ISSN 2466-2046 (Print) Asian J Beauty Cosmetol 2016; 14(1): 66-76 ISSN 2466-2054 (Online) R E S E A R C H A R T I C L E Open Access Antioxidant and Antiwrinkle Effects of Amentoflavone for Cosmetic Materials Development Songjeong Lee, Sungkwan An* Department of Biological Engineering, Konkuk University, Seoul, Korea *Corresponding author: Sungkwan An, Department of Biological Engineering, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Korea Tel.: +82 2 450 4054 Fax: +82 502 770 2278 Email: ansfgrc@konkuk.ac.kr Received February 29, 2016 Revised March 25, 2016 Accepted March 25, 2016 Published March 30, 2016 Abstract Purpose: This study aimed to investigate the potential of amentoflavone as an antiaging material for cosmetics through examining antioxidant and antiwrinkle effects. Methods: To check the antioxidant and antiwrinkle effects of amentoflavone, we performed various experiments such as in vitro antioxidant assays, quantitative real-time PCR, SA-β-galactosidase assay, and DCF-DA assay. Results: We confirmed that the cell viability of amentoflavone is increased in cytotoxic condition. Amentoflavone also had an antioxidant effect like BHT. The expression in mrna level of antioxidant enzymes such as SOD1 and CAT was increased in a dosedependent manner. In case of wrinkle, the expression in mrna level of MMP1 and MMP3 was decreased but the expression of COL1A1 was dose-dependently increased. Also, amentoflavone markedly reduced the expression of SA-β-galactosidase. Conclusion: As mentioned above, we found that amentoflavone had effects on preventing skin aging and thought that amentoflavone can be a valulable material for antiaging cosmetics. Keywords: Amentoflavone, Antioxidant, Human Dermal Fibroblast, Collagen, Senescence Introduction 플라보노이드 (flavonoids) 는과일, 채소, 곡류및허브류에널리포함된폴리페놀 (polyphenol) 성분으로건강에유익한효과들이가장많이보고되고있는식물유래영양성분 (phytonutrient) 이다. 현재 5,000가지이상의 flavonoids 가확인되어있으며일상적으로섭취하는식품에는수백종의플라보노이드가존재한다고알려져있다 (Holden et al., 2005). Flavonoids는두개의방향족인 A와 B가산화형 heterocyclic C링에 3-탄소체인으로연결되어있는기본구조를가지고있고, heterocyclic C링의산화상태나작용기에따라안토시아니딘 (anthocyanidins), 플라바놀 (flavan- 3-ols), 플라바논 (flavanones), 플라본 (flavones), 플라보놀 (flavonols) 로분류된다 (Liu, 2004). 이소플라본 (isoflavones) 은 B링이 3번대신 2번에붙은것이특징이며프로안토시아니딘 (proantocyanidins) 은플라바놀 (flavan-3-ols) 의중합체이며, flavonoid의기본적인화학구조는 Figure 1 (Song et al., 2012) 과같다. 그중 biflavonoid는플라보노이드 2분자가결합한이합체로아멘토플라본 (amentoflavone), 아가디스플라본 (agathisflavone), 쿠프레스플라본 (cupressflavone), 히노키플라본 (hinokiflavone) 등이있으며, flavonoid 2분자는같은종류인경우도있고, 다른경우도있으며, 주로겉씨식물 (gymnosperm) 에많다 (Okigawa et al., 1971). Amentoflavone의기본적인화학구조는 (Sakthivel & Guruvayoorappan, 2013; Figure 2) 와같다. 인체에는유리기에의한손상으로부터자신을보호할수있는항산화방어체계가있다. H 2 O 2 (hydrogen peroxide) 는 ROS 종류중반응성이크지않지만, 특정상태에서반응성이더욱큰 1 O 2 (singlet oxygen) 나 OH - (hydroxyl radical) 로전환될수있기때문에 H 2 O 2 에대한소거도중요한인체방어시스템중하나이다. 또한 H 2 O 2 는산화스트레스의원인을제공하며다수의유전자전사인자의발현, 칼슘과철같은이온의항상성을변화시키고, 세포의확산과분화를유발하는능력을 Copyright c Korea Institute for Skin and Clinical Sciences. