31 Journal of Investigative Cosmetology Vol. 11, No. 4, ). 인체피부조직에서세포외부기질에주로분포하는 hyaluronic acid (HA) 는 glycosaminoglycan (GAG) 의일종으로수분과의결합력이탁월

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1 대한미용학회지제 11 권제 4 호 215 J Invest Cosmetol 11(4), 39~313(215) /jic 황금 (Scutellariae Radix) 에탄올추출물의 Hyaluronic Acid 합성효과에대한연구 정영수 1, 하헌용 2, * 1 ( 주 ) 뷰티화장품, 2 세명대학교한방바이오융합과학부 The Effect of Scutellariae Radix Ethanol Extract on Hyaluronic Acid Synthesis Yeong-Su Chung 1 and Hun-Yong Ha 2, * 1 Beauty Cosmetic Co., C-413, Chungbuk T.P, 76 Yeongudanji-ro, Ochang, Chungcheongbuk-do 28116, Korea 2 School of Oriental Medicine and Bio Convergence Sciences, Semyung University, Jecheon 27136, Korea (Received November 13, 215; Revised November 24, 215; Accepted November 26, 215) Abstract Scutellariae Radix is an herbal medicine used for the treatment of febrile and dermatological diseases in oriental medicine. Hyaluronic acid (HA) is a mucopolysaccharide occurring naturally in living organisms. It is a highly hydrophilic molecule known to be associated with skin hydration and skin aging. Given the utility of Scutellariae Radix for treating dermatological diseases and febrile diseases that may cause skin dryness in humans, we investigated its effect on HA synthesis. To determine cytotoxicity, hyaluronic acid synthase 2 (HAS2) gene expression and hyaluronic acid production in HaCaT cells, MTT assay, RT-PCR and ELISA were used. HAS2 gene expression increased at all treatment concentrations of Scutellariae Radix extract. Hyaluronic acid production was higher at all concentrations of Scutellariae Radix extract than the control group. Therefore, we suggest that Scutellariae Radix can make play a role in regulating HA synthesis and may have a moisturizing effect on human skin. Keywords : HaCaT cells, Hyaluronic acid, Moisturizing, Oriental medicine, Scutellariae Radix 서론최근의글로벌화장품시장은한류문화의확산과함께동양적이고자연친화적인소재를이용한천연물유래화장품이각광을받고있다 (Choi & Kim, 26). 특히고유의전통의학인한의학을기반으로하는국내화장품트렌드는이미국내시장뿐 만아니라아시아를뛰어넘어전세계시장에서관심의대상으로부각되고있다 (Kim, 27). 이와같은한의약소재를이용하여화장품의부작용을보완하고기능성을극대화하는제품군을한방화장품으로정의하고있으며 (Eom & Kim, 24), 이들한방화장품은이미글로벌시장에서도그우수성을인정받아다양한소재가지속적으로개발되는추세로, 국내뿐만아니라해외기업들도많은관심과연구력을집중하고있다 (Kim & Hwang, * 본연구는산업통상자원부경제협력권산업육성사업의지원에의하여이루어진것임 ( 과제명 : 전통한약처방을활용한보습화장품개발및사업화 ). * Correspondence should be addressed to Dr. Hun-Yong Ha, School of Oriental Medicine and Bio Convergence Sciences, Semyung University, Jecheon 27136, Korea. Ph.: (43) , Fax: (43) , siberiahusky@hanmail.net

