## 4 Discussion  
In the present study, we first investigated the elastase/collagenase inhibitory activity of tissues and different fruit-maturity stages of _C. unshiu_. In both enzyme inhibition assays, the EJF extract showed the most enzyme inhibition activity of all tested samples. Therefore, we tried to determine potential chemical compounds from the EJF extract by a GC-MS analysis and selected four candidate chemical compounds that are related to anti-aging by a search of previous reported literatures. To confirm the four candidate compounds, we further examined these through a collagenase inhibition assay and identified three chemical compounds involved in anti-aging, namely heptadecanoic acid, D-allose, and HMF. Heptadecanoic acid, as a fatty acid, showed the highest collagenase inhibition activity of the three compounds in this study. Free fatty acids as essential structural components of biological membrane are known to play a regulatory effect on melanogenesis. [PERSON] and [PERSON] reported that the heptadecanoic acid of the free fatty acids tested showed the most inhibition activity of _Clostridium histolyticaum_ collagenase [21]. D-allose, a rare sugar, has various physiological functions with anti-cancer, anti-oxidant, anti-inflammatory properties and other health benefits [22; 23; 24]. Recently, [PERSON] et al. (2020) reported that D-allose suppresses oxidative stress and protects skin flap from the injury of ischemia/reperfusion (I/R) [25]. This could explain the anti-aging potential of D-allose, together with our result. HMF has been extensively studied for its anti-proliferative, anti-oxidant, anti-cancer, and anti-inflammatory effects [26; 27]. To our knowledge, there was no study on HMF to anti-aging, but a related study has recently been conducted by [PERSON] et al. [28]. They found that mesosilica-supported 5-HMF has a protective function on UV-induced aging of human dermal fibroblasts. Together with our results and previous reports, it is suggested that heptadecanoic acid, D-allose, and HMF participate in anti-aging processes with other unknown chemical compositions. Although the three compounds for anti-aging were identified from _C. unshiu_, there are non-polar compounds from the GC-MS analysis. It is suggested that other compounds for preventing skin aging may also be present in polar compounds. As for the correlation between the anti-aging and anti-oxidant properties of _C. unshiu_ extracts, a positive correlation was not found in this study. Some recent reports support our anti-aging results. [PERSON] et al. (2016) also did not observe a correlation between the TPC and anti-aging properties of sapota fruit [12]. They observed low levels of phenolics and undetectable flavonoids, but they detected considerable elastase and collagenase inhibitory activity. Similarly, in extracts from the bark of _Cochlospermum vitifolium_, considerable elastase inhibition activity was present, despite little anti-oxidant activity [29]. In a Trolox-equivalent anti-oxidant capacity assay, the extract from lavender and witch hazel leaves had somewhat high anti-oxidant activity but had undetectable or low elastase inhibitory activity [16]. There are contrasting reports that link anti-aging and anti-oxidant activity. Certain phenolic compounds (for example, catechin, resveratrol, epicatechin) and the flavonoids (quercetin, kaempfer, and myricetin) have been shown to have anti-elastase activity  
Figure 5: Effect of _C. unshiu_ extracts on 293T and PANC-1 cell viability. Cells were grown in the presence of _C. unshiu_ extracts. The cell viability was determined by an MTT assay in triplicate and expressed in percentage of control (100%). that is dose-dependent [30; 31]. The discrepancy in the results may be explained by whether the extracts contain different types of phenolic compounds or other bioactive components that have anti-aging properties.