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<연구논문> 한국미용학회지, 제27권제2호 (2021), pp. 370-379 http://e-jkc.org pissn 1229-4349, eissn 2765-5881 J. Kor. Soc. Cosmetol. Vol. 27, No. 2, pp. 370-379(2021) https://doi.org/10.52660/jksc.2021.27.2.370 금은화 감초 표고버섯복합추출물의세포생리활성연구 최민경 1 최미옥 2 서승희 3, * 1 동신대학교사회개발대학원뷰티미용학과, 대학원생 2 광주여자대학교미용과학과, 교수 3 동신대학교뷰티미용학과, 교수 Studies on the Cellular Physiological Activity of Lonicera japonica Glycyrrhiza uralensis Lentinula edodes Complex Extracts Min Gyeong Choi 1, Mi-Ok Choi 2, and Seung-Hee Seo 3, * 1 Graduate Student, Dept. of Cosmetology, DongShin University 2 Professor, Dept. of Beauty Science, Kwangju Women s University 3 Professor, Dept. of Cosmetology, DongShin University With ultraviolet rays, pollution and external stimuli acting as oxidation stress on the skin in line with recent changes in the global environment, modern people are increasingly relying on the frequency and dependence of cosmetics with certain effects. In particular, the characteristics of the skin of the elderly are that inflammation tends to occur due to the deterioration of the barrier function of the skin. To minimize this, research and development of cosmetics using natural materials that do not stimulate the skin and have effective effects are continuously increasing. The purpose of this study was to analyze the physiological activity of the Lonicera japonica Glycyrrhiza uralensis FISCH Lentinula edodes () complex extracts to confirm its potential as a cosmetic material. Our method are cytotoxicity test of complex extracts in the HaCaT and RAW264.7 cells. RAW 264.7 The effects of cell protection from inflammation were measure in cells to identify anti-inflammatory effects. Also, to identify antioxidant effects, DPPH radical scavenging activity test and analysis of the total phenol and total flavonoid contents., tyrosinase inhibitory activity, elastase inhibitory activity, measured cell survival rate by UV-B irradiation in HaCaT. The results of this study are as follows. In the cytotoxicity test, the complex extracts showed stable cell viability. RAW 264.7 cells treated with the complex extracts showed a superior concentration-dependent inhibitory effects on Nitric oxide (NO) synthesis than cells treated with Lipopolysaccharide (LPS). In the DPPH radical scavenging activity test, the complex extracts showed a high scavenging activity at 200 μg/ml of 50% EtOH. In the anti-inflammatory test. Moreover, the results of the analysis on the antioxidant activity of the complex extracts confirmed the phenolic and flavonoid contents. In addition, when UV-B was run on HaCaT cells and absorbance was measured, the cell protection effect was shown to increase dependent on concentrations in the complex extracts DW and 50% EtOH extracts. The tyrosinase inhibitory activity was found to increase concentration-dependent activity in 50% EtOH and 100% EtOH extracts of complex extracts, indicating that active inhibition was more effective than positive control arbutin. Elastase inhibitory activity is shown to increase concentration-dependent at complex extracts DW, 50% EtOH. Based on these results, the complex extracts were found to have excellent cellular physiological activity. Accordingly, basic data on the development of functional cosmetics are provided and systematic and continuous research on the safety of natural products is required. Keywords: Anti-imflamatory effects, Anti Oxidant effects, Elastase inhibition, Lonicera japonica Glycyrrhiza uralensis FISCH Lentinula edodes () Complex extracts, Tyrosinase inhibition I. 