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Antioxidant and Antiwrinkle Effects of Amentoflavone for Cosmetic Materials Development Figure 1. Basic structure of a flavonoid. - 가지고있다 (Barbouti et al., 2002). O 2 (superoxide anion) radical은산소를소비하는모든생물종에존재하는항산화효소인 SOD에의해제거되지못하면 H 2 O 2 로전환되며이렇게생성된 H 2 O 2 는 CAT에의해제거되지않으면세포내에서 ROS로인한산화적스트레스를유발한다. MMPs는세포외기질분해에관여하는금속성분해효소로서, 정상적인발생과정, 혈관생성및상처재생뿐만아니라, 세포간질의분해가전제조건인암의전이와관절염, 골다공증, 치막염등의질병에관여하는것으로알려져있다 (Liotta et al., 1980; Bonfil et al., 1989; Liotta & Stetler, 1990; Matrisian, 1990). 노화된피부의진피내콜라겐양의감소는 fibroblast의콜라겐합성이감소되거나, MMPs와같은효소에의한콜라겐의분해가증가되었을경우에생긴다. 또한자외선이나, ROS에지속적으로노출된피부에서의심한콜라겐감소가주름생성의중요한원인이되기도한다. 콜라겐은포유동물체내단백질의약 30% 이상을차지하고있으며특히근육, 연골, 뼈, 피부, 혈관벽, 치아등에주로분포하고있다. 콜라겐의구조는 3-나선형분자 (triple-helical molecule) 로써 2개 α1(Ⅰ)chain과 1개의 α2(Ⅰ)chain으로이루어져있으며 polypeptide 사슬은대게글리신 (glycine), 프롤린 (proline), 히드록시프롤린 (hydroxyproline) 의아미노산잔기 3개로이루어져있다. 현재까지 19 types의 collagen이밝혀져있으며, 각각의 type collagen들은우리몸을구성하는데다양하게사용되고있다 (Glimcher, 1989; Bailly et al., 1990). 이처럼콜라겐은인간의체내에서항상분해와합성이반복되는데나이가들어감에따라균형이붕괴되고합성보다는분해가더욱많아지게된다. 정상체세포는배양할때에제한된수의분열을한뒤에비가역적인증식억제상태에이르게되며이상태를세포노화 (replicative senescence) 라고한다 (Hayflick & Moorehead, 1961). 세포노화현상은 in vivo에서세포가재생능력을잃어버려발생한다고생각된다 (Campisi, 2007). 정상체세포가제한된수의분열을하는반면, 암세포는무한히분열하기때문에세포노화는개체를암으로부터보호하는역할을하는것으로도알려졌다 (Braig et al., 2005; Collado et al., 2005; Michaloglou et al., 2005). 세포노화는배양을통해서일어 Figure 2. The chemical structure of amentoflavone. 날뿐만아니라, DNA damage를일으켜서도유도할수있다. Apoptosis를일으키는것보다는조금약한 DNA damage를주면세포노화로진행되며, 강한 DNA damage를주었을때에는 apoptosis로진행하는것으로생각된다 (Luschen et al., 2000). 따라서, 본연구에서는항산화및항노화와관련된실험을통해 amentoflavone의항노화화장품소재로서의발전가능성을확인해보고자한다. Methods 1. Cell viabililty 세포생존율은 WST-1 assay를이용하였으며측정은세포내소기관인미토콘드리아의탈수소효소에의해생성되는 formazan의흡광도를측정하는원리로이루어진다. 96 well plate에 3 10 3 HDFs를접종하여 24시간배양하고, amentoflavone 는 5, 10, 15, 20, 25 µm 농도로처리하고 H 2 O 2 는 0.1, 0.2, 0.3, 0.4, 0.5 mm 처리한후 24시간동안추가배양하였다. 배양된 well에 EZ-Cytox cell viability assay kit reagent (ItsBio, Korea) 10 μl를첨가하여 1시간배양후 microplate reader (Bio-Rad, USA) 를이용하여 490 nm에서흡광도측정하였으며, 3회반복수행하여세포생존율평균값과표준편차를도출하였다. 2. Antioxidant assay in vitro 1) DPPH radical scavenging activity assay DPPH (1,1-diphenyl-2-picrylhydrazyl) radical 소거활성실험은 (Blois, 1958) 을응용하여다음과같이실행하였다. 96 well plate에 amentoflavone을 1, 2, 5, 10, 15 μm 농도로하여희석액를 100 μl씩분주한후여기에 0.2 mm의 DPPH 50 μl을넣은후 30분간방치한다. 동일한방법으로 BHT (butylated hydroxy toluene) 도 1, 2, 5, 10, 15 μm 농도로처리한다. Microplate reader (Bio-Rad, USA) 를이용하여 514 67
화장품소재개발을위한 amentoflavone 의항산화및항주름효과연구 nm에서흡광도를측정하였으며, 측정은 3회반복수행하여흡광도의평균값과표준편차를도출하였다. 2) ABTS radical scavenging activity assay ABTS [2,2-azinobis-(3-ethylbenzo-thiazoline- 6-sulfonate)] 라디칼을이용한항산화능은 potassium persulfate와의반응에의해생성된 ABTS + 유리라디칼이시료중의항산화물질에의해제거되어라디칼특유의청록색이탈색되는원리를이용한방법이다 (Re et al., 1999; Roberta et al., 1999). 7.4 mm ABTS와 2.6 mm potassium persulfate를최종농도로혼합하여실온의암실에서 12시간동안방치하여 ABTS + 를형성시킨후 732 nm에서흡광도값이 0.706 (±0.01) 이나오도록한다. 96 well plate에희석된 amentoflavone 1, 2, 5, 10, 15 μm 농도로하여 100 μl씩분주하고여기에 ABTS + 100 μl을가하여 7분동안방치한다. 