RESEARCH ARTICLE Kor. J. Aesthet. Cosmetol., Vol. 12 No. 5, , October 2014 감잎추출물의항산화및항염증효과 이정희광주여자대학교미용과학과 Anti-Oxidant and Anti-Inflammatory E

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1 RESEARCH ARTICLE 감잎추출물의항산화및항염증효과 이정희광주여자대학교미용과학과 Anti-Oxidant and Anti-Inflammatory Effects of Diospyros kaki Thumb Leaves Extracts Jung-Hee Lee Department of Beauty Science, Kwangju Women s University Abstract Oxidative stress has been implicated in cutaneous damage in various inflammatory skin diseases, including psoriasis. Flavonoids have increased lately due to their broad pharmacological effect, explained by their inhibition of certain enzymes and their antioxidant activity. Accordingly, to develop oxidant and inflammation care cosmetic ingredients, the extracts Diospyros kaki leaves were selected. In this study, we investigated effect on cell viability and anti-oxidant activity of Diospyros kaki leaves extracts by using sulforhodamine B (SRB) assay and 1,1-diphenyl-2-picryl hydrazyl (DPPH) method. The anti-inflammatory activity of Diospyros kaki leaves extracts on nitrite production and tumor necrosis factor-α (TNF-α) secretion were tested using nitric oxide (NO) colorimetric assay kit and enzyme-linked immunosorbent assay (ELISA) kit. Diospyros kaki leaves extracts showed the high cell viability over 100 % against mouse macrophage RAW cell at concentration of 10, 50, 100 and 500 μg/ml. Those exhibited anti-oxidant activity with dose dependent manner at concentration of 10, 50, 100 and 500 μg/ml. Nitrite production and TNF-α secretion were significantly decreased with dose dependant manner according to increment of treated concentration with Diospyros kaki leaves extracts (10, 50, 100 and 500 μg/ml) (p<.05). These results demonstrate that Diospyros kaki leaves extract possessing anti-oxidant and anti-inflammatory effects may be applicable to heal skin damage. Keywords: Inflammatory skin diseases, Diospyros kaki leaves, Cosmetic ingredients, Anti-oxidant, Anti-inflammatory 노화란노령화에따라서나타나는신체적, 정신적기능의퇴 화적변화로서주름살증가, 탄력성감소, 피부처짐등의현 상이동반된다 (Kim et al., 2010). 피부노화는내인성인자에 의한자연노화와장기간에걸친광노출로피부변화의현상 이오는광노화로나뉜다 (Parrado et al., 1999). 광노화의원 인인자외선은피부에존재하는자외선흡수물질에의해흡수 Corresponding author: Jung-Hee Lee, Department of Beauty Science, Kwangju Women s University, Gwangju , Republic of korea Tel: , Fax: jh4471@kwu.ac.kr Received September 17, 2014; Revised October 27, 2014; Accepted October 28, 2014; Published October 30, 2014 되며 (Adhami et al., 2008), 이로인해피부세포또는조직을손상시키는광화학적반응들과활성산소종 (reactive oxygen species, ROS) 을경유한이차적인반응들에의해홍반, 부종, 과색소침착, 일광화상세포증가, 면역억제, 광노화, 광발암등을유발한다 (Afaq & Mukhtar, 2006; Bowden, 2004) 이러한활성산소종은피부항산화제파괴, 지질과산화반응의개시, 단백질및 DNA 산화, 결합조직성분 ( 콜라젠, 히아루론산등 ) 의사슬절단및비정상적인교차결합에의한주름생성, 멜라닌생성과정등에참여하여피부노화를가속화시킨다 (Park, 2003). 또한자외선조사에의해콜라겐의합성이감소하는동시에기질금속단백분해효소 (matrix metalloproteinases, MMPs) 의발현이증가하여기질의주요구성성분인콜라겐과엘라스틴등의분해가증가되어주름살이형성되는것으로알려져있다 (Hashem et al., 2008; Yang et al., 2006). 한편일산화질소 (nitric oxide, NO) 는높은반응성을가진 719

