구아바 잎 추출물의 항산화 활성과 성분 분석 = Antioxidative Activity and Component Analysis of Psidium guajava Leaf Extracts

국문 초록 (Abstract)

본 연구는 구아바 잎 추출물의 항산화 성분 분석 및 tyroinase와 elastase 저해 효과에 관한 조사를 수행하였다. 추출물의 free radical (1,1-diphenyl-2-picrylhydrazyl, DPPH) 소거활성(FSC₅₀)은 50 % 에탄올 추출물(7.05 µg/mL) < ethyl acetate 분획(3.36) < 당을 제거시킨 플라보노이드 aglycone 분획(3.24) 순으로 증가하였다. Luminol-의존성 화학발광법을 이용한 Fe³⁺-EDTA/H₂O₂ 계에서 생성된 활성산소종(reactive oxygen species, ROS)에 대한 구아바 잎 추출물의 총 항산화능은 50 % 에탄올 추출물(OSC₅₀, 2.17 µg/mL) < ethyl acetate 분획(0.64) < aglycone 분획(0.39) 순으로, aglycone 분획에서 가장 큰 활성을 나타내었다. 구아바 잎 추출물에 대하여 rose-bengal로 증감된 사람 적혈구의 광용혈에 대한 억제 효과를 측정하였다. 구아바 잎 추출물의 경우 1 ~ 10 µg/mL의 농도에서 광용혈을 억제하였다. 특히 당을 제거시킨 플라보노이드 aglycone 분획은 1 µg/mL 농도에서 τ₅₀이 107.5 min으로 매우 큰 세포보호 효과를 나타내었다. 구아바 잎 추출물 중 ethyl acetate 분획의 당 제거 반응 후 얻어진 aglycone 분획은 TLC와 HPLC (360 nm)에서 1개의 피이크로 나타났으며, 그 성분이 quercetin 임을 확인하였다. 구아바 잎 추출물의 ethyl acetate 분획의 TLC 크로마토그램은 5개의 띠로 분리되었고, HPLC 크로마토그램도 5개의 피이크를 나타내었다. TLC와 HPLC의 띠와 피이크를 확인한 결과, HPLC의 5개의 피이크는 용리순서로 피이크 1 (10.32 %)은 quercetin 3-O-gentobioside, 피이크 2 (13.30 %)는 quercetin 3-O-β-D-glucoside (isoquercitin), 피이크 3 (11.34 %)는 quercetin 3-O-β-D-galactoside (hyperin), 피이크 4 (19.70 %)는 quercetin 3-O-α-L-arabinoside (guajavarin), 피이크 5 (45.33 %)는 quercetin 3-O-β-L-rhamnoside (quercitrin)로 확인되었다. 미백 및 주름억제 효과측정으로는 각각 tyrosinase 및 elastase의 활성 저해 효과를 측정하였다. Tyrosinase의 활성 저해 효과(IC₅₀)는 50 % 에탄올 추출물(149.67 µg/mL) < ethyl acetate 분획(30.67) < aglycone 분획(17.10) 순으로 나타났으며, elastase의 활성 저해 효과(IC₅₀)는 50 % 에탄올 추출물(6.60 µg/mL) < aglycone 분획(5.66) < ethyl acetate 분획(3.44) 순으로 나타났다. 이상의 결과들은 구아바 잎 추출물이 ¹O₂ 혹은 다른 ROS를 소광시키거나 소거함으로써 그리고 ROS에 대항하여 세포막을 보호함으로써 생체계, 특히 태양 자외선에 노출된 피부에서 항산화제로서 작용할 수 있음을 가리키며, 구아바 잎 성분에 대한 분석과 ethyl acetate 분획의 당 제거 실험 후 얻어진 aglycone 분획의 elastase 저해활성은 구아바 잎 추출물이 주름개선 기능성 화장품원료로서 응용 가능성이 있음을 시사한다.

