B2, B5 dimers, and Q trimer are found in fresh cocoa beans (Fig. 1.13). Flavan-3-ols have also been detected in mint... 

The antiatherosclerotic effect of proanthocyanidin-rich grape seed extracts was examined in cholesterol-fed rabbits. The proanthocyanidin-rich extracts [0.1% and 1% in diets (w/w)] did not change the serum lipid profile, but reduced the level of the cholesteryl ester hydroperoxides (ChE-OOH) induced by 2,2/-azo-bis(2-amidinopropane-dihydrochloride (AAPH), the aortic malonaldehyde (MDA) content and severe atherosclerosis. The immuno-histochemical analysis revealed a decrease in the number of the oxidized LDL-positive macrophage-derived foam cells on the atherosclerotic lesions of the aorta in the rabbits fed the proanthocyanidin-rich extract. When the proanthocyanidin-rich extract was administered orally to the rats, proantho-cyanidin was detected in the plasma. In an in vitro experiment using human plasma, the addition of the proanthocyanidin-rich extract to the plasma inhibited the oxidation of cholesteryl linoleate in the LDL, but not in the LDL isolated after the plasma and the extract were incubated in advance. From these results, proanthocyanidins of the major polyphenols in red wine might trap ROSs in the plasma and interstitial fluid of the arterial wall, and consequently display antiatherosclerotic activity by inhibiting the oxidation of the LDL [92]. 

An HPLC-MS method was employed to test 102 foods and found that 43 foods contained PAs (Gu et ah, 2003a Prior and Gu, 2005). A similar study detected proanthocyanidins in 49 food items out of 99 foods of plant origin (Hellstrom et ah, 2009). These foods included fmits, nuts, cereals/beans, beverages, spices, and vegetables (Gu et ah, 2003a). Fruits and tree nuts are the major dietary sources of PAs. The majority of the fruits and tree nuts contained PAs, whereas most of the vegetables and roots lacked them completely (Gu et ah, 2003a Hellstrom 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). 

Catechin extension units of proanthocyanidins react with toluene-a-thiol to yield 3,4-tran -(+)-catechin benzylthioether (—)-epicatechin in the extension units of proanthocyanidins reacts with toluene-a-thiol to form 3,4-tran -(—)-epicatechin benzylthioether and 3,4-cA-(—)-epicatechin benzylthioether (Gu et al., 2003a). The response factors of catechin and catechin benzylthioether were the same using 280 nm detection, as well as for epicatechin and epicatechin benzylthioether. Thus, catechin and epicatechin can be used as standards to quantify their benzylthioethers. For procyanidins that consisted of catechin and epicatechin, the mean DP (degree of polymerization) can be calculated using the following equation. 

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). 

Evidence of such adducts in wine fractions has been provided, as detailed in Chapter 9A. These include F-A+ (Alcalde-Eon et al. 2006 Boido et al. 2006) and F-A-A+ (Alcalde-Eon et al. 2006) adducts based on different flavanol and anthocyanin units and (F) -A+ adducts deriving from different flavanols monomers and oligomers (Hayasaka and Kennedy 2003). Proanthocyanidins arising from these reactions cannot be distinguished from those extracted from grapes. However, detection of F-A+ adducts without prior fractionation (Morel-Salmi et al. 2006) confirmed the occurrence of the acid-catalyzed interflavanic bond breaking process in wines. 

Crystal appearance and growth are slower in red wines than in white wines and also differ within red wines. Arabinogalactan-proteins and mannoproteins were the major polysaccharides in the precipitates while rhamnogalaturonan II could not be detected. The average degree of polymerisation of proanthocyanidins in the deposit was higher that that of wine proanthocyanidins, indicating that polymers were selectively associated with the tartrate crystals. A preferential association of apolar fiavonols was similarly observed, presumably as their lower solubility favours adsorption on surfaces. 

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