Conclusiones del capítulo:

The high phenolic content of grapes makes these fruits interesting as sources of antioxidants. The results from an extensive study of the antioxidant potential of grape extracts from different grape varieties have been reported (Meyer et al., 1997;

Yi et al., 1997). The selection of varieties included both red and white wine grapes as well as table grapes. The grapes were harvested at commercial maturity on the basis of the sugar content. Antioxidant potential was tested in extracts obtained after extraction of the grapes with 60% methanol in water, removal of the methanol by evaporation and final dilution with water. Total phenolic content of extracts varied from 168 to 1236 mg GAE/L in the two studies. For the red grapes, anthocyanins accounted for the main proportion of phenolic compounds, whereas flavonols dom-inated in the white grape extracts. Antioxidant activities were evaluated by a copper-initiated lecithin liposome oxidation assay using the inhibition of conjugated diene hydroperoxides and hexanal formation as the criteria (Yi et al., 1997) as well as by a copper-initiated human LDL oxidation assay similarly based on hexanal (Meyer et al., 1997). The results revealed significant differences in antioxidant activity among grape varieties with all methods. However, the relative order of antioxidant activity among the varieties depended upon the method used for evaluation. When compared at the same phenolic concentration (20 µM GAE), the grape extracts inhibited the formation of conjugated diene hydroperoxides by 25.1–67.9% and hexanal formation by 49.3–97.8% in the lecithin liposome system and by 62–91%

in the human LDL system. The relative percentage inhibition of all methods corre-lated with total phenols of the extracts.

Wang and Lin (2000) measured antioxidant activity (ORAC) and total phenolic and anthocyanin contents of thornless blackberry, strawberry and red and black rasp-berry fruits and found linear relationships between both ORAC and total phenolics and between ORAC and anthocyanins of ripe fruits. Spray-dried elderberry juice with high amounts of anthocyanin glucosides caused prolongation of the lag-phase for Cu-induced oxidation of human LDL, while the maximum oxidation rate remained unchanged (Abuja et al., 1998). For peroxyl-radical-driven LDL oxidation, however, both prolongation of lag time and reduction of maximum oxidation rate occurred.

TX69027-ch03-Frame Page 109 Wednesday, January 16, 2002 7:10 AM

Heinonen et al. (1998) studied the antioxidant activity of extracts from black-berries, highbush blueblack-berries, red raspblack-berries, strawberries and sweet cherries. The phenolic compounds were extracted by using either aqueous methanol (60%) or acetone (70%), evaporated and finally diluted with water. The antioxidant activity was determined by a human LDL oxidation assay or by a lecithin liposome oxidation assay. With the LDL system, the antioxidant activity was monitored as the inhibition of hexanal formation, while in the liposome system, both the inhibitions of hexanal and hydroperoxide formations were detected. When tested at concentrations of 20 µM GAE/L, all extracts except strawberries and blueberries completely inhibited LDL oxidation. At 10 µM GAE/L, the inhibition ranged from 53.9 to 83.9% and was in the decreasing order of blackberries > red raspberries > sweet cherries >

blueberries > strawberries. In the liposome system, the orders of antioxidant activity among the berries changed, even between the conjugated dienes and the hexanal inhibition. Inhibitions by 27.4–68.8% with the dienes and by 60.5–92.6% with the hexanal were seen at extract concentrations of 10 µM GAE/L. With conjugated diene inhibition, the antioxidant activity decreased in the order of sweet cherries > red raspberries > blackberries > blueberries > strawberries, whereas the order was sweet cherries > blueberries > red raspberries > blackberries > strawberries with hexanal inhibition. In this study, all berry extracts inhibited both LDL and liposome oxida-tion. However, the relative antioxidant activity was different when determined by the different methods, and the differences could not easily be explained by the phenolic content of the berry extracts. While the antioxidant activity in the LDL system was directly associated with anthocyanins and indirectly by flavonols of the berries, the activity was correlated with the hydroxycinnamates for the liposome method. It was concluded that berries are good sources of phenolic antioxidants, but that more research is needed to understand the antioxidant potential of phenolic compounds as they occur in nature.

Among 28 Canadian plant products, antioxidant activities of methanolic extracts of blueberries, sea buckthorn fruit and sweet cherries were determined (Velioglu et al., 1998). A method utilizing bleaching of β-carotene was applied. Both blueberries and buckthorn showed high antioxidant activities, whereas sweet cherries showed lower activity. Among all plant products tested, the anthocyanin-containing products pos-sessed high antioxidant activities. In a recent study, phenolic content and antioxidant activity of blackberries, black currants, blueberries and Saskatoon berries were com-pared (Fukumoto and Mazza, 2000). Methanol extracts (80%) of the berries were comparable in total phenolics (347–405 mg chlorogenic acid/100 g), while there were larger discrepancies in anthocyanins (149–233 mg malvidin 3-glucoside/100 g). The authors concluded that the four extracts had similar antioxidative activity when mea-sured by three different methods (β-carotene bleaching, DPPH and malonaldehyde determination by HPLC).

