EVALUATION OF ANTIOXIDANT ACTIVITY OF C. TORA WITH ROASTING

II. EVALUATION OF ANTIOXIDANT ACTIVITY OF C. TORA WITH ROASTING

A. Scavenging Activity on DPPH Radical, Superoxide Anion, and Hydroxyl Radical

Free radicals are produced in cells by cellular metabolism and by exogenous agents. The extracts of C. tora, especially roasted and unroasted samples, were evaluated for their free-radical, 2,2-diphenyl-1-piperylhydrazyl radical (DPPH . ), superoxide, and hydroxyl radical scavenging activities. Choi et al. (29)found that the methanolic extracts of the seed of C. tora have a scav- enging activity on the DPPH radical. 2-Hydroxyemodin cassiaside and ru- brofusarin gentiobioside were isolated from methanolic extract as antioxidant substances on DPPH radical.

Superoxide anion (O . 2 ), the one-electron reduced form of molecule oxygen, acts as an oxidizing or reducing agent and is capable of decomposing to form stronger oxidative species, such as singlet oxygen, hydroxyl radical, and hydrogen peroxide, that initiate the peroxidation of lipid and eventually lead to membrane damage (30). Water extracts of C. tora prepared under different roasting temperatures scavenge superoxide anion generated in the phenazine methosulfate–NADH system in a dose-dependent manner (31). The scavenging effects of extracts were in the following order: unroasted > 150jC roasted > 200jC roasted > 250jC roasted.

Yen and Chung (32)investigated the hydroxyl-radical-scavenging activity of extracts from C. tora using spin-trapping agent detected by EPR spectrometer. The hydroxyl radical rapidly reacted with the nitrone spin trap 5,5-dimethylpyrrolidine N-oxide (DMPO). Signal intensity of the DMPO- OH adduct decreased when the concentration of unroasted C. tora extracts was increased. The scavenging effects of water extracts from C. tora on hydroxyl radical were similar to the trend of scavenging action of superoxide anion. This trend is in agreement with the Finding that the antioxidant activ-

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ity of the extracts of unroasted samples was greater than that of roasted samples (33). The scavenging activity of C. tora on both hydroxyl radical and superoxide anion decreased with increasing roasting.

B. Enzymatic and Nonenzymatic Lipid Peroxidation Systems It is now clear that oxygen species such as superoxide anion hydroxyl radical,

hydrogen peroxide, and other free radicals are proposed as agents that attack polyunsaturated fatty acid in the cell membranes giving rise to lipid perox- idation (34). C. tora has been reported to have antioxidant properties in vitro that act as reducing agents, hydrogen donors, free-radical quenchers, and metal ion chelators (32). In view of this, Yen and Chung (33) evaluated the antioxidant properties of water extracts from C. tora prepared under different roasting temperatures on enzymatic microsomes and nonenzymatic lipid peroxidation systems. Their study shows that unroasted C. tora extracts had a greater inhibition effect on peroxidation of linoleic acid than that of a- tocopherol (82%). The extracts from C. tora roasted at 170jC for 80 min and at 200jC for 5 min showed equal inhibition effects on peroxidation of linoleic acid. This means that the antioxidant activity of C. tora was reduced by higher roasting temperature and longer roasting periods. The extracts of unroasted

C. tora also had good antioxidant activity in the liposome peroxidation sys- tem induced by the Fenton reaction as well as in the enzymatic microsome peroxidation system. This suggests that the oxidation of biological membrane in vivo may be inhibited. The antioxidant activity of roasted C. tora extracts decreased compared with that of the unroasted sample. These results might

be caused by several factors, including (1)the reduction of phenolic content in C. tora as a result of roasting and (2)the degradation of Maillard reaction products during overroasting, making them oxidation products without anti- oxidant activity.

Salah et al. (35)demonstrated that quercetin has better antioxidant activity than rutin in a biomembrane system. The reason may be that quer- cetin rather than rutin interacts with that bilayer membrane of phospholipid. Therefore, the antioxidant activity of antioxidants in a membrane system depends on their ability not only to donate a hydrogen atom but also to in- corporate into the membrane. a-Tocopherol scavenges the peroxyl radicals formed from the lipid peroxidation in the inner membrane (36). Therefore, it can be predicted that there are some water-insoluble components in C. tora that could be incorporated into the membrane to afford antioxidant activity.

C. Hydrogen-Peroxide-Induced Oxidative DNA Damage in Human Lymphocytes

Hydrogen peroxide is a well-known ROS produced intracellular during

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in cells. (37)As mentioned earlier, several noncell studies have demonstrated that Cassia seed was capable of reducing lipid peroxidation caused by ROS. The effect of C. tora on hydrogen-peroxide-induced oxidative DNA damage in human lymphocytes was assessed using single-cell gel electropheresis (comet assay). Water extracts of C. tora prepared under different roasting temperatures have the ability to suppress the oxidative DNA damage induced

by H 2 O 2 , albeit to different extents (38). The protective effects of extracts of C. tora were significantly decreased with an increasing roasting temperature. The lower activity of certain samples, such as those roasted at 150jC and 250jC, may be due to low concentration of active phenolic antioxidants that degraded after overroasting.