RESEARCH ARTICLE 어성초를포함한천연복합추출물의 C57BL/6 마우스에대한발모효과 홍양희 1, 배송환 2, 서형주 3 * 1 수원여자대학교미용예술과, 2 한경대학교식품생물공학과, 3 고려대학교바이오시스템의과학부 Effect of Herbl Complex Extrct Including Houttuyni cordt Thunb on Hir Growth Promotion in C57BL/6 Mice Yng Hee Hong 1, Song Hwn Be 2, Hyung Joo Suh 3 * 1 Deprtment of Beuty Art, Suwon Women s University 2 Deprtment of Food nd Biotechnology, Hnkyong Ntionl University 3 School of Biosystem nd Biomedicl Science, Kore University Abstrct Present study ws crried out to investigte the effect of herbl complex extrct including Houttuyni cordt Thunb on hir growth nd gene expression in C57BL/6 mice. Twenty mice were divided into 4 experimentl groups including norml (control), 50% herbl complex extrct (S-1), 100% herbl complex extrct (S-2) nd 5% minoxidil-treted group. The test mterils were dily pplied with 200 μl per mouse on shved dorsl skin for 3 weeks. The hir growth ws monitored by photogrph t 7, 14 nd 21 dys fter topicl ppliction. Then the chnges of hir density nd hir thickness in the hir-removed re were evluted by phototrichogrm using folliscope. Also the expression level of growth fctors relted to hir growth ws mesured by rel time polymerse chin rection. Appliction of S-2 or minoxidil-treted group stimulted the hir growth compred to vehicle tretment. Therefore hir density of S-2 or minoxidiltreted group ppliction ws incresed bout 2.5 nd 3.1 fold more thn in vehicle ppliction on 21 dy, respectively. And hir weight of both S-2 nd minoxidil-treted group were incresed bout 8.3 nd 10.0 fold more thn in vehicle spreding on 21 dy, respectively. Futhermore the gene expression of insulinlike growth fctor (IGF)-1 nd vsculr endothelil growth fctor (VEGF) were significntly up-regulted in S-2 nd minoxidil-spreded group compred to vehicle-pplied group. However the gene expression of trnsforming growth fctor (TGF)-β1 ws not significntly down-regulted in S-2 nd minoxidil-spreded group compred to vehicle-pplied group. These dt suggest tht S-2 hs potent stimulting ctivity on hir growth in C57BL/6 mice nd potentil usefulness s ingredients of hir tonic nd hir restore. Keywords: Hir re-growth, Houttuyni cordt Thunb, IGF-1, VEGF, TGF-β1 모발은피부표면에서생산된가늘고각질화된구조로서, 외 부충격에대한쿠션역할과더불어직사광선, 한냉, 마찰, 위험 등외부자극으로부터인체를보호하고, 신체에유해한비소, 수은, 