2 생체생성분자로서, 신경전달, 혈관의이완및세포매개성면역반응에관여하는데, 생체의피부가염증상태에빠지게되면 inducible NOS (inos) 가발현되어많은양의 NO를생산한다 (Choi et al., 2002). 이렇게생성된 NO와같은염증촉진매개물은피부염을더욱악화시키는것으로알려져있다 (Bruch- Gerharz et al., 1009, 1998). 따라서활성산소종이나일산화질소등에의해유발되는산화적스트레스로부터생체의항상성을유지하기위해필요한 superoxide dismutase (SOD), catalase 등생체효소가계속적으로활성되는데, 결과적으로생체내의항산화효소가고갈되면서피부염증반응이심화되는것으로알려졌다 (Zhao et al., 2000). 따라서이러한피부노화를억제하고지연시키고자주름개선및억제용화장품의개발이증가하고있다 (Seo et al., 2014). 또한이러한활성산소종으로부터발생하는손상을억제하기위해다양한항산화물질을사용해왔고이러한물질에는 BHA (butylated hydroxyanisol) 와 BHT (butylate hydroxy toluene) 등의합성항산화제나천연물에서유래된천연항산화제등이있다 [(Branen, 1975; Lee et al., 2011). 합성항산화제들의경우에는과량사용시독성을나타내는것으로보고되고있으므로보다안전하고항산화효과가뛰어난천연항산화제개발이점점더활발하게이루어지고있다 (Masaki et al., 1995). 감 (Diospyros kaki Thumb) 은우리나라를비롯한동아시아에서널리재배되고있으며, 감잎에는비타민 C, 무기질이많이함유되어있고 flavonoid, tannin 등이함유되어있다 (Joung et al., 1995). 감잎의효능으로는괴혈병, 빈혈, 동맥경화에효과적이며, 콜레스테롤저하능이보고되고있다 (Funayama & Hikino, 1979; Innami et al., 1998). 따라서본연구에서는감잎열수추출물의항산화및항염증효과를연구함으로써기능성소재로서의가능성을살펴보았다. 1. 실험재료및시약본실험에이용한감잎 (Diospyros kaki leaves) 은 2013 년충남부여군에서채취하여음건하여본연구실에보관하면서사용하였다. 감잎을분쇄한후분말 100 g에증류수 400 ml를가해열수추출한후여과지 (Whatmann No. 2) 를이용하여여과하였다. 그여액을감압농축하여농축액 12 g을얻어 -20 에보관하였다. Sulforhodamine B (SRB) 와 Butylated hydroxyanisol (BHA), dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS), 1, 1-diphenyl-2- picryl-hydrazyl (DPPH) 은 Sigma-Aldrich (St. Louis, MO, USA) 에서구입하여사용하였다. Nitric oxide (NO), colorimetric assay kit는 Biovision사 (CA, USA) 로부터구입하였으며, tumor necrosis factor-α (TNF-α), enzyme-linked immunosorbent assay (ELISA) kit는 ebioscience 사 (San Diego, CA, USA) 에서구입하였다. Antibiotic-antimycotic solution, fetal bovine serum (FBS), Dulbecco s modified Eagle s medium (DMEM), trypsin-edta는 Gibco BRL (Grand Island, NY, USA) 에서구입하여사용하였다. Tissue culture plates들은 Falcon (BD Biosciences, Franklin Lakes, NJ, USA) 에서구입하여사용하였다. 2. 세포주및세포배양 Mouse macrophage RAW 세포 (KCLB 40071, Seoul, Korea) 은한국세포주은행에서구입하였으며, 구입한세포를 DMEM 배지에 10% FBS, 1% antibiotic-antimycotic solution을첨가하여 37, 5% CO 2 조건하에서배양하였다. 3. 세포생존율의측정 (SRB Assay) 세포생존율은이미보고된 SRB 염색방법으로측정하였다 (Choi et al., 2010). 즉, 세포를 96-well plate의각 well에 개되게 100 μl씩접종하고 37, 5% CO 2 배양기에서 24시간배양하였다. 다음날세포의배양배지를제거하고감잎추출물을 10, 50, 100, 500 μg/ml로처리하여다시 24 h 동안배양한후배지를제거하고 Phosphate buffered saline (PBS) 로세포를한번세척해주었다. -20 에보관한 70% acetone 용액 100 μl를각 well 에가하여 20 에서 30분간정치한후 acetone 용액을제거한후, 건조기에서 30분간방치하였다. 