다국어 초록 (Multilingual Abstract)

In this study, the antioxidative effects, inhibitory effects on elastase and tyrosinase, and component analysis of Psidium guajava leaf extracts were investigated. The free radical (1,1-diphenyl-2-picrylhydrazyl, DPPH) scavenging activities (FSC₅₀) of extract/fractions of Psidium guajava leaf were in the order: 50 % ethanol extract (7.05 µg/mL) < ethyl acetate fraction (3.36 µg/mL) < deglycosylated flavonoid aglycone fraction (3.24 µg/mL). Reactive oxygen species (ROS) scavenging activities (OSC₅₀) of some Psidium guajava leaf extracts on ROS generated in Fe³⁺-EDTA/H₂O₂ system were investigated using the luminol-dependent chemiluminescence assay. The order of ROS scavenging activities were 50 % ethanol extract (OSC₅₀, 2.17 µg/mL) < ethyl acetate fraction (0.64 µg/mL) < deglycosylated flavonoid aglycone fraction (0.39 µg/mL). Aglycone fraction showed the most prominent ROS scavenging activity. The protective effects of extract/fractions of Psidium guajava leaf on the rose-bengal sensitized photohemolysis of human erythrocytes were investigated. The Psidium guajava leaf extracts suppressed photohemolysis in a concentration dependent manner (1 ~ 10 µg/mL), particularly deglycosylated flavonoid aglycone fraction exhibited the most prominent celluar protective effect (τ₅₀, 107.5 min at 1 µg/mL). Aglycone fraction obtained from the deglycosylation reaction of ethyl acetate fraction among the Psidium guajava leaf extracts, showed 1 band in TLC and 1 peak in HPLC experiments (360 nm). One component was identified as quercetin. TLC chromatogram of ethyl acetate fraction of Psidium guajava leaf extract revealed 5 bands and HPLC chromatogram showed 5 peaks, which were identified as quercetin 3-O-gentobioside (10.32 %), quercetin 3-O-β-D-glucoside (isoquercitin, 13.30 %), quercetin 3-O-β-D-galactoside (hyperin, 11.34 %), quercetin 3-O-α-L-arabinoside (guajavarin, 19.70 %), quercetin 3-O-β-L-rhamnoside (quercitrin, 45.33 %) in the order of elution time. The inhibitory effect of Psidium guajava leaf extracts on tyrosinase were investigated to assess their whitening efficacy. Finally, their anti-elastase activities were measured to predict the anti-wrinkle efficacy in the human skin. Inhibitory effects (IC₅₀) on tyrosinase of some Psidium guajava leaf extracts was 50 % ethanol extract (149.67 µg/mL) < ethylacetate fraction (30.67 µg/mL) < deglycosylated aglycone fraction (17.10 µg/mL). Inhibitory effects (IC₅₀) on elastase of some Psidium guajava leaf extracts was 50 % ethanol extract (6.60 µg/mL) < deglycosylated aglycone fraction (5.66 µg/mL) < ethylacetate fraction (3.44 µg/mL). These results indicate that extract/fractions of Psidium guajava leaf can function as antioxidants in biological systems, particularly skin exposed to UV radiation by scavenging ¹O₂ and other ROS, and protect cellular membranes against ROS. And component analysis of Psidium guajava leaf extract and inhibitory activity on elastase of the aglycone fraction could be applicable to new functional cosmetics for smoothing wrinkles.

