J. ENVIRON. TOXICOL. Vol. 24, No. 4, 351~357 (2009) Hairless 마우스에서대추열수추출물의피부내염증및장벽손상완화효과 최소영, 김영철 계명대학교대학원공중보건학과 Alleviative Effects of Jujube Water Extract on the Inflammation and Barrier Damage in Hairless Mice Skin So-Young Choi and Young-Chul Kim Department of Public Health, Graduate School, Keimyung University ABSTRACT To investigate the alleviative effects of jujube water extract (JWE) on the inflammation and skin barrier damage, both the irradiation of UVB and the application of squalene monohydroperoxide (Sq-OOH) were applied to the back skin of experimental animals for 4 weeks. And at the same time experimental materials were applied topically. Six weeks female SKH-1 hairless mice were divided into five groups (five animals for each group) including normal (N; saline), control (C; UVB+Sq-OOH+saline), vehicle control (VC; UVB+Sq-OOH+vehicle), positive control (PC; UVB+Sq-OOH+0.01% retinoic acid) and experimental (E; UVB+Sq-OOH+JWE) groups. The skin erythema index in the E group was significantly low compared to the C group(p 0.05). Lipid (p 0.05) and water (p 0.01) capacities in the E group were significantly high compared to the C group. In comparison with the C group, E group showed a relatively well preserved lipid lamellae in the epidermis and a relatively much less infiltration of mast cells in the dermis or hypodermis. As for the both absolute and relative weights of the spleen, PC group were significantly higher than the other groups. These results suggest that JWE have a considerably inhibitory effect on the inflammation and the skin barrier damage induced by UVB irradiation and Sq-OOH application. Key words : Jujube water extract, hairless mice, skin barrier, inflammation, lipid lamella 서 론 최근삶의질개선과함께피부미용에대한관심이높아지면서내적요인뿐만아니라자외선등에의한외적요인에의한피부노화를억제또는개 To whom correspondence should be addressed. Tel: +82-53-580-5931, Fax: +82-53-588-5233 E-mail: yckim@kmu.ac.kr 선시키기위해화장품, 식품, 의약품을비롯한다양한분야에서항노화연구가활발하게이루어지고있다. 태양광선중의자외선노출에의한피부노화를광노화라하며, 자외선은피부의각질형성세포와섬유아세포를활성화시켜유해한활성산소를생성하고일련의반응에의해항산화방어기구에불균형을가져와산화스트레스를초래한다 (Chiba et al., 2003). 또한과량의활성산소는염증반응, 홍반, 면역 351
