Fractionation of polyphenols

Alternate Protocol 2 Neutral and Acidic Fractionation of Polyphenolics 11.2.5... 

Figure 11.2.1 Fractionation of polyphenolics into non-anthocyanin and anthocyanin fractions using C18 cartridges (circles non-anthocyanin polyphenolics squares anthocyanins triangles sugars, acids, and water-soluble compounds).

Figure 11.2.2 Fractionation of polyphenolics into acidic polyphenolics and neutral polyphenolics using C18 cartridges (circles neutral polyphenolics squares acidic polyphenolics).

C18 solid-phase extraction is used to fractionate polyphenolics for their identification and characterization. This technique can eliminate interfering chemicals from crude extracts and produce desirable results for HPLC or other analytical procedures. To obtain a sufficient volume for all analyses, several separations by solid-phase extraction may be performed. The individual fractions need to be combined and dissolved in solvents appropriate for HPLC analysis. In Basic Protocol 2, the application of a current of nitrogen gas for the removal of water from the C18 cartridge is an important step in the selective fractionation of polyphenolics into non-anthocy-anin and anthocyanin fractions. After the collection of non-anthocyanin polyphenolics, no additional work is necessary to elute anthocyanins bound to the C18 solid phase if anthocyanins are not to be determined. 

The determination of polyphenolics may result in interference due to co-elution of phenolic acids and procyanidins. This problem can be eliminated by fractionation of polyphenolics into acidic and neutral polyphenolics prior to sample injection into the HPLC system. Because the fractionation techniques effectively improve the resolution of many polyphenolic peaks in the reversed-phase HPLC system, it is suggested that further characterization and identification of unknown peaks be conducted by additional methods such as mass spectrometry and nuclear magnetic resonance. 

Zawistowski, J., Biliaderis, C.G., and Murray, E.D., Isolation and some properties of an acidic fraction of polyphenol oxidase from Jerusalem artichoke (Helianthus tuberosus L.), Food Biochem., 12, 23-35, 1988b. 

Svedstrom, U. Vuorela, H. Kostiainen, R. Laakso, L Hiltunen, R. 2006. Fractionation of polyphenols in hawthorn into polymeric procyanidins, phenolic acid, and flavonoids prior to high-performance liquid chromatographic analysis. J. Chromatogr. A. 1112 103-111. 

Sample preparation by reverse-phase chromatography can be performed using a Ci8 SPE cartridge 30 mL of dealcoholized wine are loaded onto the cartridge, after rinsing with 40 mL of water PAs are recovered with lOmL of acetone/water/acetic acid 70 29.5 0.5 (v/v/v) (Lazarus et al., 1999). A method for fractionation of polyphenols in wine by reverse-phase chromatography is reported in the flow diagram in Fig. 6.20 (Sun et al., 2006). 

Figure 6.20. Fractionation of polyphenols in red wine (PA = proanthocyanidins). (Reprinted from Journal of Chromatography A, 1128, Sun et al., Fractionation of red wine polyphenols by solid phase extraction and liquid chromatography, p. 29, Copyright 2006, with permission from Elsevier.)...

Gutzeit D, Winteihaller P, Jerz G (2007) Application of preparative high-speed counter-current chromatography/electrospray ionization mass spectrometry for a fast screening and fractionation of polyphenols. J Chromatogr A 1172 40-46... 

In the last few decades, several epidemiological studies have shown that a dietary intake of foods rich in natural antioxidants correlates with reduced risk of coronary heart disease particularly, a negative association between consumption of polyphenol-rich foods and cardiovascular diseases has been demonstrated. This association has been partially explained on the basis of the fact that polyphenols interrupt lipid peroxidation induced by reactive oxygen species (ROS). A large body of studies has shown that oxidative modification of the low-density fraction of lipoprotein (LDL) is implicated... 

Polyphenols, as one of the largest and most widespread class of plant compounds, are also present in saffron stigma. In the mass spectrum of the flavonoid fraction of the methanolic extract from saffron there were ions at m/z 611 and 633 which may be attributed to protonated and sodiated quasi-molecular ions of kaempferol diglycoside. [22] Flavonoids from the water extract can be isolated and concentrated... 

Gomez-Caravaca AM, Carrasco-Pancorbo A, Canabate-Dlaz B, Segura-Carretero A and Fernandez-Gutierrez A. 2005. Electrophoretic identification and quantitation of compounds in the polyphenolic fraction of extra-virgin olive. Electrophoresis 26(18) 3538-3551. 

