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Abstract 6

Abbreviations 14

I. 서론 17

II. 이론적 배경 25

II-1. 추출법 25

II-1-1. 용매 추출법 29

II-1-2. 초임계 유체를 이용한 추출 30

II-2. 추출물의 분리 31

II-2-1. 크로마토그래피 31

II-3. 활성성분의 구조 분석 32

II-4. 미백 메커니즘 34

II-4-1. 멜라닌 34

II-4-2. Melanogenic 조절 단백질 38

II-4-3. 멜라닌 생성 자극인자 38

II-5. 활성성분 안정화 방법 40

II-5-1. 일반적인 에멀젼 41

II-5-2. 액정 44

II-5-3. 리포좀 46

II-5-4. 나노에멀젼 46

II-5-6. 전상온도 유화법 50

II-5-7. 나노에멀젼을 이용한 활성성분의 피부 전달 61

II-5-8. 제조장비 및 특성 63

II-6. 올레아놀산의 특성 65

III. 실험재료 및 방법 68

III-1. 실험 재료 68

III-2. 실험방법 74

III-2-1. 올레아놀산의 추출 및 분리정제 74

III-2-2. 세포배양 74

III-2-3. 세포생존율과 멜라닌 분비량의 측정 75

III-2-4. 티로시나아제 활성도 측정 75

III-2-5. 멜라닌 합성 단백질 발현 저해 효과 분석 76

III-2-6. mRNA 유전자 발현 저해 효과 77

III-2-7. 통계처리 79

III-2-8. 나노에멀젼의 제조 79

III-3. 기기 및 특성분석 90

III-3-1. 올레아놀산의 분석 90

III-3-2. 나노에멀젼의 분석 90

III-3-3. 올레아놀산의 봉입 효율 및 역가 분석 91

III-3-4. 나노에멀젼의 안정성 평가 92

III-3-5. 올레아놀산의 in-vitro 경피흡수 거동 93

III-4. 인체적용시험 93

IV. 결과 및 고찰 94

IV-1. 올레아놀산의 추출 및 분리 94

IV-2. 올레아놀산의 비교 분석 97

IV-3. 올레아놀산의 미백 활성 102

IV-3-1. 올레아놀산의 세포생존률과 멜라닌 분비량 측정 102

IV-3-2. 티로시나아제 활성 105

IV-3-3. 멜라닌 합성 단백질발현에 대한 올레아놀산의 효과 108

IV-3-4. 멜라닌 합성에 관여하는 유전자의 발현 115

IV-4. 올레아놀산을 함유한 폴리글리세릴 나노에멀젼의 미백 화장품으로서의 응용성 122

IV-4-1. 레시틴과 폴리글리세릴 나노에멀젼의 비교 122

IV-4-2. 경피흡수율 측정 비교 133

IV-4-3. 폴리글리세릴 나노에멀젼의 임상시험 결과 135

V. 결론 139

참고문헌 142

List of Tables

Table 1. Extraction Method from Plants 26

Table 2. Isolation and Characterization of Active Ingredients 33

Table 3. Stabilizing Method for Active Ingredients 42

Table 4. Relationship between Particle Sizes and Optical Properties of... 49

Table 5. PIT0 and SF of Emulsifier with CAPICO Method(이미지참조) 58

Table 6. ACN of Ester with CAPICO Method 59

Table 7. Materials for in-vitro Test of Oleanolic Acid 71

Table 8. Materials for Lecithin Nanoemulsion 72

Table 9. Materials for Polyglyceryl Nanoemulsion 73

Table 10. The PCR Primer Sequences 78

Table 11. Formulation for Lecithin Nanoemulsion 81

Table 12. PIT0 and SF of polyglyceryl-10 Oleate with ACN(이미지참조) 83

Table 13. PIT0 and SF of polyglyceryl-2 Oleate with ACN(이미지참조) 85

Table 14. Materials for Polyglyceryl Nanoemulsion 87

Table 15. Comparison of Oleanolic Acid from Fragaria ananassa... 98

Table 16. Result of Decreasing Melanin Levels by Eye Evaluation 136

Table 17. Result of Decreasing Melanin Levels by Mexameter 136

List of Figures

Figure 1. Synthetic pathway of melanin. 37

Figure 2. Signalling pathway of melanocortin. 39

Figure 3. Schematic manufacturing process of liposomes. 47

Figure 4. Compare transparency of PIT emulsion with traditional... 52

Figure 5. Schematic graph of PIT emulsification method. 53

Figure 6. Related graph with temperature and electric conductivity... 54

Figure 7. Schematic graph of determination for emulsifier's PIT0...(이미지참조) 55

Figure 8. Schematic graph for ester using PIT0 and SF.(이미지참조) 57

Figure 9. Determination of PIT0 and SF for polyglyceryl-10 oleate.(이미지참조) 84

Figure 10. Determination of PIT0 and SF for polyglyceryl-2 oleate.(이미지참조) 86

Figure 11. TLC fractions of extraction from the calyx of Fragaria... 95

Figure 12. ¹H NMR spectrum of oleanolic acid. 99

Figure 13. ¹³C NMR spectrum of oleanolic acid. 101

Figure 14. Inhibitory effect of oleanolic acid on melanogenesis in... 103

Figure 15. Decreasing secreted melanoma cell effect of with oleanolic... 104

Figure 16. Decreasing intracellular melanin levels effect of with... 106

Figure 17. Inhibited effect of cellular tyrosinase activity with... 107

Figure 18. Effect of oleanolic acid on intensities of tyrosinase protein... 109

Figure 19. Effect of oleanolic acid on intensities of TRP-1 protein... 110

Figure 20. Effect of oleanolic acid on intensities of TRP-2 protein... 111

Figure 21. Effect of oleanolic acid on intensities of MITF protein... 113

Figure 22. Intensities of tyrosinase, TRP-1, TRP-2, and MITF... 114

Figure 23. Effect of oleanolic acid on tyrosinase mRNA levels in... 116

Figure 24. Effect of oleanolic acid on TRP-1 mRNA levels in B16... 117

Figure 25. Effect of oleanolic acid on TRP-2 mRNA levels in B16... 118

