Antioxidant Activity of Extract from Polygonum Cuspidatum

Numerous diseases are induced by free radicals via lipid peroxidation, protein peroxidation and DNA damage. It has been known that a variety of plant extracts have antioxidant activities to scavenge free radicals. Whether Polygonum cuspidatum Sieb. et Zucc has antioxidant activity is unknown. In this study, dried roots of Polygonum cuspidatum were extracted by ethanol and the extract was lyophilized. Free radical scavenging assays, superoxide radical scavenging assays, lipid peroxidation assays and hydroxyl radical-induced DNA strand scission assays were employed to study antioxidant activities. The results indicate that the IC50 value of Polygonum cuspidatum extract is 110 μg/ml in free radical scavenging assays, 3.2 μg/ml in superoxide radical scavenging assays, and 8 μg/ml in lipid peroxidation assays, respectively. Furthermore, Polygonum cuspidatum extract has DNA protective effect in hydroxyl radical-induced DNA strand scission assays. The total phenolics and flavonoid content of extract is 641.1 ± 42.6 mg/g and 62.3 ± 6.0 mg/g. The results indicate that Polygonum cuspidatum extract clearly has antioxidant effects. superoxide radical is normally formed first. It produces other kinds of cell-damaging free radicals and oxidizing agents. In this way, its oxidizing effects can be magnified (7). Phenolics have been known to possess a capacity to scavenge free radicals. They are commonly found in both edible and nonedible plants, and have multiple biological effects, including antioxidant activity (8, 9). The antioxidant activity of phenolics is principally due to their redox properties, which allow them to act as reducing agents, hydrogen donors and singlet oxygen quenchers. In addition, they have a metal chelation potential (10).


INTRODUCTION
Ultraviolet light, ionizing radiation, chemical reactions and metabolic processes can induce the production of reactive oxygen species in the cells.These reactive oxygen species cause lipid peroxidation, protein peroxidation, DNA damage and cellular degeneration in the cells.The accumulation of these damages can induce numerous diseases including cardiovascular disease, ageing, cancer, inflammatory diseases and a variety of other disorders (1,2,3,4,5,6).Reactive oxygen species primarily consist of superoxide radical anion (•O 2 -), hydroxyl radicals (•OH), singlet oxygen ( 1 O 2 ) and hydrogen peroxide (H 2 O 2 ).The damaging action of the hydroxyl radical is the strongest among free radicals.In cellular oxidation reactions, barks (11).Larson has proposed that they play an important preventive role in the development of cancer, heart disease and ageing-related diseases.
The importance of the antioxidant constituents of plant materials in the maintenance of health and protection from ageing-related diseases has intrigued scientist for a long time.We have screened the antioxidant activity of a variety of wild plants and other substances used in traditional Oriental medicine, using 1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging assays.The results indicate that roots of Polygonum cuspidatum Sieb.et Zucc (Polygonaceae) exhibits a DPPH free radical scavenging effect.Polygonum cuspidatum has been traditionally used for treatment of various inflammatory diseases, hepatitis, tumors and diarrhea (12).Recently, its extracts have been reported to possess the antiviral activity against hepatitis B virus (13), have potent estrogenic activities (14), inhibit bacterial DNA primase (15), and inhibit acyl-coenzyme A-cholesterol acyltransferase activity (16).Nevertheless, it is unknown whether Polygonum cuspidatum Sieb.et Zucc has antioxidant activity on superoxide radical scavenging, lipid peroxidation and DNA damage.The content of total phenolics and flavonoids of Polygonum cuspidatum Sieb.et Zucc has not been described.In the present study, we have studied the antioxidant properties of Polygonum cuspidatum.

Preparation of plant extract
The plant extract was prepared as previously described in Chang et al (17) and Hsu (18).Dried powder of Polygonum cuspidatum (5 g) were extracted in 50 ml 50% ethanol solution at 25 o C for 30 min with shaking.The extract was centrifuged at 15000 rpm for 3 min and supernatant was collected.The supernatant was concentrated in a rotary evaporator and then lyophilized.

