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1 ª Ÿ (Korean J. Medicinal Crop Sci.) 19(3) : (2011) 고로쇠수액나노입자의항산화활성및미백효과의증진 ½ *Á *Á **Á½w ***Á½ x***á x***á **** x ****Á **Á x *, ** * w w œw, ** w œw, *** y t, **** l Enhancement of Antioxidant Activities and Whitening Effect of Acer mono Sap Through Nano Encapsulation Processes Ji Seon Kim*, Yong Chang Seo*, Woon Yong Choi**, Hack Soo Kim***, Bo Hyeon Kim***, Dae Hyeon Shin***, Chang Soon Yoon****, Hye Won Lim****, Ju Hee Ahn** and Hyeon Yong Lee*, ** *Medical & Bio-Material Research Center and College of Bioscience and Biotechnology, Kangwon National University, Chuncheon , Korea. **College of Bioscience and Biotechnology, Kangwon National University, Chuncheon , Korea. ***SOMANG Cosmetics, Namdong-gu, Incheon , Korea. ****Shebah Biotech Co., Hallym Business Incubator/Industry-University, Coop Center #301, Chuncheon , Korea. ABSTRACT : In this study, we investigated antioxidant activities and whitening effects of Acer mono sap by encapsulation of nanoparticles. Acer mono sap was through ultra high pressure process and then encapsulated by lecithin. Nano-encapsulated The nanoparticles of Acer mono sap showed highest free radical scavengering effect as 89.7% in adding sample (1.0 / ), compared to sap of non-encapsulation. It was showed strong inhibition effect on melanin production test by Clone M-3 cells as 47.8%. High inhibitory of tyrosinase was also measured as 85.8% by adding lecithin nano-particle of 1.0 /. The nano-particles also showed 14.8% of low cytotoxicity against human normal fibroblast cells in adding 1.0 / of the highest concentration. These results indicate that Acer mono sap may be a source of cosmetic agents capable of improving whitening effect and antioxidant activites. Key Words : Acer mono Sap, Ultra High Pressure, Antioxidant, Whitening Effect v, ü w v ü v. tv» d sü w» mw tv ƒ s v ùküš,, l v yw» w, w e Á» x wš v y y j» ùkü» w (Maeda and Fukuda, 1991). w z w, tyrosinase tyrosine» w dihydroxyindole y w (Aroca et al., 1993). x tyrosinase y ƒ y w š, tyrosinase w hydroquinone, 4-hydoroxyanisole, kojic acid, azelaic acid ù, w (Tomita et al., 1990; Cabane et al., 1994; Jeong et al., 1995; Hwang et al., 2002). w v», y y ƒ ùkü. v w wš y w v Ÿ y x w w. ü v w œ w ñ ù yw w y»» ü» w, w y ƒ,» w y p ƒ ü Corresponding author: (Phone) ( ) hyeonl@kangwon.ac.kr Received 2011 May 12 / 1st Revised 2011 June 14 / 2nd Revised 2011 June 21 / Accepted 2011 June

2 ½ Á Á Á½w Á½ xá xá Á x Á Á x ƒw y s,,, DNA, z T s m g, qk y y, xx, y ƒ j (Park et al., 2007; Halliwell, 1991). w, y ƒ ü w y» w w (Park, 1997). w ü y, y w y y w w w z ƒ d. w w w y» w w ƒ y w š. š ù tš 100~1,800 m w tù ù w,, sw. ù š ù, w š w üw w. ù š ù (Acer mono), š ù (A. mono for. rubripes), š ù (A. dkamotoanum), š ù (A. truncatum), ¼š ù (A. mono for. dissectum), š ù (A.mono var. savatieri), š ù (A. mono var. horizontale), š ù (A. mono for. connivens), lš ù (A. mono var. ambiguum) 9 t wš (Lee, 1982). š ù e w wš t. ü w ew ü š tù š ù t, ù ù, ù, ù, ù, ù (Lee et al., 2010). 