참고문헌 (Reference)

1 S. N. Park, Seoul National Univ. 1989

2 박수남, "함초(나문재 및 퉁퉁마디) 추출물의 항산화 작용에 관한 연구(I)" 대한화장품학회 33 (33): 145-152, 2007

3 박수남, "쇠뜨기 추출물의 항산화 작용에 관한 연구(I)" 대한화장품학회 33 (33): 61-67, 2007

4 박수남, "쇠뜨기 추출물의 성분 분석과 엘라스타제 활성 저해 효과 연구(Ⅱ)" 대한화장품학회 33 (33): 139-144, 2007

5 G. F. Vile, "UVA radiation- induced oxidative damage to lipid and protein in vitro and in human skin fibroblasts is dependent on iron and singlet oxygen" 18 : 721-, 1995

6 L. H. Kligman, "UVA induced biochemical changes in hairless mouse skin collagen: a contrast to UVB effects" 209 : 1992

7 M. Meckes, "Terpenoids isolated from Psidium guajava hexane extract with depressant activity on central nervous system" 10 : 600-, 1996

8 S. N. Park, "Skin aging and antioxidant" 23 : 75-, 1997

9 M. Wlaschek, "Singlet oxygen may mediate the ultraviolet A in induced synthesis of interstitial collagenase" 104 : 194-, 1995

10 R. M. Tyrrell, "Singlet oxygen involvement in the inactivation of cultured human fibroblast by UVA and near visible radiations" 49 : 407-, 1989

11 K. Scharffetter-Kochanek, "Singlet oxygen induces collagenase expression in human skin fibroblasts" 331 : 304-, 1993

12 X. Lozoya, "Quercetin glycosides in Psidium guajava L. leaves and determination of a spasmolytic principle" 25 (25): 11-, 1994

13 C. S. Foote, "Photosensitized oxidation and singlet oxygen; consequences in biological systems" Acdemic press 2 : 85-, 1976

14 K. Scharffetter-Kochanek, "Photoaging of the connective tissue of skin: its prevention and therapy, antioxidants in disease mechanism and therapy" 38 : 639-, 1997

15 박수남, "O2으로 유도된 사람 적혈구의 광용혈에 있어서 대두의 아이소플라본인 제니스테인의 보호작용" 한국식품과학회 35 (35): 510-518, 2003

16 L. M. Pery, "Medicinal Plants of East and Southeast Asia" MIT Press 1980

17 H. Arima, "Isolation of antimicrobial compounds from guava (Psidium guajava L.) and their structural elucidation" 66 : 1727-, 2002

18 I. Emerit, "Free radicals and aging of the skin, eds L. Emerit and B. Chance, Free radicals and aging, 328, Basel, Birkhauser (1992)"

19 E. H. Witt, "Evidence for UV light as an oxidative stressor in skin" Oxidative Stress in Dermatology 29-, 1993

20 T. Okuda, "Ellagitannins of the casuarinaceae, stachyuraceae and myrtaceae" 21 (21): 2871-, 1982

21 R. G. Belemtougri, "Effects of two medicinal plants Psidium guajava L. (Myrtaceae) and Diospyros mespiliformis L. (Ebenaceae) leaf extracts on rat skeletal muscle cells in primary culture" 7 (7): 56-, 2006

22 A. Oikarinen, "Connective tissue alterations in skin exposed to natural and therapeutic UV-radiation" 2 : 15-, 1985

23 S. Begum, "Chemical constituents from the leaves of Psidium guajava" 18 : 135-, 2004

24 S. N. Park, "Antioxidative properties of baicalein, component from Scutellaria baicalensis Georgi and its application to cosmetics (I)" 14 (14): 657-, 2003

25 J. W. Choi, "Antioxidative and cellular protective effects of Jeju plant extracts against reactive oxygen species (Ⅰ)" 32 (32): 181-, 2006

26 J.W. Choi, "Antioxidative and cellular protective effects of Jeju native plant extracts against reactive oxygen species (I), J. Soc. Cosmet" 32 (32): 181-, 2006

27 J. Y. Kim, "Antioxidative and antiagiang effect of Jeju native plant extracts (Ⅱ)" 32 (32): 181-, 2006

28 S. Kimura, "Acceleration of fibrinolysis by the N-terminal peptide of alpha 2-plasmin inhibitor" 66 (66): 157-, 1985

29 A. Oikarinen, "A biochemical and immunohistochemical study of collagen in sun-exposed and protected skin" 6 : 24-, 1989