동일한방법으로 BHT도 1, 2, 5, 10, 15 μm 농도로처리한다. Microplate reader (Bio-Rad, USA) 를이용하여 732 nm에서흡광도를측정하였으며, 측정은 3회반복수행하여흡광도의평균값과표준편차를도출하였다. 3) Hydrogen peroxide scavenging activity assay 활성산소는피부노화를일으키는대표적인물질이기때문에, in vitro 실험을통해 H 2 O 2 제거능을확인하는것으로시험물질의항산화능을확인할수있다 (Long et al., 1999). Amentoflavone을각각 1, 2, 5, 10, 15 μm 농도로하여 500 μl씩분주하고여기에 40 mm의 H 2 O 2 를 500 μl을넣은후 10 분간방치한다. 동일한방법으로 LA (L-ascorbic acid) 도 1, 2, 5, 10, 15 μm 농도로처리한다. 96 well plate에일정량분주하여 microplate reader (Bio-Rad, USA) 를이용하여 230 nm 에서흡광도를측정하였으며, 측정은 3회반복수행하여흡광도의평균값과표준편차를도출하였다. 4) DCF-DA cellular ROS detection assay 세포내활성산소종농도변화를측정하기위해 HDFs (2 10 5 cells/well) 를 60 mm 배양접시에접종하여 24시간배양후, amentoflavone을 5, 10, 15 μm 농도로각각처리하고 H 2 O 2 처리후추가로 24시간을배양하였다. 세포내활성산소종을측정하기위한 dye인 DCF-DA (dichlorofluorescein diacetate) 를 10 μm 첨가하여 30분배양후세포를수확하여 PBS를첨가하여세포를풀어준다음 flow cytometer (BD Biosciences, USA) 를이용하여 ROS의변화량을측정하였다. Amentoflavone의활성산소종제거효능을검증하기위해 ROS scavenger 역할을하는 NAC (N-acetyl-L-cystein) 도동일한방식으로처리한후측정하였다. 3. Quantitative real-time PCR (qrt-pcr) H 2 O 2 와 amentoflavone에의해 HDFs 내에서일어나는 COL1A1, MMP1, MMP3, IL-6, TNF-α, CAT, SOD1 유전자변화를정량하기위하여 quantitative real-time PCR (qrt-pcr) 을수행하였다. Amentoflavone 및 H 2 O 2 가처리된 HDFs의 RNA 추출을위해 60 mm 세포배양접시에 HDFs (2 10 5 cells/well) 로 seeding하고, amentoflavone을 5, 10, 15 μm 농도로처리하고 3시간전처리후, H 2 O 2 를처리하여 24시간배양하였다. 세포배양이끝난세포를 trizol reagent (Invitrogen, USA) 를이용하여용해후 0.2 ml chloroform (Biopure, Canada) 를첨가하여상온에방치한다. 12,000 rpm, 4 조건으로 20분간원심분리하여단백질이포함된하등액과 mrna가포함된상등액을분리한다. 상등액은 0.5 ml isopropanol을첨가하여 10분간상온에방치한다음 12,000 rpm, 4 조건으로원심분리하여 RNA를침전시키고 75% ethanol을이용하여세척후 ethanol을제거하고상온에서건조시킨다. 건조된 mrna는 diethylpyrocarbonate (DEPC, Biopure) 를 water로녹여실험에사용하였으며, 추출된 RNA는 nanodrop (Nanodrop, USA) 을이용하여 260 nm, 280 nm의 ratio 1.8 이상의순도의 RNA만을실험에사용하였다. 추출된 RNA를이용하여 PCR tube에 1 μg RNA, 0.5 ng oligo dt18, DEPC water를 total 10 μl로제조후 70 에서 10분간처리하여 RNA 변성을유도한다음 M-MLV reverse transcriptase (Enzynomics, Korea) 을이용하여 37 에서 1시간반응시켜 cdna를합성하였다. cdna를주형으로 qrt-pcr을이용하여 amentoflavone에의해세포내에서일어나는전사적인유전자발현변화를정량적으로확인하였다. qrt-pcr은 HOT FIREPol EvaGreen PCR Mix Plus (Solis BioDyne), 1 pmole forward primer, 1 pmole reverse primer, 10 ng cdna를혼합하여반응액을만들어 Linegene K (BioER, China) 를이용하여반응을진행하였다. 반응의세부과정은 94 에서 3분간 denaturation 후, denaturation (94, 30초 ), annealing (58, 30초 ), polymeration (72, 30초 ) 을 40 cycle 수행하여 PCR을진행하였다. PCR의유효성은 melting curve로검증하였고, 각유전자의발현은 β-actin의발현을표준화하여비교분석하였다. 4. Senescence associated β-galactosidase assay 세포노화 (senescence) 는 β-galactosidase를염색하는방법인 SA-β-gal (senescence associated β-galactosidase) assay를이용하여측정하였다 (Dimri et al., 1995). SA-β -gal assay는 senescence detection kit (Biovision, USA) http://www.e-ajbc.org 68