97%ƒ š ù (Korea Forest Service, 2002). ù ( Š ) ù š (Ahn, 1975,) w ƒ š ù š ù w ƒ y, w wš š (Kim et al., 1991; Yoon et al., 1992; Ahn, 1975). ù š ù w ¾š ƒwš w yƒ š ù ew w ƒ w., 30~40 l ƒ ww ù, š ù ƒ wš 2002 ƒ xƒ ƒ y w š. š t Ca, Mg, K, Na» t w, w y z z ù (Kim, 2005). w sü z w, w y ùkü» w ù s». ù l (10 9 ) f yw p ùkü š, w p y w» xw w» (Kim, 2006). ù enw» y mw ƒ», p 100 w t s w w s w ey ƒw. ù üv s e nw». s ü en z w w p s w s xk y ù wš g w en y ùküš w ƒ w š (Kim and Kwak, 2004)., sü enƒ w š p s w ù yw s ü enw y ƒ q. w š ù š mw» y t ƒ mwš, ù ƒ w y w» w» wš ww. 1. s Human dermal fibroblasts CCD-986sk w s w(kclb) l k w. w melanocyte Clone-M3 (KCLB No ) w s w l k w. w s w l w CCD- 986sk Clone-M3 cell ƒƒ DMEM RPMI % fetal bovine serum (FBS, Hyclone), 1% penicillinstreptomycin ƒw 37æ, 5% CO2 w w w. x Sigma Chemical w w. 2. x x š, š ~4 x x w. œ w ù ù y w w w. œ 12, ¾ 15 m w. œ e l 1m ü w (Moon and 192

3 š ù w y y z Kwon, 2004). w š þ k w š x w w w w. 3. ù y œ v j ƒ w lecithin 50 chloroform (minimum, 99%, USA)» w multilayer x k. ƒ w y k z layerƒ x v j š š 50 š, q» (VCX-500, SONIC, USA) w 20 2 ³ y g ù w (Kang et al., 2005). 4. ù d š ù j» ³, j» s d w DLS (Dynamic light scattering, Brookhaven Instrument Co.) w. TEM» w phosphotungstic acid solution w negative staining w. ù ƒ z, formvar/carbon gq grid r EFTEM (LEO 912AB OMEGA, Carl Zeiss, Germany), 120 kv w (Saxena et al., 2005). 5. s d s 3-(4,5-dimethythiazo-2-yl)-2,5-dipheny-tetrazoliumbromide (MTT) w s d w Mosmann xw w (Mosmann, 1983). CCD-986sk s 2 104cells/well 96 well plate w z, ƒ well 70-80% confluency n w CO2» 24 w. MTT (5 /ml) ƒwš 4 z mw d w, 10 acid-isopropanol (0.04 N HCl in isopropanol) ƒw z t formazan w microplate reader (Molecular Devices, Sunnyvale, CA, USA) 565 Ÿ d w. s MTT w formazan j k y w w. 6. Tyrosinase z k Tyrosinase z dopachrome w d w. 150 mushroom tyrosinase-150 unit, mm L-tyrosine, M hepes buffer (ph 6.8), š 300 ethanol y (1 / ) z 15 z ƒƒ Ÿ 475 d w r. Tyrosinase w. Tyrosinase Inhibition (%) = (D - C) - (B - A) / (D - C) 100 A B ƒƒ ƒ ƒ z Ÿ, C D ƒƒ (» ) z Ÿ. tyrosinase z 100 ùkú w w, 0 x w w w. 7. Clone M-3 s l melanin d melanocyte Clone-M3 s melanin w. Melanin tv» d w melanocyte ü» melanosome w ƒ Ÿ Ÿ d w w (Lim et al., 2006). Clone M-3 s z 3 wš w s PBS wš, ƒ well 1 1 N NaOH ƒw z w s ww melanin ù w, ƒ Ÿ ƒ q t ƒ Ÿ, 400 Ÿ d w w. 8. y d y š w ù mm sodium phosphate buffer (2.5, ph 6.6) 1% potassium ferricyanide 2.5 yw k z yw 50æ 20 incubation k 2.5 trichloroacetic acid (10%, w/v) ƒw 650 g 10 w. w 5 k 5 1% ferric chloride 1 ƒ k z UV -vis Spectrophotometer (852A Diode Array Spectrophotometer, Hewlett Packard) w 700 Ÿ d w (Oyaizu, 1986). 9. DPPH y d š ù w y y d k 0.5 ƒƒ 50, 100, 250, 500 / w, L-ascorbic acid w. z 0.15 mm DPPH 2 ƒw.» w 10 vortexingw, 30 k. û DPPH d w» w UV-vis spectrophotometer w 517 Ÿ d w. 10. m š š ù v y z Microsoft excel student t-test w w. w ƒ y x w 3 193

