## 2 Results
### Effect of (2'S)-Columbianetin on the UVA-Mediated Expression of MMP-1 and Collagen  
The expression levels of MMP-1 and type I pro-collagen in UVA-irradiated HDFs were analyzed at the gene and protein level. In addition, levels of collagen were also determined. UVA irradiation at a dose of 10 J/cm\({}^{2}\) notably increased the mRNA expression of MMP-1 in HDFs while decreasing expression of type I pro-collagen (Figure 4A). Parallel effects were observed in the protein levels of MMP-1 and type I pro-collagen (Figure 4B). However, UVA-induced changes in the MMP-1 and type I pro-collagen were ameliorated by (2'S)-columbianetin treatment in a concentration-dependent manner. UVA exposure resulted in decreased levels of collagen along with its precursor type I pro-collagen (Figure 4B). The collagen level was increased by the presence of (2'S)-columbianetin in a similar fashion to that of type I pro-collagen.  
Figure 2: Effect of (2’S)-columbianetin on viability of human dermal fibroblasts (HDFs) and nitrogen species (NO) generation in UVA-irradiated HDFs. (**A**) HDFs were incubated for 48 h following treatment with changing doses of (2’S)-columbianetin. Effects of (2’S)-columbianetin on the viability of HDFs was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzolium bromide (MTT) assay. Viability of the cells was calculated from absorbance values and given as the relative percentage of untreated control cells (0 uM). Values are means \(\pm\) SD (\(n=3\)). \({}^{**}\)\(p<0.01\) compared to untreated control group (0 uM). (**B**) HDFs were exposed to UVA radiation (10 J/cm\({}^{2}\)) and treated with given concentrations of (2’S)-columbianetin. After 24 h incubation, nitric oxide generation was quantified as the nitrate amount in the cell culture medium. Values are means \(\pm\) SD (\(n=3\)). \({}^{**}\)\(p<0.01\) compared to non-irradiated blank group; \({}^{*}\)\(p<0.05\), \({}^{**}\)\(p<0.01\) compared to UVA-irradiated untreated control group.  
Figure 4: Effect of (2\({}^{\prime}\)S)-columbianetin on the mRNA (**A**) and protein (**B**) expression levels of the MMP-1 and type I\(\alpha\)1 pro-collagen in UVA-irradiated HDFs after 24 h from treatment analyzed by Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR) and Western blotting, respectively. \(\beta\)-actin was used as internal loading control. RT-qPCR expression quantity was shown relative to the untreated UVA-irradiated control, calculated by \(\mathbf{2^{-(\Lambda\Lambda\mathrm{CI})}}\) method. Western blot bands were densitometrically quantified and plotted as relative percentage of UVA-irradiated untreated control group. Values are means \(\pm\) SD (\(n=3\)). \(\lx@note{footnote}{\(p<0.01\) compared to UVA-irradiated untreated control group.}\)  
Figure 3: Effect of (2\({}^{\prime}\)S)-columbianetin on the cellular release of MMP-1 (**A**) and type I\(\alpha\)1 pro-collagen (**B**) in UVA-irradiated HDFs. The contents of MMP-1 and type I\(\alpha\)1 pro-collagen were calculated from UVA-irradiated cell culture media following 24 h incubation with different concentrations of (2\({}^{\prime}\)S)-columbianetin using ELISA. Values are means \(\pm\) SD (\(n=3\)). \(\lx@note{footnote}{\(p<0.01\) compared to non-irradiated blank group; \({}^{\star}p<0.05\), ** \(p<0.01\) compared to UVA-irradiated untreated control group.}\)