pissn 1229-1153 J. Fd Hyg. Safety Vol. 28, No. 4, pp. 354~359 (2013) http://dx.doi.org/10.13103/jfhs.2013.28.4.354 Journal of Food Hygiene and Safety Available online at http://www.foodhygiene.or.kr 발효된홍삼미강대두의혼합비율에대한항산화와항노화효과 손정현 하배진 * 신라대학교의생명과학대학생명공학과 Antioxidative and Antiaging Effects of Fermented Soybean, Rice Bran, and Red Ginseng by Mixed Ratios Jeong-Hyeon Son and Bae-Jin Ha* Department of Biotechnology, College of Medical Life Science, Silla University, Baekyangdaero 700 beon-gil, Sasang-gu, Busan 614-735, Korea (Received August 8, 2013/Revised September 10, 2013/Accepted November 26, 2013) ABSTRACT - This study was performed to elucidate the antioxidative and antiaging activities of mixed rates 2:6:2(262), 6:2:2(622) and 4:4:2(442) of fermented soybean, fermented rice bran, fermented red ginseng based on the comparision with their separate results of our three previous studies. The antioxidative and antiaging effects of 262, 622 and 442 mixed ratios were evaluated by the determination of superoxide radical scavenging activities, hydroxy radical scavenging activities, linoleic acid inhibition activtiy, elastin synthesis activity, and cell viability of B16F10. The material of 442 ratio showed the higher effects than those of 262 and 622 ratios, and presented the higher effect than the separate material of red ginseng in the antioxidative and antiaging activities. Therefore, this study suggested that the material of 442 ratio in the production of red gineng- containing cosmetics could be preferred as a useful cosmetic ingredient for antioxidation and antiaging. Key words: fermentation, soybean, ginseng, rice bran, antiaging 노화란모든신체기관과세포에걸쳐일어나는기능, 구조, 생화학적변화라할수있다. 피부의노화는시간이흘러감에따라자연적으로섬유아세포의작용과세포수가감소하여 collagen, elastin, fibroin 등의세포외기질단백질의합성양이줄어들고, 이에따른피부의구조가느슨해지거나, 탄력이감소하며세포내수분양이손실되며나타나는생리적노화 (chronological aging) 와바람, 온도, 습도, 담배, 자외선등의외부인자에의해활성산소종이발생하게되고이때발현되는염증성사이토카인이촉진되어 activator protein-1 (AP1) 과 nuclear factor κb (NF-κB) 의활성화에인한염증작용이증가되며피부를구성하는여러지질, 단백질, 효소등이손상되어발생하는외인성노화 (extrinsic aging) 으로나누어진다 1,2). 피부노화의대표적인증상은주름의발생으로, 이는피부진피조직의교원질중콜라겐의많은감소에의해서발생하게된다 3). 피 *Correspondence to: Bae-Jin Ha, Department of Pharmaceutical Engineering, College of Medical Life Science, Silla University, 1-1 San, Gwaebup-dong, Sasang-gu, Busan 617-736, Korea Tel: 82-51-999-5466, Fax: 82-51-999-5684 E-mail : bjha@silla.ac.kr 부에서발생한활성산소종이세포막을공격하여산화가일어나고이때발생한산화된지질에의해세포막이손상되어정상적인피부기능을상실하게된다. 또한여러가지효소적인항산화활동의균형이흐트러지게되며, 이러한과정의지속적인반복은주름을유발하게되는원인이된다 4). 인삼 (ginseng) 은두릅나무과 (Araliaceae) 에속하며느리게자라는다년생초이다 5). 이를고온에쪄서말린것을홍삼이라하며, 인삼의대표적인성분은진세노사이드 (ginsenoside, ginseng+glycoside) 로서다른식물에서발견되는사포닌과는다른독특한구조및활성을가지는것으로알려져있다 6). 또한홍삼에서만나타나는 ginsenoside Rg2, Rg3, Rh1, Rh2 등의특이성분은암예방, 암세포성장억제, 혈압강하, 뇌신경세포보호, 항혈전, 항산화작용등이있다고보고되어져있다 7). 미강은현미를도정할때나오는부산물로단백질 11-15%, 탄수화물 34-62%, 섬유질 7-11%, 지질 15-20% 그리고회분 7-10% 등으로구성되어영양적으로우수한것으로평가되고있다 8). 또한미강에는 α-tocopherol, α- Tocotrienol, γ-tocopherol, γ-tocotrienol 및 γ-oryzanol 등 354