Lipid peroxidation assay
The brains of young adult male Balb/c mice were dissected and homogenized with a homogenizer in ice-cold Tris-HCl buffer (20 mM, pH 7.4) to produce a 1/10 homogenate.The homogenate was centrifuged at 12000g for 15 min at 4 o C, and the supernatant was used for in vitro lipid peroxidation assay.An aliquot of liposome (1 ml) was incubated with the test samples (final concentrations were 1, 5, 10, 20, 50 and 100 μg/ml, respectively) in the presence of 10 mM FeSO4 and 0.1 mM ascorbic acid at 37 o C for 1 h.The reaction was ended by the addition of 1.0 ml of trichloroacetic acid (TCA; 28%, w/v) and 1.5 ml of TBA (1%, w/v), followed by heating at 100 o C for 15 min.The absorbance of the malondialdehyde (MDA)-TBA complex was measured at 532 nm.(+)-catechin was used as positive controls.The inhibition ratio (%) was calculated as % of inhibition = [(absorbance of controlabsorbance of test sample)/absorbance of control] x 100% (17).

DNA strand scission assay
The assay was carried out as described previously by Keum et al. with minor modifications (22).The reaction mixture (30 ml) contained 10 mM Tris-HCl, 1 mM EDTA buffer (pH 8.0), ΦX174 RF1 supercoiled DNA (0.6 μg) and H 2 O 2 (0.04M).Additionally, different amounts of extract of Polygonum cuspidatum (to get final concentrations of 10, 100, 1000, 5000 and 10000 μg/ml) were added prior to H 2 O 2 addition.Hydroxyl radicals were generated by irradiation of the reaction mixtures at a distance of 5 cm with a 12 W UV lamp (short wave).The mixture was incubated at room temperature for 2 min and the reaction was ended by addition of loading buffer (0.25% bromophenol blue tracking dye and 40% sucrose).The mixtures were analyzed by 0.8% submarine agarose gel electrophoresis (50eV, 1.5 h) as described by Chang et al (17).

Total flavonoid of determination
Samples (1 mg) were added in 1ml of 80% ethanol.An aliquot of 0.5 ml was added to test tubes containing 0.1 ml of 10% aluminum nitrate, 0.1 ml of 1 M potassium acetate and 4.3 ml of 80% ethanol.The absorbance of the supernatant was measured at 415 nm after 40 min at room temperature.Total flavonoid concentration was calculated using quercetin as standard.(23).

Total phenols determination
Total phenolics content was determined according to the Folin-Ciocalteu method (24), using gallic acid as standards.Extract powders (1 mg) were dissolved in 1 ml 50% methanol solution.Extract solution (0.5 ml) was mixed with 0.5 ml of 50% Folin-Ciocalteu reagent.After of 2-5 min, 1.0 ml of 20% Na 2 CO 3 was added to the mixture and incubated for 10 min at room for temperature.The mixture was centrifuged at 150 g for 8 min and the absorbance of the supernatant was measured at 730 nm.The total phenolic content was expressed as gallic acid equivalents (GAE) in milligrams per gram sample (17).