It is estimated that the mean total intake of polyphenols in the Spanish diet ranges from 2,590 to 3,016 mg/person/day including polyphenols soluble in aqueous-organic solvents, plus insoluble condensed and hydrolyzable tannins. Fruits and vegetables provide a daily intake of 700-1,000 mg of polyphenols/person/diet a major fraction of this (600 mg/person/day) is associated with DF (Saura-Calixto and others 2007). 

Gardana, C., Scaglianti, M., Pietta, P. and Simonetti, P. (2007) J. of Pharmaceutical and Biomedical Anal., Analysis of the polyphenolic fraction of propolis from different sources by liquid chromatography-tandem mass spectrometry, 45, 390-399. 

Zaprometov, M. N., N. V. Zagoskina and T. F. Koretskaya. Effect of some precursors on the formation of phenolic compounds in tea plant tissue cultures. Fiziol Rast 1976 23 1274. Higashi-Okai, K., S. Otani and Y. Okai. Potent suppressive activity of pheophytin A and B from the non-polyphenolic fraction of green tea (Camellia sinensis) against tumor promotion in mouse skin. Cancer Lett 1998 129(2) 223-228. 

Monophasic action potential. Glycerin/ ethanol extract of the fresh leaf, administered intragastrically to dogs at a dose of 30 mg/kg, increased duration of ventricular monophasic action potential (16%) . Mutagenic activity. Ethanol (100%) extract of the fresh leaf, on agar plate, was active on Salmonella typhimurium T1530 . Natural-killer cell enhancement. Fixed oil of embryos, administered orally to adults, was inactive . Nonsaponifiable fraction of fruit fixed oil and seed oil, administered to rats at a dose of 0.3% of diet, were active. The polyphenol stripped seed oil was inactive . 

Gonthier, M.P., Cheynier, V., Donovan, J.L., Manach, C., Morand, C., Mila, I., Lapierre, C., Remesy, C., and Scalbert, A., Microbial aromatic acid metabolites formed in the gut account for a major fraction of the polyphenols excreted in urine of rats fed red wine polyphenols, J. Nutr., 133, 461, 2003. 

Ethyl acetate is used on the C18-adsorbed anthocyanins to remove polyphenolic compounds. More efficient polyphenolic recovery will be accomplished if residual water is removed from the cartridge with a nitrogen gas stream for 2 to 3 min before application of ethyl acetate. After washing the column with ethyl acetate, water should not be added because some anthocyanins could be eluted and lost. Ethyl acetate removal of polyphenolics is particularly recommended if the anthocyanin fraction is to be subsequently analyzed by electrospray mass spectroscopy. 

In this protocol, polyphenolics are fractionated into neutral and acidic fractions to prevent interference among polyphenolics in HPLC analysis. Phenolic acids are completely ionized at pH 7.0 and un-ionized at pH 2.0. This property allows for fractionation of neutral polyphenolics at pH 7.0 and acidic polyphenolics at pH 2.0. Two individually preconditioned Cl8 cartridges, one for neutral polyphenolics and the other for acidic polyphenolics, are used for this separation (Figure 11.2.2). 

The Basic Protocol describes the reversed-phase HPLC analysis of polyphenolic compounds isolated into nonanthocyanin and anthocyanin fractions by solid-phase extraction. The Alternate Protocol describes the HPLC separation of acidic and neutral polyphenolic fractions. Fractionated samples are used because significant amounts of interfering compounds are extracted along with polyphenolics from plant materials. Solid-phase extraction with C18 Sep-Pak cartridges (vnitu.2) is used to selectively eliminate undesired components from crude extracts, and may minimize the effects of sample cleanup or preparation on the integrity of polyphenolics. The isolation and purification step using solid-phase extraction of polyphenolics will make possible the efficient analysis of individual polyphenolics by reversed-phase HPLC. 

REVERSED-PHASE HPLC ANALYSIS OF POLYPHENOLICS SEPARATED INTO NONANTHOCYANIN AND ANTHOCYANIN FRACTIONS... 

Figure 11.3.12 HPLC chromatograms of polyphenolics in Concord grape extract detected at 280 nm. (A) All polyphenolics, including anthocyanins. (B) Nonanthocyanin polyphenolics after fractionation. Peak identification 1, cis-caftaric acid 2, frans-caftaric acid 3, procyanidin B3 4, c/s-coutaric acid 5, frans-coutaric acid 6, epicatechin 7, quercetin galactoside 8, quercetin glucoside. Reproduced from Oszmianski and Lee (1990) with permission from the American Society for Enology and Viticulture.

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