In Vitro Antioxidant Capacity and Neuronal Cell Toxicity of Roots of Ostericum koreanum Maximowicz

Ostericum koreanum maximowicz (Umbelliferae), a medicinal herb in Korean Oriental Medicine, has been applied to treat cold, headache, neuralgia and arthralgia. The ethyl acetate fraction of O. koreanum root was subjected to in vitro antioxidant activity with different methods for free radical scavenging activities. In addition, the cell viability and nitric oxide release assays were performed here for the first time in neuroblastoma (Neuro-2a) cell cultures. Among all the tested methods, the ethyl acetate fraction was expressed very active, exhibiting a good Trolox equivalent values and IC50, comparable to that of the commercial antioxidants, Trolox and ascorbic acid, respectively. The results showed that there was a reduction of cell viability by the fraction in a concentration dependent manner. These results suggest that O. koreanum shows good antioxidant activities in vitro by inhibiting free radicals. These findings provide a rationale for the in vivo testing. Also, the major constituents behind the antioxidant mechanisms of this fraction warrant further study.


Plant material
O. koreanum roots, cultured in Bongwha Alpine Medicinal Plant Experiment Station (BAMPES; Gyeongsangbook, Republic of Korea), were authenticated.A voucher specimen was deposited in the herbarium of BAMPES, with the registration number NK-2007-10.

Extraction and fractionation
Air-dried roots of O. koreanum (1.5 kg) were cut into small pieces, pulverized and extracted three times with 4.5 L of methanol at room temperature for 24 h.The supernatant was collected by filtration and the solvent was evaporated under in vacuum at 40 °C (~ 74 g).The extract (70 g) was sequentially partitioned into hexane (11 g), ethyl acetate (10 g), n-butanol (14 g) and water (35 g) portions.The most active ethyl acetate fraction was used for bioassays.

Statistical analysis
The data are expressed as the mean ± SD of three measurements.Statistical analysis was performed using Tukey's test.p < 0.05 was considered statistically significant.

Results and Discussion
In this study, we designed to evaluate the antioxidant and free radical scavenging activities of ethyl acetate fraction of methanol extract of O. koreanum roots.From results, the O. koreanum roots ethyl acetate fraction showed potent antioxidant activity based on TEAC, ORAC and DPPH and other free radical scavenging tests in a dose dependent manner.The O. koreanum roots were found to be active in TEAC, ORAC and DPPH as 0.516, 2.884 and 1.514 mmole Trolox equivalents per grams, respectively (Table 1).The ethyl acetate fraction showed the most efficient against TEAC followed by DPPH and ORAC.From this, the O. koreanum was weak in scavenging the ORAC compared to others.According to the TEAC method, this fraction acted as a direct free radical scavenger, indicating that this fraction had significant antioxidant activity.The scavenging data suggests that the components within the fraction is capable of scavenging free radicals via a mechanism of electron/hydrogen donation and should be able to protect susceptible matrices from free radical-mediated oxidative degradation.
• and NO • with the IC 50 being 23.582, 66.310, 170.582 and 60.779 micrograms per mL and the activity is comparable to that of ascorbic acid.The extract also strongly chelated Fe 3+ ions at 7.846 micrograms per mL of IC 50 (Table 2).In the PMS-NADH-NBT system, superoxide anion, derived from dissolved oxygen from the coupling reaction of PMS-NADH, reduces NBT.Then, scavenging of H 2 O 2 may be attributed to electrons donation to H 2 O 2 , thus neutralizing it to water.Although H 2 O 2 itself is not very reactive, it can sometimes cause cytotoxicity by giving rise to OH • in the cell.The O. koreanum fraction was evaluated for their ability to scavenge OH • using the deoxyribose degradation assay and nitric oxide radicals using sodium nitroprusside (SNP) as a NO • donor.The chelation of ferrous ions, in which ferrozine quantitatively forms complexes with Fe 2+ .In the presence of chelating agents, the formation of this complex is disrupted, thereby impeding the formation of the red colour imparted by the complex as well.Considering the results obtained, it may be anticipated that the ethyl acetate fraction of O. koreanum methanol extract have antioxidant activity, shown here by the scavenging of O 2 • and NO • and ferrous ion chelating efficacies.
To determine the effects of roots of ethyl acetate fraction of O. koreanum on cell viability, the N2a cells were exposed to O. koreanum (2.5 ~ 30 µg/mL) for an incubation time of 1 h.In Figure 1, the MTT test after 1 h of incubation with O. koreanum roots does not indicate any significant viability difference at 2.5 and 5.0 micrograms per mL in treated N2a cell cultures in comparison to control.A significant decreasing of viability is observed in roots of O. koreanum ethyl acetate fraction at 7.5 ~ 30 micrograms per mL treated N2a cells in comparison to control.NO determination was performed after 1 h of incubation in the presence of O. koreanum (2.5 ~ 30 µg/mL).The treatment with 7.5 micrograms per mL of O. koreanum ethyl acetate fraction produces NO decrease significantly when compared to control, but other doses did not show any significant changes in NO release.From this result that the minimal doses of O. koreanum have no toxicity effects.

Conclusion
This study demonstrates that the various free radical scavenging mechanisms of O. koreanum ethyl acetate fraction may be attributed to its strong abilities as a hydrogen donor.Also, the study verified the effectiveness of O. koreanum on cell viability and nitric oxide release in cell culture model.As a result, the medicinal claims of O. koreanum being used in the treatment of various diseases may be in part due to the antioxidant activity having potential in scavenging of free radicals come out for the therapeutic value.Further isolation of antioxidant constituents and in vivo antioxidant studies are warranted.

Figure 1 .
Figure 1.Effect of O. koreanum on cell viability and nitric oxide release in neuro-2a cells.Values are means ± SD of three measurements.* p < 0.05, ** p < 0.01, *** p < 0.001 (ANOVA/Tukey) compared with untreated normal NO • ) scavenging and ferrous ion chelating efficacies.Moreover, the effects of ethyl acetate fraction of O. koreanum roots on cell viability and nitric oxide (NO) release in neuro-2a (N2a) cells were analyzed.

Table 1 .
The antioxidant activities of ethyl acetate fraction of O. koreanum.