352 J. ENVIRON. TOXICOL. Vol. 24, No. 4 억제, DNA 손상및피부암을일으키는주요요인으로작용한다 (Mukhtar and Elmets, 1996). 피부의각질층지질은피부장벽대의역할인각질층내수분유지와함께외부수분의유입을막는기능을담당하며상피세포증식조절, 세포간응집과표피탈락에도관여한다 (Blanken et al., 1989). 과량의자외선노출은피부의각질층지질을산화시켜피부장벽의손상을초래함으로써피부의유분과수분유지기능의저하와함께피부노화를일으킨다. Squalene monohydroperoxide (Sq-OOH) 는과산화된 squalene 의초기생성물질로써자외선노출에의해피부에서생성된다. Hairless 마우스에서 Sq-OOH 도포는피부거침, 각질층의수분손실, 주름형성을야기하는것으로보고되었다 (Chiba et al., 1999). Retinoid 는세포분화를촉진하고생체에필요한각종단백질의생합성기능을가지고있어주름개선을위한기능성화장품의원료로널리상용화되고있다. Retinol 은생체내에서 retinoic acid 로변환되고, retinoic acid 는비타민 A 유도체로써세포내활성산소를제거하여피부노화억제작용을한다 (Weiss et al., 1988). 그러나 retinoid 를국소도포제로사용하는많은환자들은홍반, 건조, 작열감, 소양감등을나타내는레티노이드피부염과같은부작용을겪기도한다 (Fisher et al., 1991). 이에따라최근에는약품 (pharmaceuticals) 의기능이첨가된기능성화장품또는약용화장품 (cosmeceuticals) 의개념이도입되어보다안전하고효과가좋은항산화제를약용식물, 천연물에서탐색하려는노력이활발하게이루어지고있다 ( 최신욱등, 2004). 대추는노화방지에효과가있는신비의생약또는식품으로인식되어져오랜기간한방에서사용되어왔으며, 품종마다약간의함량차이가있으나일반적으로당질이 50% 이상함유되어있으며일반과실보다비타민의함량이높고 epicatechin 과같은 polyphenol 성분이다량존재한다 (Choi et al., 1990). 대추는위경련, 불면증, 소화불량, 대장하혈, 청혈그리고지각과민등에개선효과가있는것으로알려져있다. 또한항산화작용, 간보호작용, 항암작용, 진정작용, 항알러지작용이보고되어져있다 ( 남진희, 2004). 이처럼대추의생리활성및성분에관한많은연구들이있으나, 피부의노화개선또는예방효과에관한연구는현재까지확인되지않고있다. 이에본연구는 hairless 마우스에 UVB 조사와 Sq- OOH 도포를반복적으로실시하여피부에염증과피부장벽손상을유발시키고이와병행하여대추열수추출물을도포하여염증및피부장벽손상완화효과와이들관련인자들의변화양상을알아보았고안전성확인측면에서주요면역장기인흉선과비장의무게변동을알아보았다. 1. 시약및기기 재료및방법 Hydrogen peroxide 는 Junsei 사 (Japan) 의제품을, hematoxylin, bovine serum albumin, retinoic acid, polyethylene glycol 은 Sigma 사 (USA) 의제품을, squalene 은 Acros 사 (USA) 의제품을사용하였으며그외일반시약들은특급품을, 조조바오일 (jojoba oil) 은 Desert Whale 사 (USA) 의제품을사용하였다. 실험기기중자외선조사장치는 UVB sunlamp (UVM-225D, Mineralight Lamp UVP, USA) 를, 자외선측정장치는 UV-radiometer (HD 9021, Delta OHM, Italy) 를사용하였고홍반도및유분함량, 수분함량측정은각각 mexameter (MX18, CK electronic GmbH, Germany), sebumeter (SM815, CK electronic GmbH, Germany), corneometer (CM825, CK electronic GmbH, Germany) 를이용하였다. 시료추출은초고속감압저온추출기 (COSMOS-660, 경서기계산업, 한국 ) 를사용하였다. 조직표본관찰은 light microscope (CX41, Olympus, Japan) 를사용하였다. 2. 시료조제 경남밀양에서재배한유기농대추를 600 g 구입하여물 6L 를가하여초고속감압저온추출기로열수추출한다음진공농축용기에담아 2L 로농축하고파우치진공포장후냉장보관하여실험에사용하였다. 동결건조시켜측정한대추추출물의수율 (yield rate) 은 35.63% 였다. 시료는대추추출물 10 ml, jojoba oil 1 ml, 100% 에탄올 2.5 ml, 물 6.5 ml 비율로혼합하여대추추출물시료로사용하였다. 3. 실험동물및처치 실험동물은 6 주령의 SKH-1 hairless mouse (Charles-River, Japan) 를분양받아 1 주일간사육실에서적