Antioxidant and Antiwrinkle Effects of Amentoflavone for Cosmetic Materials Development 를사용하였으며, HDFs (2 10 5 cells/well) 로 60 mm 배양접시에접종하여 24시간동안배양후 amentoflavone을 5, 10, 15 μm 농도로각각처리하고 H 2 O 2 를처리후 24시간배양하였다. 배양된 HDFs는배지를제거하고 1 ml PBS로세척후 fixing solution 0.5 ml 을첨가하여상온에서 15분간방치하여고정시킨다. 고정된 HDFs를 staining solution 혼합액 (staining solution 470 μl, staining supplement 5 μl, 20 mg/ml X-gal in dimethylformamide 25 μl) 를 0.5 ml씩첨가하고 37 에서 24시간동안배양하여세포염색을한다. 염색된세포는 PBS로세척후광학현미경 (Olympus, Japan) 을통하여염색된세포수를측정하여세포의비율을분석한다. Results and Discussion 1. Inhibition of cell damage by amentoflavone HDFs에시험물질인 amentoflavone의세포독성을확인하기위해농도별로 amentoflavone을처리한결과, amentoflavone 5, 10, 15, 20, 25 µm 처리시 99.2%, 97.9%, 92.2%, 87.9%, 77.6% 의세포생존율을가지는것을확인할수있었다. 본연구에서는세포생존율 90% 이상을기록하는 amentoflavone 5, 10, 15 μm을이후실험농도로적용하였다 (Figure 3A). HDFs에 H 2 O 2 를사용하여산화적스트레스를유도하고세포생존회복율의변화를확인하였다. HDFs에 H 2 O 2 를 0.1, 0.2, 0.3, 0.4, 0.5 mm 농도로처리할경우세포생존율은각각 89.6%, 72%, 66.6%, 55.3%, 42.7% 을나타냈으며, H 2 O 2 적용농도는세포생존율이 70% 이상인 0.2 mm를사용하기로결정하였다 (Figure 3B). H 2 O 2 의 HDFs 세포손상에대한 amentoflavone의세포보호효과를확인하기위해 HDFs에 amentoflavone을 5, 10, 15 µm의농도로 3시간전처리후 H 2 O 2 를 0.2 mm을처리하여세포생존율의변화를확인하였다. Amentoflavone은 5, 10, 15 μm 처리시각각 75.5%, 92.6%, 97.1% 로농도의존적으로세포생존율이증가함을확인하였다 (Figure 3C). 2. Antioxidant effects of amentoflavone DPPH radical 소거작용은항산화성물질과반응하여수소원자를받아들임으로써산화를억제시켜항산화능의정도를측정하는방법이다. H 2 O 2 에의한세포생장저해및사멸을억제하는 amentoflavone의항산화효과를확인한결과, amentoflavone을 1, 2, 5, 10, 15 µm 처리시 16.4%, 48.2%, 77.9%, 98.1%, 98.7% 로농도의존적으로항산화효과가증가하여항산화표준물질로알려진 BHT와유사한결과를나타냄 Figure 3. Inhibition of cell damage by amentoflavone. (A) Cytotoxicity of amentoflavone on HDFs. (B) Cytotoxicity of H 2 O 2 on HDFs. (C) The effect of amentoflavone on cell viability in H 2 O 2 -treated HDFs. The Student s t-test was performed to determine statistical significance (*p<.05). 을확인하였다 (Figure 4A). ABTS radical 소거작용은산화방지제에의해 ABTS + 라 디칼의흡광도를저해시키는원리를이용하는방법이다. Amentoflavone 의항산화효과는 positive control 그룹인 BHT 를 1, 2, 5, 10, 15 µm 처리시 85.4%, 99.8%, 99.2%, 99.7%, 100.7% 로나타났으며, amentoflavone 은 26.4%, 82.1%, 91.3%, 98.6%, 100.4% 로농도의존적으로상승하였 으며 DPPH 결과와마찬가지로 BHT 와유사한항산화효과를 나타내었다 (Figure 4B). 69
화장품소재개발을위한 amentoflavone 의항산화및항주름효과연구 Figure 4. Antioxidant effects of amentoflavone. (A) Electron donating ability from amentoflavone. (B) ABTS + radical cation scavenging activity of amentoflavone. (C) Hydrogen peroxide scavenging ability of amentoflavone. (D) The intracellular ROS levels in H 2 O 2 -treated HDFs. The Student s t-test was performed to determine statistical significance (*p<.05). 한편, amentoflavone의 H 2 O 2 에대한항산화효과에관련해서 positive control 그룹인 LA는 1, 2, 5, 10, 15 µm 처리시 14.7%, 74.4%, 104.1%, 110.8%, 125.1% 로나타났으며, amentoflavone은 9.8%, 31.9%, 45.2%, 108.1%, 110.8% 로나타났다. Amentoflavone이저농도에서는그효율이 LA보다좋지못하였으나, 농도가높아질수록비슷하거나유사한효과가나타난것을확인하였다 (Figure 4C). HDFs에서활성산소종측정방법인 DCF-DA assay는세포내의산화과정을통해가장보편적으로사용되는활성산소종측정방법이다. Amentoflavone이 H 2 O 2 에의해세포내발생하는 ROS의억제효능을확인한결과, HDFs에 H 2 O 2 를 0.2 mm 처리시 H 2 O 2 를처리하지않았을때보다 ROS가 6.8배정도증가하였으나 amentoflavone를 5, 10, 15 μm 처리시 ROS는 6 배, 4배, 2.3배로농도의존적으로억제되었다. 그결과 H 2 O 2 에의해생성된세포내 ROS를 amentoflavone이효과적으로제거함을확인하였으며본실험의양성대조군인 ROS 소거능력이뛰어난 NAC 30 μm 처리시, 유사한효과를갖는것을확인함으로써 amentoflavone의 ROS scavenging effect가유의적임을알수있었다 (Figure 4D). 