In a study of 92 Finnish edible and nonedible plant materials, a total of 15 berry samples were screened for antioxidant activity (Kähkönen et al., 1999). The activity was compared with the total phenolic content of the samples. Inhibition of methyl linoleate oxidation was used as a measure of antioxidant activity of berry acetone extracts and total phenolics determined by the Folin-Ciocalteau method as GAE/g dry matter of extracts. Berry extracts were high in antioxidant activity; among the

Flavonoids from Berries and Grapes 111

species tested, crowberry, cloudberry, whortleberry, cowberry, aronia, rowanberry and cranberry had the highest activities. The lowest antioxidant activities were seen for red currant, black currant, strawberry and raspberry, whereas bilberry and goose-berry had intermediate antioxidant activities. No significant correlation was obtained between total phenolic content and antioxidant activity in this study. The authors conclude that the antioxidant activity of an extract cannot be predicted from the content of total phenols. They ascribe this partly to varying response of the phenolic subgroups in the Folin-Ciocalteau method and to the fact that the antioxidant activity of phenols depends upon their chemical structure.

A commercial V. myrtillus anthocyanin extract inhibited lipid peroxidation (IC₅₀

= 50.28 µg/ml) and scavenged superoxide anions (IC50 < 25 µg/ml) and hydroxyl radicals (Martín-Aragón et al., 1998). Pigments isolated by high-speed counter-current chromatography from commercial black currant (Ribes niger) extract exerted antioxidant activities comparable to those of pure anthocyanins on a molar basis (Degenhardt et al., 2000). The analyses were performed using the TEAC method at pH 7.4, a pH value reflecting the physiological condition.

With the ORAC method, comparisons between various studies can be performed (Table 3.6). At present, the availability of data is still rather limited (Wang et al., 1996; Prior et al., 1998; Kalt et al., 1999a). When ranked on a fresh weight basis, lowbush blueberries have the highest ORAC values, followed by highbush blueberry, bilberry, rabbiteye blueberries, red raspberry, strawberry and red and white grapes.

Judged on a dry matter basis, the ranking is bilberry, lowbush blueberry, highbush blueberry, rabbiteye blueberry, strawberry and red and white grapes. It is interesting to note that for blueberries, germ plasm is available with higher activity than the principal varieties in commercial usage today.

In a study with acetone/acetic acid extracted berries of different cultivars within four different Vaccinium species, ORAC values comparable to those of strawberry pulp were obtained (Prior et al., 1998). Berries from six commercial cultivars of V. corymbosum L. (Northern Highland) averaged 107.2 µmol TE/g on a dry weight basis—the cultivars ranged from 63.2 to 182.8 µmol TE/g. Berries from three commercial cultivars of V. ashei Reade (rabbiteye) had ORAC values ranging from 85.0 to 206.5 µmol TE/g, the average being 136.2 µmol TE/g. Among cultivars not commercially available, even higher ORAC values were obtained. The average for five cultivars of V. corymbosum L. was 173.4 µmol TE/g, and for five cultivars of V. angustifolium (lowbush), it was 229.8 µmol TE/g. These ORAC values were comparable to the ORAC value of 282.3 µmol TE/g obtained for wild bilberries, V. myrtillus L., in the same study.

Prior et al. (1998) reported that ORAC values of blueberries increased with maturity of the fruits, while various growing places throughout the United States did not affect the ORAC values. A linear relationship between ORAC values, i.e., antioxidant capacity as determined by the ORAC method, was obtained with the anthocyanin (r_xy = 0.77) and total phenolics (r_xy = 0.92) content.

During storage of fresh strawberries, raspberries and highbush and lowbush blueberries at 0, 10, 20 and 30°C for up to 8 days, the antioxidant capacity was found to be stable or even to increase (Kalt et al., 1999a). The antioxidant capacity was strongly correlated with the content of total phenolics (r = 0.83) and anthocyanins

TX69027-ch03-Frame Page 111 Wednesday, January 16, 2002 7:10 AM

(r = 0.90) in the berries. The ORAC values obtained for strawberries and blueberries deviated considerably from values reported previously using the same method (Wang et al., 1996; Prior et al., 1998). The authors explain this as being due to genetic and environmental factors influencing both total phenolics and anthocyanin contents as well as antioxidant capacities of the fruits.