서론 *Corresponding author: Seung-Hee Seo Tel : +82-61-330-3297 E-mail : ssh@dsu.ac.kr 접수일 (2020 년 10 월 21 일 )/ 수정일 (2020 년 12 월 10 일 )/ 채택일 (2021 년 3 월 12 일 ) 최근지구의환경변화로인한자외선, 공해, 외부자극등의문제들이피부에산화성스트레스를유발하여각종피부질환을겪는환자의수가급증하고있다. 이러한현상과함께현대 370

금은화 감초 표고버섯복합추출물의세포생리활성연구 371 인들의외모관리에대한인식이높아지면서건강한피부를위해적극적인피부관리행동을하는사람들이증가하고있는추세이다. 그중에서도화장품을이용한피부관리방법은가장일반적이고대중화된방법이다. 최근에는이러한화장품 (Cosmetics) 의개념이단순히피부를보호하는차원에서벗어나, 특정효능이부여된기능을갖고피부의문제를개선및예방하는차원의개념으로인식이바뀌어가고있으며, 이에따른기능성화장품에대한연구가활발히진행되고있다 (Jung, 2018). 이를반영하듯현대인들의기능성화장품사용빈도와의존도가증가하고있으나, 그부작용사례또한무시할수없는실정이다. 특히, 환경및대기오염, 스트레스및우울감증가, 잘못된식생활습관등다양한내 외적요인으로인해피부의문제를호소하는사람들이증가하고있는가운데, 이를최소화하기위해피부에자극이없고유효한효능을가진꽃, 나무, 뿌리, 버섯등천연소재를활용한연구및화장품개발이지속적으로증가하고있는추세이다 (Kim et al., 2019; Jeon et al., 2010). 기존에는천연소재와관련하여단일천연물질의효능에대한연구들이다수진행되었으며, 선행연구결과들을바탕으로최근에는상호상생작용하는복합추출물에대한연구들이진행되고있다. 아직까지는복합추출물에대한명확한정의가내려진바없으나, 복합물을합성하게되면천연물의유효성분들의최대효과를기대해볼수있으며, 다양한성분으로구성되어있으므로성분의조성이효능에크게영향을미친다고알려져있다 (Jiang, 2005; Xue & Roy, 2003). 이에, 본연구는선행연구를바탕으로약리적인효능이있고독성이없는여러천연소재중피부개선및노화예방에효과적이라알려진천연물을스크리닝하였으며, 그중화장품성분으로도많이활용되고있는금은화, 감초, 표고버섯을선정하여복합추출물로서의효능을살펴보고자하였다. 금은화 (Lonicerae Flos) 는인동과 (Caprifoliaceae) 에속하는인동덩굴의꽃으로항염증, 항산화, 면역증강및혈전형성억제작용등이보고되었으며 (Elliott, 1982), 감초는다년생초본으로피부탄력및주름생성을억제하고세포손상에대한보호효과가있으며 (Lee, 2005), 피부에적용하였을때광노화를유발하는효소의발현을저하시켜항노화효과및주름생성억제와피부탄력등의효과가보고된바있다 (Lee, 2015). 표고버섯은자실체와균사체에서항암작용, 면역증강작용, 성인병예방등의효과가있다고밝혀짐에따라식품에서의활용뿐만아니라의약품의소재로서의활용성또한주목받고있다 (Park et al., 1998). 복합추출물에관한선행연구를살펴보면애기달맞이꽃, 상황버섯, 감초의약리활성및세포활성의효과가우수하게나타남으로써화장품소재로써활용가치가높으며 (Kim et al., 2014), Kwon (2018) 의선학초단일추출물및복합추출물의항노화, 항산화효과에관한연구에서는단일추출물과비교하였을때효능이유사하거나다소감소하는경향도나 타나복합추출물을사용함에있어배합비율의정량적연구에대한후속연구의필요성을제기하였다. 또한, 금은화, 계지, 오미자, 목단피, 우방자, 향유등총 6가지의천연물로한약복합추출물을제조하여효능을살펴본 Kim (2019) 의연구결과, 항염증및항산화효과에서우수한효과를보임으로써화장품및건강기능식품분야에서의활용가능성을확인하였다. 더불어 Kim & Yoon (2013) 의복합추출물을이용한바다제비집과병풀복합발효추출물의피부진정효과연구와 Cho et al. (2016) 의고욤잎, 감잎및뽕잎복합추출물의항아토피효과등의복합추출물에관한다양한연구가진행되고있지만, 복합추출물의화장품소재로써이용가능성에관한연구는아직미비한실정이다. 따라서, 본연구는항노화, 항염증및항산화등에서효과가검증된금은화 감초 표고버섯 (Lonicera japonica Glycyrrhiza uralensis FISCH Lentinula edodes ()) 복합추출물의용매조성별추출물의효과를확인한후인체적용실험을통해우수한피부개선효과를선행연구로써확인하였으며 (Choi et al., 2020), 이를바탕으로 복합추출물의화장품소재로서의이용가능성에대한기초적데이터로활용하고자본연구를수행하였다. II. 재료및방법 1. 세포배양본실험에서사용된 HaCaT(Human Keratinocyte cell line) 세포는 C대학교약학대학 (Gwangju, Korea) 으로부터분양받았다. RAW 264.7 세포는한국세포주은행 (Seoul, Korea) 에서분양받아실험에사용하였으며 FBS 10% 와 Penicillin/streptomycine 1% 가함유된 Roswell Park Memorial Institute 1640 배지를사용하였다. HaCaT는 FBS 10% 와 penicillin/streptomycine 1% 함유된 Dulbecco's modified Eagle's medium 배지를이용하여 37 C의온도와 CO 2 5% 로세포를배양하여사용하였다. 2. 금은화 감초 표고버섯 () 복합추출물제조본연구에사용되는금은화와감초는본초원 ( 현진제약, 서울시동대문구제기동 ) 에서구입하였고, 표고버섯은정남진장흥농협 ( 전남장흥군장흥읍행원리 ) 에서구입하여사용하였다. 금은화, 감초, 표고버섯각각 10g씩총 30g을혼합하여 Distilling Water(DW), 50% Ethanol(EtOH), 100% Ethanol(EtOH) 총 3 가지용매각각 2L씩 80 o C에서 3시간 2회가열추출하였다. 이후 Filter paper로여과를거쳐감압농축하여분말상태의추출물을얻었다.

