ANTIOXIDANT EFFECTS OF HERBAL EXTRACTS AND THEIR FOOD APPLICATION

Herbal extracts are considered as a good sources of antioxidant compounds. This work describes antioxidant effect of 15 kinds of herbs. Three different antioxidant assays were used, Trolox equivalent antiradical activity (TEAC), test based on deoxyribose oxidation and DPPH method. The TEAC values ranged from 0.38mM to 0.77 mM. Deoxyribose assay showed antioxidant activity of selected extracts expressed as the inhibition of formation of oxidative products of deoxyribose from 2.68 to 50.05 %. The DPPH method values ranged from 6.47 to 73.80 %. Extracts of Prunus spinosa L., Euphrasia rostkoviana Hayne were the best antioxidants confirmed by all methods. Scrophularia nodosa L. extract showed the weak antioxidant effect determined by all selected methods. Phenolic content was in relation to the antioxidant effect of herbs, very high significancy between DPPH and polyphenols content was found. To improve selected properties of apple juice (taste, smell, functional properties) by addition of medicinal herbs was achieved in this work. Antioxidant effect of apple juice variants with herbs additions determined by DPPH method was high and ranged from 73 to 78.49 %. Herbal extracts can be utilised in selected combinations to improve sensory and functional properties of some kinds of beverages. doi:10.5219/75


INTRODUCTION
Dietary habits influence the risk of developing a variety of diseases, especially cancer and heart diseases.Epidemiologic observations and laboratory studies have indicated that tea consumption may have beneficial effects in reducing certain types of cancer in some populations (Mukhtar, Ahmad, 2000).The traditional Chinese medicinal plants associated with anticancer demonstrated significantly stronger antioxidant activity and contained much more phenolic compounds than common vegetables and fruits.A positive and significant correlation existed between antioxidant activity and total phenolic content, revealing that phenolic compounds were the dominant antioxidant components in the tested medicinal herbs.Phenolic acids, flavonoids, tannins, coumarins, lignans, quinones, stilbenes, and curcuminoids were identified as major types of phenolic compounds.The investigation of the inter-relationship between phenolic compounds and antioxidant/anticancer activity will be a promising field to understand and elucidate possible mechanisms for the functionality of traditional Chinese medicines for cancer prevention and treatment (Cai et al., 2004).
Plant extracts contain polyphenols (natural antioxidants), which are believed to be effective nutrients in the prevention of oxidative stress-related diseases such as cancer and heart diseases.These extracts, possibly mainly due to their phenolic content, retard oxidative degradation of lipids (Proestos et al., 2008 In the present, fresh fruit and vegetable juices are very popular and their improvement with natural functional (herbal) additives to increase their nutritional and sensory value is one of the trends in the soft industry.
The aims of this work were to compare antioxidant effect of water herbal extracts evaluated by different assays -TEAC (Trolox Equivalent Antiradical Capacity) and deoxyribose test (dR test), DPPH and to test their use to enrich soft drinks (apple juice).

Herbal extracts preparation
Dry and homogenized herbs (2 g) were extracted with 200 ml of boiled destilled water during 5 min of infusion time.Extracts were filtrated and used for measure.
After that time 1 ml of the radical solution was mixed with 60 ml deionized water, 50µl of herbal extract was added and mixed rigorously.Decrease of absorbancy at 734 nm (UV -1601 Shimadzu, Tokio, Japan) during 600 s was observed.Adjusted value of extract in 600 s was expressed by the calibration curve of Trolox concentration (6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid).

DPPH
The method of BRAND -WILLIAMS et al. (1995) was modified and used.Herbal extract (0.1 ml) was added to 3.9 ml DPPH˙ ethanolic solution (25 mg.l −1 ), mixed and absorbancy decrease was measured at 515 nm during 10 min of reaction time -spectrofotometrically.

TOTAL POLYPHENOLS (TP)
The contents of total phenolic compunds in water extracts were estimated using the Folin-Ciocalteau reagent.Tanin was used as the standard solution.

STATISTICAL ANALYSIS
Statistical processing was performed with use of nonparametric methods (Kendall΄s and Spearman΄s correlation analysis).Datas were computed in R language with use of R software environment for statistical computing.

