UVA 조사된 각질형성세포에서 큰금계국 메탄올추출물(CLME)의 피부 광노화에 대한 보호 효과를 알아보기 위해 항산화능, collagenase와 elastase 활성저해능, hyaluronic acid 및 ROS 생성량, apoptosis 생성을 확인하였다. 항산화 활성 실험에서 CLME의 총 폴리페놀 함량은 351.6 TAE mg/g, 총 플라보노이드 함량은 382.5 RE mg/g 이었다. 1,000 μg/mL에서 CLME의 전자공여능은 94.8% 였고 SC50은 83.63 μg/mL였다. 이를 통하여 CLME는 우수한 항산화 활성을 지님을 확인하였다.
시험관내 collagenase와 elastase 활성 저해효과에서, CLME는 1,000 μg/mL에서 각각 73.2%, 79.4% 저해하였고, 양성대조군인 ascorbic acid는 각각 69.7%, 40.5% 저해하였다. IC50을 산출한 결과 CLME는 각각 460.5 μg/mL, 529.2 μg/mL으로 산출되었다. 양성대조군인 AA의 collagenase IC50은 448.3 μg/mL으로 산출되었으며 elastase의 IC50은 1,000 μg/mL 이상으로 확인되어 CLME의 collagenase와 elastase 활성저해능이 우수하였음을 확인하였다.
MTT assay를 이용한 HaCaT 각질형성세포에서의 CLME의 세포독성을 확인한 결과, 최대허용농도는 100 μg/mL 이었고 양성대조군인 ascorbic acid는 200 μg/mL 이상인 것으로 확인되었다. 10, 20, 30 mJ/cm2 UVA 조사된 각질형성세포에서 세포생존률은 각각 22.8%, 28.7%, 33.1% 감소하였고, 자외선 조사 후 CLME 100 μg/mL 처치시 각각 14.4%, 12.3%, 16.9% 세포생존률을 유의하게 증가시켰다 (p < 0.05).
30 mJ/cm2 조사된 HaCaT 각질형성세포에서 ROS 생성량은 34.1% 증가하였고, 자외선 조사 후 CLME 100 μg/mL 처치시 24.2% 유의하게(p < 0.001) 감소시켜 양성대조군인 ascorbic acid(17.2%, p < 0.001)보다 억제능이 우수하였다. 30 mJ/cm2 조사된 HaCaT 각질형성세포에서 세포자멸사는 332.9% 증가하였고, 자외선 조사 후 CLME 100 μg/mL 처치시 25.9% 유의하게(p < 0.001) 감소시켜 양성대조군인 ascorbic acid(22.3%, p < 0.001)보다 억제능이 우수하였다. 30 mJ/cm2 UVA 조사된 HaCaT 각질형성세포에서 hyaluronic acid 생성량은 22.8% 감소하였고, 자외선 조사 후 CLME 100 μg/mL 처치시 27.3% 유의하게(p < 0.001) 증가하여 양성대조군인 ascorbic acid(24.9%, p < 0.001)보다 생성능이 우수하였다.
이상의 연구 결과를 종합해 보면, 큰금계국 메탄올추출물은 높은collagenase와 elastase의 활성저해능과 우수한 항산화능을 가짐을 확인하였다. 또한, UVA 조사한 HaCaT 각질형성세포에서 양성대조군인 ascorbic acid 보다 우수한 ROS 생성 저해능, apoptosis 억제능, hyaluronic acid 생성능을 가짐을 확인하여 큰금계국 메탄올추출물은 자외선 조사에 의한 광노화 보호 및 개선 효과를 확인하였다. 따라서 큰금계국 메탄올추출물은 피부 광노화 개선용 천연물질 기능성 화장품 소재로서의 활용 가능성이 있을 것으로 사료된다.

In order to investigate the protective effects of Coreopsis lanceolate L. methanol extract (CLME) on UVA-induced photoaging in HaCaT keratinocytes, reactive oxygen species (ROS), apoptosis, and hyaluronic acid production were investigated in UVA-irradiated HaCaT human keratinocytes. In addition, antioxidant, collagenase and elastase activities were investigated in a cell-free system.
Total polyphenolics and flavonoids in the CLME were 351.6 TAE mg/g and 382.5 RE mg/g, respectively. At concentrations of 1,000 μg/mL, the electron-donating ability of CLME was 94.8% and SC50 was calculated to be 83.63 μg/mL. These findings confirmed that CLME has excellent antioxidant activity.
Collagenase and elastase activity inhibition of CLME at 1,000 μg/mL was 73.2% and 79.4%, respectively. The IC50 of collagenase and elastase activity was calculated to be 460.5 μg/mL and 529.2 μg/mL, respectively, which were similar to or even superior to positive control ascorbic acid (AA) (448.3 μg/mL and over 1,000 μg/mL, respectively).
As determined with an MTT assay, the maximum permissible level for treating CLME to HaCaT keratinocytes was shown to be 100 μg/mL. The treatment with 100 μg/mL CLME decreased ROS production by 24.2% (p < 0.001), which was higher than that of AA (17.2%, p < 0.001), in 30 mJ/cm2 UVA-irradiated HaCaT cells. The treatment with 100 μg/mL CLME decreased apoptosis production by 25.9% (p < 0.001), which was higher than that of AA (22.3%, p < 0.001), in 30 mJ/cm2 UVA-irradiated HaCaT cells. The treatment with 100 μg/mL CLME increased HA production by 27.3% (p < 0.001), which was higher than that of AA (24.9%, p < 0.001), in 30 mJ/cm2 UVA-irradiated HaCaT cells.
The results obtained in this study showed that CLME had ability of decrease in ROS and apoptosis production and increase in HA production, which were even superior to AA, in UVA-irradiated HaCaT human keratinocytes. In addition, CLME had excellent antioxidant activity and inhibition of collagenase and elastase in a cell-free system. Therefore, it is likely that CLME could be used as a natural cosmetic material for the prevention and alleviation of skin photoaging.