372 J. Kor. Soc. Cosmetol. Vol. 27, No. 2, 2021 3. 금은화 감초 표고버섯 () 복합추출물의세포및생리활성효과측정 1) 세포독성측정세포독성은 복합추출물을 RAW 264.7와 HaCaT를통해확인하였다. 12시간배양한후 DW, 50% EtOH, 100% EtOH을 50, 100, 200 μg/ml 농도로제조하여 48시간배양한후 MTT 용액을 5 mg/ml의농도로첨가한후 1시간동안배양하였다. 상등액을제거하고남은 formazan 생성물을 DMSO로용해시켜 540 nm의파장에서흡광도를측정하였다. HaCaT와 RAW 264.7에서세포독성이나타나지않은 Butein을양성대조군으로사용하였다 (Seo et al., 2015). 2) RAW 264.7에서염증으로부터세포보호효과측정 복합추출물 DW, 50% EtOH, 100% EtOH을각각 50, 100, 200 μg/ml 농도로희석한후 RAW 264.7에 3시간배양한후 Lipopolysaccharide (LPS) 를 1 μg/ml의농도로제조하여투입하였다. 24시간배양후세포에서배양액으로부터분비된 Nitric oxide(no) 를 Griess 시약으로반응시켜 570 nm의파장에서흡광도를측정하였다. RAW 264.7에서 Nitrite 생성억제효과가뛰어나다고알려진 Butein을양성대조군으로사용하였다 (Sung & Lee, 2015). 3) DPPH radical 소거활성측정 2,2-diphenyl-1-picrylhydrazyl(DPPH) radical 소거활성측정방법은 DPPH radical 용액을 MeOH과혼합하여 0.3 mm의농도로제조하였으며 복합추출물용액을 3가지농도로제조하여 DPPH 용액과 1:9로혼합후암실에서 30분간반응시켰다. 이후, 517 nm의파장에서흡광도를측정하여 DPPH radical 소거율을계산하였다. 값을대입하여총페놀함량을산출하였다. 6) 총플라보노이드함량측정본연구의 복합추출물의총플라보노이드함량을알아보기위해 1 ml의 diethylene glycol을투입하고농도별로녹인 복합추출물 100 μl를투입하여잘혼합한후 1 N NaOH 100 μl를처리하여반응을지켜보았다. 이후 96 well plate에 200 μl씩분주하여 420 nm의파장에서흡광도를측정하였으며표준물질은 Quercetin을사용하여표준곡선에의한기울기와절편값을구한후 복합추출물의흡광도값을대입하여계산하였다. 7) Tyrosinase 저해활성측정 복합추출물의 Tyrosinase 활성측정은 Jung & Kim (2009) 의실험방법을변형하여실시하였다. 0.1 M Sodium phosphate buffer (ph 6.5) 110 μl에농도에따른 복합추출물을 10 μl씩분주한다음 mushroom tyrosinase 10 μl를넣는다. 그후 7.5 mm tyrosine을 20 μl씩넣고 37 o C에서 20분간반응을시키고 20 o C에서 5분간방치하여반응을중단시켰다. 475 nm에서흡광도를측정하였으며양성대조군으로 arbutin을사용하였다. 8) Elastase 저해활성측정 복합추출물의 Elastase 활성측정은 Jung & Kim (2009) 의실험방법을변형하여실시하였다. 100 mm Tris-HCI buffer (ph8.0) 160 μl에농도별시료를 10 μl씩넣고 2.9 mm N- Succinyl-Ala(3)-P-nitroanilide를 20 μl씩분주뒤 0.2 unit으로조제한 Elastase( 효소액 ) 를 10 μl씩넣어최종양을 200 μl로맞춰 25 o C에서 20분간방치후 20 o C에서 5분간반응을중단시켜 410 nm에서흡광도를측정하였다. 양성대조군은 ursoilc acid를사용하였다. 4) UV-B 조사세포보호효과측정 HaCaT 세포에서파장별 UV-B 조사에의한세포생존율을측정하기위해 HaCaT를 12시간배양하여추출물을농도별로 3시간전처리한후 20 mj/cm 2 파장의 UV-B를조사하였다. 이후 24시간동안세포를배양하여 MTT assay 처리후 540 nm의파장에서흡광도를측정하였다. 5) 총페놀함량측정총페놀함량측정은농도에따른 복합추출물 25 μl에 10% Folin-Ciocalteau s phenol reagent를 500 μl 처리하여실온에서 5분동안반응시킨후 sodium carbonate (Na 2CO 3) 용액 500 μl를첨가하여 30 o C에서 90분간정치시켰다. 이후 725 nm 의파장에서흡광도를측정하였으며표준물질은 caffeic acid를사용하여표준곡선에의한기울기와절편값을구한후흡광도 4. 통계처리본연구의통계처리는 GraphPad Prism, version 3.03 (GraphPad Software Inc., San Diego, CA) 을사용하였으며각실험군의결과는평균치와표준오차로나타내었고각실험군간의결과는 ANOVA test를사용하여분석하였으며유의적인차이가나타나는항목에대해서검정하였다. 실험군간의차이는 95% 수준 (p<0.05) 에서유의한것으로하였다. III. 결과및고찰 1. 세포독성 복합추출물의용매조성별세포독성을알아보고자

