Polyphenols in Cereals and Their Positive and Negative Role in H

Polyphenols in Cereals and Their Positive and Negative Role in Human and Animal Nutrition.

ABSTRACT

Phenolic compounds have been repeatedly implicated as potent antioxidants with the potential therapeutic value esp. of flavonoids in reduced risk for cardiovascular disease and cancer. Other groups as phenolic acids and high molecular tannins have negative flavour and protein digestibility properties. There are manifested the dominant polyphenols (flavonoids, coumarins, phenolcarboxylic acids, anthocyanidins) in wheat, barley, oat, rye, triticale and buckwheat caryopses isolated and identified by means of chromatographic methods and spectral analyses and discussed their preventive beneficial and antinutrient effects as dietary constituents regarding the differences in their structure among cereals. In this respect differ barley and buckwheat from other cereals. The variability of the content of total polyphenols in the barley caryopses is influenced by the genotype of variety, the conditions of ripening of growth and the deterioration of caryopses in the ageing process.

INTRODUCTION

The polyphenols constitute a large group of secondary plant metabolites being ubiquitous among higher plants.They represent a large group of naturally occurring compounds which recently have attracted considerable attention as dietary constituents with potential importance for human health.This work focuses on such groups of them as the flavonoids, coumarins, anthocyanins, phenolic acids and high molecular tannins are. Flavonoids have a common skeleton of diphenylpyranes and are often hydroxylated in the positions 3,5,7,3´,4´, and 5´. The presence or absence of a hydroxyl group in the 3 position determines the division into the two main subgroups of flavonoids: 3-hydroxyflavonoids (flavonols, flavanonols and catechins) and flavonoids with an unsubstituted 3-position (flavones and flavanones). Due to the enormous variation in substitution types and substitution patterns (hydroxylation-, methoxylation-, sulphation- and glycosidation patterns in addition to C-methylation, C-glycosylation and prenylation) more than 4.000 flavonoids have been described, and the number of characterised substances is continually increasing. From in vitro data and biochemical knowledge, it appears that the flavonoids may be regarded as dietary constituents which may have beneficial effect of a preventive nature in relation to cancer, coronary heart disease, antioxidant effect (radical scavenging), immunomodulating and antiiflammatory effects, modulation of other enzyme activities (inhibition), antiviral, antibacterial and antifungal effects, effect on blood vesels and counteraction of vascular permeability (M e l t z e r, M a l t e r u d, 1997). In reverse the phenolic acids being characterised as possesing sour, bitter, astringent and phenol-like flavours,contributing to the flavour of flours, and affecting the mixing properties of doughs. The high molecular tannin fractions soluble in water (m.w.3.000-5.000) decrease the digestibility of proteins in relation to monogastric animals and in human diet.

The outer layer coats of the seeds of plants contain different polyphenolic compounds, e.g. flavonoids, phenolic acids, coumarins, anthocyanins. Their content and composition is strongly influenced by different factors (W e i d n e r et al., 1996). Phenolic compounds, especially free phenolic acids are regulating factors that retard precocious germination processes. It was found a positive correlation between dormancy levels and the contents of phenolic acids in the process of after-ripening, supporting the idea of involvement of phenolic compounds in the control of dormancy and sprouting in cereal caryopses. Many other factors influence the flavonoid content of plants, among others the conditions of growth, ripeness and season. Growth beneath glass reduces the levels of flavonoids, and in general, processed foods have 50% lower concentrations than fresh.

This study focuses on the composition of polyphenolic complexes contained in some chosen cereals and their comparison, and the amounts of polyphenols which are in the grains contained regarding their positive and negative properties in human and animal nutrition.

MATERIAL AND METHODS

The major polyphenolic compounds were isolated and identified in barley and triticale grains and compared with the phenolic constituents that had been found in the wheat, oat and the rye. Special attention was paid to the constituents of the buckwheat as the source of rutin in human nutrition.

The barley grains were obtained from the harvest 1996 and 1997: cvs. Forum, Akcent and Amulet - from the Chrlice and Krásné Údolí localities, the grains of the triticale cvs. Dagro, Presto, Lasko, Grado, Salvo, Bolero, BR-2, MAH-384, MAH-484 Malno, KS-12 and UH-74 Korm were obtained from the Breeding Station Úhřetice.