아연등의중금속을체외로배출하는기능등다양한역할을담당하고있으며, 생명유지에중요한기관은아니지만건강상태의척도이자외모를결정짓는신체의중요한일부분이다 *Corresponding uthor: Hyung Joo Suh, School of Biosystem nd Biomedicl Science, Kore University, 145 Anm-ro, Seongbuk-gu, Seoul, 136-713, Republic of Kore Tel.: +82 2 3290 5639, Fx: +82 2 940 2849, E-mil:suh1960@kore.c.kr Received Mrch 22, 2015; Revised June 24, 2015; Accepted June 26, 2015; Published June 30, 2015 321
(Grtner & Hitt, 2007; Stenn, 2003). 모발은두피기저부에있는기질세포의계속적인증식으로피부가함입된구조인모낭에서생성되고, 다양한성장단계를포함한모발주기를가진다 (Reynolds et l., 1991). 모발은정상모발의 90% 를차지하는성장기 (ngen), 생장정지와모근이위축되는퇴행기 (ctgen), 모구가건조되고곤봉모가되는휴지기 (telogen) 그리고탈락기 (exogen) 의 4단계의모발주기에따라성장과탈모를반복한다 (Sitoh et l., 1970). 특히, 퇴행기동안많은모낭의세포사멸 (poptosis) 이진행되어휴지기에들어가면서모낭의크기가줄어들게된다 (Buhl et l., 1990). 탈모는일련의노화현상으로인식되어왔으나, 최근여러유전적인요인과함께스트레스, 서구화된식습관및영양불균형등의다양한원인에의해탈모가진행됨이밝혀지고있다 (Peters et l., 2006; Trüeb, 2002). 탈모의대표적인기전은남성호르몬인 testosterone이 5α-reductse에의하여 dihydrotestosterone (DHT) 으로전환되어두피의모낭을위축시켜탈모를유발하는것이다. 나이가들면 DHT가증가되어모낭세포의단백질합성이지연되고이로인해휴지기모낭의비율이증가되면서탈모의진행이빠르게진행된다고볼수있다 (Adchi & Kno, 1970). 이러한탈모증상에는미녹시딜 (minoxidil) 도포제와경구로복용하는피나스테라이드 (finsteride) 제제가남성용탈모증의주된치료제로사용되고있다. 미녹시딜은 1970년대초에고혈압치료를위한혈관확장제로개발되었으나, 부작용으로다모증이보고되면서발모촉진제로사용되고있다 (Zppcost, 1980). 미녹시딜의발모효과에관한작용기전은아직명확하게밝혀져있지않지만혈관확장을통한영양공급증가및 potssium chnnel opening 효과등이모발성장에관여하는것으로생각되고있다. 또한 1% 미녹시딜은 vsculr endothelil growth fctor (VEGF) 발현을유도하고, 고혈압치료제로사용된만큼혈관내피세포및평활근세포를자극하여혈액순환을개선하고모낭상피세포 (folliculr kertinocyte) 를포함한모발성장에사용되는세포들의성장을촉진시킴으로써 (Hmmoto & Mori, 1989) 발모를촉진하는것으로여겨지고있다. 피나스테라이드는전립선비대증치료제로개발되었다가현재는탈모치료제로사용되고있으며 (Shpiro & Price, 1998), 5-α-redutse의저해제로혈중 DHT 농도를낮추어발모효과를나타낸다고알려지고있다 (Dllob et l., 1994; Imperto-McGinley et l., 1974). 하지만미녹시딜의경우체중증가, 부종, 심장박동증가, 협심증, 피부염, 가려움증, 홍반및피부건성화와같은이상반응이보고되고있고 (Hgemn et l., 2005; Mcky & Isles, 1981), 피나스테라이드의경우모발성장촉진효과를계속유지시키기위해서는지속적인약물복용이요구되며남성의경 우성기능장애, 임신한여성이복용할경우기형아출산과같은부작용이유발됨이보고되어있다. 이에최근에는이러한단점을보완하여지속적인사용에도안전하고효과가있는제품에대한요구에대응하여전통적으로탈모예방및발모에효능이있는것으로알려진한방처방및한약재를비롯한천연물에대한연구가활발히진행되고있다 (Irwig, 2012; Rogers & Avrm, 2008). 토코트리에놀 (tocotrienol) 의섭취가두피의지질과산화및산화적스트레스를감소시켜발모를촉진하는것으로보고하였으며 (Beoy et l., 2010), 수종의천연물과 Lctobcillus plntrum 균주를이용한발효추출물의도포가모발생장에관여하는 cytokine을조절하여 C57BL/6 mouse의모발성장을촉진하는것으로보고하였다 (Joo, 2011). Kim et l. (2004) 은화학요법제인 cytrbine에의한자발적인탈모실험모델에서측백나무 (Thuj orientlis) 의추출물의도포가염증성 cytokine과염증유발인자의조절을통해 DBA/1J mouse의모발성장을촉진한것으로보고하였다 (H & Prk, 2013). 