Antioxidative and Antiaging Effects of Fermented Soybean, Rice Bran, and Red Ginseng by Mixed Ratios 355 이함유되어있어항산화작용과혈청콜레스테롤을낮춰주는기능을가진다고보고되어져있다 9). 대두는한국에서간장, 된장등전통발효식품의형태로다양한영양소의공급원이되고있으며, 최근대두발효식품과단백가수분해물의다양한생리활성능력이보고되면서식품소재로널리쓰인다 10). 대두에포함되어있는페놀물질에는 chlorogenic acid, isochlorogenic acid 와 caffeic acid 이있으며이소플라본또한강한활성을가지는플라보노이드류이다. 이러한물질들은발효과정에서미생물에의해여러대사물질로변화됨이보고되어져있으며, 청국장으로발효한이후항산화활성이증가됨이보고되어져있다 11). 또한본연구실에서선행되어진연구보고에따르면발효된홍삼, 미강, 대두의각각개별에대한발효최적조건확립및항산화, 미백, 주름에대해실험을실행하였으며그결과숙성된홍삼에서는항산화효과, 지질과산화억제효과, collagen 생성촉진효과, collagenase 활성저해효과를확인하였으며 12), 미강에서또한높은항산화활성및지방산산화억제활성, 멜라닌생성에관여하는 mushroom tyrosinase 활성저해능을확인하였다 13). 발효된대두또한그렇지않은대두에비해항산화활성, 미백활성, linoleic acid 억제활성및 tyrosinase 억제활성을보였다 14). 따라서본연구는개별로뛰어난활성을가지는홍삼, 미강, 대두의 3가지비율로혼합을하여개별효과보다더욱더뛰어난항노화효과와주름개선효과를가지는기능성화장품의원료를개발하기위해 in vitro 수준에서의항산화효과와주름개선효과를연구하였다. 재료및방법 균주및성장조건 Bacillus subtilis 168 (ATCC 33234, KCTC 2217) 는한국미생물보존센터 (KCCM) 에서분양받았으며, 35 o C 조건으로 Luria-Bertani broth (LB Difco Laboratories, Detroit, MI) 에서배양하였다. 을 4시간동안침지후상등액을채취하여 40 o C에서농축시킨미강발효추출물을동결건조하여사용하였다. 홍삼시료제조본실험에서사용한수삼은금산에서제배한 5년근을구입하여사용하였다. 구입한수삼 50 g을오쿠 (OC-7700R) 로 7시간동안숙성시킨뒤, 80% 에탄올에 4시간동안침지시킨후상등액을채취하여 40 o C에서농축한후동결건조하여사용하였다. 시료의혼합위의 3가지발효후추출된시료를대두 : 미강 : 홍삼순으로 2:6:2, 6:2:2, 4:4:2 비율로혼합하여사용하였다. Superoxide Radical 소거효과 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) 1 mm 50 µl와 esterase (600 unit/ml) 50 µl를혼합한뒤 37 o C에서 20분간반응시켜 2',7'-dichlorodihydrofluorescein (DCFH) solution을만든후 DCF측정법을사용하여서평가하였다 15). 96 well plate에농도별각시료 10 µl를넣고 50 mm 칼륨인산완충용액 130 µl를넣은뒤 20 mm menadion 10 µl 와칼륨인산완충용액으로 100배희석한 DCFH solution 50 µl를넣고 5분간섞어준다. Elisa (Microtiter plate reader, Molecular Devices Co., Sunnyvale, CA., USA) 로 485/530 nm에서 fluorescence를 5분간격으로 30분간측정하였다. Hydroxy Radical 소거효과 DCFDA (1 mm) 와 esterase (600 unit/ml) 를혼합한뒤 37 o C에서 20분간반응시켜 DCFH solution을만들어 DCF 측정법을이용하여실시하였다 16). 96 well plate에농도별각시료를넣고 10 mm FeSO 4 와 1.35 mm H 2 O 2 를섞은혼합액을넣어준다. 100배희석한 DCFH solution를넣고 5분간섞어준다. Elisa (Microtiter plate reader, Molecular Devices Co., Sunnyvale, CA., USA) 로 485/530 nm 에서 Fluorescence를 10분간격으로 40분간측정하였다. 대두시료제조 20 g의대두에 Bacillus subtilis를 600 nm 흡광도가 1.0 일때 1% 를접종하여 40 o C에서 36시간동안고체발효하였다. 대두발효시료에 80% 에탄올을 4시간동안침지후상등액을채취하여 40 o C에서농축시킨대두발효추출물을동결건조하여사용하였다. 미강시료제조 20 g의미강을멸균수 200 ml을혼합하여 Bacillus subtilis 를 600 nm 흡광도가 1.0일때 1% 를접종하여 40 o C에서 36 시간동안고체발효하였다. 미강발효시료에 80% 에탄올 지질과산화억제효과자동산화가잘되는물질인리놀산을이용하여자동산화를억제하는효과를실험한것으로 10 mm 리놀산용액과시료를농도별로첨가한후 4 o C, 24시간보관후지질과산화정도를 thyocyanate법에의하여실시하였다 17). 상기의혼합액에 75% 에탄올을첨가하여교반한후 30% NH 4 SCNsolution과 20 mm FeCl 2 을첨가하여 3분간방치한후발색시켜 Elisa (Microtiter plate reader, Molecular Devices Co., Sunnyvale, CA., USA) 로 500 nm에서흡광도를측정한다.
