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This paper summarizes our research for herbal extracts with potent antioxidant activity obtained from a large scale screening based on superoxide radical (O2•−) scavenging activity followed by characterization of antioxidant properties. Firstly, scavenging activity against O2•− was extensively screened from ethanol extracts of approximately 1000 kinds of herbs by applying an electron spin resonance (ESR)-spin trapping method, and we chose four edible herbal extracts with prominently potent ability to scavenge O2•−. They are the extracts from Punica granatum (Peel), Syzygium aromaticum (Bud), Mangifera indica (Kernel), and Phyllanthus emblica (Fruit). These extracts were further examined to determine if they also scavenge hydroxyl radical (•OH), by applying the ESR spin-trapping method, and if they have heat resistance as a desirable characteristic feature. Experiments with the Fenton reaction and photolysis of H2O2 induced by UV irradiation demonstrated that all four extracts have potent ability to directly scavenge •OH. Furthermore, the scavenging activities against O2•− and •OH of the extracts of P. granatum (peel), M. indica (kernel) and P. emblica (fruit) proved to be heat-resistant.
The results of the review might give useful information when choosing a potent antioxidant as a foodstuff. For instance, the four herbal extracts chosen from extensive screening possess desirable antioxidant properties. In particular, the extracts of the aforementioned three herbs are expected to be suitable for food processing in which thermal devices are used, because of their heat resistance.
Superoxide radical (O2•−) is known to be harmful to cellular components and to function as a precursor of other reactive oxygen species (ROS), such as singlet oxygen (1O2) and hydroxyl radical (•OH).(1–6) A dismutation reaction can result in the formation of hydrogen peroxides (H2O2) and O2 from the reaction of O2•− with water.(4) A reactive non radical, H2O2 is very important because it can penetrate biological membranes. Although H2O2 itself is not very reactive, it can convert into more reactive species such as •OH, the most reactive and harmful ROS, by ultraviolet irradiation, Fenton like reactions and the metal ion catalyzed Haber-Weiss reaction.(5,6) Electron spin resonance (ESR) using the spin trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) is a technique for the direct detection of unpaired electrons such as O2•− and •OH, both of which are identified by hyperfine coupling constants assigned to the spin adducts such as DMPO-OOH (an adduct from DMPO and O2•−) and DMPO-OH (an adduct from DMPO and •OH).(7)
Foodstuffs possess two major functions. That is, the primary function is nutritional feature (life support), and the secondary function is gustational feature (taste, flavor, and texture). Recently, antioxidant potency has been received much attention as one of the tertiary function of foodstuffs. For instance, human studies with de-alcoholized red but white wine showed short-term cardiovascular benefits, and the specific components of the de-alcoholized wine that are active on cardiovascular endpoints are the polyphenols found in red wine, especially resveratrol.(8–13) In addition to its potent antioxidant properties,(14,15) it has recently been reported that resveratrol mimics the anti-ageing effects of calorie restriction in lower organisms, and ameliorates insulin resistance, increases mitochondrial content, and prolongs survival in mice fed a high-fat diet.(16) Based on these backgrounds, we conducted a large scale screening to search for edible herbal extracts with potent antioxidant activity.(17,18) In this review, we summarize our research for herbal extracts with potent antioxidant activity obtained from a large scale screening based on O2•− scavenging activity followed by characterization of antioxidant properties.
More than a thousand of herbal ethanol extracts were prepared as shown in Table 1. The assay used in this study was essentially identical to that described in our previous papers.(19–21) In brief, 50 µl of 2 mM hypoxanthine (HPX), 30 µl of dimethyl sulfoxide (DMSO), 50 µl of sample dissolved in DMSO, 20 µl of 4.5 M DMPO, and 50 µl of 0.4 U/ml xanthine oxidase (XOD) were placed in a test tube and mixed. In the primary screening, all of the herbal extracts were served at a fixed concentration of 25 µg/ml in the reaction mixture. The mixture was transferred to an ESR spectrometry cell, and the DMPO-OOH spin adduct was quantified 100 s after the addition of XOD. When a spin trapping agent, DMPO, was added to a solution of the HPX-XOD reaction system, an ESR signal with the hyperfine coupling constants of aN = 1.37 mT, aHβ = 1.10 mT, aHγ = 0.12 mT was observed. This signal was assigned to the spin adduct, DMPO-OOH, by the hyperfine coupling constants.(7) The reduction of the signal intensity of DMPO-OOH is likely reflected by the ability to scavenge O2•−. Polyphenol contents of some herbal extracts were determined by Folin-Denis method,(8) and results were expressed as garlic acid equivalency.