이밖에도탈모치료에이용되는한방처방전또는민간요법에근거한다양한종류의한약재나천연물의추출물을이용한발모제의개발에대한연구가보고되어천연물로부터발모효과가우수한제제의개발이가능할것으로시사하였다 (Lee & Hwng, 2009; Ryu & Shin, 2011; Ryu et l., 2013). 어성초 (Houttuyni cordt Thunb) 는삼백초에속하는다년생약초로써비린내가나는식물로알려져있다. 어성초는전통약용작물로잘알려져있으며, 대표적인생리활성물질로는정유 (essentil oil) (Lu et l., 2006), 퀘세틴 (quercitin), 하이페린 (hyperin) 의 flvonoids (Meng et l., 2006), lkloids (Kim et l., 2001) 등이다량존재하는것으로알려져있다. 이런이유로본연구에서는발모및탈모방지에효과가있다고알려진어성초, 상엽, 자초근등을포함하는천연복합추출물을제조하여모발성장을조절하는성장인자인 insulin-like growth fctor (IGF)-1, vsculr endothelil growth fctor (VEGF), trnsforming growth fctor (TGF)-β1의발현를통하여발모효과를분석하고자하였다. 1. 시료의준비본시험에사용한어성초, 상엽, 자초근, 상백피, 녹차, 감초, 박하잎, 질경, 자소엽, 동백씨및인삼씨는경동시장 (Seoul, Kore) 에서구입한후냉장보관하여사용하였다. 어성초를포함한복합물 1.0 kg ( 어성초 350 g, 상엽, 자초근, 상백피, 녹차와감초는각각 100 g, 박하잎, 질경, 자소엽, 동백씨및인삼 322
어성초포함복합추출물에대한발모효과 씨는각각 50 g씩사용 ) 의 10배에해당되는물을첨가하여 95-100 에서 6 h 추출후추출액을여과포를이용하여여과하였다. 여과액을진공회전농축기를사용하여 2배농축하여농축액을사용하였다. 2. 실험동물및시료의도포 6주령의 C57BL/6 수컷마우스는대한바이오링크 (Chungcheongbuk-do, Kore) 에서구입하였으며, 온도 23± 3, 상대습도 50±10%, 12 h 조명주기조건하에서식이와식수는자유롭게섭취토록하였으며 7일간의적응기간후실험에사용하였다. 추출물을도포하는군별로 5마리씩 4군을실험하였으며, 총 20마리의실험동물사용하였다. 펜토바비탈 (Entobr, Hnlim Phrm, Co., LTD, Seoul, Kore) 50 mg/kg을복강주사하여마취시킨다음이발기를이용하여마우스의등부위의털을제거하였고, 피부속에남아있는모낭과미세한털을제거하기위하여제모제 (Niclen, Ildong Phrm ceuticl, Co., LTD, Seoul, Kore) 를피부에도포하여 5 min간방치한후제모주걱을이용하였다. 제모된 C57BL/6 수컷마우스는미지근한물로피부에남아있는제모제를씻어내고, 24 h 회복기를거친뒤음성대조군 (Control), 실험군 (S-1, -2), 미녹시딜군으로분리하여각각의약물을매일 1회제모된마우스의등부위에마리당 200 μl씩도포하였다. 음성대조군은생리식염수를, 실험군은어성초포함복합추출물 50% 희석액 (S-1) 과 100% 원액 (S-2) 을, 양성대조군인미녹시딜군은 5% 미녹시딜용액을 3주간도포하였다. 모든동물실험은고려대학교동물실험윤리위원회의연구윤리지침에따라진행하였다 (KUIACUC-2014015-2). 3. 육안적평가에의한발모효과의분석실험시작후 1, 2, 3주차에털이자라는상태를확인하기위하여가볍게마취후디지털카메라를이용해사진촬영하였다. 털이자란상태를육안적으로판단하여점수화하고군별로비교하였다 ; 0%-19% (1점), 20%-39% (2점), 40%-59% (3점), 60%-70% (4점), 80%-100% (5점) (Ryu & Shin, 2011). 10명의평가원에의해서평가하여기록하였으며, 각평가원들의점수를합산하여각군별로평균치를산정하였다. 또한털의길이및무게변화관찰을위해실험 3주차에마리당 10개씩등부위의털을뽑아와트만종이에평평하게잘펴서의료용카메라를이용하여촬영한후 2개부위의일정한면적을정하여 Folliscope (Ver. 2.8, Led M, Seoul, Kore) 로측정하였다. 4. 피부조직의효소활성도측정실험 3주차에피부조직을절취하여빙냉하에서미세절편 으로만들고, 그중일정량을칭량한후, 피부조직의 4배량의인산완충용액 (phosphte buffer solution) 을가하여마쇄기 (homogenizer) 를이용하여균질액을만들었다. 