3. Effects of amentoflavone on antioxidant and antiwrinkle gene expression 노화억제기전에있어대표적항산화효소인 SOD1의유전자발현변화를확인한결과, H 2 O 2 를 0.2 mm 처리시대조군에비해 0.57배감소하였으나 amentoflavone 5, 10, 15 μ M 처리시 0.72배, 0.80배, 0.86배로회복됨을확인하였다 (Figure 5A). CAT mrna의경우, H 2 O 2 를 0.2 mm 처리시대조군에비해 0.55배감소하였으나 amentoflavone을 5, 10, 15 μm 처리시 0.62배, 0.75배, 0.81배로회복됨을확인하였다 (Figure 5B). 한편, 주름과관련된 MMP1의유전자발현변화결과는 H 2 O 2 를단독으로 0.2 mm 처리시 H 2 O 2 와 amentoflavone 을모두처리하지않은대조군에비해 3.6배이상증가하는것으로나타났으나 amentoflavone을 5, 10, 15 µm로처리하였을때 MMP1의발현양이 2.4배, 2.3배, 1.7배로감소하였다 (Figure 5C). 또한 MMP3 mrna의경우, H 2 O 2 만 0.2 mm 처리시 H 2 O 2 와 amentoflavone을모두처리하지않은대조군에비해 4.7배이상증가하는것으로나타났으나 amentoflavone 을 5, 10, 15 µm로처리하였을때 MMP3의발현양이 3.4배, 2.6배, 1.4배로감소하였다 (Figure 5D). http://www.e-ajbc.org 70
Antioxidant and Antiwrinkle Effects of Amentoflavone for Cosmetic Materials Development Figure 5. Effects of amentoflavone on antioxidant and antiwrinkle gene expression. (A) The effect of amentoflavone on SOD1 expression in H 2 O 2 -treated HDFs. (B) The effect of amentoflavone on CAT expression in H 2 O 2 -treated HDFs. (C) The effect of amentoflavone on MMP1 expression in H 2 O 2 -treated HDFs. (D) The effect of amentoflavone on MMP3 expression in H 2 O 2 -treated HDFs. (E) The effect of amentoflavone on COL1A1 expression in H 2 O 2 -treated HDFs. The Student s t-test was performed to determine statistical significance (*p<.05). H 2 O 2 산화적스트레스처리시세포는다양한신호전달에의해콜라겐합성이저해되는데 amentoflavone의콜라겐합성에미치는영향을확인한결과, H 2 O 2 를 0.2 mm 처리시 COL1A1 mrna의발현은대조군에비해 0.30배감소하였다. 그러나 amentoflavone을 5, 10, 15 µm로전처리하고 H 2 O 2 를 0.2 mm 처리시 COL1A1 mrna의발현양은 0.51 배, 0.72 배, 0.80배로나타나 amentoflavone이농도의존적으로 COL1A1 의유전자발현을증가시키는것을확인할수있었다 (Figure 5E). 4. Antiaging effects of amentoflavone Amentoflavone를처리하지않고 H 2 O 2 0.2 mm을처리시 HDFs의 SA-β-galactosidase의비율은 amentoflavone과 H 2 O 2 를모두처리하지않은대조군에비해 66.4% 로증가하였다. 그러나 amentoflavone을 5, 10, 15 μm 농도로전처리하고 H 2 O 2 를 0.2 mm로처리한경우 HDFs의 SA-β-galactosidase 비율은 52.7%, 41.9%, 26.1% 로현저히감소하는것을확인하였다 (Figure 6). 71
화장품소재개발을위한 amentoflavone 의항산화및항주름효과연구 Figure 6. The protective effect of amentoflavone in H 2 O 2 -induced senescent HDFs. The Student s t-test was performed to determine statistical significance (*p<.05). Conclusion 피부에노화를일으키는다양한이론중 ROS 는세포막의지 질, 효소, 구조단백질, DNA 와같은분자의손상은물론, 질 병과노화를가속시킨다. 피부의진피층에존재하는 HDFs 에 ROS 종류인 H 2 O 2 를처리하였으며, 본연구에서는항노화기능 성화장품원료로서의가능성을확인하기위해항암, 항염효과 가우수한 biflavonoid 의종류중하나인 amentoflavone 를통 해항산화, 항노화연구를검증하였다. Amentoflavone 은인간진피섬유아세포 (human dermal fibroblast; HDFs) 에대한독성은없었으며, H 2 O 2 로인한세포 독성상황에서도 amentoflavone 을처리시세포생존율이증가 함을확인함으로써 amentoflavone 이세포사멸억제에기여한 다는것을확인할수있었다. 또한, amentoflavone 의항산화효과를확인하기위하 여 DPPH assay 및 ABTS assay 를수행한결과, 두실험모 두농도의존적으로항산화효과가증가하여대조군인 BHT 와유사한효과를나타냄을확인하였다. H 2 O 2 assay 에서도역 시대조군인 LA 와유사한항산화효과를나타내었다. ROS detection assay 와관련하여, H 2 O 2 에의해증가된 ROS 는 amentoflavone 5, 10, 15 µm 처리시 6.0 배, 4.0 배, 2.3 배로 농도의존적으로억제되었고 15 µm 농도에서 ROS 제거능력 은 NAC 30 µm 과유사한수치로나타났다. 항산화효과를유전자수준에서도확인해본결과, 대표적인 항산화효소인 SOD1 mrna 및 CAT mrna 역시 H 2 O 2 에의 해유전자발현이감소하였으나, amentoflavone 처리시농 도의존적으로유전자발현이상승하였다. 이러한결과를통해 amentoflavone 의항산화효과뿐만아니라, 항산화소재로서 의가능성도확인할수있었다. 항노화연구와관련하여 H 2 O 2 처리시발현이증가하는 MMP1, MMP3는 amentoflavone 처리시유전자발현이감소한반면, H 2 O 2 에의해발현이감소한 COL1A1 유전자는 amentoflavone 처리시발현이증가하였다. 