Figure 26. Effect of oleanolic acid on MITF mRNA levels in B16... 119

Figure 27. Intensities of tyrosinase, TRP-1, TRP-2, and MITF... 121

Figure 28. C-SEM image of lecithin nanoemulsion. 123

Figure 29. HPLC analysis of capsulation efficiency of oleanolic... 124

Figure 30. Particle size analysis of lecithin nanoemulsion. 125

Figure 31. C-TEM image of polyglyceryl nanoemulsion. 127

Figure 32. HPLC analysis of capsulation efficiency of oleanolic... 128

Figure 33. Particle size analysis of polyglyceryl nanoemulsion. 129

Figure 34. Stability of polyglyceryl nanoemulsion 130

Figure 35. HPLC analysis of oleanolic acid in polyglyceryl nano... 131

Figure 36. Particle size analysis of oleanolic acid in polyglyceryl... 132

Figure 37. Skin penetrating ratio in 12 hr 134

Figure 38. Change of melanocyte number treated by oleanolic acid... 137

Figure 39. Picture of skin test result treated with polyglyceryl... 138

List of Schemes

Scheme 1. Principles of extraction from plant. 27

Scheme 2. Scheme of standard compound isolation from plant extract. 28

Scheme 3. Flowchart of formulation design for CAPICO. 60

Scheme 4. Structure of oleanolic acid. 66

Scheme 5. Manufacture process of lecithin nanoemulsion. 82

Scheme 6. Scheme of manufacture process of polyglyceryl nano... 89

초록보기

 Natural products with non-toxic and environmentally friendly properties are good sources for skin whitening and brightening cosmetic agents. Strawberries (Fragaria ananassa) and their parts are used as cosmetic agents because they contain high levels of bioactive substances. In the present study, this researcher isolated and identified compounds from Fragasia ananassa calyx. The representative compound of them is oleanolic acid. Oleanolic acid has multiple biological activities, including anti-tumor, anti-angiogenic, anti-inflammatory, anti-oxidant, and pro-apoptotic effects. However, the influence of oleanolic acid on melanin synthesis in B16 F10 melanoma cells has not been investigated.

This researcher investigated the effect of oleanolic acid on melanin biosynthesis in B16 F10 melanoma cells stimulated with α-melanocyte stimulating hormone (α-MSH). Oleanolic acid mediated melanogenesis inhibition was studied by measuring intracellular and secreted melanin levels and by using Western blot and semiquantitative reverse transcriptation polymerase chain reaction (RT-PCR) analysis.

Oleanolic acid suppressed melanin release and expression, resulting in a significant dose-dependent decrease in secreted and intracellular melanin levels and cellular tyrosinase activity. Furthermore, it inhibited the expression of melanogenesis associated factors, including tyrosinase, tyrosinase-related proteins-1 (TRP-1) and tyrosinase-related proteins-2 (TRP-2), and microphthalmia associated transcription factor (MITF), in α-MSH stimulated B16 F10 melanoma cells. The results of the present study can contribute to the development of cosmetic agents for the skin whitening and brightening. These effects of oleanolic acid will likely have a wide range of applications in the cosmetic industry and/or clinical practice in the future.

In this study, oleanolic acid was stabilized by polyglyceryl surfactant from natural origin with only a simple stirring operation, and compared with lecithin nanoemulsion that was manufactured with high cost facility. The transdermal transition rate of 0.4% oleanolic acid polyglyceryl nanoemulsion was 94.90%, and it was similar to lecithin nanoemulsion of 91.26%. Above 80% oleanolic acid in polglyceryl nanoemulsion was penetrated to skin within 4hours compare with that 60% of oleanolic acid in lecithin nanoemulsion penetrated. It means polyglyceryl nanoemulsion has quickly release property comparing with slow release property of lecithin nanoemulsion. In the result of in-vivo clinical trial test, the decreased numerical value of melanocyte treated with 0.4% oleanolic acid contained polyglyceryl nanoemulsion was 43.87 at 8th weeks. This result was 57.36% higher than non-treated control as 27.89 at 8th weeks.

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