December 2009 Choi and Kim : 대추열수추출물의피부장벽손상완화효과 353 응시킨후실험전기간동안사료와물은자유로이공급하였다. 사육실은온도 22±1 C, 상대습도 50 ±5%, 조명주기 12 시간씩명암을유지하였다. 실험동물은실험 4 주째되는날 4 시간전부터절식시킨다음에테르로마취한후피부는적출하여 10% 중성포르말린용액에고정하였다. 면역학적안전성을확인하기위하여적출한흉선과비장은생리식염수로씻어내고여과지로수분을제거한후전자저울로무게를측정하였다. 실험군은정상군 (normal group, N): 아무런처치를하지않은군, 대조군 (control group, C): saline 도포군, 용매대조군 (vehicle control group, VC): 용매 (100% 에탄올, jojoba oil, 물 ) 도포군, 양성대조 (positive control group, PC): 0.01% retinoic acid 도포군, 실험군 (experimental group, E): 대추열수추출물도포군, 총 5 개군으로나누어실험하였다. 각실험군은 5 마리의마우스로구성하였다. 모든실험은동물실험윤리위원회 (Institutional Animal Care and Use Committee, IACUC) 의표준작업지침서 (Standard Operating Procedures, SOP) 에따라수행되었다. 4. 피부장벽손상유발및시료도포 자외선조사장치의광원은 302 nm 의 UVB 를방출하는 sunlamp 를사용하였다. 자외선조사량은 UV-radiometer 로측정하였으며, 마우스를자외선조사용 cage 에가둔후등부위에균등하게 80 mj/cm 2 의광량으로격일간격으로 1 주일에 3 회, 4 주동안조사하였다. 피부손상을촉진하기위하여 Sq-OOH 를 1 일 2 회, 주 6 일, 4 주동안매회 100 μl 씩도포하였다. 광노화유발과병행하여대추열수추출물 (0.855 g/kg BW/day), 용매, saline 은 1 일 2 회, 주 6 일, 4 주동안매회 200 μl 씩도포하였다. Retinoic acid 는 polyethylene glycol 에 0.01% 로희석하여 1 일 2 회, 주 6 일, 4 주동안매회 200 μl 씩도포하였다. 5. 피부홍반도, 유분함량및수분함량측정 홍반도는 mexameter (MX18) 를, 유분함량은 sebumeter (SM815) 를, 수분함량은 corneometer (CM825) 를사용하여비침습적방법으로실험기간동안주 1 회측정하였다. 6. 피부의조직학적관찰 1) Toluidine blue 염색관찰절취한피부조직을실온에서 10% 중성포르말린용액에 24 시간고정한후통상적인방법으로수세, 탈수, 투명, 침투과정을거친다음파라핀으로포매하고 4 μm 두께로절편을만들어 Toluidine blue 염색후진피층및피하층내 mast cell 의분포양상및탈과립정도를광학현미경으로관찰하였다. 2) Oil red O 염색관찰절취한피부조직을실온에서 10% 중성포르말린용액에 24 시간고정한후 10% formal-calcium 에 1 주일동안 oxidation 처리한다음 30% sucrose 용액에 cryo-protection 한후냉동절편기로 10 μm 두께의냉동절편을제작하여 Oil red O 염색후표피각질층내지방과 lipid lamella 분포양상을광학현미경으로관찰하였다. 7. 자료분석 통계적분석은 SPSS 14.0 for windows (SPSS Inc., USA) 를이용하여각실험군간의차이를비교하기위하여 Student s t-test 로분석하였고, 통계적유의수준은 p 0.05 이하로하였다. 1. 피부홍반도변동 결과및고찰 홍반량의변동을측정한결과는 Table 1 과같다. 대조군은정상군보다홍반량이실험전기간동안유의하게높았으며용매대조군, 양성대조군및대추추출물도포군은대조군에비해낮게나타났다. 실험 28 일후에양성대조군과대추추출물도포군은대조군에비해각각 8.8%, 13.3% (p 0.05) 낮았다. 자외선에노출되면피부의초기반응으로염증이생기는데호중구가염증부위에제일먼저이주하고염증성세포의침윤이일어나며 (Soter, 1990), 염증반응이심한경우비만세포의증가와함께탈과립으로방출된 histamine 은혈관확장을초래함으로써혈류량증가로인한홍반 (erythema) 과부종 (edema) 이일어난다 (Pathak, 1972). 본연구에서대추열수추출물은염증반응을경감시키는효과가있는것