4 ½ Á Á Á½w Á½ xá xá Á x Á Á x Fig. 1. The nanoparticles of lecithin by TEM. TEM micrograph of nanoparticle from Acer mono sap by lecithinencapsulation. Scale of bar is 200. x w Mean ± SD t w ƒ s³e student t-test w p = 0.05 w. š 1. ù y w y y z w š ù s w W/ O xk s. š ù x k y w» w TEM w w (Fig. 1). ù negative staining w w x. w phosphotungstic acid j sw ù w. ƒ ù y w» w DLS d w Fig. 2 ù kü. DLS d, p s w š ù 150~200 x. n ƒ ƒ w 100~500 ü, ³ w j» swš š ù ƒ. s³ 200 tv j» š w ù z ƒ w x š q. 2. ù s x š ù 1.0 / w s w s mw. Fig. 3 CCD-986sk s w ùkü š 1.0 / p š ù ƒ 14.8% š 22.1% w û s. 20% w ùkù š ù s HEK293 s w e Fig. 2. Size distribution of Acer mono sap nanoparticles with lecithin using dynamic light scattering (DLS). Fig. 3. Cytotoxicity of the Acer mono sap and nano particle of Acer mono sap at 1.0 /. Mean values±sd from triplicate separated experiments are shown. Mean with difference letter (A-B) within same concentration are significantly different at p < 0.05 and mean with difference letter (a-c) within same sample are significantly different at p < LE: Acer mono sap by lecithinencapsulation, sap: Acer mono sap. s w 40% w p s w š ù s j w, w (Ji et al., 2008), w. 3. Tyrosinase z k Tyrosinase v w z tyrosinase wy d w š ù z dw. ù ƒƒ w tyrosinase w y Fig. 4 ùkü., x ƒ ƒw tyrosinase w y ƒw w, 1 / 85.8% w ùkþ. ƒ ù z 194

5 š ù w y y z Fig. 4. Tyrosinase inhibitory activity of the nano-particl of Acer mono sap. Mean values±sd from triplicate separated experiments are shown. Mean with difference letter (A) within same concentration are significantly different at p < 0.05 and mean with difference letter (a-d) within same sample are significantly different at p <0.05. LE: Acer mono sap by lecithin-encapsulation, sap: Acer mono sap. 300 / ƒ ù tyrosinase w 60% ù, š ù 200 / 73.6% w ùkü (Lee et al., 2010).š ƒ s p w tryosinase wy 5% ùkù, p y x w ee q. w z tyrosinase y z ƒ š, š p ù» w y ƒeƒ. 4. Clone M-3 s l melanin d š ù Clone M-3 s melanin e w» w 100% v w d w. Melasolv w 59% wy. Fig. 5 š ù 1.0 / 47.8% w y š 36.9% wy. š ƒ s p w w 2% ùkù. š ù, y ù œ mw s ƒ z w ùkü. mw ù œ mw z s ü n ƒ y w. Fig. 5. Melanin contents inhibitory activity of the nanoparticles of Acer mono sap in Clone M-3 cells. Mean values±sd from triplicate separated experiments are shown. Mean with difference letter (A-C) within same concentration are significantly different at p < 0.05 and mean with difference letter (a-b) within same sample are significantly different at p < LE: Acer mono sap by lecithinencapsulation, sap: Acer mono sap; lecithin: only lecithin particles. 5. y š ù ƒƒ w ƒw z y j y d w Table 1. y d Ÿ e e ƒ y ùkü, y ƒ Ÿ eƒ ùkù. x ƒ ƒw y ƒw w, 1.0 / p ù O.D , š O.D d. p s w š ù ù y š y w ù y, š z y p l p š z 1.5 / 0.8 Ÿ w y ùkü (Kim et al., 2003), š ù ƒ w y y ƒ, ù œ w z y ý». 6. DPPH y d DPPH e d w y y d w ù. DPPHƒ ascorbic acid, tocopherol, polyhydroxy w yw w ù ƒ w x w y k w d w š p ù œ z 1 / 89.7% e 195