또한, SA-β-galactosidase의세포노화실험에서농도의존적으로노화된세포의비율이현저히감소하였다. 이러한결과는 amentoflavone이 H 2 O 2 에의해유도된세포노화를효과적으로억제함을보여주는것이다. 이로써 amentoflavone이 ROS종의하나인 H 2 O 2 의산화적스트레스로부터피부를효과적으로보호하며, 항노화화장품소재로서충분한가능성이있음을보여주는것이라할수있다. Acknowledgements 본논문은교육과학기술부중견도약연구지원사업 ( 과제번호 20110028646) 과보건복지부보건의료연구개발사업 ( 과제번호 HN13C0075) 의지원에의한것이며, 이에감사드립니다. References Ahn BT, Shin SA, Kim JG, Park WH. Inhibitory Effect of Amentoflavone of Selaginella tamariscina on MMP-9 Expression through NF-κB and AP-1 in Macrophage Raw 264.7 cells. Korean Journal of Oriental Physiology & Pathology, 21: 243-249, 2007. Bailly C, Dreze S, Asselineau D, Nusgens B, Lapiere CM, Darmon M. Retinoic acid inhibits the production of collagenase by human epidermal keratinocytes. Journal of Investigative Dermatology, 94: 47-51, 1990. Banerjee T, Van der Vliet A, Ziboh VA. Downregulation of COX-2 and inos by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line. Prostaglandins, Leukotrienes and Essential Fatty Acids, 66: 485-492, 2002. Barbouti A, Doulias PT, Nousis L, Tenopoulou M, Galaris D. DNA damage and apoptosis in hydrogen peroxideexposed Jurkat cells: bolus addition versus continuous generation of H 2 O 2. Free Radical Biology & Medicine, 33: 691-702, 2002. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature, 181: 1199-1200, 1958. Bonfil RD, Reddel RR, Ura H, Reich R, Fridman R, Harris CC, Klein-Szanto JP. Invasive and metastatic potential of a v-ha-ras-transformed human http://www.e-ajbc.org 72
Antioxidant and Antiwrinkle Effects of Amentoflavone for Cosmetic Materials Development bronchial epithelial cell line. Journal of the National Cancer Institute, 81: 587-594, 1989. Braig M, Lee S, Loddenkemper C, Rudolph C, Peters AHFM, Schlegelberger B, Stein H, Dorken B, Jenuwein T, Schmitt CA. Oncogene induced senescence as an initial barrier in lymphoma development. Nature, 436: 660-665, 2005. Campisi J, d Adda di Fagagna F. Cellular senescence: when bad things happen to good cells. Nature Reviews Molecular Cell Biology, 8: 729-740, 2007. Collado M, Gil J, Efeyan A, Guerra C, Schuhmacher AJ, Barradas M, Benguria A, Zaballos A, Flores JM, Barbacid M, et al. Tumour biology: senescence in premalignant tumours. Nature, 436: 642, 2005. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O. A biomarker that identifies senescenthuman cells in culture and in aging skin in vivo. Proceedings of the National Academy of Sciences of the United States of America, 92: 9363-9367, 1995. Glimcher MJ. Mechanism of calcification: role of collagen fibrils and collagen-phosphoprotein complexes in vitro and in vivo. The Anatomical Record, 224: 139-153, 1989. Guruvayoorappan C, Kuttan G. Inhibition of tumor specific angiogenesis by amentoflavone. Biochemistry, 73: 209-218, 2008. