Purification polyphenolics

The chemical stability of Toyopearl HW-40 resin to organic eluents allows this material to be used for a variety of applications, including the purification of synthetic functionalized surfactants, polyphenolics, and phenolic glycosides (50,51). 

After extraction, an extract purification stage is generally reconunended. This is most often done by liquid-solid exchange using resins such as Sephadex, Amber-lite XAD-7, or Cjg mini-columns. ° All the polar compounds are first trapped on the resin, and then in succession the sugars, acids, and other polar compounds (excluding polyphenolic compounds), polyphenolic compounds (excluding anthocyanidins), and anthocyanidins are respectively eluted with acidified water (HCl 0.01% v/v), ethyl acetate, and acidified methanol (HCl 0.01% v/v). 

Gandia-Herrero, R, Garefa-Carmona, R, and Escribano, J., Purification and characterization of a latent polyphenol oxidase from beet root Beta vulgaris L.), J. Agric. Food Chem., 52, 609, 2004. 

Cabanes J, Escribano J, Gandia-Herrero F, Garcia-Carmona F nd Jimenez-Atienzar M. 2007. Partial purification of latent polyphenol oxidase from peach (Prumis persica L. Cv. Catherina). Molecular properties and kinetic characterization of soluble and membrane-bound forms. J Agric Food Chem 55(25) 10446-10451. 

Chazarra S, Garcia-Carmona F and Cabanes, J. 2001. Evidence for a tetrameric form of iceberg lettuce (Lactuca sativa L.) polyphenol oxidase purification and characterization. J Agric Food Chem 49(10) 4870-4875. 

Perez-Gilabert M, Morte A, Honrubia M and Garcia-Carmona F. 2001. Partial purification, characterization, and histochemical localization of fully latent desert truffle (Terfezia claveryi Chatin) polyphenol oxidase. J Agric Food Chem 49(4) 1922-1927. 

Sajo M Mar, Nunez-Delicado E, Garcia-CarmonaF, Sanchez-Ferrer A. Partial purification of a bannana polyphenol oxidase using Triton X-l 14 and PEG 800G for removal of polyphenols. J Agric Food Chem 1998 46 4924-4930. 

Moore NL, Mariam DH, Williams AL, Dashek WV. Substrate specificity, de novo synthesis and partial purification of polyphenol oxidase derived from the wood-decay fungus, Coriolus versicolor. J Indust Microbiol 1989 4 349-364. 

Polyphenols were eluted with various mixtures of water-methanol (8 2, 7 3, 6 4 and 5 5 v/v). Further purification was achieved on a cellulose layer, and on an ODS SPE cartridge. The TLC conditions used for the measurements are listed in Table 2.38. The RF values measured on the HPTLC plates are compiled in Table 2.39. 

Methods used for the detection of PAs in cmde or partially purified extracts can also be adapted for post-column analysis after fractionation (see below). Direct quantitative analysis of PAs in crude grape phenolic extracts is often impossible due to the complex sample matrix. Thus, fractionation or purification is often necessary before analysis. The Folin-Ciocalteu and Pmssian Blue assays are widely used for the quantification of total polyphenols in plants [27,28]. These methods are not specific for PAs due to the reaction of other phenolic compounds with these reagents. 

Removing Interfering Polyphenols. The most troublesome problem encountered was high polyphenol content. If not removed, this dark-colored wood extractive decreased resolution during chromatographic separations, quickly render expensive chromatographic media (e.g., HPLC columns) nearly useless, and often precipitated enzymes during subsequent purification steps (14,15), We found it best to remove the bulk of this material prior to the first concentration step. 

Bond, T.A., Lewis, J.R., Davis, A., and Davies, A.P., Analysis and purification of catechins and their transformation products, in Methods in Polyphenol Analysis, Santos-Buelga, C. and Williamson, G., Eds., Royal Society of Chemistry, Cambridge, 2003, p. 11. 

Lazarus, S.A. et al.. Analysis and purification of proanthocyanidin oligomers. Methods Polyphenol Anal, 267, 2003. 

Since considerable amounts of potential interfering materials can be extracted along with the polyphenolics, an isolation/purification step is often required to eliminate components that may interfere with analysis. The fractionation techniques presented in Basic Protocol 2 and Alternate Protocol 2, using solid-phase extraction to minimize the effects of sample preparation/cleanup on the integrity of the extract, will make possible the identification and quantification of individual polyphenolics by HPLC (unit ii j), MS, and NMR. 

Besides solid-phase extraction, column chromatography is also often used for cleanup and purification of polyphenolics from plant material. Ionic adsorbants (polyvinylpyrrolidone or PVP, polyamides, and Sephadex LH-20) and Amberlite XAD-2 resin have been used to isolate and purify polyphenolics from crude extracts. For the separation of polyphenolics from plant material, column chromatography using Sephadex LH-20, a gel-filtration matrix, is often used with various eluting solvents (Park and Lee, 1996). The most widely used solvents for column chromatography are aqueous methanol and aqueous ethanol. 

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. 

Zawistowski, J., Biliaderis, C.G., and Murray, E.D., Purification and characterization of Jerusalem artichoke (Helianthus tuberosus L.) polyphenol oxidase, Food Biochem., 12, 1-22, 1988a. 

Microorganisms Plant/animai cellular materials Retention of microbiological solids in activated sludge processing concn of viral/bacterial cell cultures separation of fermentation products from broth retention of cell debris in fruit juices, etc. retention of cellular matter in brewery/distillery wastes Polyphenol ics Synthetic water-soluble polymers Concn/purification of lignosulphonates Concn of PVA/CMC desize wastes... 

A practical method for concentration and purification of wine is to perform an ultrafiltration followed by dialysis of the sample by low cutoff membranes (i.e., 3.5 kDa). If quantitative recovery is not required, it is possible to remove most polyphenols by passing the sample through a Ci8 cartridge (Curioni et al., 2008). A method for quantitative recovery of proteins from wine and to remove phenols is summarized in Table 10.4 (Vincenzi et al.,2005). 

Many chemical reactions carried out in supercritical fluid media were discussed in the first edition, and those developments are included in total here after some recent work is described. In the epilogue (chapter 13) of the first edition we made reference to one of the author s work in enzyme catalyzed reactions in supercritical fluids that was (then) soon to appear in the literature. The paper (Hammond et al., 1985) was published while the first edition was in print, and as it turned out, there was a flurry of other activity in SCF-enzyme catalysis many articles describing work with a variety of enzymes, e.g., alkaline phosphatase, polyphenol oxidase, cholesterolase, lipase, etc., were published starting in mid 1985. Practical motivations were a potentially easier workup and purification of a product if the solvent is a gas (i.e., no liquid solvent residues to contend with), faster reaction rates of compounds because of gas-like transport properties, environmental advantages of carbon dioxide, and the like. 

Since the 1950s, many efforts have been made to isolate polyphenols directly from black tea, and the structures of the major phenolic compounds characteristic of fermented tea have been elucidated." ° However, the composition of black tea polyphenols is so complex that the minor phenolic substances, which cumulatively account for a substantial portion of black tea polyphenols, are difficult to separate even by high-performance liquid chromatography (HPLC). This difficulty associated with the purification is mainly due to the presence of uncharacterized substances that are detected as a broad hump on the baseline during HPLC analysis but do not produce any clear spots on thin-layer chromatography. These snbstances are probably a complex mixture of catechin oxidation products with higher molecular sizes, and usually account for the majority of black tea polyphenols. 

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