356 Jeong-Hyeon Son and Bae-Jin Ha Increasing Rate of Elastin Synthesis 피부의주름개선효과를검정하는 elastin 합성효과를보기위하여수행하였고, 실험에사용한섬유아세포인 Raw264.7 cell 은한국세포주은행에서분양받았으며 10% 의 fetal bovine serum (FBS, Bio whittaker) 와 1% 의 penicillin (Bio whittaker) 이첨가된 dulbecco's modified eagle medium (DMEM, Bio whittaker) 으로 37 o C, 5% CO 2 조건에서배양한후, 새로운 serum free 배지의적정농도범위에서단계적으로희석한시료를가하고, 다시 24시간동안 CO 2 incubator에서배양한다. 배양액을가지고 FASTIN TM assay (Biocolor,Belfast,Ireland) 사용방법대로 elastin 합성효과를관찰한다. sample이나 standard를침전시약과동량으로섞어서 10분간방치한후 10,000 g에서 10분간원심분리한다. 원심분리후상층액은제거하고 Fastin Dye Reagent 1 ml을첨가하여 90 min 동안반응시킨뒤 10,000 g에서 10분간원심분리하여 elastin-dye complex를분리한다. 상층액은제거하고 Dye Dissociation Reagent 250 µl를넣고섞은뒤 Elisa (Microtiter plate reader, Molecular Devices Co., Sunnyvale, CA., USA) 를이용하여 513 nm에서흡광도를측정한후, α-elastin 표준곡선에대입한뒤 elastin 농도를산정한다. MTT를이용한세포독성측정 MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) 는 Sigma Chemical Co. (St. Louis, MI, USA) 제품을사용하였다. 실험에사용한 melanoma cell 인 B16F10 은한국세포주은행에서분양받았으며 10% 의 fetal bovine serum (FBS, Bio whittaker) 와 1% 의 penicillin (Bio whittaker) 이첨가된 dulbecco's modified eagle medium (DMEM, Bio whittaker) 으로 37 o C, 5% CO 2 조건에서 48시간배양한후 96-well plate에세포현탁액을 180 µl 씩분주하고, phosphatebuffer of saline (PBS, Bio whittaker) 로 sample을희석한후농도별로 20 µl 씩첨가하였다. 37 o C, 5% CO 2 조건에서 48시간동안추가배양한후배양액을제거하고 MTT (5 mg/ ml in PBS) 용액을 100 µl 씩가하고 37 o C, 5% CO 2 에서 4시간동안추가배양하였다. 이후 DMSO 100 µl를넣고 37 o C, 5% CO 2 에서 10~15분동안반응시킨후발색정도를 Elisa (Microtiter plate reader, Molecular Devices Co., Sunnyvale, CA., USA) 를이용하여 540 nm에서흡광도를측정한다. 통계처리본실험에대한모든실험결과는평균치와표준편차로나타내었고, 통계적유의성은 SPSS 12.0 (SPSS Inc., Chicago, IL, USA) 를이용한 one-way ANOVA 로검정하였으며, 사후검증으로 Duncan's post-hoc test를실시하였고유의성은 p < 0.05로하였다. 결과및고찰 Superoxide Radical 소거효과활성산소는체내생물학적반응에서만들어지며정상적인경우 superoxide dismutase (SOD) 에의해과산화수소로전환되지만, 과잉생산이될경우체내에서독성을나타내게되어여러가지질환을발생시키기도한다 18,19,20). Superoxide radical 소거실험결과는 Table 1과같다. 442 > 622 > 262 순으로 84%, 83%, 60% 의소거활성을보였으며, 농도의존적인 superoxide radical 소거활성을보였다. 가장높은활성을보인 442는표준물질로사용된 vitamin C와유사한활성을보였으며, 이는본연구실에서선행되어진개별연구결과인발효된홍삼 70% 12), 발효된미강 89% 13), 발효된대두 89% 14) 와비교했을때 superoxide radical 결과에서 84% 의소거활성을보여미강과대두와는비슷하지만홍삼과비교하였을때혼합비율에서더높은연구결과가나타났다. Hydroxy Radical 소거효과 Hydroxy radical은반응성이가장큰 free radical로 DNA 손상을일으켜세포독성이나돌연변이를유발하여체내에서전반적으로가장강한독성을나타내는 free radical이다 21,22). Hydroxy radical 소거율의결과는 Table 2와같다. 100 µl/ml 농도에서각각 85%, 75%, 84% 로표준물질인 vitamin C에비해매우높은항산화능을보였으며, 특히 262와 442가우수한 free radical 소거효과를보였다. 