RESULTS
The results showed that the IC50 values (the concentration required to inhibit radical formation by 50%) of Polygonum cuspidatum extract were about 110 μg/ml (Fig 1).The IC 50 values of (+)-catechin and ascorbic acid were about 35 μg/ml and about 50 μg/ml, respectively.Compared to (+)catechin and L-ascorbic acid, they exhibited a similar curve of antioxidant activity.However, the IC 50 value of Polygonum cuspidatum extract was lower than that of (+)-catechin and L-ascorbic acid.This result demonstrated that Polygonum cuspidatum extract has inhibitory activity against the DPPH radical.
The figure 2 shows the superoxide scavenging activity of Polygonum cuspidatum extract with (+)-catechin as control.The results showed that the IC 50 value of Polygonum cuspidatum was about 3.2 μg/ml, while the IC 50 value of (+)catechin was about 40 μg/ml.Almost all superoxide radicals were inhibited by 20 μg/ml Polygonum cuspidatum extract.The superoxide scavenging activity of Polygonum cuspidatum extract was remarkably higher than that of (+)-catechin.Therefore, Polygonum cuspidatum seems to be a potential source of superoxide radical scavenging.This result demonstrates that Polygonum cuspidatum extract has a significant superoxide scavenging activity.
The lipid peroxidation suppressing activity of Polygonum cuspidatum extract is shown in the Figure 3, with (+)-catechin as control.The result shows that the IC 50 values of Polygonum cuspidatum extract were about 8 μg/ml (Fig. 3).The IC 50 value of (+)-catechin was about 17 μg/ml.The lipid peroxidation suppressing activity of Polygonum cuspidatum extract was higher than that of (+)-catechin.Therefore, Polygonum cuspidatum seems to be a potential lipid peroxidation suppressor.This result demonstrated that Polygonum cuspidatum extract has a significant lipid peroxidation suppressing activity.
The protective effect of DNA of Polygonum cuspidatum extracts is shown in the Figure 4   Plant phenolics are widely distributed in the tissues of plants as well as play a pivotal role in the highly effective free radical scavengers and antioxidant activity.The phenolic and flavonoid total contents in Polygonum cuspidatum extract were 641.1 ± 42.6 mg/g and 62.3 ± 6 mg/g, respectively (Table 1).These results imply that Polygonum cuspidatum extract contains a high quantity of phenolics and flavonoids.