금은화 감초 표고버섯복합추출물의세포생리활성연구 373 Fig. 1. Effects of DW, 50% EtOH, 100% EtOH extracts on cell viability by MTT assay in the RAW 264.7 cells. Data are presented as the mean values ± S.D. of 3 experiments. Fig. 3. Effects of DW, 50% EtOH, 100% EtOH extracts on nitrite production in the RAW264.7 cells. After injecting LPS (1 μg/ ml) into the RAW 264.7 cell, the amount of NO secreted from the culture fluid in the cell was measured. Data are presented as the mean values ± S.D. of 3 experiments. Butein was used as a positive control. ***p<0.001 VS LPS treated Groups. 세포생존율에영향을미치지않은것으로나타났다 (Fig. 2). 따라서, D.W, 50% EtOH 추출물, 100% EtOH 추출물에서 50, 100, 200 μg/ml 농도까지세포독성이없는것을확인하였다. Fig. 2. Effects of DW, 50% EtOH, 100% EtOH extracts on cell viability by MTT assay in the HaCaT cells. Data are presented as the mean values ± S.D. of 3 experiments. RAW 264.7세포와 HaCaT 각질형성세포에 복합추출물 D.W, 복합추출물 50% EtOH, 복합추출물 100% EtOH을각각 50, 100, 200 μg/ml 농도로처리하였다. 그결과 RAW 264.7에서농도별복합추출물과아무것도처리하지않은대조군과비교하였을때 91.93~96.86% 로세포생존율에영향을미치지않았다 (Fig. 1). HaCaT 세포에서도 89.58~103.86% 로 2. RAW 264.7에서의염증으로부터세포보호효과 복합추출물 D.W, 50% EtOH, 100% EtOH 50, 100, 200 μg/ml에 LPS를투입하여 nitric oxide (NO) 의양을살펴보았으며, RAW 264.7 세포에서 nitrite 생성억제효과가있다고알려진 Butein을양성대조군으로사용하였다. 복합추출물 D.W 추출물 50, 100, 200 μg/ml은각각 14.4±0.6 μm, 13.8±0.1 μm, 14.81±0.5 μm로유의미한결과는없었으며 50% EtOH 추출물 50, 100, 200 μg/ml은각각 16.4±0.6 μm, 15.4±0.01 μm, 9.4±0.1 μm로나타났으며 100% EtOH 추출물은각각 Table 1. DPPH radical scavenging effect of complex extracts DPPH radical scavenging (%) Exp. 1 Exp. 2 Exp. 3 Mean±SD D.W 50% EtOH 100% EtOH 50 μg/ml 27.106 22.357 29.820 26.428±3.78 100 μg/ml 31.868 33.676 28.015 31.187±2.89 200 μg/ml 53.938 53.601 51.377 52.972±1.39 50 μg/ml 25.092 23.386 22.032 23.503±1.53 100 μg/ml 40.934 39.570 37.702 39.402±1.62 200 μg/ml 74.451 73.620 72.840 73.637±0.81 50 μg/ml 20.330 20.954 20.038 20.441±0.47 100 μg/ml 32.418 31.431 32.384 32.077±0.56 200 μg/ml 52.564 49.860 48.813 50.412±1.94

374 J. Kor. Soc. Cosmetol. Vol. 27, No. 2, 2021 Fig. 4. DPPH radical scavenging effect of DW, 50% EtOH, 100% EtOH extracts. Data are presented as the mean values ± S.D. of 3 experiments. 12.8±0.02 μm, 6.9±0.3 μm, 1.1±0.03 μm로농도가증가할수록 NO의양이감소하는것으로나타났다 (Fig. 3). Fig. 5. In HaCaT, 20 mj/cm 2 of wavelengths, which represent about 40% of cell survival compared to control by UV-B irradiation, were set as wavelengths for measuring cell protection effects on composite extracts. Data are presented as the mean values ± S.D. of 3 experiments. ***p<0.001 VS Control Groups. 