표준물질 vitamin C와비교하였을때 vitamin C의농도가 100 µl/ml 일때 262는 10배낮은 10 µl/ml에서약 3% Table 1. Scavenging Effect of Rate Mixed Soybean, Rice Bran, Red Ginseng Fermented on Superoxide Radical. 262 : soybean, rice bran, red ginseng mixed rate 2:6:2, 622 : soybean, rice bran, red ginseng mixed rate 6:2:2, 442 : soybean, rice bran, red ginseng mixed rate 4:4:2. Vitamin C was used as positive control, a, b, c, d, e, f are different group by one-way ANOVA with Duncan s post-hoc test Concentration (µl/ml) O 2 scavengingactivity(%) 262 622 442 Vitamin C 100 60.54 ± 2.13a 83.23 ± 2.47a 84.72 ± 2.64a 76.41 ± 0.36b 10 61.17 ± 5.28d 60.41 ± 3.74d 70.52 ± 3.07bc 69.39 ± 1.32c 1 41.60 ± 4.34f 41.03 ± 4.08e 51.54 ± 4.06e 53.36 ± 2.97f
Antioxidative and Antiaging Effects of Fermented Soybean, Rice Bran, and Red Ginseng by Mixed Ratios 357 Table 2. Scavenging Effect of Rate Mixed Soybean, Rice Bran, Red Ginseng Fermented on Hydroxy Radical. 262 : soybean, rice bran, red ginseng mixed rate 2:6:2, 622 : soybean, rice bran, red ginseng mixed rate 6:2:2, 442 : soybean, rice bran, red ginseng mixed rate 4:4:2. Vitamin C was used as positive control, a, b, c, d, e, f are different group by one-way ANOVA with Duncan s post-hoc test Concentration (µl/ml) OH scavenging activity (%) 262 622 442 Vitamin C 100 85.94 ± 2.42a 75.13 ± 4.26b 84.07 ± 0.69a 65.66 ± 1.26c 10 62.96 ± 4.78c 55.45 ± 0.82d 72.57 ± 2.61b 32.35 ± 0.23f 1 51.03 ± 2.83d 44.30 ± 2.83e 63.83 ± 2.84c 24.17 ± 1.57f Table 3. Inhibitory Effect of Rate Mixed Soybean, Rice Bran, Red Ginseng Fermented on Linoleic acid anti-oxidantion. 262 : soybean, rice bran, red ginseng mixed rate 2:6:2, 622 : soybean, rice bran, red ginseng mixed rate 6:2:2, 442 : soybean, rice bran, red ginseng mixed rate 4:4:2. Vitamin C was used as positive control, a, b, c, d, e, f, g are different group by one-way ANOVA with Duncan s post-hoc test Concentration (µl/ml) Inhibition of anti-oxidation by using linoleic acid (%) 262 622 442 Vitamin C 100 42.83 ± 2.07g 43.30 ± 1.20e 56.57 ± 1.97e 83.78 ± 1.09a 10 44.72 ± 0.78ef 47.60 ± 1.05de 48.03 ± 2.60d 82.43 ± 1.36a 1 36.20 ± 2.00e 43.71 ± 0.58e 42.39 ± 1.60c 66.79 ± 1.02b 낮은, 442는 1µL/mL에서약 2% 더낮은 hydroxy radical 소거효과를나타내었으며, 다른 radical 소거활성에비해매우뛰어난 hydroxy radical 소거효과를나타내었다. 이는본연구실에서선행되어진개별연구결과인발효된홍삼 75% 12), 발효된미강 82% 13), 발효된대두 79% 14) 와비교했을때약 3%~10% 가량증가한것으로나타나혼합비율에서의 hydroxy radical 소거효과가더뛰어나다는것이나타났다. 지질과산화억제효과생체막의주성분인인지질의구성성분인 linoleic acid는 free radical의반응에의해자동산화반응을일으키기쉬우며, 자동산화가일어나게되면 DNA 손상에영향을미치게된다 23,24). Linoleic acid의자동산화능을이용한 thiocyanate법으로항산화활성을측정한결과는 Table 3과같다. 442 > 622 > 262 순으로각각 56%, 43%, 42% 의자동산화저해활성을보였으며농도의존적으로억제효과가나타났다. 