The sample 100 g amounts of the ground grains of each variety were pre-extracted for 12 hours in the Soxhlet apparatus with petroleum ether. The extraction of the pre-extracted material was made with 80% aqueous ethanol for 12 hours. The extract was thickened under reduced pressure and then left to dry over calcium chloride in dessicator. The evaporation residue of the aqueous ethanol extract (4g) was ground and mixed with a small quantity of polyamide powder. The separation was carried out by the polyamide column chromatography (CC), by the two-dimensional paper chromatography (PC) and by one-dimensional thin layer chromatography (TLC). The powder of the mixture of the evaporation residue and the polyamide powder was put on the washed polyamide column (70 x 4.5 cm, granulation 0.315-0.500 mm). Elution was performed for the first time with water until the Molisch reaction to the saccharides was negative (aprox.2.000 mL) and then with methanol (aprox.2.000 mL).the methanolic eluate was thickened on the water bath to the 50 mL volume at a temperature of 50o C, clarified by centrifugation and then separated with the two-dimensional descending PC (Whatman No.4) in the following eluting systems: S1: 1-butanol-acetic acid-water (4:1:2 V/V/V) and S2: acetic acid-water (15:85 V/V). The separated substances were eluted from the two-dimensional chromatograms by means of methanol, the eluates were thickened to small volumes and each compound was re-chromatographied by means of TLC (Polygram Cel 300 MN and Cellulose F Merck sheets, 200x200 mm, layer thickness 0.1 mm , in S2 and S3: tert.butanol-water-acetic acid 3:1:1 V/V/V or DC - aluminium pre-coated silica gel 60 F254 Merck sheets, 200x200 mm, layer thickness 0.2 mm in S4: chloroform-methanol-n-propanol-water 9:12.1:8 V/V/V/V, lower layer acidified with formic acid). The separated compounds were eluated with methanol (for UV spectroscopy) and measured by UV spectroscopy methods.

Spectral analyses: Spectra of separated compounds were measured on the Specord UV-VIS spectrophotometer (Carl Zeiss Jena) in methanolic solutions and after addition of diagnostic reagents natriummethanolate, aluminium chloride, hydrochloric acid, natrium acetate and hydroboric acid causing tha characteristic bathochromic and hypsochromic shifts.

Acid hydrolysis: Methanolic solution of flavonoid glycoside was after addition of 6 mL 6% HCl heated in the boiling bath under reflux condenser for 45 min. After cooling the hydrolysate was applied on the polyamide column (10 x 100 mm) and eluated with 50 mL water and then with 50 mL methanol. The saccharides in the thickened water eluate were identified by means of co-chromatography with authentic samples, the flavonoid aglycones chromatographically and spectrally.

Detection of saccharides: Saccharides in the thickened water eluate were identified by co-chromatography with authentic markers using PC (Whatman No.1, S1) and detection was made by benzidine and anthrone agents, anilinium hydrogensulphate, aniline diphenylamine-trihydrogenphosphoric acid and neotetrazolium blue agent (Lachema Brno).

Authentic markers: As authentic preparates for co-chromatography and comparison of spectral data were used rutin dihydrate (Lachema Brno) and trihydrate (Fluka Chemie AG and Galena Opava), quercetin dihydrate (Fluka Chemie AG, Lachema Brno), apigenin (Carl Roth KG and Fluka Chemie AG), luteolin (Carl Roth KG ).

Accelerated ageing (AA): Seeds were weighed and placed on a screen tray which was inserted into an inner box containing 50 mL of water. The inner chamber was placed into an accelerated ageing chamber and the seeds were aged at 41o C for 48 hours. The AA was performed after T e K r o n y (1995).

Determination of total polyphenol content (TP):Weighed grinded seeds (approx. 20 g barley) were extracted in the Soxhlet apparatus with ethanol-water mixture (80:20 V/V) for 20 hours. The extract was adjusted to 250 mL volume and from this volume 5 mL aliqouts were pipetted into 50 mL volumetric flasks. After dilution with 80% water-ethanolic solution to the approx. 30 mL it was 2.5 mL of Folin-Ciocalteau´s phenol reagent (Fluka Chemie AG) added, agitated and mixed. Then was 7.5 mL of 20% sodium carbonate solution added, the volume adjusted with distilled water till the mark and after thorough agitation it was left to stand for 2 hours for a quantitative formation of blue colour. The same procedure was used for blank where instead of 5 mL of sample solution 5 mL 80% ethanol was used. After two hours standing the solutions were centrifugated on the Janetzki T 30 centrifuge at 2.000 cycles per minute for 12 minute period. Absorbancy values were measured on the Spekol 11 spectrophotometer against blank at l =765 nm wave length and expressed as gallic acid.