이균질액을원심분리기를이용하여 4 에서 12,000 rpm으로 20 min간원심분리하여그상층액을자동생화학분석기 (Konelb 20XT, Thermo Fisher Scientific Inc., Finlnd) 를이용하여 lkline phosph tse (ALP) 및 γ-glutmyl trnspeptidse (γ-gt) 를분석하였다. 5. 실시간정량적중합효소연쇄반응을이용한분자생물학적분석모발성장인자로알려진 IGF-1, VEGF와탈모유발인자인 TGF-β1의발현정도를알아보기위하여연쇄반응 (rel time polymerse chin rection, PCR) 을이용해분석하였다 (H & Prk, 2013). 분석조직으로부터 trizol (Tkr Bio., Shig, Jpn) 을이용하여 RNA를분리하고, intron사의 RT premix kit를사용하여 cdna를합성하였다. 합성된 cdna는 2 SYBR Premix Ex Tq (Tkr bio., Shig, Jpn) 을사용하여 MJ mini therml cycler system (Biord, Hercules, CA, USA) 으로측정하였다. 시료는 95 에서 30 s간반응후 95 에서 5 s, 60 에서 30 s씩 40회반응시켰다. 각유전자의상대적정량을위해 glycerldehyde-3-phosphte dehydrogense (GAPDH) 의발현정도를이용하여보정하였다. 6. 통계처리 SPSS 18.0 for windows (SPSS Inc., Chicgo, IL, USA) 통계프로그램을이용하여일원배치분산분석 (one-wy ANOVA) 을실시하였다. 각그룹간의차이를검정하기위하여 Duncn s multiple rnge test를이용하여사후분석을실시하였다. 본연구의모든측정항목에대한평균 (men) 과표준오차 (stndrd error, SE) 를산출하였으며, 통계학적인유의성검증은 p<.05로하였다. 1. 어성초포함복합추출물의육안적발모효과평가 C57BL/6 수컷마우스는사람과유사한검은색의체모를가져모발의성장주기를색으로쉽게판정할수있다는장점을가지고있기때문에모발연구에서가장널리사용되고있는모델이다. 6주령부터모발주기가휴지기로들어가는유전적인특성이잘알려져있기때문에이시기에실험을시작하면많은장점이있다 (Tobin et l., 1998). 323
Control S-1 S-2 Minoxidil 1 week 2 week 3 week Figure 1. Time-dependent chnges of hir re-growth in C57BL/6 mice treted with herbl complex extrct including Houttuyni cordt Thunb for period of 3 weeks. Control; group of sline solution tretment, S-1; group of 50% herbl complex extrct including Houttuyni cordt Thunb tretment, S-2; group of 100% herbl complex extrct including Houttuyni cordt Thunb tretment, Minoxidil; group of 5% minoxidil tretment. 제모한후어성포포함천연복합추출물을매일도포하면서털이자라는양상을육안적으로관찰하고, 디지털카메라를이용하여기록하였다 (Figure 1). 시료도포 1주차에는모든군에서발모현상을관찰할수없었으나, 2주차에서는양성대조군인미녹시딜군과어성초포함천연복합추출물을 100% 도포한 S-2군에서제모되었던등피부의발모가시작되었으며, 어성초포함천연복합추출물을 50% 희석하여도포한 S-1군에서는 1마리는확연히발모되는현상이나타났으나, 나머지 4마리에서는발모현상을확인할수없었다. 또한생리식염수를도포한음성대조군은 1마리에서일부피부가흑색을띄었으나나머지 4마리는 S-1군과마찬가지로선홍빛을띄었다. 3주차에는미녹시딜군과 S-2군은전체적으로발모가진행되었으며, S-1군은일부쥐에서의발모현상이나타났으나편차가있었으며, 음 성대조군에서는발모의진행상황이미미하였다. 털이자란상태를육안적으로판단하여점수화하여비교한결과 S-1군 (3.14 점 ) 과 S-2군 (4.69 점 ) 그리고미녹시딜군 (4.87 점 ) 은모두대조군 (2.13 점 ) 보다통계적으로유의하게높은발모상태를보였으며 (p<.05), 그중에서도 S-2군과미녹시딜군은점수가가장높았으며유의적으로도차이가없었다 (Tble 1). 2. 