2 31 Journal of Investigative Cosmetology Vol. 11, No. 4, ). 인체피부조직에서세포외부기질에주로분포하는 hyaluronic acid (HA) 는 glycosaminoglycan (GAG) 의일종으로수분과의결합력이탁월하여피부의수분유지에매우중요한역할을하는것으로알려져있다 (Necas et al., 28). 피부의보습과관련하여중요한역할을하는 HA는노화가진행됨에따라자연스럽게감소되며, 자외선이나건조한공기등에지속적으로노출될경우에도감소되는경향을보이는것이일반적이다 (Kim et al., 27). 이와같이다양한원인으로인한피부조직내 HA 의감소는피부의탄력저하를유발하고, 그로인한주름유발및거친피부를형성하게하는중요한요인으로작용하게된다 (Lee et al., 213). 따라서피부의노화및주름의생성을억제할목적으로 HA의형성또는감소를억제하는연구가다양하게시도되고있다 (Lee et al., 213; Song et al., 213). 황금 (Scutellariae Radix) 은꿀풀과 ( 脣形科, Labiatae) 에속한다년생초본인속썩은풀의뿌리를건조한것으로한반도와중국을포함한동아시아지역에 1여종이분포하고, 그중 2여종이약용으로사용된다. 한약재황금은신농본초경 ( 神農本草經 ) 에중품 ( 中品 ) 으로처음수록된기록이있으며, 그성질이차갑고쓴맛이있어청열조습 ( 淸熱燥濕 ), 사화해독 ( 瀉火解毒 ), 지혈안태 ( 止血安胎 ) 등의작용이있는것으로알려져있다 (Zhao & Xiao, 29). 황금의주요약리성분으로는 baicalin, baicalein, wogonoside, chrysin, apigenin, scutellarin 등이알려져있으며, 그중 baicalein이황금의지표성분으로사용된다 (Choi, 1998; Zhao & Xiao, 29). 황금의주요약리효과로는항균, 항염, 간장보호, 면역조절, 항종양, 콜레스테롤저하등이알려져있다 (Lee et al., 211). 이와같이황금은주로열성질환을치료하고열로인한건조성증상을완화하는작용이있으며, 전통적으로금궤당귀산 ( 金櫃當歸散 ), 궁소산 ( 芎蘇散 ), 당귀육황탕 ( 當歸六黃湯 ) 등의혈허로인한건조증을치료하는처방과형개연교탕 ( 荊芥連翹湯 ), 황금탕 ( 黃芩湯 ), 서각승마탕 ( 犀角升麻湯 ) 등각종피부과질환을치료하는한의처방에응용되어왔다 (Bong et al., 214). 그러나현대적인연구의분야는황금의항균활성또는항염및항산화등의약리적효과에중점을두고진행되어왔으며, 피부미용과관련된연구들이시도된예가거의없다. 따라서한의학적활용방법을근거로황금의인체피부에대한영향과보습효과에대한과학적근거를확보하기위하여본연구를수행하였다. 한방바이오융합과학부천연물실험실에보관하여사용하였다. 약재는 1 g을측정하여 7% (v/v) 에탄올용매에담가냉침하였으며, 72시간동안 3회반복하였다. 이용액을여과지로여과하여 bottle에포집하였으며, 감압농축기로농축한이후에 3일간동결건조를실시하였다. 추출물의추출수율은 38.27% 였다. 2. 실험조건실험에사용한 HaCaT 세포주는미국 ATCC로부터분양받아사용하였으며, HaCaT 세포는 Dulbecco s modified Eagle s medium (DMEM, Gibco) 에 1% fetal bovine serum (FBS, Gibco) 과 1% penicillin-streptomycin (PS, Gibco) 이함유된배지에서 37 C, 5% CO 2 조건에서배양하였다. 3. 세포배양및독성시험세포를 /ml 농도로 96-well plate에 seeding 하고, 24시간후 serum-free DMEM으로교체해준후황금추출물을처리하였다. 24시간동안배양한후 media를걷어내고 2 μl의 3- (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, 5 mg/ml) 을넣어주고 CO 2 배양기에서 2시간배양하였다. 1 μl의 DMSO로결정을용해시킨후에 57 nm에서흡광도를측정하였다. 생존율은 control과비교하여 % 로표시하였다. 세포에처리한샘플에포함된 dimethly sulfoxide (DMSO) 는최종농도.1% 로조정하였다. 4. RT-PCR assay 세포를 /ml 농도로 6-well plate에 seeding하였다. 24시간후에 serum-free DMEM으로교체해준후에황금추출물을처리하였으며, 최종 DMSO 농도는.1% 로맞추었다. 24시간동안배양한후에 easyblue (intron) 로 RNA를추출하였다. RNA농도와순도 (OD26/OD28) 를측정한후에 2 μg RNA로 power cdna synthesis kit (intron) 를이용하여 cdna 합성을하였다. PCR은 premix PCR kit (Solgent, Korea) 를이용하였고, PCR product는 1.5% agarose gel에전기영동하여밴드를확인하였다. All-trans-retinoic acid (ATRA, sigma) 1 μm을양성대조군으로사용하였다. Primer는 Sim et al. (27) 의 sequence를사용하였다 (Table 1). 5. HA-ELISA assay 재료및방법 1. 시료의제조본연구에사용된한약재황금 (Scutellariae Radix) 은 ( 주 ) 퓨어마인드 (Yongchoen, Korea) 에서구입하였으며, 시료는세명대 세포를 /ml 농도로 6-well plate에 seeding하였다. 24시간후에 serum-free DMEM으로 2번 washing 해준후에 serumfree DMEM를새로교체하고샘플을처리하였으며, DMSO는.1% 로맞추어주었다. 샘플처리후 24시간이지난뒤에 35 μl의 media를걷어내었고, 다시 15분후에동량을걷어내었으