자동산화억제효과는표준물질인 vitamin C 에비해활성이다소떨어지는것으로나타났으며, 기존에본연구실에서선행되어진개별연구결과인발효된홍삼 57% 12), 발효된미강 79% 13), 발효된대두 62% 14) 와비교하였을때또한낮은억제율을나타냈으며, 다른 radical 소거활성에비해서는활성이조금떨어지는것으로나타났다. Increasing Rate of Elastin Synthesis 콜라겐이외의탄력섬유인 elastin은주름형성에있어서많은관여를한다 25,26). 또한 UV에의해피부표피의각질형성세포 (keratinocyte) 에서도 elastase의전구체인 tropoelastin 의 mrna 발현이증가한다 27). Elastin synthesis를측정한결과는 Table 4와같다. 262, 622, 442 모두 100 Table 4. Inhibitory Effect of Rate Mixed Soybean, Rice Bran, Red Ginseng Fermented on elastin synthesis. 262 : soybean, rice bran, red ginseng mixed rate 2:6:2, 622 : soybean, rice bran, red ginseng mixed rate 6:2:2, 442 : soybean, rice bran, red ginseng mixed rate 4:4:2. a, b, c, d, are different group by one-way ANOVA with Duncan s post-hoc test Concentration Inhibition of anti-aging by using elastin synthesis (%) (µl/ml) 262 622 442 100 42.67 ± 4.71a 42.17 ± 2.12a 41.33 ± 1.89a 10 28.67 ± 1.89bc 32.78 ± 0.69b 31.22 ± 0.77b 1 22.11 ± 2.22d 27.33 ± 5.19cd 25.00 ± 2.36bcd Table 5. Cell Viability of Rate Mixed Soybean, Rice Bran, Red Ginseng Fermented on melanoma cell. 262 : soybean, rice bran, red ginseng mixed rate 2:6:2, 622 : soybean, rice bran, red ginseng mixed rate 6:2:2, 442 : soybean, rice bran, red ginseng mixed rate 4:4:2. a, b, c, d, e, f, g are different group by one-way ANOVA with Duncan s post-hoc test Concentration Cell Viability (%) (µl/ml) 262 622 442 100 19.89 ± 1.56g 23.76 ± 1.17f 37.85 ± 1.17cd 10 34.94 ± 0.20de 32.73 ± 2.93e 44.48 ± 0.48b 1 36.05 ± 2.15de 40.06 ± 1.56c 55.80 ± 0.39a µl/ml 의농도에서약 42% 로비슷하게나타났으며, 농도의존적인 elastin synthesis를보였다. 따라서 elastin 합성의증가는 elastase의활성을저해하며주름형성이나각질형성억제에도도움을줄수있을것으로보여진다. MTT를이용한세포독성측정 B16F10 cell은피부의색소를형성하는세포인 melanin cell의악성종양으로 melanin이과잉생산되어색소의피부침착에기인하여 B16F10 cell의성장을억제하면 tyrosinase
358 Jeong-Hyeon Son and Bae-Jin Ha 활성과 tyrosinase protein 발현을억제하여 melanin 생성을저해한다 28,29). B16F10의세포활성을측정해본결과는 Table 5와같다. 262에서가장낮은세포활성도인 19% 를나타내었고, 622와 442 순으로세포활성도를나타내었으며, 농도의존적인세포활성도를나타내었다. 요약 본연구는발효된대두, 미강, 홍삼 3종을배합을하여시너지효과를확인함으로써개별시료보다더욱더좋은혼합물을만들기위해서시행되었다. 3가지혼합비율 262, 622, 442를비교하였을때 442 비율이항산화활성과항노화활성이뛰어난것으로나타났다. 또한 442 혼합비율은개별재료의효과와비교하였을때개별홍삼보다높은시너지효과를보여주었다. 그러므로홍삼을원료로하여화장품을제조할때는홍삼을단독으로사용하는것보다미강과대두와혼합된 442 혼합비율재료가항산화활성과주름개선에더높은활성을가지는기능성화장품의중요원료로선호될수있다. 앞으로이물질에서주요한유효성분을분리, 정제하여항산화검증과항노화검증의필요성이있다고사료된다. 참고문헌 1. Saliou, C., Kitazawa, M., McLaughlin, L., Yang, J. P., Lodge, J. K., Tetsuka, T.,... & Packer, L.: Antioxidants modulate acute solar ultraviolet radiation-induced NF-kappa-B activation in a human keratinocyte cell line. Free Radic Biol Med 26, 174-183 (1999). 2. Kang, S., Cho, S., Chung, J. H., Hammerberg, C., Fisher, G. J., & Voorhees, J. J.: Inflammation and Extracellular Matrix Degradation Mediated by Activated Transcription Factors Nuclear Factor-κB and Activator Protein-1 in Inflammatory Acne Lesions in Vivo. Am J Pathol 166, 1691-1699 (2005). 