털길이및털무게제모한 C57BL/6 수컷마우스등부위발모상태에서털을뽑아털길이와털무게를관찰한결과 (Tble 2), 실험 3주차에서의털길이는양성대조군인미녹시딜군 (6.56 mm) > S-2군 (5.28 mm)=s-1군 (3.28 mm) > 음성대조군 (2.11 mm) 순으로등부위털이긴것으로관찰되었으며통계적으로도각군간의 Tble 1. Comprison of hir re-growth fter topicl ppliction of herbl complex extrct including Houttuyni cordt Thunb for 3 weeks Contents Control S-1 S-2 Minoxidil Hir re-growth score 2.13 0.21 c 3.14 0.72 b 4.69 0.52 4.87 0.13 Vlues re men stndrd error (SE) of 5 mice. Vlues with different superscripts (, b, c) in the sme week re significntly different (p<.05) by ANOVA nd Duncn s multiple rnge tests. Hir regrowth scoring index: 0% 19% (1), 20% 39% (2), 40% 590% (3), 60% 79% (4), 80% 100% (5). Control; group of sline solution tretment, S-1; group of 50% herbl complex extrct including Houttuyni cordt Thunb tretment, S-2; group of 100% herbl complex extrct including Houttuyni cordt Thunb tretment, Minoxidil; group of 5% minoxidil tretment. 324
어성초포함복합추출물에대한발모효과 ALP (IU/mg protein) γ-gt (IU/mg protein) 600 500 400 300 200 100 0 72 60 48 36 24 12 0 c 유의적인차이를보였다 (p<.05). 털무게에있어서는미녹시딜 군 (56.73 mg) > S-2 군 (46.84 mg) > S-1 군 (21.32 mg) > 대조군 (5.64 mg) 순으로나타났으며통계적으로도유의적인차이를 보였다 (p<.05). c 3. 피부조직의효소활성도변화 b c Figure 2. Chnges of ALP nd γ-gt ctivity in the skin of mice. Test compounds were topiclly pplied to the bcks of C57BL/6 mice for 3 weeks. Vlues re men± stndrd error (SE) of 5 mice. Vlues with different superscripts (, b, c) in the sme week re significntly different (p<.05) by ANOVA nd Duncn s multiple rnge tests. ALP; Alkline phosphtse, γ-gt; γ-glutmyl trnspeptidse, Control; group of sline solution tretment, S-1; group of 50% herbl complex extrct including Houttuyni cordt Thunb tretment, S-2; group of 100% herbl complex extrct including Houttuyni cordt Thunb tretment, Minoxidil; group of 5% minoxidil tretment. 피부조직내 ALP 의효소활성도를측정한결과는 Figure 2 에 나타내었다. 양성대조군인미녹시딜군이 498.70 IU/mg 으로 b IGF-1 gene expression (rtio of control) 6 5 4 3 2 1 0 가장높은활성을보였으며, S-2 군은 453.50 IU/mg 으로역시 높은활성을보였으며두군사이에통계적으로유의적인차이 는없었다. 그러나미녹시딜군, S-1 군과 S-2 군은음성대조군 에비해각각 55.1%, 41.1%, 24.1% 로통계적으로유의하게차 이를보였다 (p<.05). b 피부조직내 γ-gt 의효소활성도를측정한결과는 Figure 2 에 나타내었다. 미녹시딜군 (52.42 IU/mg) 과 S-2 (48.30 IU/mg) 군 은대조군 (34.72 IU/mg) 에비해각각 15.0%, 14.0% 가증가하 여통계적으로유의하게높은활성을나타내었으나 (p<.05), S-1 군 (39.15 IU/mg) 군에서는음성대조군과통계적으로유의성이 없었다. γ-gt 활성은미녹시딜군 > S-2 군 > S-1 군 = 대조군순으 로통계적으로유의하게높은활성을보였다 (p<.05). b Figure 3. Effects of herbl medicines on the expression of IGF-1 by rel time polymerse chin rection. GAPDH ws used s comprtive