DISCUSSION
Antioxidant activity of Polygonum cuspidatum extract has been revealed by free radical scavenging assays, superoxide radical scavenging assays, lipid peroxidation assays and hydroxyl radical-induced DNA strand scission assays.In addition, Polygonum cuspidatum extract has high phenolic and flavonoid contents.This study shows that Polygonum cuspidatum extract clearly has antioxidant effects.
In cellular oxidation reactions, superoxide radicals are normally formed first, and their effects can be magnified because they produce other kinds of free radicals and oxidizing agents (7).Additionally, xanthine oxidase is one of the main enzymatic sources of those reactive oxygen species in vivo.In the current study, superoxide radicals scavenging property has been found in Polygonum cuspidatum extract.The IC 50 value of Polygonum cuspidatum extract is about 3.2 μg/ml, whereas the IC 50 value of (+)-catechin is about 40 μg/ml.The maximum inhibitory The maximum inhibition concentration of Polygonum cuspidatum is approximately 20 μg/ml.However, the maximum inhibition concentration of (+)catechin is higher than 100 μg/ml.It is noteworthy that the superoxide scavenging activity of Polygonum cuspidatum extract is superior to that of (+)-catechin.The IC 50 value of Polygonum cuspidatum (3.2 μg/ml) is larger than that of Paeonia suffruticosa (50 μg/ml) (7), but less than Polygonum aviculare L. (0.8 μg/ml) (18).These results show that Polygonum cuspidatum is an important source of superoxide radicals scavenging.
In this study, lipid peroxidation of mouse brain homogenates was induced by ferric ion plus ascorbic acid.Lipid peroxidation scavenging activity has been found in Polygonum cuspidatum extract.The IC 50 values of Polygonum cuspidatum extract were about 8 μg/ml.The IC 50 value of (+)-catechin was about 17 μg/ml.The maximum inhibitory effect of Polygonum cuspidatum was about 75%.The maximum inhibition concentration of Polygonum cuspidatum was approximately 10 μg/ml.The maximum inhibition concentration of (+)-catechin was approximately 50 μg/ml.The inhibitory effect of Polygonum cuspidatum was higher than (+)-catechin.It is also higher than described previously for Polygonum aviculare L. (16 μg/ml) (18).This result indicates that Polygonum cuspidatum extract is a good source of lipid peroxidation scavenging.
The cellular damage resulting from hydroxyl radicals is strongest among free radicals.Hydroxyl radicals can be generated by biochemical reaction.Superoxide radical is converted by superoxide dismutase to hydrogen peroxide, which can subsequently produce extremely reactive hydroxyl radicals in the presence of transition metal ions such as iron and copper or by UV photolysis.Hydroxyl radicals can attack DNA to cause strand scission.Incubation of ΦX174 RF1 supercoiled DNA with H 2 O 2 and then UV radiation resulted in complete conversion of supercoiled DNA to the open circular form.In this study, the administration of Polygonum cuspidatum extract to the reaction mixture substantially decreased the DNA strand scission induced by both H 2 O 2 and UV radiation specially at high concentration.It shows a dose-dependent protection of DNA under oxidative stress.Nearly complete protection was found at a dose of 5000 μg/ml.However, the effect of DNA protection of Polygonum cuspidatum was lower than those described by Acacia confusa (1000 μg/ml) (17) and Polygonum aviculare L. (1000 μg/ml) (18).These results reveal that Polygonum cuspidatum extract is a DNA protector.
Phenolics are found in large quantities in the plant kingdom, and they have been proposed to have multiple biological functions, including antioxidant activity (27,28,29).Our results showed that Polygonum cuspidatum extract contains 641.1 ± 42.6 mg/g phenolics.The Polygonum cuspidatum extract contained a higher amount of phenolics than the bark (470.6 ± 43.9 mg/g phenolics) and heartwood extracts (529.7 ± 14.4 mg/g phenolics) of Acacia confusa based on Folin-Ciocalteu procedures (17).However, the phenolics concentration of Polygonum cuspidatum extract was lower than that described for Polygonum aviculare L. extract (677.4 ± 62.7 mg/g phenolics).
Nevertheless, Polygonum cuspidatum is a significant source of phenolics.The results in this study suggest that the effectiveness of the antioxidant activity of Polygonum cuspidatum extract is probably related to their phenolic contents, and the observed antioxidant activities of the extract may be due to the hydroxyl groups in phenolics (30).A similar finding has been demonstrated in the plant extracts of Eucommia ulmoides (Du-zhong), Acacia confusa and Polygonum aviculare L., where the phenolics concentration have correlated with their antioxidant activities (17,18,31).The flavonoid concentration in Polygonum cuspidatum was 62.3 ± 6 mg/g, lower than Polygonum aviculare L. (112.7 ± 13 mg/g), but higher than propolis (23).The higher content of total phenolics and flavonoids in Polygonum cuspidatum have been correlated with hepatitis B virus inhibition (13), bacterial DNA primase inhibition (15), acyl-coenzyme A acyltransferase activity (16), inhibition of copper catalyzed oxidation for low-density lipoprotein (32), inhibition of platelet clotting and arachidonate metabolism, liver injury from peroxidized oil (33), having cancer-chemopreventive activities (34), and estrogenic activities (14,35).Taken together, this study indicates that Polygonum cuspidatum extract clearly has antioxidant effects.
, with ΦX174 RF1 supercoiled DNA as control (lane 1).UV illumination alone did not cause DNA strand cleavage (lane 2).The treatment of supercoiled DNA with UV plus H 2 O 2 led to the conversion of the DNA to open circular form (lane 3).High concentration of Polygonum cuspidatum extract led to the maintenance of the DNA to super coiled form (lanes 6-8), showing almost complete protection at a dose of 5000 μg/ml.This protective effect of DNA exhibits dose-dependence.This result reveals a DNA protective activity from oxidative stress of Polygonum cuspidatum.

TABLE 1
Total phenolic content and total flavonoid content of ethanolic extract fromPolygonum cuspidatum