Apoptosis inhibitory, pharmacological activities of Magnolia biondii and an application for anti-inflammatory cosmetic : 신이화의 apoptosis 억제, 약리활성 및 항염증 화장품 응용에 관한 연구

다국어 초록 (Multilingual Abstract)

In order to apply Magnolia biondii extracts to Korean herbal cosmetics, pharmacological activities, safety and stability of cosmetics added with Magnolia biondii were tested. In detail, pharmacological, anti-cancer and anti- inflammatory effects of the extracts of Magnolia biondii were evaluated under different extraction conditions such as hot water and 80% ethanol. In the DPPH-scavenging assay, 56.7% scavenging ability of Magnolia biondii water extract was higher than 42.4% scavenging ability of Magnolia biondii ethanol extract at 100ppm. From the measurement on xanthine oxidase inhibition of Magnolia biondii water extract and Magnolia biondii ethanol extract were 76.9% and 61.3% at 1,000ppm concentration, respectively. In the test of super oxide dismutase (SOD)-like activity, ascorbic acid showed quit more activity with 99.3% in 5,000ppm, while Magnolia biondii water extract and Magnolia biondii ethanol extract were 84.2% and 43.8%, respectively. In the whitening effect, water extract and ethanol extract of Magnolia biondii showed 62.0% and 42.6% at 5,000ppm, respectively. 98.2% nitrite scavenging ability of Magnolia biondii water extract was higher than 92.1% nitrite scavenging ability of Magnolia biondii ethanol extract at 1,000ppm concentration. Magnolia biondii extract were showed cytotoxic effects in murine melanoma B16F10. In anti-inflammatory test, Magnolia biondii extract significantly inhibited dose-dependently generation of nitric oxide and iNOS protein which was LPS-induced macrophage Raw 264.7. In the Apoptosis test of HaCaT, Magnolia biondii extract inhibited generation of DNA fragmentation, caspase-3,-8, TUNEL assay on UVB(50mJ/cm2) with a dose-dependent manner. The results of stability test showed that cosmetics containing the water extract and ethanol extract of Magnolia biondii were very stable for both temperature and artificial sun lamp. In addition to, pH and viscosity of emulsion containing Magnolia biondii extracts were also stable during 28days. From the result of human patch test, emulsion containing Magnolia biondii extracts confirmed safety. These results indicate that the extract of Magnolia biondii has anti-inflammatory activities as a cosmeceuticals compared to commercials.

국문 초록 (Abstract)

신이화를 한방 화장품에 응용하고자 약리활성 및 화장품 첨가시 안정성 및 안전성에 관하여 검토하였다. 신이화의 추출조건에 따른 약리활성, 항암, 항염증 측정을 하기 위하여 신이화를 열수, 80%에탄올로 추출하였다. 신이화 추출물의 전자공여능 측정 결과 100ppm에서 열수추출물은 56.7%, 에탄올 추출물은 42.4% 이상의 전자공여능을 나타내었다. Xanthine oxidase 저해능은 500ppm의 농도에서 열수추출물은 76.9%, 에탄올 추출물은 61.3%의 효과를 나타내었다. SOD 유사활성 측정결과는 5,000ppm의 농도에서 대조군 ascorbic acid 99.6%에 비하여, 열수추출물, 에탄올추출물 모두 84.2%, 43.8%로 낮게 나왔다, 미백에 관여하는 tyrosinase 저해활성을 측정한 결과 시료농도 500ppm에서 열수추출물은 62.0% 나타내었으며, 에탄올 추출물은 42.6% 효과를 나타내었다. 아질산염 소거능 실험에서는 1,000ppm의 신이화 열수추출물이 98.2%의 효과를 나타내었고, 에탄올 추출물에서는 92.1% 효과를 나타내었다. Mouse melanoma B16F10에 대한 성장 억제능을 확인할 수 있었다. 항염 실험에서 신이화 추출물이 LPS로 자극된 Mouse macrophage RAW 264.7 cells에서 nitric oxide, iNOS을 농도 의존적으로 저해하였다. human skin keratinocyte HaCaT cells 의 UVB(50mJ/cm2) 실험결과 DNA fragmentation, caspase-3, -8, TUNEL assya 실험모두 농도 의존적으로 손상도가 감소하였으며, 화장품 안정성 측정결과 온도안정성과 광안정성 모두 안정함을 나타내었고, 28일 동안 측정한 pH, 점도에서도 수치상의 큰 변화 없이 안정함을 나타내었다. 신이화를 첨가한 화장품의 패치 테스트 결과 실험자 모두에게 음성 반응이 나타나 피부에 안전성을 확인 할 수 있었다. 신이화 추출물을 이용하여 실험한 결과 항염증 화장품 소재로서의 기능성을 확인할 수 있었다.

