Plant extracts phenolics

Enrichment of processed food with plant material or plant extracts rich in polyphenols has two aspects in relation to human nutrition and human health. Food protected against oxidation has better keeping quality and will stay healthy longer since formation of toxic oxidation products, like cholesterol oxides, is being prevented (Britt et al., 1998). The other aspect is the beneficial effects of the intake of polyphenols on human health. Both of these aspects are, however, related to the availability of the phenolic substances. 

CALDWELL c R (2001) Oxygen radical absorbance capacity of the phenolic compounds in plant extracts fractionated by high-performance liquid chromatography, ,4Biochem, 293, 232-8. 

Kahkonen MP, Hopia AI, Vurela HJ, Rauha J-P, Pihlaja K, Kujala TS and Heinon M. 1999. Antioxidant activity of plant extracts containing phenolic compounds. J Agric Food Chem 47 3954-3962. 

LC-NMR plays a central role in the on-line identification of the constituents of crude plant extracts (Wolfender and others 2003). This technique alone, however, will not provide sufficient spectroscopic information for a complete identification of natural products, and other hyphenated methods, such as LC-UV-DAD and LC-MS/MS, are needed for providing complementary information. Added to this, LC-NMR experiments are time-consuming and have to be performed on the LC peak of interest, identified by prescreening with LC-UV-MS. NMR applied to phenolic compounds includes H NMR,13 C NMR, correlation spectroscopy (COSY), heteronuclear chemical shift correlation NMR (C-H HECTOR), nuclear Overhauser effect in the... 

A number of spectrophotometric methods for the quantification of phenolic compounds in plant materials have been developed. Based on different principles, these assays are used to determine various structural groups present in phenolic compounds. Spectrophotometric methods may quantify all extractable phenolics as a group (Marshall and others 2008), or they may determine a specific phenolic substance such as sinapine (Ismail and Eskin 1979) or a given class of phenolics such as phenolic acids (Brune and others 1989). 

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. 

The method is more sensitive than the biuret method and has an analytical range from 10 ju,g to 1.0 mg of protein. Using the method outlined below this is equivalent to sample concentrations of between 20 mg l-1 and 2.0 g l-1. The relationship between absorbance and protein concentration deviates from a straight line and a calibration curve is necessary. The method is also subject to interference from simple ions, such as potassium and magnesium, as well as by various organic compounds, such as Tris buffer and EDTA (ethylenediamine-tetraacetic acid). Phenolic compounds present in the sample will also react and this may be of particular significance in the analysis of plant extracts. 

In earlier times, thin-layer chromatography (TLC), polyamide chromatography, and paper electrophoresis were the major separation techniques for phenolics. Of these methods, TLC is still the workhorse of flavonoid analysis. It is used as a rapid, simple, and versatile method for following polyphenolics in plant extracts and in fractionation work. However, the majority of published work now refers to qualitative and quantitative applications of high-performance liquid chromatography (HPLC) for analysis. Llavonoids can be separated. 

Once a suitably polar plant extract is obtained, a preliminary cleanup is advantageous. The classical method of separating phenolics from plant extracts is to precipitate with lead acetate or extract into alkali or carbonate, followed by acidification. The lead acetate procedure is often unsatisfactory since some phenolics do not precipitate other compounds may coprecipitate and it is not always easy to remove the lead salts. 

Hostettmann, K. et al., On-line high-performance liquid chromatography ultraviolet-visible spectroscopy of phenolic compounds in plant extracts using post-column derivatization, J. Chromatogr., 283, 137, 1984. 

Fiamegos, Y.C. et al.. Analytical procedure for the in-vial derivatization-extraction of phenolic acids and flavonoids in methanolic and aqueous plant extracts followed by gas chromatography with mass-selective detection, J. Chromatogr. A, 1041, 11, 2004. 

Achilli, G. et ak, Identification and determination of phenolic constituents in natural beverages and plant extracts by means of a coulometric electrode array system, J. Chromatogr., 632, 111, 1993. 

Much data on the structure of flavonoids in crude or semipurified plant extracts have been obtained by HPLC coupled with MS, in order to obtain information on sugar and acyl moieties not revealed by ultraviolet spectrum, without the need to isolate and hydrolyze the compounds. In the last decade, soft ionization MS techniques have been used in this respect, e.g., thermospray (TSP) and atmospheric pressure ionization (API). However, the most used methods for the determination of phenols in crude plant extracts were the coupling of liquid chromatography (LC) and MS with API techniques such as electrospray ionization (ESI) MS and atmospheric pressure chemical ionization (APCI) MS. ESI and APCI are soft ionization techniques that generate mainly protonated molecules for relatively small metabolites such as flavonoids. 

Stankevicius, M. Akuneca, L Jakobsone, L Maruska, A. Analysis of phenolic compounds and radical scavenging activities of spice plants extracts. Food Chemistry and Technology. 2010 44(2) 85-91. 

Rauha, J. P., Remes, S., Heinonen, M., Hopia, A., Kahkonen, M., Kujala, T., Pihlaja, K., Vuorela, H., Vuorela, P. (2000). Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds. Int. J. Food Microbiol, 56, 3-12. 

Polyphenols are compounds that have more than one phenolic hydroxyl group attached to one or more benzene rings. The term is somewhat misleading since it tends to make people think of polymers of individual phenol molecules. Of course such polymers exist. Phenolic compounds are characteristic of plants and as a group they are usually found as esters or glycosides rather than as free compounds. It is important to realize this if you want to extract phenols from plant tissues. 

A third method relies on the precipitation of proanthocyanidins with formaldehyde. First, the total phenolic content is measured using the Folin-Ciocalteu reagent as described before. A 0.5 mole equivalent of phloroglucinol (1.3) is added for every gallic acid equivalent in the extract. To 2 mL of this plant extract and phloroglucinol is added 1 mL of a 2 5 HC1 /H20 solution and 1 mL of an aqueous solution of formaldehyde (13 mL of 37% formaldehyde diluted to 100 mL in water). After an overnight incubation at room temperature, the unprecipitated phenols are estimated in the supematent by the Folin-Ciocalteu method. The precipitate contains the proanthocyanidins and the known amount of phloroglucinol, which is always quantitatively precipitated. 

The vast literature associated with flavanoid chemistry precludes a discussion here but two valuable reviews have been published. The first reviews a number of spectroscopic techniques used for flavonoid analysis, with a strong emphasis on NMR spectroscopy (plus also mass spectrometry, vibrational spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, X-ray crystallography, and circular dichrosim (CD)) . The second review deals with NMR methods that have been successful in the characterization of phenolic acids and flavonoids from plant extracts that have not been separated or isolated as single components. The emphasis of the article is 2-D NMR methodology and a variety of experiments such as total correlated spectroscopy (TOCSY), COSY, nuclear Overhauser enhancement spectroscopy (NOESY) and heteronuclear multiple quantum correlation (HMQC) are discussed . 

Pomponio, R., Gotti, R., Hudaib, M., and Cavrini, V. 2002. Analysis of phenolic acids by micellar electrokinetic chromatography application of Echinacea purpurea plant extracts. J. Chromatogr. A 945, 239-247. 

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