mer) ATT GTT GCC ATC AAT GAC CC (2 mer) AGT AGA GGC AGG GAT GAT GT (2 mer) 393 546 Cell viability (%) 14 12 1 8 6 4 2.1% 1 μm 1% DMSO ATRA DMSO 1 5 1 2 Scutellariae Radix (μg/ml) Figure 1.

3 Chung Y-S & Ha H-Y : Hyaluronic Acid Synthesis of Scutellariae Radix 311 Table 1. Primer sequences of target genes used in the PCR Gene Direction Sequence (5 3 ) Size (bp) HAS2 GAPDH Forward Reverse Forward Reverse GCT ACC AGT TTA TCC AAA CG (2 mer) GTG ACT CAT CTG TCT CAC CG (2 mer) ATT GTT GCC ATC AAT GAC CC (2 mer) AGT AGA GGC AGG GAT GAT GT (2 mer) Cell viability (%) % 1 μm 1% DMSO ATRA DMSO Scutellariae Radix (μg/ml) Figure 1. Effects of test materials on viability of HaCaT cells. DMSO: dimethyl sulfoxide, ATRA: all-trans-retinoic acid. Values are mean±sd of triplicate experiments. 며, 다시 15 분후에 1 차례더동량을걷어내었다. 15, g 에 서 5 분간원심분리하고상층액을걷어내어 ELISA 를수행할때 까지 - 2 C 에서보관하였다. ELISA kit 는 HA-ELISA kit (Echelon, USA) 를이용하였으며제조사에서제공한방법에의해진 행하였다. ATRA 1 μm 을양성대조군으로사용하였다 HAS2 GAPDH Figure 2. HAS2 mrna expression in HaCaT cells. 1: 1 μm ATRA, 2: 1 μg/ml Scutellariae Radix ethanol extract, 3: 5 μg/ml Scutellariae Radix ethanol extract, 4: 1 μg/ml Scutellariae Radix ethanol extract, 5: 2 μg/ml Scutellariae Radix ethanol extract. 효능평가및제품화에있어서배합비율을결정하는데유용한자료가될수있을것으로사료된다. 6. 통계분석통계는 Student s t-test를이용하였으며유의성기준을 p-value 가.5 미만일경우로설정하였다. 결과및고찰 1. MTT assay 분석 7% 에탄올황금추출물의세포독성을확인하기위하여 MTT assay 분석을수행하였다. 추출물을 1, 5, 1, 2 μg/ml로처리한범위에서그생존율을확인하였다. 각농도별생존율은양성대조군인 ATRA에비하여 1 μg/ml에서 11.4%, 5 μg/ml 에서 15.7%, 1 μg/ml에서 17.81%, 2 μg/ml에서 % 로 2 μg/ml의농도에서세포생존율이약간감소하였으나, 뚜렷한세포독성을나타내지는않았다 (Fig. 1). 이로써황금이일정농도에서인체유래의각질세포에뚜렷한독성작용을나타내지않아비교적안전한물질임을확인하였으며, 황금에대한 2. 황금의 HAS2 유전자발현증가 7% 에탄올황금추출물의 HAS2 유전자의발현여부를확인하기위하여 RT-PCR을시행하였다. 모든농도의 7% 에탄올황금추출물처리군에서양성대조군인 ATRA에비해 HAS2 유전자의발현이뚜렷하게나타났다 (Fig. 2). 이러한결과는황금의에탄올추출물이인체유래각질세포에서 HAS2 유전자를발현시켜보습효과와관련된 HA의합성을유도한다는것을뒷받침하며, 향후농도에따른정량적인반응확인이필요할것으로사료된다. 3. HA-ELISA assay 7% 에탄올황금추출물을 5, 1, 2 μg/ml 처리했을때 HA의생성량을확인하기위해 ELISA kit를사용하였다. 24시간동안시료를처리한뒤 HA-ELISA kit를이용하여 15분이경과한시험군에서 HA의생성량을측정한결과, 각농도별로 , , μg/ml로각각측정되었으며 (Fig. 3), 3 분이경과한시험군에서는각각 65.2, , μg/ml