3. DPPH ridical 소거활성 복합추출물의항산화효과를알아보기위해 DPPH ridical 소거활성을살펴보았다. D.W 50, 100, 200 μg/ml 의농도로처리하였을때 26.4±3.7%, 31.1±2.8%, 52.9±1.3% 로나타났으며 50% EtOH 50, 100, 200 μg/ml는각각 23.5±1.5%, 39.4±1.6%, 73.6±0.8% 이며 100% EtOH 50, 100, 200 μg/ml는각각 20.4±0.4%, 32.0±0.5%, 50.4±1.9% 로 D.W, 50% EtOH, 100% EtOH 추출물에서농도의존적으로 DPPH radical 소거활성이높아지는것으로나타났다 (Table 1, Fig. 4). Fig. 6. In order to measure the cell survival rate by UV-B irradiation by wavelength, HaCaT was cultured for 12 hours and the extract was treated three hours before each concentration, and UV-B of 20 mj/ cm 2 wavelength was investigated. The absorption was measured at 540 nm wavelength after processing MTT assay by culturing cells for the next 24 hours. Data are presented as the mean values ± S.D. of 3 experiments. ***p<0.001 VS UVB-induced Groups. 4. UV-B 조사세포보호효과 HaCaT에서 UV-B 선량별조사시나타나는 HaCaT 세포보호효과에미치는영향을확인해보고자실험을진행하였다. HaCaT 세포에 UVB를각각 5, 10, 20, 50 mj/cm 2 로조사하여진행된실험결과에서 UVB 조사량이증가할수록세포의생존율이감소하는것을확인할수있었다. 이와같은결과를바탕으로 UVB 20 mj/cm 2 의파장을복합추출물에대한세포보호효과측정용파장으로설정하였다 (Fig. 5). 그후같은방식으로세포를배양하고흡광도를측정한결과, D.W 50, 100, 200 μg/ml 처리하였을때각각 31±5.9%, 36±5.7%, 42±9.1% 로나타나농도의존적으로세포보호효과가증가하는것으로나타났으며, 50% EtOH 추출물에서도 40±9.8%, 44±10.1%, 50±5.2% 로세포보호효과가증가하는것을확인하였다. 반면, 100% EtOH 추출물은 43±10.8%, 40±9.8%, 39±8.8% 로세포보호효과는보였으나농도의존적으로세포보호효과가증가하지않고거의일정한효과를보였다 (Fig. 6). Fig. 7. Calibration curve of caffeic acid. R 2 : 0.994 5. 총 phenolic 함량농도에따른 D.W, 50% EtOH, 100% EtOH 추출물의총페놀함량은 caffeic acid를표준물질로사용하였으며표준곡선의흡광도값과비교하여페놀함량을산출하였다 (Fig. 7).

금은화 감초 표고버섯복합추출물의세포생리활성연구 375 Table 2. Total phenol content complex extracts Total phenol content (equivalent of caffieic acid μg/ml) D.W 50% EtOH 100% EtOH Exp. 1 Exp. 2 Exp. 3 Mean±SD 0.5 mg/ml 27 28.86667 28.66667 28.18±1.025 1 mg/ml 47.4 47.06667 47.33333 47.27±0.176 2 mg/ml 93.86667 91.46667 89.8 91.71±2.044 0.5 mg/ml 26.4 32.66667 24.06667 27.71±4.447 1 mg/ml 54.93333 52.33333 55 54.09±1.521 2 mg/ml 106.4667 102.9333 105.9333 105.11±1.905 0.5 mg/ml 33.26667 32.2 30 31.82±1.666 1 mg/ml 63.33333 60.73333 61.33333 61.80±1.361 2 mg/ml 142.2 140.2667 142.5333 141.67±1.223 Fig. 8. Total phenolic content of DW, 50% EtOH, 100% EtOH extracts. The data results calculated the phenol content compared to the absorbance value of the standard curve. Data are presented as the mean values ± S.D. of 3 experiments. Fig. 9. Calibration curve of Quercetin. R 2 : 0.996 caffeic acid 대비총페놀함량은 D.W 0.5 mg/ml, 1 mg/ ml, 2 mg/ml에서각각 28.18±1.03 μg/ml, 47.27±0.18 μg/ml, 91.71±2.04 μg/ml로나타나농도의존적으로유의미하게나타났으며, 50% EtOH은 0.5 mg/ml, 1 mg/ml, 2 mg/ml에서각각 27.71±4.45 μg/ml, 54.09±1.52 μg/ml, 105.11±1.91 μg/ml 로농도에따라페놀함량이유의적으로증가하였다. 100% EtOH의 0.5 mg/ml, 1 mg/ml, 2 mg/ml에서는농도별로 31.82±1.67 μg/ml, 61.80±1.36 μg/ml, 141.67±1.223 μg/ml로나타난것으로보아총페놀함량이농도에따라유의적으로증가하였음을알수있다 (Table 2, Fig. 8). Fig. 10. Total flavonoid content of DW, 50% EtOH, 100% EtOH extracts. The data results calculated the flavoniod content compared to the absorbance value of the standard curve. Data are presented as the mean values ± S.D. of 3 experiments. Table 3. Total flavonoid content complex extracts Total flavonoid (equivalent of Quercetin μg/ml) Exp. 1 Exp. 2 Exp. 3 Mean±SD D.W 73.06 65.03 72.73 70.27±4.543 50% EtOH 83.67 86.68 88.11 86.14±2.296 100% EtOH 78.1 76.15 99.15 84.47±12.75