control. Vlues re men±stndrd error (SE) of 5 mice. Vlues with different superscripts (, b) in the sme week re significntly different (p<.05) by ANOVA nd Duncn s multiple rnge tests. IGF; insulin-like growth fctor, GAPDH; glycerldehyde-3-phosphte dehydro gense, Control; group of sline solution tretment, S-1; group of 50% herbl complex extrct including Houttuyni cordt Thunb tretment, S-2; group of 100% herbl complex extrct including Houttuyni cordt Thunb tretment, Minoxidil; group of 5% minoxidil tretment. 주로혈관신생에관여하는 ALP 는진피유두에밀집된모 세혈관에영양을공급하여발모작용을원활하게해주는지표 로서 ALP 측정으로발모작용을간접적으로추측할수있으며, non-growth 부위에비해 re-growth 부위에서 ALP 활성이 Tble 2. Comprison in the hir length nd hir weight fter topicl ppliction of herbl complex extrct including Houttuyni cordt Thunb for 3 weeks Contents Control S-1 S-2 Minoxidil Hir length (mm) 2.11 0.21 c 3.28 0.01 b 5.28 0.21 b 6.56 0.05 Hir weight (mg) 5.64 0.83 d 21.32 2.21 c 46.84 1.67 b 56.73 0.87 Vlues re men stndrd error (SE) of 5 mice. Vlues with different superscripts (, b, c, d) in the sme week re significntly different (p<.05) by ANOVA nd Duncn s multiple rnge tests. Control; group of sline solution tretment, S-1; group of 50% herbl complex extrct including Houttuyni cordt Thunb tretment, S-2; group of 100% herbl complex extrct including Houttuyni cordt Thunb tretment, Minoxidil; group of 5% minoxidil tretment. 325
VEGF A gene expression (rtio of control) 5 4 3 2 1 0 c bc b TGF-β1 gene expression (rtio of control) 2.4 2.0 1.6 1.2 0.8 0.4 0.0 NS Figure 4. Effects of herbl medicines on the expression of VEGF by rel time polymerse chin rection. GAPDH ws used s comprtive control. Vlues re men±stndrd error (SE) of 5 mice. Vlues with different superscripts (, b, c) in the sme week re significntly different (p<.05) by ANOVA nd Duncn s multiple rnge tests. VEGF; vsculr endothelil growth fctor, GAPDH; glycerldehyde-3- phosphte dehydrogense, Control; group of sline solution tretment, S-1; group of 50% herbl complex extrct including Houttuyni cordt Thunb tretment, S-2; group of 100% herbl complex extrct including Houttuyni cordt Thunb tretment, Minoxidil; group of 5% minoxidil tretment. 현저히증가함이보고되고있다 (Iid et l., 2007). 본실험의 피부조직내 ALP 의효소활성도측정결과에서 S-2 군의양성 대조군인미녹시딜군과통계적으로유의적인차이를보이지않 아서어성초포함천연복합추출물에의한 ALP 증가가발모효 과에영향을주었을것으로생각된다. 