목차 (Table of Contents)

• Ⅰ. Introduction = 1
• Ⅱ. Materials and Methods = 3
• 1. Materials = 3
• 1) Plant Materials = 3
• 2) Preparation of Mognolia biondii extracts = 3
• 3) Chemicals, Reagents and instrumental analysis = 5
• 2. Methods = 7
• 1) Antioxidation activity Test = 7
• (1) DPPH scavenging assay = 7
• (2) Xanthine oxidase inhibition effect = 8
• (3) Superoxide dismutase (SOD)-like activity = 9
• 2) Nitrite-scavenging ability = 10
• (1) Nitrite-scavenging ability = 10
• 3) Whitening effect = 11
• (1) Tyrosinase inhibition assay = 11
• 4) Antimicrobial susceptibility test = 12
• (1) Bacterial strains = 12
• (2) Microbial culture = 12
• (3) Agar disc diffusion method = 13
• 5) Anti-cancer activity = 14
• (1) Cell lines = 14
• (2) Cell culture = 15
• (3) Antitumor activity on MTT assay = 15
• (4) Cell viability on MTT assay = 16
• 6) Anti-inflammatory effect = 17
• (1) Inhibition rate of Nitric oxide in macrophage cells = 17
• (2) iNOS analysis with western blot = 18
• 7) Apoptosis of HaCaT = 19
• (1) Magnolia biondii extract on DNA fragmentation patterns in HaCaT cells = 19
• (2) Protein analysis of western blot = 21
• (3) Change modality of HaCaT cell with TUNEL assay = 22
• 8) Cosmetic manufacturing method = 23
• (1) Manufacturing method of cream = 23
• 9) Stability test of cream = 24
• (1) Measurement of pH = 24
• (2) Measurement of viscosity = 24
• (3) Incubation (0, 25, 40 ℃) test = 25
• (4) Cycle chamber test = 25
• (5) Photo stability = 25
• 10) Safety test of human skin = 27
• (1) Patch test = 27
• 11) Statistical analysis = 28
• Ⅲ. Results and Discussion = 29
• 1. Result of antioxidation activity Test = 29
• 1) DPPH scavenging assay = 29
• 2) Xanthine oxidase inhibition effect = 31
• 3) Superoxide dismutase (SOD)-like activity = 33
• 2. Result of nitrite-scavenging ability = 35
• 1) Nitrite-scavenging ability = 35
• 3. Result of whitening effect = 37
• 1) Mushroom tyrosinase inhibition effect = 37
• 4. Result of antimicrobial susceptibility test = 39
• 1) Agar disc diffusion method = 39
• 5. Result of anti-cancer activity = 41
• 1) Antitumor activity on MTT assay = 41
• 2) Cell viability on MTT assay = 43
• 6. Result of anti-inflammatory effect = 45
• 1) Inhibition rate of Nitric oxide in macrophage cells = 45
• 2) iNOS analysis with western blot = 47
• 7. Result of apoptosis of HaCaT = 49
• 1) Magnolia biondii extract on DNA fragmentation patterns in HaCaT cells = 49
• 2) Protein analysis of western blot = 51
• 3) Change modality of HaCaT cell with TUNEL assay = 53
• 8. Result of stability test = 55
• 1) Measurement of pH = 55
• 2) Measurement of Viscosity = 55
• 3) Incubation (0, 25, 40 ℃) test = 59
• 4) Cycle chamber test = 59
• 5) Photo stability = 61
• 9. Safety test of human skin = 63
• 1) Patch test = 63
• Ⅳ. References = 65
• Abstract = 71
• 초록(국문) = 73