Antioxidant effects of selected herbal extracts compared to different assays
Antioxidant effect of selected water extracts from herbs which are commonly cultivated in gardens, or in nature was observed and is shown in the table 1. Water as extraction solvent was used because of polyphenols characteristics, safety and also Buřičová and Réblová  (2008) found that the extraction of substances with the antioxidant activity was markedly more efficient using hot water than ethanol at laboratory temperatures.Determination of antioxidant activity by ABTS radical showed the extracts of euphrasia (0.77 mM), silverweed (0.77 mM) and black thorn (0.60 mM) to be the best sources of antioxidants.Similarly use of DPPH radical showed the best antiradical effect at euphrasia (73.8 %) and black thorn (73.4 %) and the lowest at figwort (6.47 %). Assay based on oxidative damage of deoxyribose (dR) confirmed the black thorn as very strong antioxidant (50.05 %).
Figwort was confirmed as one of the weakest source of antioxidants compared to all selected antioxidant assays.The effect of individual extracts in the activity order is different considering different components presence (table 1).The reason could be the presence of compounds with different reactivity in given reaction medium and their different ability to eliminate present type of radical.Antioxidant effect of herbal extracts is described mainly due to polyphenolic compounds, which concentration and presentation is different in individual herbs.As rich sources of phenolics were estimated euphrasia, thorn and gowan.Shi et al. (2009) describes that chlorogenic acid isomers are the key phenolic compounds responsible for antioxidant activity of the extract from Prunus mume flowers.
Antioxidant effect of ascorbic acid (2 g) determined by DPPH method was high (94.6 %) even compared to euphrasia, which was classiffied as one of the best antioxidant (73.8 %) sources.BHT was tested in low amounts (0.45 g BHT) and at this sample weight was determined 52 % inhibition of DPPH radical (fig.1).Antioxidant effect of selected fenolic acids (not shown) determined by dR-test was found to be high (54.42%) at rosmarinic and gallic (53.48 %) acids.

Application of selected herbal extracts in aplle juices
Sensory evaluation showed that pure (100 %) apple juice was very sweet and sensorically not very well accepted.Some authors (Buřičová, Réblová, 2008) recommend selected plants (Rosaceae, Lamiaceae, Tiliaceae) for consumption in various kinds of beverages or as extracts to increase the nutritional value of different foods and diets.The aim of work was to improve properties of apple juice (taste, smell, functional properties) by addition of selected medicinal herbs in purpose of sensory and health improvements of juices.By non-parametric testing it was found that with use of Friedman test concentrated apple juice (E variant) was Regarding to the antioxidant effect of individual variants can be showed (table 2) that the best antioxidant effect was determined at variant A (raspberry and linden-78.49%) .Antioxidant effect of our juice variants was high and ranged from 73 to 78.49 %, probably because of use of good antioxidative extracts from herbs and good antioxidant effect of apple juice as well.Also Buřičová and Réblová (2008) state that considerable antioxidant activities were found in the extracts of plants from Rosaceae family (e.g.blackberry), Lamiaceae (oregano, mint), Tiliaceae (linden) and appear to be good and safe sources of antioxidants.
Dry matter content and acidity were found to be the highest at concentrated apple juice (variant E) and decreased with the additions of herbal extracts.
Mendelova et al. ( 2009) tested to improve sensory parameters of concentrated apple and sour cherry juices by mixing them in different proportions.

CONCLUSION
Determination of antioxidant activity by ABTS radical showed the extracts of euphrasia, silverweed and black thorn to be the best sources of antioxidants.Similarly use of DPPH radical showed the best antiradical effect at euphrasia and black thorn, assay based on oxidative damage of deoxyribose (dR) confirmed the black thorn as very strong antioxidant.Figwort was confirmed as one of the weakest source of antioxidants compared to all selected antioxidant assays.While all herbal extracts showed some antioxidant effect, achieved results in individual kinds of herbs compared to different methods indicate some differences in the activity (effect) order evaluated by different methods.The reason could be the presence of compounds with different reactivity in given reaction medium and their different ability to eliminate present type of radical.
To improve properties of apple juice (taste, smell, functional properties) by addition of selected medicinal herbs (10 %) was achieved in this work.Antioxidant effect of apple juice variants with herbs additions was high and ranged from 73 to 78.49 %.Herbal extracts can be utilised as potential food additives to avoid oxidative changes in the food products and in selected combinations to improve sensory and functional properties of some kinds of beverages.

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Efficiency of plant extracts as antioxidants was calculatedcontrol absorbancy of DPPH solution (time = 0 min) A At -absorbancy of solution after reaction with antioxidant (time = 10 min) ).