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Experimental Cell Research, 25: 585-621, 1961. Holden JM, Bhagwat SA, Haytowitz DB. Development of a database of critically evaluated flavonoids data: application of USDA s data quality evaluation system. Journal of Food Composition and Analysis, 18: 829-844, 2005. Hwang IS, Lee JY, Jin HG, Woo ER, Lee DG. Amentoflavone stimulates mitochondrial dysfunction and induces apoptotic cell death in Candida albicans. Mycopathologia, 173: 207-218, 2012. Hwang JH, Choi HM, Woo ER, Lee DG. Antibacterial effect of amentoflavone and its synergistic effect with antibiotics. Journal of Microbiology and Biotechnology, 23: 953-958, 2013. Ishola IO, Chatterjee M, Tota S, Tadigopulla N, Adeyemi OO, Palit G, Shukla R. Antidepressant and anxiolytic effects of amentoflavone isolated from Cnestis ferruginea in mice. Pharmacology Biochemistry and Behavior, 103: 322-331, 2012. Jung HJ, Sung WS, Yeo SH, Kim HS, Lee IS, Woo ER, Lee DG. Antifungal effect of amentoflavone derived from Selaginella tamariscina. Archives of Pharmacal Research, 29: 746-571, 2006. Lee CW, Na YJ, Park NH, Kim HS, Ahn SM, Kim JW, Kim HK, Jang YP. Amentoflavone inhibits UVBinduced matrix metalloproteinase-1 expression through the modulation of AP-1 components in normal human fibroblasts. Applied Biochemistry and Bootechnology, 166: 1137-1147, 2012. Lee EJ, Shin SY, Lee JY, Lee SJ, Kim JK, Yoon DY, Woo ER, Kim YM. Cytotoxic activities of amentoflavone against human breast and cervical cancers are mediated by increasing of PTEN expression levels due to peroxisome proliferator-activated receptor γ activation. Bulletin of the Korean Chemical Society, 33: 2219-2223, 2012. Lee JS, Sul JY, Park JB, Lee MS, Cha EY, Song IS, Kim JR, Chang ES. Fatty acid synthase inhibition by amentoflavone suppresses HER2/neu (erbb2) oncogene in SKBR3 human breast cancer cells. Phytotherapy Research, 27: 713-720, 2013. Liotta LA, Steler-Stevenson WG. Metalloproteinases and cancer invasion. Seminars in Cancer Biology, 1: 99-106, 1990. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S. Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature, 284: 67-68, 1980. Liu RH. Potential synergy of phytochemicals in cancer prevention: mechanism of action. The Journal of Nutrition, 134: 3479-3485, 2004. Long LH, Evans PJ, Halliwell B. Hydrogen peroxide in human urine: implications for antioxidant defense and redox regulation. Biochem. Biochemical and Biophysical Research Communications, 263: 605-609, 1999. Lüschen S, Ussat S, Scherer G, Kabelitz D, Adam- Klages S. Sensitization to death receptor cytotoxicity by inhibition of fas-associated death domain 73
화장품소재개발을위한 amentoflavone 의항산화및항주름효과연구 protein(fadd)/caspase signalling. Requirement of cell cycle progression. The Journal of Biological Chemistry, 275: 24670-24678, 2000. Matrisian LM. Metalloproteinases and their inhibitors in matrix remodeling. Trends in Genetics, 6: 121-125, 1990. Michaloglou C, Vredeveld LC, Soengas MS, Denoyelle C, Kuilman T, van der orst CM, Majoor DM, Shay JW, Mooi Wj, et al. BRAFE600-associated senescencelike cell cycle arest of human naevi. Nature, 436: 720-724, 2005. Okigawa M, Hwa CW, Kawano N, Rahman W. Biflavones in Selaginella species. Phytochemistry, 10: 3286-3287, 1971. Park NH, Lee CW, Bae JH, Na YJ. Protective effects of amentoflavone on Lamin A-dependent UVB-induced nuclear aberration in normal human fibroblasts. Bioorganic & Medicinal Chemistry Letters, 21: 6482-6484, 2011. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26: 1231-1237, 1999. Sakthivel KM, Guruvayoorappan C. Amentoflavone inhibits inos, COX-2 expression and modulates cytokine profile, NF-κB signal transduction pathways in rats with ulcerative colitis. International Immunopharmacology, 17: 907-916, 2013. Song HH, Khiev P, Chai HS, Lee HK, Oh SR, Choi YH, Chin YW. Flavonoids from Thyrsanthera suborbicularis and Their NO Inhibitory Activity. Natural Product Sciences, 18; 273-278, 2012. Wilsky S, Sobotta K, Wiesener N, Pilas J, Althof N, Munder T, Wutzler P, Henke A. Inhibition of fatty acid synthase by amentoflavone reduces coxsackievirus B3 replication. Archives of Virology, 157: 259-269, 2012. Zhang J, Liu Z, Cao W, Chen L, Xiong X, Qin S, Zhang Z, Li X, Hu CA. Amentoflavone inhibits angiogenesis of endothelial cells and stimulates apoptosis in hypertrophic scar fibroblasts. Burns, 40: 922-929, 2014. http://www.e-ajbc.org 74
Antioxidant and Antiwrinkle Effects of Amentoflavone for Cosmetic Materials Development 국문초록화장품소재개발을위한 amentoflavone의항산화및항주름효과연구 이송정, 안성관 * 건국대학교생물공학과, 서울, 한국 목적 : 본연구는항산화및항주름효과검증을통해 amentoflavone의노화예방화장품소재로서의발전가능성을확인하는데그목적이있다. 방법 : amentoflavone의항산화및항주름효과를확인하기위해 in vitro antioxidant assays, quantitative real-time PCR, SAβ-galactosidase assay, DCF-DA assay를수행하였다. 결과 : amentoflavone은 0.2 mm의 H 2 O 2 처리시, 15 μm농도까지농도의존적으로세포생존율을증가시켰으며, 양성대조군인 BHT와유사한항산화효과를나타냈다. 유전자수준에서대표적항산화효소인 SOD1 mrna와 CAT mrna의발현을농도의존적으로증가시켰다. 콜라겐분해와관련된 MMP1 mrna와 MMP3 mrna의발현은농도의존적으로감소시켰으나콜라겐합성과관련된 COL1A1은농도의존적으로증가시켰다. 또한 0.2 mm의 H 2 O 2 에의해유발된진피섬유아세포의노화를농도의존적으로감소시키는것으로나타났다. 결론 : 이상의결과들을통해, amentoflavone이농도의존적으로 H 2 O 2 에의해손상된인간진피섬유아세포에대한항산화, 항주름효과가있음이확인되었다. 그러므로 amentoflavone은노화예방효능을가진천연유래화장품원료로서활용가능하리라고사료된다. 핵심어 : Amentoflavone, 인간진피섬유아세포, 항산화, 항주름, 노화예방 본논문은교육과학기술부중견도약연구지원사업 ( 과제번호 20110028646) 과보건복지부보건의료연구개발사업 ( 과제번호 HN13C0075) 의지원에의한것이며, 이에감사드립니다. 75
화장품소재개발을위한 amentoflavone 의항산화및항주름효과연구 中文摘要化妆品开发原料 amentoflavone 的抗氧化及抗皱纹效果研究 李松靜, 安成官 * 建国大学校生物工学科, 首尔, 韩国 目的 : 通过鉴定抗氧化及抗皱纹效果, 确认 amentoflavone 作为抗皱化妆品原料的发展可能性 方法 : 通过 In vitro antioxidant assays,quantitative real-time PCR,SA-β-galactosidase assay,dcf-da assay 等方法研究 amentoflavone 抗氧化及抗皱纹效果 结果 : 对 0.2 mm H 2 O 2 处理的皮肤真皮层存在的 HDFs, 用 amentoflavone 处理时, 随着 amentoflavone 的浓度增加而提高了细 胞生存率, 与阳性对照品 BHT 有类似的抗氧化效果 从基因角度上看,H 2 O 2 抑制代表性抗氧化酶 SOD1 mrna 和 CAT mrna 的 表达, 而随着 amentoflavone 的浓度增加而提高了代表性抗氧化酶 SOD1 mrna 和 CAT mrna 的表达 分解胶原蛋白相关的酶 MMP1 mrna 和 MMP3 mrna 随着 amentoflavone 的浓度增加而减少, 而合成胶原蛋白相关的 COL1A1 随着 amentoflavone 的 浓度增加而增加 0.2 mm H 2 O 2 诱发的真皮细胞的老化随着 amentoflavone 的浓度增加而减少 结论 : amentoflavone 对 H 2 O 2 所损伤的人体真皮纤维细胞具有抗氧化及抗皱效果 因此 amentoflavone 可以用作预防抗皱效果 的天然化妆品原料 关键词 : Amentoflavone, 人体真皮纤维细胞, 抗氧化, 抗皱, 预防老化 http://www.e-ajbc.org 76