In order to use Corni fructus and its major bioactive matrial ursolic acid as functional food materials, we investigated the biological activities of ethanol extracts from Corni fructus (CFEE) and, apoptosis induce mechanisms of ursolic acid from Corni fructus.
1. The hydrogen-donating activity of CFEE was increased in a dose dependent manner showing 64 and 74% at 300 and 500 μg/mL concentration, respectively. NO productions in RAW264.7 marcrophage cells treated with CFEE were increased in dose dependent manners. CFEE significantly inhibited the growth of MCF-7 human breast cancer cells in dose and time dependent manners. CFEE of 500 μg/mL concentration inhibited the proliferation by over 60% in the MCF-7 cells when treated for 72 h.
2. Cell viability was decreased and cell death rate was increased in both dose- and time-dependent manners in cells treated with 10, 100, 300, and 500 μg/mL CFEE for 24, 48, and 72 h. Proliferation was also inhibited more than 60% in cells treated with CFEE at the 100 μg/mL concentration for 48 h. In addition, the morphology of cells treated with CFEE at the 100 and 500 μg/mL concentrations was distorted with shrunken cell masses and lower cell numbers compared to the control cells. In the cells treated with CFEE, the formation of apoptotic bodies and nuclear condensation were observed in dose dependent manners. CFEE also increased DNA fragmentation values at the 100 and 500 μg/mL concentrations. The apoptosis induced by CFEE was connected to the proteolytic activation of caspase-3. When CFEE was administered at 100 and 300 mg/kg, ip, for 7 consecutive days in mice inoculated with sarcoma-180 cancer cell, the life span of the mice was found to be longer than that of the control mice that did not receive the extract.
3. Anti-proliferation effects of CFEE were investigated in the RC58T/h/SA#4 cells treated with environmental hormones including dioxin and bisphenol A. The proliferation was decreased at the concentration over 500 μg/mL of the ethanol extract. The environmental hormones such as dioxin and bisphenol A increased the growth of RC58T/h/SA#4 cells in the charcoal-treated FBS (cFBS) medium. The proliferation was highest at concentration of 1 nM and 0.1 μM for the tested dioxin and bisphenol A, respectively. CFEE showed inhibition of the proliferation in a dose-dependent manner at the tested concentrations (10, 100, 300, and 500 μg/mL) in the RC58T/h/SA#4 cells treated with the environmental hormones. The anti-proliferation was the highest at 500 μg/mL concentration among the tested ethanol extracts.
4. In the present study, ursolic acid significantly inhibited the growth
of RC58T/h/SA#4 cells in dose- and time-dependent manners. Ursolic acid induced cell death as evidenced by an increased proportion of cells in sub-G1 phase, the formation of apoptotic bodies, nuclear condensation, and DNA fragmentation. After ursolic acid treatment at concentrations above 40 μM, the activities of caspase-3, -8, and -9 were significantly increased compared that of control. Ursolic acid modulated the upregulation of Bax (pro-apoptotic) as well as the downregulation of Bcl-2 (anti-apoptotic). Ursolic acid also stimulated Bid cleavage, which indicates that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thus, the apoptotic effect of ursolic acid was involved in extrinsic and intrinsic signaling pathways. In addition, ursolic acid increased the expression of the caspase- independent mitochondrial apoptosis factor (AIF) in RC58T/h/SA#4 cells.
These results suggest that, Corni fructus and ursolic acid could exert potential anti-oxidant effects, immuneactivities and anti-cancer activities, which can be used as fuctional food materials.