Isolation of polyphenolics

Extraction and Isolation of Polyphenolics Basic Protocol 1 Ultrasound-Assisted Aqueous Methanol Extraction of 11.2.1... 

Polyphenolics constitute a wide range of chemical compounds composed of aromatic ring(s) with one or more hydroxyl substituents, including their functional derivatives. Methods for extraction and isolation of polyphenolics from plant material are described in this unit. Extraction and isolation are the first important steps for separation, characterization, and quantification of polyphenolics from plant material. Polyphenolics are often most soluble in organic solvents less polar than water. The solubility is dependent on the polar properties of the polyphenolics. The correct selection of the extracting solvent is not as simple as it may seem. Aqueous methanol is a popular choice of solvent (see Background Information). 

Aliotta, G., Cafiero, G., Fiorentino, A., Oliva, A., and Temussi, F. 2002. Olive oil mill wastewater isolation of polyphenols and their action on radish and wheat germination in vitro. Allelopathy J. 9, 9-17... 

Before the extraction and isolation of polyphenols, samples containing these compounds must be collected, preserved, and properly prepared. 

Altiok E, Baycin D, Bayraktar O, Ulku S (2008) Isolation of polyphenols from the extracts of olive leaves Olea europaea L.) by adsorption on silk fibroin. Sep Purif Technol 62 342-348... 

Recent scientific investigations of natural polyphenols have demonstrated their powerful antioxidant property (Niki et al, 1995). Several classes of polyphenols have been chemically identified. Some of these are grape polyphenols, tea polyphenols, soy polyphenols, oligomeric proanthocyanidines (OPA) and other natural polyphenols of the flavone class. Rice bran polyphenols are different from the above in that they are p-hydroxy cinnamic acid derivatives such as p-coumaric acid, ferulic acid and p-sinapic acid. Tricin, a flavone derivative, has also been isolated from rice bran. 

Hanamura, T., Hagiwara, T., and Kawagishi, H., Structural and functional characterization of polyphenols isolated from acerola Malpighia emarginata DC) fruit, Biosci. Biotechnol. Biochem., 69, 280, 2005. 

Thousands of polyphenols from fruits (grapes, apples, etc.), vegetables (horse beans), and teas have been identified, many having good coloring properties, especially anthocyanins and some flavonoids. Well-documented reviews discuss the coloring capacities of some polyphenols including procyanidins. - Detailed presentations of anthocyanin and flavonoid properties and analysis are included in Sections 2.3, 4.3, and 6.3. The soluble proanthocyanidins of the colored horse bean Viciafaba L. seed coats were isolated and separated by solvent partition. 

Males et al. [103] used aqueous mobile phase with formic acid for the separation of flavonoids and phenolic acids in the extract of Sambuci flos. In a cited paper, authors listed ten mobile phases with addition of acids used by other investigators for chromatography of polyphenolic material. For micropreparative separation and isolation of antraquinone derivatives (aloine and aloeemodine) from the hardened sap of aloe (Liliaceae family), Wawrzynowicz et al. used 0.5-mm silica precoated plates and isopropanol-methanol-acetic acid as the mobile phase [104]. The addition of small amounts of acid to the mobile phase suppressed the dissociation of acidic groups (phenolic, carboxylic) and thus prevented band diffusions. 

There is the often-overlooked group of polyphenols containing the derivatives of hydro-xycinnamic and hydroxybenzoic acids such as caffeic, chlorogenic, and gallic acids (Figure 29.8), which occur in food and exhibit certain antioxidant activity. For example, some caffeic acid esters isolated from propolis from honeybee hives showed antiinflammatory... 

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. 

Anderson RA, Broadhurst CL, Polansky MM, Schmidt WF, Khan A, Flanagan VP, Schoene NW, Graves DJ. 2004. Isolation and characterization of polyphenol type-A polymers from cinnamon with insulin-like biological activity. J Agric Food Chem 52 65-70. 

Park, E.B., Lee, J.S., and Choi, E.H., Isolation and characteristics of polyphenol oxidase from Jerusalem artichoke tuber, Han guk Sikp um Kwahakhoechi, 23, 414—419, 1991. 

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. 

Ellagitannins are among the most diverse and structurally complex class of polyphenolics in plants. They are important contributors to color, flavor and stability in fruits as well as oaked wine and distilled spirits. Recently, consumption of ellagitannins has also been linked to potential health benefits. The extraction, isolation, and identification... 

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