3. Yaar, M., & Gilchrest, B. A.: Aging versus photoaging: postulated mechanisms and effectors. J Invest Dermatol 3, 47-51 (1998). 4. Yaar, M., & Gilchrest, B. A.: Photoageing: mechanism, prevention and therapy. Br J Dermatol 157, 874-887 (2007). 5. Wang, C. Z., & Yuan, C. S.: Potential role of ginseng in the treatment of colorectal cancer. Am J Chin Med 36, 1019-1028 (2008). 6. Choi, K. T.: Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginseng CA Meyer. Acta Pharmacol Sin 29, 1109 (2008). 7. Lee, S. H., Kang, J. I., & Lee, S. Y.: Saponin Composition and Physico-Chemical Properties of Korean Red Ginseng Extract as Affected by Extracting Conditions. J Korean Soc Food Sci Nutr 37, 256-260 (2008). 8. Kim, S. R., Ahn, J. Y., Lee, H. Y., Ha, T. Y.: Various properties and phenolic acid contents of rices and rice brans with differentmillig fractions. Korean J. Food Sci. Technol 36, 903-936 (2004). 9. Bae, S. M., Kim, J. H., Jo, C. W., Jung, T. J., Yuk, H. S., Byeon, M. W., Lee, S. C.: Effect of γ-irradiation on the Antioxidant Activity of Rice Hull, Rice Bran and Barley Bran. J. Korean Soc. Food Sci. Nutr 31, 246-250 (2002). 10. Song, H. N., Jung, K. S.: Quality Characteristics and Physiological Activities of Fermented Soybean by Lactic Acid Bacteria. Korean J. Food Sci. Technol. 38, 475-482 (2006). 11. Park, J. W., Lee, Y. J., Yoon, S.: Total flavonoids and phenolics in fermented soy products and their effects on antioxidant activities determined by different assays. Korean J. Food Culture. 22, 353-358 (2007). 12. Kim, M. J., Kwon, R. H., Jang, M. W., Ha, B. J.: Antioxidant and Anti-wrinkle Effects of Steamed Three Ginseng Extracts. J. Soc. Cosmet. Scientists Korea. 38, 155-162 (2012). 13. Chae, G. Y., Kwon, R. H., Jang, M. W., Kim, M. J., Ha, B. J.: Whitening and Antioxidative Effect of Rice Bran Fermented By Bacillus subtilis. J. Soc. Cosmet. Scientists Korea. 37, 153-159. (2011). 14. Chae, G. Y., Bae, J. H.: The Comparative Evaluation of Fermented and Non-fermented Soybean Extract on Antioxidation and Whitening. Toxicol Res. 27, 205-209 (2011). 15. Kang, H. S., Son, K. H., & Choi, J. S.: Scavenging effect of Korean medicinal plants on the peroxynitrite and total ROS. Kor. J. Pharmacogn. 9, 73-79 (2003). 16. Fujita, Y., Uehara, I., Morimoto, Y., Nakashima, M., Hatano, T., & Okuda, T.: Studies on inhibition mechanism of autoxidation by tannins and flavonoids. II. Inhibition mechanism of caffeetannins isolated from leaves of Artemisia species on lipoxygenase dependent lipid peroxidation]. Yakugaku zasshi. 108, 129 (1988). 