312 Journal of Investigative Cosmetology Vol. 11, No. 4, 215 Scutellariae Radix (24 h) 15 min Scutellariae Radix (24 h) 45 min 9 8 9 8 7 6 7 6 5 5 4 4 3 3 2 2 1 1 V 5 1 2 ATRA V 5 1 2 ATRA Figure 3.

V: vehicle group, 5: 5 μg/ml Scutellariae Radix ethanol extract treated group, 1: 1 μg/ml Scutellariae Radix ethanol extract treated group, 2: 2 μg/ml Scutellariae Radix ethanol extract Values are

Effect of Scutellariae Radix ethanol extract on hyaluronic 45 min. mean±sd (n =6). The value with an asterisk is significantly different from the vehicle group by Student s t-test (*p<.5, p<.1).

4 312 Journal of Investigative Cosmetology Vol. 11, No. 4, 215 Scutellariae Radix (24 h) 15 min Scutellariae Radix (24 h) 45 min V ATRA V ATRA Figure 3. Effect of Scutellariae Radix ethanol extract on hyaluronic 15 min. V: vehicle group, 5: 5 μg/ml Scutellariae Radix ethanol extract treated group, 1: 1 μg/ml Scutellariae Radix ethanol extract treated group, 2: 2 μg/ml Scutellariae Radix ethanol extract Values are mean±sd (n=6). The value with an asterisk is significantly different from the vehicle group by Student s t-test (*p<.5, p<.1). Figure 5. Effect of Scutellariae Radix ethanol extract on hyaluronic 45 min. V: vehicle group, 5: 5 μg/ml Scutellariae Radix ethanol extract treated group, 1: 1 μg/ml Scutellariae Radix ethanol extract treated group, 2: 2 μg/ml Scutellariae Radix ethanol extract Values are mean±sd (n =6). The value with an asterisk is significantly different from the vehicle group by Student s t-test (*p<.5, p<.1) Scutellariae Radix (24 h) 3 min 정되었다 (Fig. 5). 이상과같이측정 3분경과한샘플군에서 HA의생성율이가장높게나타났으며, 황금추출물의처리농도별로는 1 μg/ ml, 5 μg/ml, 2 μg/ml의순으로 HA의생성량이높게나타났다. 이와같은결과는황금을보습기능성을목적으로사용하고자할때농도의설정에중요한지표가될수있을것으로판단되며, 2 μg/ml에서 HA의발현량이오히려약간감소되는것으로미루어 1 μg/ml와 2 μg/ml 사이에서최적의농도를설정하기위한추가적인연구가필요할것으로사료된다. 2 1 V ATRA Figure 4. Effect of Scutellariae Radix ethanol extract on hyaluronic 3 min. V: vehicle group, 5: 5 μg/ml Scutellariae Radix ethanol extract treated group, 1: 1 μg/ml Scutellariae Radix ethanol extract treated group, 2: 2 μg/ml Scutellariae Radix ethanol extract Values are mean±sd (n=6). The value with an asterisk is significantly different from the vehicle group by Student s t-test (*p<.5, p<.1). 로나타났다 (Fig. 4). 또한 45 분이경과한시험군에서는 HA 의 생성량이각농도별로 69.42, , μg/ml 로각각측 결론본연구에서는황금 (Scutellariae Radix) 에탄올추출물의 HA 합성효과를확인하고천연물유래화장품원료로서의가능성을파악하고자 HaCaT 세포를사용하여 MTT, RT-PCR, HA-ELISA assay를수행하였다. 주요결과는다음과같다. HaCaT 세포에서양성대조군인 ATRA와비교하여뚜렷한독성을나타내지않아피부및외용제의원료로서비교적안전한물질로판단하였다. HAS2 유전자발현평가에서는시료를처리한모든농도에서유전자의발현이확인됨으로서, 황금에탄올추출물이 HA의합성과연관된유전자의발현을통하여각질세포의수분함유량을증가시키고이를통하여보습효과를유도