Table 1 shows a list of all herbs tested and herbs that showed 80% or more reduction of signal intensity of DMPO-OOH measured by ESR-spin trapping method at a fixed concentration of 25 µg/ml. Fig. 1 shows the representative ESR spectra of solvent control and ethanol extract with either poor or potent O2•− scavenging activity. Based on the data, we picked up four edible herbal extracts for further analyses. They are No.A-058 (Peel of Punica granatum), No.C-037 (Bud of Syzygium aromaticum), No.I-077 (Kernel of Mangifera indica), and No.I-079 (Fruit of Phyllanthus emblica). As shown in Fig. 2, these four extracts contained abundantly polyphenols, whilst herbs with poor O2•− scavenging activity (5% or less reduction of signal intensity of DMPO-OOH at a fixed concentration of 25 µg/ml) contained a little amount of polyphenols. The results indicate that the O2•− scavenging activity was apparently reflected by the polyphenols content.
ESR analyses of •OH from Fenton reaction and from photolysis of H2O2 by UV-irradiation were conducted. The assays used in this study were essentially identical to those described in a previous paper.(21) In brief for the former assay, 50 µl of 2 mM H2O2 dissolved in 0.1 M phosphate buffer (pH 7.4), 50 µl of 8.9 mM DMPO dissolved in pure water, 50 µl of sample dissolved in pure water and 50 µl of 0.2 mM FeSO4 dissolved in pure water were placed in a test tube and mixed. Each mixture was transferred to an ESR spectrometry cell and the DMPO-OH spin adduct was quantified 113 s after the addition of FeSO4. For the latter assay, a reaction mixture consisting of 440 µl of 100 mM H2O2 prepared in 25 mM phosphate buffer (pH 7.4), 10 µl of 111.25 mM DMPO dissolved in pure water and 50 µl of sample dissolved in pure water was exposed to 254 nm UV irradiation at 4 W for 1 min at a distance of 12 cm. Then the ESR spectrum of DMPO-OH was measured. When DMPO was added to a solution of the Fenton reaction system, the spin adduct DMPO-OH was formed. A reduction in the signal intensity of DMPO-OH by the addition of selected four herbal extracts likely reflects an ability to scavenge •OH. This was also confirmed by the assay of photolysis of H2O2 by UV-irradiation. In the assay, the ESR signal of DMPO-OH was reduced by adding any of the four herbal extracts, depending on the concentration. This indicates that any of the extracts has the ability to directly scavenge •OH.
As for the heat resistance of the antioxidant potency of the four herbal extracts, the effect of heat (100°C) exposure on the O2•−- and •OH-scavenging activity of the extracts was examined. While the O2•−-scavenging activity of 6.25 µg/ml L-ascorbic acid as a reference agent, at which concentration L-ascorbic acid exerted scavenging activity comparable to 25 µg/ml herbal extract, was completely inactivated after heat exposure for 30 min, the activities of 25 µg/ml extracts from P. granatum (peel), M. indica (kernel) and P. emblica (fruit) were reduced by only about 20% even after heat exposure for 120 min. The extract of S. aromaticum (bud) was relatively heat-labile compared with the other three herbal extracts, as its activity was reduced by almost 40% after heat exposure for 60–120 min. Similar tendency was also observed in the •OH-scavenging activity of the four herbal extracts exposed to heat (100°C).
These results indicate that the four herbal extracts chosen from extensive screening possess desirable antioxidant properties. In particular, the extracts of P. granatum (peel), M. indica (kernel) and P. emblica (fruit) are expected to be suitable for food processing in which thermal devices are used, because of their heat resistance.