376 J. Kor. Soc. Cosmetol. Vol. 27, No. 2, 2021 Table 4. Total tyrosinase content complex extracts Total tyrosinase content (equivalent of arbutin μg/ml) D.W 50% EtOH 100% EtOH Exp. 1 Exp. 2 Exp. 3 Mean±SD 0.5 mg/ml 25.225 29.657 21.816 25.56±3.931 1 mg/ml 11.588 19.088 13.974 14.88±3.831 2 mg/ml 22.157 18.066 18.407 19.54±2.270 0.5 mg/ml 37.839 40.567 48.408 42.27±5.487 1 mg/ml 55.11 66.702 68.407 63.41±7.235 2 mg/ml 75.566 83.407 84.089 81.02±4.736 0.5 mg/ml 38.405 41.473 40.451 40.11±1.562 1 mg/ml 51.135 61.704 60.681 57.84±5.829 2 mg/ml 71.591 76.364 64.772 70.91±5.826 6. 총 flavonoid 함량 본실험에서는 복합추출물의플라보노이드함량을알아보기위하여항산화의효능이있는 Quercetin을사용하여작성한표준곡선에의해기울기와절편값을구한후시료의흡광도값을대입하여계산하였다 (Fig. 9). 표준물질 Quercetin 대비하여 D.W, 50% EtOH, 100% EtOH 추출물의플라보노이드함량은각각 70.27±4.543 μg/ml, 86.14±2.296 μg/ml, 84.47±12.75 μg/ml로확인되었다 (Table 3, Fig. 10). 7. Tyrosinase 활성 Fig. 11. Tyrosinase inhibition of DW, 50% EtOH, 100% EtOH extract and Arbutin. Data are presented as the mean values ± S.D. of 3 experiments. 복합추출물의 Tyrosinase 활성을알아보기위하여양성대조군으로는 arbutin를사용하여비교하였다. D.W 0.5 mg/ml, 1 mg/ml, 2 mg/ml 농도에서각각 25.57±3.93%, 14.88±3.83%, 19.54±2.27% 로유의미한결과는없었으며 50% EtOH 0.5 mg/ml, 1 mg/ml, 2 mg/ml은각각 42.27 ±5.49%, 63.41±7.23%, 81.02±4.74% 로농도에따라 Tyrosinase 저해활성도가유의미하였고 100% EtOH 0.5 mg/ml, 1 mg/ml, 2 mg/ml은각각 40.11±1.56%, 57.84±5.83%, 70.91± 5.83% 로농도의존적으로증가하는것으로나타나양성대조군 arbutin에비하여 Tyrosinase 활성억제가효과적인것으로확인되었다 (Table 4, Fig. 11). Table 5. Total elastase content complex extracts Total elastase content (equivalent of ursolic acid μg/ml) D.W 50% EtOH 100% EtOH Exp. 1 Exp. 2 Exp. 3 Mean±SD 0.5 mg/ml 7.0055 12.5686 12.9121 10.83±3.315 1 mg/ml 15.0412 14.0797 15.1786 14.77±0.598 2 mg/ml 23.0769 23.0082 18.3378 21.47±2.716 0.5 mg/ml 6.8681 3.2280 7.8984 5.99±2.453 1 mg/ml 14.3544 14.7665 17.1017 15.41±1.481 2 mg/ml 21.0165 20.3297 21.3599 20.90±0.524 0.5 mg/ml 7.9670 9.8901 8.7912 8.88±0.965 1 mg/ml 13.8049 15.2473 16.8956 15.32±1.546 2 mg/ml 13.5989 10.7829 8.5852 10.99±2.513