Determination of m-dihydroxyl phenolic groups in catechin, resorcin and phloroglucin type polyphenols (CRP): 25 mL aliquots were evaporated on the water bath to dryness, then redissolved in 15 mL methanol, quantitatively displaced into 25 mL volumetric flasks. Into every flask it was 6 mL 3M HCl in methanol added and after agitation the volume was adjusted till the mark. Then it was 1 mL of p-dimethylaminocinnamaldehyde (p-DMASA) reagent (Merck-Schuchardt, Hohenbrunn bei Mü nchen) added and after thorough agitation the solution was left to stand for 30 min. Finally the absorbancy on the Spekol 11 spectrophotometer was measured against blank at l =638 nm wave length and expresed as phloroglucin (sample C6C3(OH)3.2H2O, The British Drug House, Ltd.).

RESULTS AND DISCUSSION

O- and C- glycosides of flavones are characteristic for cereals, esp. that derived from apigenin and luteolin. In rye and triticale have been also described glycosides of quercetin (Tab.1).

Also the composition of polyphenolic complex of barley seeds is showing on their strong antioxidant activity ( M a i l l a r d et al., 1996). Depending on the variety, the antioxidant activity of barley is in relationship with the content of the three main phenolic groups - flavan-3-ols ( more than 85 %), hydroxycinnamic acids ( approx. 10 %) and flavones ( less than 5 % from the total content of polyphenol). The major antioxidant activity posses flavan-3-ols. In the barley seeds is antioxidant eficiency caused mainly by flavan-3-ols and flavan-3,4-diols, resp. (more than 85 %), i.e. with gallocatechin and (-)-epicatechin and with leucoanthocyanidins of procyanidin and prodelphinidin type which could be converted by oxidation to anthocyanidins or condense to high molecular weight phlobaphene and condensed tannin fractions. Gallocatechin could originate by the (-)-epicatechin oxidation (hydroxylation), prodelphinidin from procyanidin. 10 % of the total polyphenol content form phenolcarboxylic acids: p-hydroxybenzoic acid (its hydroxylation leads to gallic acid and esterification to m-galloylgallic acid), vanillic acid, o-hydroxycinnamic acid, ferulic acid, sinapic acid and chlorogenic acid (Tab.2).Barley dormant caryopses are very rich in free phenolic acids, as it is shown in Tab.1 - the predominant free and bound acid in all cereals is caffeic acid, for barley are typical protocatechuic, isoferulic and vanillic, oat ferulic and mandelic and rye isoferulic and veratric and triticale sinapic acids. The buckwheat is rich in rutin and quercetin with very favourable protective effect on blood vessels and counteraction on vascular permeability.

Tab.1. Dominant polyphenols in cereals.

The average TP and esp. CRP contents in barley caryopses differ from other cereals, e.g. triticale (TP content in barley caryopses varied from 85.7 to 168.5 mg/100 g dry matter and CRP content from 9.5 to 44.8 mg/100 g dry matter, resp., in triticale the corresponding values were 43.96-60.7 and 0.57-1.05 mg/100 g dry matter, resp.) showing considerable antioxidant activity of barley grains. The TP content in the barley caryopses is significantly dependent on the given variety and is influenced by the locality, specific weather features of the given year and the conditions of ageing (Tab. 3).

Tab.2. Phenolcarboxylic acids in cereals.

b-barley, w-wheat, o-oat, r-rye, t-triticale, bw-buckwheat

Tab.3.The average total polyphenol content (TP) and the catechin, resorcin and phloroglucin type polyphenol content (CRP) in barley caryopses (influence of variety, year and ageing)

Significance levels: values with different letters significantly differ at 0.01 according to Tukey´s HSD test

ACKNOWLEDGEMENTS : This work was supported by the grant GA ČR No. 521/96/0616.

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Source: Lachman, J. - Hosnedl, V. - Pivec, V. - Orsák, M., 1998. Polyphenols in Cereals and Their Positive and Negative Role in Human and Animal Nutrition. Referát na mezinárodní konferenci "Cereals for Human Health and Preventive Nutrition." Brno, 7.-11.7.1998, organized by Agricultural Research Institute Kroměříž, Ltd., Mendel University of Agriculture and Forestry Brno, Food Research Institute Prague. Proceedings: 118 - 125.