1% 초산용액에녹인 0.4% SRB 100 μl를각 well에가하여실온에서 30분간반응시킨다음, SRB를제거하고 1% 초산용액으로 5회세척한후 24시간건조, 고정시켰다. 건조시킨 plate에 10 mm Tris-base용액 100 μl을가하여세포에결합한 SRB를용해시켜 562 nm 에서 VERSAmax microplate reader (Molecular Devices, PaloAlto, CA, USA) 를사용하여 plate의각 well의흡광도를측정하였다. 동시에 620 nm 파장에서흡광도를측정하여배경색에대한대조군으로사용하였다. 결과는대조군의 % 로계산되어졌다. 4. 항산화활성측정 DPPH 방법에의한항산화활성은 Blois 방법에따라측정하였다 (Blois, 1958). 감잎추출물 0.2 ml에 0.4 mm DPPH 3.8 ml을가하고 10초간혼합한후, 실온에서 30분간반응하고 517 nm 파장에서흡광도를측정하였다. 음성대조군은시료대신에탄올을동량첨가하여실험하였고, 양성대조군으로는합성항산화제인 BHA를이용하여동일한방법으로실시하였다

3 감잎추출물의항산화및항염증효과 Figure 1. Cell viability of Mouse macrophage RAW cell treated with extracts of Diospyros kaki leaves. The extracts of Diospyros kaki leaves treated with concentration of 10, 50, 100, 500 μg/ml. The data are displayed with mean±standard deviation (n=3). 항산화능은시료첨가구와무첨가구의흡광도를구하여백분율 로표시하였다. 5. NO 생성저해작용의측정 NO 의농도는배양액내의 nitrite 농도를보고된방법에따 라 Griess Reagent System 을이용하여측정하였다 (Huang et al., 2011). RAW 세포를 cells/ml 로 96 well plate 에분주하여 12 h 부착시킨후 LPS 를 10 μg/ml 이되 도록가하였다. 감잎추출은 LPS 와동시에배양액에 10, 50, 100, 500 μg/ml 의농도를가한다음 37, 5% CO 2 incubator 에서 24 h 배양하였다. 배양액과동량의 Griess Reagent 를가 하고 10 min 간상온에서반응시킨후 540 nm 에서흡광도를 측정하였다. sodium nitrite 의농도별표준곡선을이용하여배 양액의 NO 농도를결정하였다. 6. 세포배양액내의 TNF-α 측정 RAW 세포를 96 well plate 에 cells/well 이 되도록분주하고 12 h 동안배양한후 LPS (10 μg/ml) 와동시 에감잎추출물을 10, 50, 100, 500 μg/ml 의농도로처리하고 24 h 동안배양한후세포배양액을얻은다음배양액에함유된 TNF-α 을 ELISA kit 을이용하여측정하였다 (Thaher et al., 2011). 7. 통계처리 모든결과값은세번의독립적인실험에의해얻어진값들의 평균 ± 표준편차로나타내었다. 대조군과실험군사이의통계학 적유의성검정은 Tukey test 를사용하면서 ANOVA 검정을적 용하였으며, p<.05 인경우통계적으로의미있는차이가있다 고판정하였다. Figure 2. Antioxidant activity of the extracts of Diospyros kaki leaves. BHA tested at a concentration of 1 μg/ml. The data are displayed with mean ± standard deviation (n=3). 1. 세포생존율 RAW 세포에대한감잎추출물의세포독성을보기위 하여감잎추출물을 10, 50, 100, 500 μg/ml 의농도로처리 한후 SRB assay 를실시하여 cell viability 를측정하였다. 실험 결과각각 100.5±4.6%, 101.2±8.0%, 100.0±1.6%, 99.9± 5.1%, 로세포독성을나타내지않았다 (Figure 1). 2. 항산화활성 감잎추출물의 DPPH 라디칼소거능을알아보기위해합성 항산화제로잘알려진 BHA 와비교하였다. 그결과 Figure 2 과 같이감잎추출물의 DPPH 라디칼소거능은농도에의존적으 로증가했으며, 500 μg/ml 의농도에서 BHA 와유사한효과를 나타내었으며, 나머지농도 (10, 50, 100 μg/ml) 에서는 BHA 보다낮은효과를나타내었다. 전의보고에서많은식물체들 은항산화화합물을보유하고있으며, 특히플라보노이드화합 물을함유하고있는것으로알려져있다 (Jeong & Noh, 2008). 본연구에서감잎추출물은항산화활성이높은것으로나타났 으며, 이는감잎중에플라보노이드에기인한것으로사료된다. 따라서향후감잎추출물로부터항산화물질을나타내는단일 화합물을규명하는연구가더필요하다. 3. NO 생성에미치는영향 활성산소의일종으로최근염증유발에중요한역할을하는 것으로알려진 NO 는높은반응성을가진생체생성분자로서, NOS (Nitric oxide synthase) 에의해 L-arginine 으로부터생 성되는데, 특히 TNF-α 와같은염증성사이토카인의자극이있 을때발현된다 (Park & Jung, 2013). 염증반응에관여되는핵 심적인분자인 NO 는염증매개물로써염증반응을가속화시켜 721