5 Chung Y-S & Ha H-Y : Hyaluronic Acid Synthesis of Scutellariae Radix 313 할수있을것으로판단되었다. HA-ELISA kit 를통한 HA 합성 효과평가에서도각각 15 분, 3 분, 45 분에합성량을확인한결 과, 3 분에측정한 1 μg/ml 처리군평균값이 μg/ml 으 로가장높게나타났다. 이와같은결과는황금에탄올추출물을단독혹은복수의 천연물과함께화장품의원료소재로사용할때유의성있는효과를나타낼수있을것으로기대되며, 실험결과를토대로황금의최적사용농도를결정하기위한추가적연구와추출방법에따른효능평가가뒷받침될경우한방소재천연물화장품원료로서의개발과활용이가능할것으로판단된다. 참고문헌 Bong DR, Kim MH, Lee GY, Hwang EH, Kim JH, Lee GS: Medicinal efficacy of Scutellaria Radix, Coptidis Rhizoma, and Phellodendri Cortex in heat-clearing and dampness-eliminating formulas in dongeibogam. Kor Herb Med Inf 2(2) : 77-88, 214. (Korean) Choi SK: A basic study on the storage of major cultural herbal medicine materials, Scutellariae Radix. J Basic Sci 9(1) : 11-16, (Korean) Choi YJ, Kim JD: A study on the propensity to consume oriental herbal cosmetics. J Soc Cosmet Sci Korea 32(4) : , 26. (Korean) Eom JN, Kim JD: An empirical study on the oriental herbal cosmetics purchase behaviors in women in the metropolitan area. J Soc Cosmet Scientists Korea 3(1) : 93-12, 24. (Korean) Kim JD: The prospects and current situation of oriental herbal cosmetics. J Kor Soc Fashion Beau 5(1) : 1-7, 27. (Korean) Kim KT, Kim YH, Kim JG, Han CS, Park SH, Lee BY, Kim KH: Preparation of oligo hyaluronic acid by hydrolysis and its application as a cosmetic ingredient. J Soc Cosmet Scientists Korea 33(3) : , 27. (Korean) Kim SY, Hwang SJ: An investigation of cultural identity through the packaging of Korean cosmetics -in collaboration with Korean herbal medicine cosmetics-. J Kor Soc Des Cult 2(4) : 97-18, 214. (Korean) Lee KS, Park MH, Cheon ME, Hong JW, Cho SI: Review of pharmacological effects of Scutellaria baicalensis and its bioactive compounds. Kor J Orient Preventive Med Soc 15(2) : 69-99, 211. (Korean) Lee PJ, Kim HT, Yoon KS, Park HC, Ha HY: The effect of Astragalus membranaceus methanol extract on hyaluronic acid production in HaCaT cells. J Kor Med Ophthalmol Otolaryngol Dermatol 26(1) : 75-81, 213. (Korean) Necas J, Bartosikova L, Braunder P, Kolar J: Hyaluronic acid (Hyaluronan): a review. Veterinarni Medicina 53(3) : , 28. Sim GS, Lee DH, Kim JH, An SK, Choe TB, Kwon TJ, Pyo HB, Lee BC: Black rice (Oryza sativa L. var. japonica) hydrolyzed peptides induce expression of hyaluronan synthase 2 gene in HaCaT keratinocytes. J Microbiol Biotechnol 17(2) : , 27. (Korean) Song HJ, Mu HJ, Lee SH: Effect of ferulic acid isolated from Cnidium officinaale on the synthesis of hyaluronic acid. J Soc Cosmet Scientists Korea 39(4) : , 213. (Korean) Zhao ZZ, Xiao PG: Encyclopedia of medicinal plants. WPCSH, Shanghai, pp , 29.

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,