17. Kim, I. D., Kwon, R. H., Heo, Y. Y., Jung, H. J., Kang, H. Y., Ha, B. J.: Supercritical extraction of oriental Herb : Anti-aging and anti-wrinkle effects, Korean J. Biotechnol. Bioeng. 23, 529 (2008). 18. Bulkley, G. B.: The role of oxygen free radicals in human disease processes. Surgery. 94, 407 (1983). 19. Yoon, W. J., Lee, J. A., Kim, J. Y., Oh, D. J., Jung, Y. H., Lee, W. J., & Park, S. Y.: Antioxidant activities and anti-inflammatory effects on Artemisia scoparia. Kor. J. Pharmacogn. 37, 235-240 (2006). 20. Halliwell, B., & Gutteridge, J. M.: Free radicals in biology and medicine, Oxford university press. UK, 3ed. pp. 267 (1999) 21. Manian, R., Anusuya, N., Siddhuraju, P., Manian, S.: The antioxidant activity and free radical scavenging potential of two different solvent extracts of Camellia sinensis (L.) O. Kuntz, Ficus bengalensis L. and Ficus racemosa L, Food Chem. 107, 1000 (2008). 22. Hyon, J. S., Kang, S. M., Han, S. W., Kang, M. C., Oh, M. C., Oh, C. K., Kim, D. W., Jeon, Y. J., Kim, S. H.: Flavonoid component changes and antioxidant activities of fermented citrus grandis osbeck peel, J. Korean Soc. Food Sci. Nutr. 38, 1310 (2009). 23. Kim, W.S., Lee, S.J.: Studies on the Electrochemical Proper-
Antioxidative and Antiaging Effects of Fermented Soybean, Rice Bran, and Red Ginseng by Mixed Ratios 359 ties for Rancidity of Linoleic Acid. Korean j. FOOD & NUTR, 13, 360-364 (2000). 24. De Kok, T. M. C. M., Zwingman, I., Moonen, E. J., Schilderman, P. A. E. L., Rhijnsburger, E., Haenen, G. R. M. M., & Kleinjans, J. C. S.: Analysis of oxidative DNA damage after human dietary supplementation with linoleic acid. Food Chem Toxicol. 41, 351-358 (2003). 25. Antonicelli, F., Bellon, G., Debelle, L., & Hornebeck, W.: ElastinElastases and InflammAging. Curr. Top. Dev. Biol. 79, 99-155 (2007) 26. S. seite, H. Zucchi, D. Septier, S. Igondjo-Tchen, L. Senni, and G. Godeau.: Elastin Changes during chronogical and photoaging: the important role of lysozyme. J. Eur. Acad. Dermatol. Venereol. 20, 980 (2006). 27. Tsuji, N., Moriwaki, S., Suzuki, Y., Takema, Y., & Imokawa, G.: The Role of Elastases Secreted by Fibroblasts in Wrinkle Formation: Implication Through Selective Inhibition of Elastase Activity. Photochem Photobiol. 74, 283-290 (2001) 28. Kim, Y.Y., Kim, I.S., Park, O.J., Kim, Y.M.: EGCG induces Apoptosis under Hypoxic State in B16F10 Melanoma Cancer Cells. Korean Society of Life Science. 21, 251-256 (2010). 29. Oh, W. K., Kim, K. B., Lim, J. Y., Lee, S. K., Kwon, Y. D., Yeom, S. R., Song, Y. S.: Effects of Dokhwalkisaeng-tang on Melanin Synthesis Inhibition and Gene Expression in B16F10 Melanoma Cells. Korean J. Oriental Physology & Pathology. 23, 63-75 (2009).