Flavonoids from Machilus japonica stems and their inhibitory effects on LDL oxidation.

Stems of Machilus japonica were extracted with 80% aqueous methanol (MeOH) and the concentrated extract was successively extracted with ethyl acetate (EtOAc), normal butanol (n-BuOH), and water. Six flavonoids were isolated from the EtOAc fraction: (+)-taxifolin, afzelin, (-)-epicatechin, 5,3'-di-O-methyl-(-)-epicatechin, 5,7,3'-tri-O-methyl-(-)-epicatechin, and 5,7-di-O-methyl-3',4'-methylenedioxyflavan-3-ol. The chemical structures were identified using spectroscopic data including NMR, mass spectrometry and infrared spectroscopy. This is the first report of isolation of these six compounds from M. japonica. The compounds were evaluated for their diphenyl picryl hydrazinyl scavenging activity and inhibitory effects on low-density lipoprotein oxidation. Compounds 1 and 3-6 exhibited DPPH antioxidant activity equivalent with that of ascorbic acid, with half maximal inhibitory concentration (IC50) values of 0.16, 0.21, 0.17, 0.15 and 0.07 mM, respectively. The activity of compound 1 was similar to the positive control butylated hydroxytoluene, which had an IC50 value of 1.9 µM, while compounds 3 and 5 showed little activity. Compounds 1, 3, and 5 exhibited LDL antioxidant activity with IC50 values of 2.8, 7.1, and 4.6 µM, respectively.

Enhancement of the skin-protective activities of Centella asiatica L. Urban by a nano-encapsulation process.

Aqueous extracts of Centella asiatica L. Urban were encapsulated by an edible biopolymer, gelatin, which has no effect on their cosmetic activities. The nanoparticles were w/o-type spherical liposomes that had an average diameter of 115.0nm. The encapsulation efficiency was estimated to be approximately 67%, which was relatively high for these aqueous extracts. The nanoparticles showed lower cytotoxicity (10%) in human skin fibroblast cells than the unencapsulated crude extract (15%) at 1.0mg/ml, this was possibly because a smaller amount of the extract was present in the nanoparticles. The nanoparticles efficiently reduced the expression of matrix metalloproteinase (MMP)-1 in UV-irradiated cells from 136.1% to 77.6% (UV-irradiated control) and inhibited hyaluronidase expression (>60%) at a concentration of 0.5mg/ml, which was higher than the levels produced by the unencapsulated crude extracts. The nanoparticles had a very high flux through mouse skin and also remained at relatively large concentrations in the derma when compared to the unencapsulated crude extracts. These results clearly indicate that the skin-protective activities of C. asiatica were significantly improved through the nano-encapsulation process. These findings also imply that a crude extract can be used and have the same efficacy as purified compounds, which should reduce the purification process and production costs.

Effects of ginsenoside Rg2 on the ultraviolet B-induced DNA damage responses in HaCaT cells.

Our previous study demonstrated the increase in the repair of UVB damage by mRg2, a mixture of ginsenosides containing 60% Rg2 in NIH3T3 cells. In the present study, the effects of purified Rg2 on the repair and apoptosis in ultraviolet B (UVB)-exposed HaCaT cells were investigated on gene expression levels. When cells were exposed to UVB and post-incubated in normal medium for 24 h, the cell viability decreased to about 50% of that in nontreated control. When Rg2 was post-incubated, however, the UVB-induced cytotoxicity was significantly prevented in an Rg2 concentration- and time-dependent manner. The apoptotic nuclear fragmentation resulting from UVB exposure was also significantly protected by the Rg2 post-incubation. Microarray analysis showed that the genes stimulated by the Rg2-alone treatment include those involved in p53 signaling pathway such as GADD45alpha, GADD45beta, and cell communication genes. RT-PCR analysis showed that the Rg2-alone treatment slightly upregulated the p53 and GADD45 transcript and protein levels by about 1.5-fold as compared with the nontreated control. The mRNA levels of p53 and GADD45 in cells exposed to UVB and post-incubated with Rg2 for 24 h decreased in an Rg2 concentration-dependent manner as compared with that post-incubated in normal medium. However, the mRNA level of the UVB-exposed cells post-incubated with 5 microM retinol was essentially the same as that post-incubated in normal medium. Time course experiment showed that the mRNA levels of p53 and GADD45 in UVB-exposed cells were upregulated by post-incubation with 50 microM Rg2 until 6 and 9 h, respectively, and then gradually decreased until 24 h. By Western blot analysis, it was also revealed that the Rg2 post-incubation decreases the expression of p53, phospho-p53, GADD45, and ATM in UVB-exposed cells. Time course analysis also indicated that these decreased expressions were due to the earlier upregulation of p53 and GADD45 proteins. When UVB-exposed cells were post-incubated with Rg2 for 24 h after UVB exposure, the level of remaining cyclobutane pyrimidine dimers decreased in both Rg2 concentration- and time-dependent manner. All these results suggest that Rg2 protects cells against UVB-induced genotoxicity by increasing DNA repair, in possible association with modulation of protein levels involved in p53 signaling pathway.

Effects of mRg2, a mixture of ginsenosides containing 60% Rg2, on the ultraviolet B-induced DNA repair synthesis and apoptosis in NIH3T3 cells.

Ginseng has been used worldwidely as a traditional medicine of Asian countries for treatment of various diseases including cancer. The purpose of this study was to determine the effect of ginseng saponin mRg2, a mixture of ginsenosides containing 60% Rg2, on the repair and apoptosis of ultraviolet B (UVB)-exposed NIH3T3 cells. When cells were exposed to UVB and then incubated with normal growth medium for 48 h, cell viability, as determined by trypan blue exclusion assay decreased to about 25%. However, when mRg2 was included in the postincubation medium, the UVB-induced loss of cell viability was significantly reduced as compared with that postincubated in normal growth medium. 4,6-diamidino-2-phenylindole (DAPI) staining showed that postincubation of the UVB-exposed cells in medium containing mRg2 significantly reduced the apoptotic nuclear fragmentation. Interestingly, when cells were preincubated with mRg2 for 24 h and then exposed to various doses of UV, the amount of repair synthesis significantly increased as compared with those in cells exposed to UVB alone. Western blot analysis indicated that the mRg2 postincubation after UVB exposure potentiated the level of p53 and p21. The level of Triton nonextractable proliferating cell nuclear antigen (PCNA) also remained elevated by mRg2 postincubation. All these results suggest that mRg2 protects cells against UVB-induced genotoxicity by increasing DNA repair and decreasing apoptosis, in possible association with the modulation of protein levels involved in cell cycle arrest or progression.