금은화 감초 표고버섯복합추출물의세포생리활성연구 377 Fig. 12. Elastase inhibition of DW, 50% EtOH, 100% EtOH extract and Ursolic acid. Data are presented as the mean values ± S.D. of 3 experiments. 8. Elastase 활성 복합추출물의 Elastase 활성을알아보기위하여양성대조군으로는 Ursolic acid를사용하였다. D.W 0.5 mg/ml, 1 mg/ml, 2 mg/ml 농도에서각각 10.83±3.32%, 14.77± 0.60%, 21.47±2.72% 로나타나농도에따라 Elastase 활성이유의미하게확인되었으며 50% EtOH 0.5 mg/ml, 1 mg/ml, 2 mg/ml은각각 5.99±2.45%, 15.41±1.48%, 20.90±0.52% 로농도의존적으로증가하는것으로나타났다. 100% EtOH 0.5 mg/ml, 1 mg/ml, 2 mg/ml은각각 8.88±0.96%, 15.32±1.55%, 10.99±2.51% 로 100% EtOH에서는유의미한결과는나타나지않았다 (Table 5, Fig. 12). 나이가들어감에따라세포의분열속도가느려지면서인체를둘러싼피부는각질층의천연보습인자와효소, 세포외기질 (extracellular matrix) 등의기능이저하되고, 이에따라피부는점차거칠어지고건조해지며, 점점더얇아지고주름생성이촉진된다 (An, 2014). 뿐만아니라, 산업이발달해감에따라환경및대기오염이심각해지고, 스트레스및우울, 잘못된식생활습관등의다양한내 외적요인에따라여러피부의문제들이발생하고있다. 이에항노화, 항산화에관련된연구가활발히진행되고있으며, 소비자들의화장품에대한안전성요구가증가함으로써독성이없는천연소재를이용한기능성화장품시장의관심이집중되고있다 (Sim, 2012). 이에본연구는선행연구를통해항산화, 미백, 항염증등에서의효능이있다고알려진천연소재중화장품성분으로도널리활용되고있는금은화, 감초, 표고버섯을선정하였으며, 금은화 감초 표고버섯 (Lonicera japonica Glycyrrhiza uralensis FISCH Lentinula edodes ()) 복합추출물을제조하여인체적용실험을선행하였다 (Choi et al., 2020). 그결과, 복합추출물의안면피부개선효과가우수한것을확인하였으며, 이를바탕으로세포생리활성실험을통해화장품소재로서의활용가능성을확인하고자추출용매조성별 DW, 50% EtOH, 100% EtOH의 복합추출물을제조하여그효과를비교하고자하였다. 먼저, RAW 264.7과 HaCaT 세포에서세포독성검증을확인한결과, 복합추출물처리후매우안정적인세포생존율을보였다. 이는단일추출물로서의금은화가과산화수소 (hydrogen peroxide, H 2O 2) 로유발되는활성산소를제거하여 HaCaT 세포에서농도에따라세포보호효과가증가한다는것을확인하였으며 (Seo, 2014), Kang (2008) 는감초추출물 200 mg/ml 농도 5 μl, 10 μl에서 95% 이상의세포생존율을확인하였다. 표고버섯추출물관련한 Seo (2018) 의연구에서는 MTT assay를통해표고버섯추출물 10, 25, 50 μg/ml에서독성이없는것으로나타남으로써본연구의 복합추출물의세포독성검증결과를지지한다. 복합추출물의염증으로부터세포보호효과를규명하기위해염증물질인 LPS를대식세포인 RAW 264.7에 복합추출물을동시처리하여 LPS로부터 NO를농도의존적으로억제하는것을확인하였다. 이는, 염증세포와 proinflammatory cytokine, 상피세포의 apoptosis 억제효과가우수하고, 마우스의알러지성피부염에대한금은화추출물의효능연구에서금은화추출물이 IFN-γ cytokine과 IL-4를억제함으로써항알러지효능이있다는 Kim (2010) 의연구와관련이깊을것으로사료되며, Yoo (2008) 의연구에서는금은화가 NO 생성의염증반응억제하는것으로나타났다. 이와같은선행연구결과는 추출물이염증으로부터세포보호효과를보이며항염증효과를나타낸다는본연구결과와같은맥락이다. 또한, Min (2011) 은금은화의총페놀함량과 DPPH, hydroxyl radical, superoxide anion의활성도를측정함으로써금은화의항산화효능을규명하였으며, 금은화의항염증작용이 apigenin, quercetin, luteolin 등의 flavonoid 성분에기인한것을확인하였다 (Moon et al., 1999). 감초는고분자의페놀성화합물이다량존재하는데 (Ishikawa et al., 1999), Jung (2018) 의연구결과, 68.11 μg GAE/mL의폴리페놀을함유한다고나타났으며, 감초추출물의 mouse melanoma B16F10 cell에서최고농도 400 μg/ml 까지처리한결과시료무첨가군에비해 tryrosinase 활성을저해하는것으로나타났다. 또한, 감초는 liquiritin 및 liquiritigenin와같은플라보노이드를함유하고있다고알려져있으며, 주요성분인 flavone의 triterpenoid에의해항염증작용이있는것으로확인된바있다 (Kim, 2014). 표고버섯은 Kim et al. (2017) 의연구에서페놀성분과항산화활성실험에서우수한효과를보였으며또한, Han et al. (2015) 의연구에서표고버섯용매추출물의 DPPH radical에서소거활성과항산화및항균효과의실험에서효능을확인하였다. 이상의선행연구결과들은 복합추출물의항산화및항염증효과에관한연구결과를지지한다. UV-B 조사시 복합추출물의세포보호효과는 Seo (2014) 의연구에서 HaCaT 세포에 UV-B 조사시증가하던

