Extraction and 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). 

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

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. 

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. 

Extraction and isolation techniques of tamarind xyloglucans and polyphenols... 

Many naturally occurring plant extracts are reputed to possess anti-irritant properties and have been recommended for use in cosmetic formulations. These include such diverse mixtures as tea tree oil, borage seed oil, Paraguay tea extract. Kola nut extract, oil of rosemary, and lavender oil. It is, however, difficult to standardize plant extracts and there may be a great deal of lot-to-lot variability in constituents. Understandably, this makes identification and isolation of any specific active constituent complex and laborious. The extracts may be oily or hydrophilic and contain compounds such as a-bisabolol, xanthines, polyphenols, and phytosterols.There is great potential in the use of plant extracts for irritation and sensitization reduction. This has been established within the cosmetic industry, and interest here has stimulated activity into reducing variability by more consistent cultivation techniques and more standardized extraction methods. 

This method is also used to measure ex vivo low-density lipoprotein (LDL) oxidation. LDL is isolated fresh from blood samples, oxidation is initiated by Cu(II) or AAPH, and peroxidation of the lipid components is followed at 234 nm for conjugated dienes (Prior and others 2005). In this specific case the procedure can be used to assess the interaction of certain antioxidant compounds, such as vitamin E, carotenoids, and retinyl stearate, exerting a protective effect on LDL (Esterbauer and others 1989). Hence, Viana and others (1996) studied the in vitro antioxidative effects of an extract rich in flavonoids. Similarly, Pearson and others (1999) assessed the ability of compounds in apple juices and extracts from fresh apple to protect LDL. Wang and Goodman (1999) examined the antioxidant properties of 26 common dietary phenolic agents in an ex vivo LDL oxidation model. Salleh and others (2002) screened 12 edible plant extracts rich in polyphenols for their potential to inhibit oxidation of LDL in vitro. Gongalves and others (2004) observed that phenolic extracts from cherry inhibited LDL oxidation in vitro in a dose-dependent manner. Yildirin and others (2007) demonstrated that grapes inhibited oxidation of human LDL at a level comparable to wine. Coinu and others (2007) studied the antioxidant properties of extracts obtained from artichoke leaves and outer bracts measured on human oxidized LDL. Milde and others (2007) showed that many phenolics, as well as carotenoids, enhance resistance to LDL oxidation. 

T. Nakatani, K. Ohtani, S. Sakanaka, CS059 M. Kim and I. Matsui-Yuasa. Protective effect of green tea extract and tea polyphenols against the cytotoxicity of 1,4-naphthoquinone in isolated rat hepatocytes. Biosci Biotech Biochem 1997 61(11) 1901-1905. 

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. 

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. 

More than 5000 different phytochemicals have been identified, but it is believed that thousands more have yet to be discovered (Liu, 2003). Based on their structural characteristics, they may be classified into several groups including polyphenols, terpenoids, and alkaloids (Manach et ah, 2009). As evidence continues to emerge linking phytochemicals to health promotion and disease prevention, there is increased interest in extraction, isolation, and analysis of these compounds from a variety of plant sources. These procedures have been optimized for several different types of phytochemicals and are used in the discovery of compounds from new sources. 

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... 

In this study, therefore, flow injection analysis-chemiluminescence (FIA-CL) methods to evaluate quenching effects of functional foods against ROS such as singlet oxygen ( 02) and hydroxyl radicals (OH) were developed. The applicabilities of these methods were confirmed by evaluating quenching effects of grape seed extracts and their isolated polyphenols. 

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