본실험에서또다른효소인 γ-gt 는글루타티온이나트리 펩타이드로부터시스테인을분리하여케라틴단백질을합성하 는작용을촉진하는효소로서모발성장주기에서신생혈관형성 의지표로서알려져있으며모발성장기동안높은활성을나타 내고, 휴지기동안크게감소한다. 성장기가유도될때더많이 발현되어발모표지성분중하나로제시되고있다 (Hmd & Suzuki, 1996). 본실험에서피부조직내 γ-gt 활성은 S-1 군은대조군과비교하여통계적으로유의한차이가없었지만 S-2 군은대조군과비교시통계적으로유의적인증가를보여 용량의존적으로발모효과에영향을주었을것으로생각된다. 4. 모발성장인자관련유전자발현에미치는영향 어성초포함천연복합추출물에의해촉진된모발의성장이 IGF-1 와관련이있는지확인하기위해시험물질도포개시후 3 주차에채취한조직을사용하여 IGF-1 의발현을확인하였다 (Figure 3). IGF-1 의발현의증가가미녹시딜군과 S-2 군에서 음성대조군에비해통계적으로유의하게나타났다 (p<.05). 미 녹시딜에의한 IGF-1 의발현은음성대조군에비해 3.73 배증 가하였고 S-2 군은 3.47 배증가하여두군사이에서는유의적 인차이는없었다. Figure 5. Effects of herbl medicines on the expression of TGF-β1 by rel time polymerse chin rection. GAPDH ws used s comprtive control. Vlues re men±stndrd error (SE) of 5 mice. NS: not significnt. VEGF; vsculr endothelil growth fctor, GAPDH; glycerldehyde-3- phosphte dehydrogense, Control; group of sline solution tretment, S-1; group of 50% herbl complex extrct including Houttuyni cordt Thunb tretment, S-2; group of 100% herbl complex extrct including Houttuyni cordt Thunb tretment, Minoxidil; group of 5% minoxidil tretment. 어성초포함천연복합추출물의발모촉진효과가혈액순환과 관련이있는지확인하기위해시험물질도포개시후 3 주차에 조직을사용하여 VEGF 의발현을확인하였다 (Figure 4). 그결 과 S-2 군에서 VEGF 의발현이양성대조군인미녹시딜군보다 높은수준의발현량을보였으며, 두군간의통계적으로도유의 적인차이를보였다 (p<.05). 또한 TGF-β1 발현량을측정한결 과 (Figure 5), 미노시딜군에서낮은수준의발현량을보였으나 대조군을포함한모든실험군에서통계적으로유의적인차이는 없었다. 본실험에서육안적인모발의성장, 털의길이및무게의변 화뿐만아니라모발성장인자인 IGF-1, VEGF 와 TGF-β1 의 변화를관찰해본결과모든부분에서실험군인 S-2 군이양성 대조군인미녹시딜군의변화에뒤쳐지지않는효과를보였다. IGF-1 은 in vitro 에서배양된모낭및상피세포의성장을촉 진하며모발세포의세포사멸을방지하며모발성장을촉진한다 고알려져있다. 또한 IGF trnsgenic 동물에서모발의성장이 유의성있게증가한다는보고가있다 (Tng et l., 2003). 또 한 VEGF 는혈관내피를성장시켜혈액순환을개선하여모발 의성장과모근세포의분화를강화시키는인자로알려져모발 관련연구에사용되는지표인자중하나이다 (Li et l., 2003). IGF-1 은모발성장을조절하는가장중요한성장인자의하나 로모유두세포에서 IGF-1 생성에남성호르몬이직접적으로 영향을미치는것으로알려져있다. IGF-1 은간엽제세포에서 만생성되고상피계세포에그수용체가존재하기때문에, 모낭 의경우모유두세포에서분비된 IGF-1 가상피계세포의증식 을촉진하고모낭조직의길이를현저히증가시키는것으로알 326
어성초포함복합추출물에대한발모효과 려져있다. VEGF는새로운모발의성장기로유도하는인자로휴지기에신생혈관생성을촉진시키는인자로작동한다 (Ozeki & Tbt, 2003). 실시간정량적중합효소연쇄반을을이용한분자생물학적분석을통한관찰에서모발성장인자인 IGF-1과 VEGF의활성도가양성대조군인미녹시딜군과비교하여통계적으로유의한차이를보이지않아서어성초포함천연복합추출물이모발성장의중요한기전으로작용했을가능성을시사하고있다. 모발의성장에영향을미치는내분비계물질중 TGF-β1은성장기인모발을퇴행기로진입하게하여상피세포의증식을억제하는쪽으로작용하는것으로알려졌으며성장을저해하는기전을통해탈모를유발하며, 모발이퇴행기로전환될때증가되는인자이다 (Tsuj et l., 2003). 모낭의기질내에유도되어기질세포의세포사멸을일으켜성장기의모낭을퇴행기로유발한다. 