4 Figure 3. Inhibitory effects of the extracts of Diospyros kaki leaves on NO production in LPS-stimulated RAW264.7 Cells. RAW264.7 cells were treated with or without LPS (10 μg/ml) and then with extracts of Diospyros kaki leaves and incubated for 24 h. The nitrite concentrations in medium were determined by NO Detection Kit. Results of three independent experiments were averaged mean value of three independent experiments (mean±standard deviation). *Significantly different from control (p<.05). **Significantly different from LPS (p<.05). Con, without LPS. 생체의질환을더욱악화시킨다 (Gallin & Snyderman, 1999; Tinker & Wallace, 2006). 특히건선 (psoriasis) 과같은만성 적인염증성질환은이러한염증매개물이높게발현되어있는 특징이있다 (Iversen et al., 1997). 따라서염증반응을억제시 키기위해서는 NO 와같은염증매개물을효과적으로억제시키 는것이필수적이다. 본연구에서 LPS 를이용하여 RAW 세포의 NO 생성에대한감잎추출물의효과를측정한결과 (Figure 3), RAW 세포만배양한대조군에서 NO 의농도 는 1.4±1.4 μm 로매우낮게측정되었으며, LPS 를처리한군 에서 NO 의농도는통계적으로대조군과유의성을나타내면서 40.2±6.9 μm 로증가되었다 (p<.05). 감잎추출물을 10, 50, 100, 500 μg/ml 처리한실험군은각각 29.6±3.2 μm, 25.7 ±1.9 μm, 14.2±3.2 μm, 7.4±0.5 μm 로측정되어농도의존 적으로 NO 생성이억제되는것을관찰할수있었고 LPS 를처 리한군과비교시유의한감소를나타내었다 (p<.05). 이러한감 잎추출물의 NO 저해효과는감잎추출물이함유한 flavonoid 계열 (Joung et al., 1995) 의화합물에의한것으로사료된다. 3. TNF-α 분비에미치는영향 TNF-α 분비에대한감잎추출물의영향을살펴본결과를 Figure 4 에나타내었다. RAW264.7 세포만배양한대조군에서 TNF-α 의농도는 1.2±0.1 ng/ml 로나타났다. 그러나 LPS 를처리한군에서 TNF-α 의농도는 3.9±0.1 ng/ml 로유의하 게증가됨을알수있었다 (p<.05). 감잎추출물을 10, 50, 100, 500 μg/ml 처리한군에서는각각 3.5±0.2 ng/ml, 3.1±0.1 ng/ml, 2.1±0.5 ng/ml, 1.2±0.1 ng/ml 로측정되어농도 Figure 4. Effects of the extracts of Diospyros kaki leaves on expression of TNF-α in macrophage cell lines. Raw cells were treated with LPS (10 μg/ml) or 10, 50, 100, and 500 μg/ ml of extracts of Diospyros kaki leaves. They were cultured for 24 h later and assayed TNF-α. The values are expressed as mean±s.d. of triplicate tests. *Significantly different from control (p<.05). **Significantly different from LPS (p<.05). Con, without LPS. 의존적으로 NO 생성이억제되는것을관찰할수있었다. 감잎 추출물 50, 100, 500 μg/ml 의농도로처리하였을때 LPS 처 리군에비해유의한감소를나타내었다 (p<.05). 이러한감잎추 출물의 NO 저해효과는감잎추출물이함유한 flavonoid 계열 (Joung et al., 1995) 의화합물에의한것으로사료된다. 산화적스트레스는피부염과같은여러가지염증성피부질 환에있어표피손상을더욱악화시키는것으로알려져있다. 따 라서인체에무해하면서도보다강한항산화효과및항염증효 과를나타내는물질을개발하려는연구는오래전부터진행되 어왔다. 본연구에서감잎추출물의항산화력과항염증효과를 조사하였다. 감잎추출물의항산화력을측정한결과농도에의 존적으로 DPPH 를소거하는항산화활성을증가시켰다. 또한 RAW 세포에서 LPS 자극에의해증가된 NO 및 TNF-α 등염증매개물을유의적으로억제시키는효과가있었다. 따라 서감잎추출물은염증성피부질환을개선하는데유용한물질 로활용될수있는효과적인조성물이라사료된다. Adhami VM, Syed DM, Khan N, Afaq F. Phytochemicals for prevention of solar ultraviolet radiation induced damages. Photochem. Photobiol., 84: ,

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Journal of Life Science 2011, Vol. 21. No μ μ

Journal of Life Science 2011 Vol. 21. No. 8. 1120~1126 ISSN : 1225-9918 DOI : http://dx.doi.org/10.5352/jls.2011.21.8.1120 μ μ μ α β Journal of Life Science 2011, Vol. 21. No. 8 1121 μ μ 1122 생명과학회지 2011,