6 ½ Á Á Á½w Á½ xá xá Á x Á Á x Table 1. Reducing power of Acer mono sap and its nanoparticles by lecithin. Reducing power (O.D.) Sample Concentration (mg/ml) LE 1.566±0.035 Aa 1.620±0.065 Aa 1.698±0.046 Aa 1.715±0.048 Ab 1.885±0.050 Ac * sap 1.570±0.040 Aa 1.618±0.051 Aa 1.658±0.046 Ab 1.705±0.049 Ab 1.814±0.014 Bb Lecithin 0.340±0.065 Ba 0.375±0.014 Ba 0.315±0.065 Ba 0.340±0.048 Ba 0.321±0.049 Ca *Mean values±sd from triplicate separated experiments are shown. Mean with difference letter (A-C) within same concentration are significantly different at p < 0.05 and mean with difference letter (a-b) within same sample are significantly different at p <0.05. LE: Acer mono sap by lecithin-encapsulation, sap: Acer mono sap, Lecithin: Only lecithin particle. ( š y : A103017). LITERATURE CITED Fig. 6. DPPH radical scavenging activity on Acer mono sap nanoparticles. Mean values±sd from triplicate separated experiments are shown. Mean with difference letter (A) within same concentration are significantly different at p < 0.05 and mean with difference letter (a-c) within same sample are significantly different at p < LE: Acer mono sap by lecithinencapsulation, sap: Acer mono sap. ùkü (Fig. 6), ù 83.4% w ƒeƒ (Seo et al., 2008). w, w y z ù š g œ 200 / 26% w, š ù 2.5 y y w (Kang et al., 2010). š w y y j w, y ƒ x z š (Tobin et al., 1994; Bernadette et al., 1998) w š ù ƒ v ü y w» w ùký d. w Ahn WH. (1975). Color index and coloring substances in Korean sugar maple, Acer mono Max. syrup. Journal of Korea Forest Research. 26:7-12 Aroca P, Urabe K, Kobayashi T, Tsukamoto K and Hearing VJ. (1993). Melanin biosynthesis patterns following hormonal stimulation. Journal of Biological Chemistry. 268: Bernadette EK, Marianne D and Bernhard P. (1998). Protective effect against sunburn of conbined systemic ascorbic acid and α-tocopherol. Experimental Dermatology. 38: Cabane J, Chazara S and Garcia CF. (1994). Kojic acid, a cosmetic skin whitening agent, is a slow-binding inhibitor of catecholase activity of tyrosinase. Journal of Pharmacy and Pharmacology. 46: Halliwell B. (1991). Drug antioxidant effect. A basis for drug selection? Drugs. 42: Hwang JS, Shin HJ, Noh HS, Choi HJ, Ahn SM, Park DS, Kim DH, Lee BG, Chang IS and Kang HH. (2002). The inhibitory effects of 3,4,5-Trimethoxy cinamate thymol ester(tcte, Melsolv ) on Melanogenesis. Journal of the Society of Cosmetic Scientist of Korea. 28: Jeong SW, Lee NK, Kim SJ and Han DS. (1995). Screening of tyrosinase inhibitor from plants. Journal of Food Science and Nutrition. 27: Kang HI, Kim JY, Kwon SJ, Park KW, Kang JS and Seo KI. (2010). Antioxidative effects of peanut sprout extracts. Journal of the Korean Society of Food Science and Nutrition. 39: Kang KC, Lee CI, Pyo HB and Jeong NH. (2005). Preparation and characterization of nano-liposomes using phosphatidylcholine. Journal of Industrial and Engineering Chemistry. 11: Kim CM, Jung DL and Sheo HJ. (1991). A study on the ingredients in the sap of Acer mono Max. and Betula costata T. in Mt. Jiri area. Journal of the Korean Society of Food Science and Nutrition. 20: Kim DM and Kwak HS. (2004). Nano food materials and approchable development of nanofunctional dairy products 196

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