즉 TGF-β1의양이증가하면털의기질세포와외모근초세포들이세포사멸을유도하며이는휴지기모낭이증가하며성장기모낭이퇴행기모낭으로바뀌게되고모낭재생이불완전하여재생된모낭의크기가축소하여쉽게빠지며가늘고짧은모발이생성된다 (Lee & Lee, 2007). 본연구에서는 TGFβ1는 S-2군이음성대조군에비해약간높게발현되었는데, 통계적으로는유의적인차이는보이지않았다. 본실험결과 S-2군에서음성대조군과비교하여 IGF-1과 VEGF의효소발현의통계적으로유의적인증가를보였으며, 이와같은결과는육안적, 조직학적실험결과와부합하는것으로어성초포함천연복합추출물이피부세포의 IGF-1과 VEGF 발현을증가시킴으로써모낭의성장을촉진시키고발모에도움을주는것으로생각된다 본연구는천연자원으로써어성초를포함한천연복합추출물을이용하여발모효과를알아보고자하였다. 생후 6주령의 C57BL/6 수컷마우스를이용하여 3주동안경피도포후육안적관찰과 IGF-1, VEGF의성장인자와 TGF-β1 분석을통해분자생물학적인효과를분석하였다. C57BL/6 수컷마우스에시료도포후 1주차에서는미녹시딜군과어성초포함천연복합추출물 100% 를적용한 S-2군에서제모되었던등부위피부의발모가시작되었으며, 3주차에서는미녹시딜군과 S-2군은전체적으로발모가진행되었으며, S-1군은일부쥐에서의발모현상이나타났으나편차가있었으며, 음성대조군에서는발모의진행상황이미미하였다. 털이자란상태를육안적으로판단하여점수화하여비교한결과 S-1군과 S-2군그리고미녹시딜군은모두대조군보다통계적으로유의하게높은발모상태를 보였으며, 그중에서도 S-2군과미녹시딜군은점수가가장높았으며유의적으로도차이가없었다. 제모한 C57BL/6 수컷마우스등부위발모상태에서털을뽑아털길이와털무게를관찰한결과실험 3주차에서의털길이와털무게모두에서미녹시딜군 >S-2군 >S-1군 > 대조군순으로등부위털이길고무거운것으로관찰되었으며통계적으로도각군간의유의적인차이를보였다. 또한 IGF-1와 VEGF의발현의증가가미녹시딜군과 S-2군에서음성대조군에비해통계적으로유의하게나타내었다. 결론적으로, 위의결과로보아어성초포함천연복합추출물은미녹시딜과같이 C57BL/6 수컷마우스에서모발의성장에영향을끼치는물질인 ALP, γ-gt, IGF-1, VEGF 성장인자들을조절하여모낭및주변조직의성장을촉진하고혈액순환을개선하므로발모및탈모관련제품에응용하여관련산업분야에적용하는데기초적인자료가될것으로사료된다. 하지만이번연구는동물실험이라는한계가있기때문에앞으로임상적인실험과부작용에대한추가적인연구가필요할것이다. Adchi K, Kno M. Adenyl cyclse in humn hir follicles: its inhibition by dihydrotestosterone. Biochem. BIophy. Res. Comm., 41: 884-890, 1970. Beoy LA, Woei WJ, Hy YK. Effects of tocotrienol supplementtion on hir growth in humn volunteers. Tro. Life Sci. Res., 21: 91-99, 2010. Buhl AE, Wldon DJ, Miller BF, Brunden MN. Differences in ctivity of minoxidil nd cyclosporin A on hir growth in nude nd norml mice. comprisons of in vivo nd in vitro studies. Lb. Invest., 62: 104-107, 1990. Dllob AL, Sdick NS, Unger W, Lipert S Geissler LA, Gregoire SL, Nguyen HH, Moore EC, Tnk WK. The effect of finsteride, 5-reductse, on sclp skin testosterone nd dihydrotestosterone concentrtions in ptients with mle pttern boldness. J. Clin. Endocrinol. Metb., 79: 703-706, 1994. Grtner LP, Hitt JL. Color textbook of histology (3rd), Elsevier, pp339-340, 2007. Hgemn T, Schlutter-Bhmer B, Allm JP, Bieber T, Novk N. Positive lymphocyte trnsformtion test in ptient with llergic contct dermtitis of the sclp fter short-term use of topicl minoxidil solution. 327
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