The Evaluation on the Effectiveness as a Cosmetic Material of Ascidian shell Extract Using Zebrafish model

Abstract

The extracts of AS contain in alloxanthin, halocynthiaxanthin, astaxanthin and 13 kinds of carotenoids. The aim of the study was to assess the anti-oxidant activity and cell viability of AS. The anti-oxidant activity was determined by using DPPH radical inhibition activity and superoxide dismutase (SOD)-like activity. The results of cell viability assay showed that the extracts from AS were cytotoxic at concentrations above $5.0mg/m{\ell}$. This study was designed to examine inflammation induced by LPS, protection effect by UVB and the toxicity of Ascidian shell extract(ASE) as a functional cosmetic ingredient. Evaluation of embryo toxicity resulted in embryo coagulation and mortality when treated at 5.0, 10.0, $20.0mg/m{\ell}$. At the lowest concentration of $1.0mg/m{\ell}$, hatchability resulted in 100.0 % rate. The results of arrhythmia measurement in larvae showed similarity to the evaluation of embryo toxicity. This result demonstrated that toxicity is present at concentrations greater than $5.0mg/m{\ell}$. The protective effect of ASE on LPS and UVB-induced in the zebrafish was investigated. Intracellular reactive oxygen species(ROS) generated by the exposure of zebrafish to LPS, UVB-radiation were significantly decreased after treatment with ASE at $0.1mg/m{\ell}$. As a result, ASE similarly reduced UVB-induced ROS generation and cell death in live zebrafsih. Therefore, it is suggested that ASE has anti-Inflammatory effects and can possibly be used as a functional substance for skin protection in the future.

Fig. 1. The effects of Ascidian shell extract on the growth of HaCaT cell line. Serum-starved HaCaT cells were incubated with Ascidian shell extract for 24 hours at the indicated concentration of Ascidian shell extract. The cell viability was estimated using WST-1 assay.

Fig. 2. The effects of Ascidian shell extract on the growth of B16F10 cell line. Serum-starved B16F10 cells were incubated with Ascidian shell extract for 24 hours at the indicated concentration of Ascidian shell extract. The cell viability was estimated using WST-1 assay.

Fig. 3. DPPH radical scavenging activity(%) of Ascidian shell extract.

Fig. 4. SOD-like activity (%) of Ascidian shell extract.

Fig. 5. Coagulation rate (%), and Hatching rate (%) of Ascidian shell extract.

Fig. 6. Arrhythmia test of Ascidian shell extract on zebrafish larvae. The heartbeat of zebrafish larvae after having been treated with Ascidian shell extract were counted.

Fig. 7. Protective effect of ASE against UVB-induced ROS generation in zebrafish. The embryos were exposed to UVB (50 mJ/㎠) and treated with ASE. After incubation, the embryos were stained with DCF-DA and intracellular ROS were detected by spectrofluorometry (A) and fluorescencemicroscopy (B).

Fig. 8. Protective effect of ASE against UVB-induced cell death in zebrafish. The embryos were exposed to UVB (50 mJ/㎠) and treated with ASE. After incubation, the embryos were stained with acridine orange and cell death was detected by spectrofluorometry (C) and fluorescence microscopy (D).

Fig. 9. Protective effect of ASE against LPS-induced ROS production in zebrafish. After incubation, the embryos were stained with DCF-DA and intracellular ROS were detected by spectrofluorometry (E) and fluorescence microscopy (F).

Fig. 10. Protective effect of ASE against LPS-induced cell death in zebrafish. After incubation, the embryos were stained with acridine orange and cell death were detected by spectrofluorometry (G) and fluorescence microscopy (H).