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Protective effect of a freshwater algae Spirogyra sp. against skin aging induced by ultraviolet-B irradiation

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Abstract
Spirogyra sp. a freshwater algae, was collected during Januray to March in Kongju, South Korea. The lyophilized sample was extracted by 70% ethanol and obtained the 70% ethanol crude extract (SPE). The free radical scavenging activities and protective effects against skin aging induced by UV-B irradiation of SPE were evaluated in vitro on HaCaT cell line. The results suggests that SPE have protective effects against skin aging. Then, SPE was fractionated by organic solvets based on polarity. The ethyl actate fraction (SPEE) was found to contain a higher phenolic content than the other fractions, so it was selected for futher studies. The protective effects of SPEE against skin aging induced by UV-B irradiation were evaluated in vitro on HaCaT cell line and in vivo on zebrafish model. Anti-aging compounds of SPEE were seperated by one step using preparative centrifugal partition chromatography (CPC). This study suggests that Spirogyra sp. was rich in phenolic content and have strong radical scavenging activities and anti-UV-B irradiation activity. It has the potential to be used as a promising ingredient in cosmetic industry.
Author(s)
왕뢰
Issued Date
2015
Awarded Date
2016. 2
Type
Dissertation
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000007551
Alternative Author(s)
Wang, Lei
Department
대학원 해양생명과학과
Advisor
전유진
Table Of Contents
ABSTRACT . 1
LIST OF FIGURES . 2
LIST OF TABLES . 8
1. INTRODUCTION . 9
2. METERIALS AND METHODS . 15
2.1. Chemicals and reagents . 15
2.2. Sample collection and pre-processing . 16
2.3. Extraction of 70% crude ethanol extract 16
2.4. Sample preparation . 18
2.5. Determination of total phenolic content of samples . 18
2.6. Determination of DPPH radical scavenging activity of samples 19
2.7. Determination of alkyl radical scavenging activity of samples 19
2.8. Determination of hydroxyl radical scavenging activity of samples 20
2.9. Determination of hydrogen peroxide scavenging activity of samples 20
2.10. Determination of ABTS radical scavenging activity of SPE by on-line High Performance Liquid Chromatography (HPLC) system 21
2.11. Cell culture 22
2.12. Determination of cytotoxicity of samples in HaCaT cells 22
2.13. Determination of intracellular ROS generation induced by UV-B irradiation in HaCaT cells 23
2.14. Determination of cytoprotective effect of samples against UV-B irradiation in HaCaT cells 23
2.15. Fractionation of SPE using different solvents . 24
2.16. Determination of ABTS radical scavenging activity of SPEE by on-line HPLC system . 24
2.17. Determination of apoptotic body formation by nuclear staining with Hoechest 33342. . 25
2.18. Cell cycle analysis . 26
2.19. Origin and maintenance of parental zebrafish 26
2.20. Determination of UV-B protection effect of SPEE in vivo / in zebrafish mode 27
2.21. Optimisation the Centrifugal Partition Chromatographic solvent system 27
2.22. Seperation of active compounds from SPEE by CPC . 28
2.23. Analysis of fractions collected by CPC using HPLC . 29
2.24. NMR spectroscopy of the target compounds 29
2.25. Statistical analysis . 30
3. RESULTS AND DISCUSSION 31
3.1. Sample extraction - 70% crude ethanol extract (SPE) 31
3.2. Total phenolic content of SPE . 31
3.3. Free radical and hydrogen peroxide scavenging activity of SPE 33
3.4 . On-line HPLC analysis of SPE 39
3.5. Cytotoxicity of SPE in HaCaT cells 41
3.6. Intracellular ROS scacvenging activity of SPE in HaCaT cells . 43
3.7. Cytoprotective effect of SPE against UV-B irradiation in HaCaT cells . 45
3.8. Fractionation of SPE using different solvents . 47
3.9. Total phenolic contents of each fractions of SPEE . 49
3.10. Free radicals and hydrogen peroxide scavenging activities of SPEE . 51
3.11. On-line HPLC analysis of SPEE . 57
3.12. Cytotoxicity analysis of SPEE in HaCaT cells . 59
3.13. Intracellular ROS sacvenging activity of SPEE in HaCaT cells 61
3.14. Cytoprotective effect of SPEE against UV-B irradiation in HaCaT cells 63
3.15. Apoptotic body formation induced by UV-B irradiation in HaCaT cells . 65
3.16. Cell cycle analysis . 67
3.17. Determination of ROS generation induced by UV-B irradiation in zebrafish 69
3.18. Optimisation of the Centrifugal Partition Chromatographic solvent system 71
3.19. Seperation bioactivity compounds from SPEE by CPC 73
3.20. Analysis of the fractions collected by CPC using HPLC 75
3.21. Free radicals and hydrogen peroxide scavenging activities of the tragets fractions collected by CPC. 78
3.22.NMR spectroscopy of the target compound . 84
4. CONCLUSION 87
REFERENCES 88
ACKNOWLEDGEMENT . 94
Degree
Master
Publisher
제주대학교 대학원
Citation
왕뢰. (2015). Protective effect of a freshwater algae Spirogyra sp. against skin aging induced by ultraviolet-B irradiation
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General Graduate School > Marine Life Sciences
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