378 J. Kor. Soc. Cosmetol. Vol. 27, No. 2, 2021 DNA 분절현상과핵응축현상이금은화추출물을처리하였을때농도의존적으로감소시킴을확인하였다. 또한, Lee (2015) 의연구에서감초의유효성분인 liquiritigenin, isoliquiritigenin 이 UVB 조사로인한손상에서세포보호효과가있으며 elastase의저해활성이세포내의활성산소종을소거하고주름생성및피부탄력을억제시켜세포손상에대한보호작용을한다고보고한바있다. 이와같은결과는 D.W, 50% EtOH 추출물, 100% EtOH 추출물에서 UV-B 조사시세포보호효과와 elastase 활성을저해하는것으로나타난본연구결과와관련이깊을것으로사료된다. Lee (2001) 은감초로부터 Tyrosinase 억제활성을하는물질을분리하여미백효과를규명하였으며, Hankook shinyak Corp (2010) 에서는표고버섯균사체에서추출한다당체가피부의미백및보습화장품성분으로유효하게사용할수있음을밝혔다. Jung (2018) 은항산화활성효과가우수하다고알려진상백피추출물보다표고버섯균사체배양물에서 Tyrosinase를저해하는효과가더우수하다고밝혔다. 본연구역시, 50% EtOH 추출물과 100% EtOH 에서 Tyrosinase 저해효과를확인하였으며, 이와같은연구결과들을바탕으로 복합추출물의미백기능성화장품소재로써활용가능성을유추해볼수있다. 따라서, 본연구를통해 복합추출물은독성이없으며, 항산화및항염증효과, 미백효과등이있다는것을확인하였으며, 이는천연소재를활용한기능성화장품개발에소재활용의가능성을시사한다. 하지만, 복합추출물이단일추출물보다더효과적일것이라는것은단일추출물과의비교연구가진행되지않아유추하기어렵다. 추후, 후속연구로써복합추출물에대한다각적인세부연구를통해객관적인데이터가필요할것으로사료되며, 이러한연구들을바탕으로복합추출물의화장품소재로서의효율성및실용화가능성을판단해볼수있을것으로사료된다. IV. 결론 본연구는 복합추출물의세포독성, UVB에서의세포보호효과, 항산화, 항염증등의생리활성탐색하여화장품소재로의활용가능성을살펴보고자하였다. 본연구의결과는다음과같이도출되었다. 첫째, 복합추출물의 RAW 264.7와 HaCaT에서세포독성측정결과 D.W, 50% EtOH, 100% EtOH의 50, 100, 200 μg/ml 농도에서매우안정적인세포생존율을보였다. 둘째, 복합추출물의염증으로부터세포보호효과측정결과 50% EtOH, 100% EtOH 추출물 50, 100, 200 μg/ml 농도에서 LPS 단독처리하였을때양성대조군인 Butein 과비교하였을때농도의존적으로 NO(Nitric oxide) 가억제됨 을확인하였다. 셋째, 복합추출물의 DPPH radical 소거활성측정결과 D.W, 50% EtOH, 100% EtOH 50, 100, 200 μg/ ml에서농도의존적으로 DPPH radical 소거활성이높아지는것으로나타났다. 넷째, HaCaT에서 UV-B 조사에의한세포독성에대하여 D.W, 50% EtOH, 100% EtOH에서세포보호효과가증가하는것을확인하였다. 다섯째, 복합추출물을농도별로항산화효능을갖는표준물질과비교한결과총페놀함량과플라보노이드함량이유의미하게나타났다. 이와같은결과는 복합추출물이화장품개발에있어항산화물질로써천연소재활용가능성을시사한다. 여섯째, 복합추출물의 Tyrosinase, elastase 활성측정결과 Tyrosinase 활성에서는 50% EtOH, 100% EtOH 추출물에서표준물질인 Arbutin 보다높은활성저해효과를나타냈으며, elastase 활성에서는 D.W, 50% EtOH 추출물에서농도의존적으로 elastase 활성효과가우수한것으로나타났다. 이상의결과를종합해보면, 복합추출물은세포독성, UVB에서의세포보호효과, 항염증, 항산화효과뿐만아니라 Tyrosinase 활성및 elastase 활성저해효과또한우수한것을확인함으로써화장품소재로서의활용가능성을확인하였다. 따라서, 복합추출물은천연유래기능성화장품소재로서적합할것으로사료되며, 이와같은연구결과를바탕으로기능성화장품개발에대한기초자료로써활용되기를기대한다. 향후, 복합추출물의성분분석및단일추출물과의비교연구등세부적인후속연구를통해뷰티산업및화장품시장의활성화에도움이되기를바란다. References An, E. J. (2014). Study on the anti-aging and skin aging, and officials to recognize women by age group behavior. Master's thesis, Konkuk University, Seoul. Cho, B. O., Yin, H. H., Shin, J. Y., Fang, C. Z., Chang, C. D., & Jang, S. I. (2016). Anti-Atopic Effects of Mixed Extracts from Date Plum, Persimmom, and Mulberry Leaves. Journal of the Korean Society of Food Science and Nutrition, 45(4), 501-509. Choi, M. G., Kang, E. J., & Seo, S. H. (2020). Facial skin enhancement effect of Lonicera japonica Glycyrrhiza uralensis FISCH- Lentinula edodes complex extract. Journal of the Korean Society of Cosmetology, 26(5), 1179-1190. Elliott, M. Jr. (1982). Effects of flavonoids and transitional metal cations on antigen-induced histamine release from human basophils. Biochem. Pharmacol, 31(7), 1449. Hankook shinyak Corp. (2010). Moisturizing and/or whitening cosmetic composition comprising polysaccharide extracted from

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