354 J. ENVIRON. TOXICOL. Vol. 24, No. 4 Table 1. Changes in skin erythema index of SKH-1 hairless mice Days 2 238.73±17.42 285.62±14.12* a) 259.67±9.53 260.37±12.27 261.38±10.25 6 241.54±22.39 498.12±13.33*** a) 471.44±25.72*** a) 485.37±34.68*** a) 450.91±25.16*** a) 12 253.64±23.57 342.43±15.36** a) 327.67±26.42* a) 332.25±30.10* a) 279.71±15.36* b) 22 278.38±12.64 362.78±30.54** a) 330.16±55.32 342.48±25.61* a) 320.33±29.76 28 273.85±15.66 330.32±10.81** a) 296.71±25.46 301.26±13.57 286.25±13.83* b) N: Normal group, C: Control group, VC: Vehicle control group, PC: Positive control group, E: Extract group. Values are mean±sd of 5 mice. Unit: AU (Arbitrary unit) a) Significantly different from the normal group by t-test (*; p 0.05, **; p 0.01, ***; p 0.001); b) Significantly different from the control group by t-test (*; p 0.05) Table 2. Changes in skin lipid capacity of SKH-1 hairless mice Days 2 10.31±0.83 7.12±4.30 10.10±4.22 14.47±3.25* b) 14.52±6.13 6 7.21±4.34 2.50±2.05 2.50±1.54 4.00±1.50 4.50±5.52 12 6.50±3.52 2.50±1.00 3.00±1.10 4.25±1.75 4.50±2.98 22 6.00±2.01 2.00±1.56* a) 2.25±1.86* a) 3.50±0.90* a) 5.00±2.16 28 5.50±1.08 1.75±1.00** a) 2.00±1.03** a) 3.25±0.82* a) 3.75±1.01* b) N: Normal group, C: Control group, VC: Vehicle control group, PC: Positive control group, E: Extract group. Values (Unit: μg/cm 2 ) are mean±sd of 5 mice a) Significantly different from the normal group by t-test (*; p 0.05, **; p 0.01); b) Significantly different from the control group by t-test (*; p 0.05) 으로나타났다. 2. 피부유분함량과수분함량변동 유분함량의변동을측정한결과는 Table 2 와같다. 용매대조군, 양성대조군및대추추출물도포군은대조군에비해시간이경과함에따라높았다. 실험 28 일후대조군 (p 0.01), 용매대조군 (p 0.01), 양성대조군 (p 0.05) 은정상군에비해모두유의하게낮았으나대추추출물도포군은유의한차이가없었으며대조군과비교할때용매대조군, 양성대조군, 대추추출물도포군은각각 14.3%, 85.7%, 114.3% (p 0.05) 높았다. 수분함량의변동을측정한결과는 Table 3 과같다. 실험전기간동안모든군은정상군에비해수분함량이낮았다. 실험 28 일후에대추추출물도포군은대조군 (p 0.01), 용매대조군 (p 0.05), 양성대조군 (p 0.05) 에비해유의하게높았다. 과량의 UVB 조사와 Sq-OOH 도포는표피의각 질형성세포와진피의섬유아세포에작용하여 superoxide anion radical (O 2 - ) 과 singlet oxygen ( 1 O 2) 과같은활성산소를생성시키고 (Chiba et al., 2003), 활성산소는진피결합조직의손상과함께표피의피부장벽을붕괴시켜유분및수분함량의감소를가져온다 (Schurer et al., 1991). 본연구에서대추열수추출물은피부장벽손상을완화시키는효과가있는것으로나타났다. 3. 피부의조직학적변화 1) Toluidine blue 염색관찰진피및피하층내 mast cell 의분포양상과탈과립정도를관찰한결과는 Fig. 1 과같다. 정상군은 mast cell 이적게관찰되었고탈과립이거의없는반면, 대조군은 mast cell 이현저히많이관찰되었고탈과립정도가심하게나타났다. 양성대조군과대추추출물도포군은대조군에비해 mast cell 수가적게관찰되었고탈과립정도도경미하게나타났다. 염
December 2009 Choi and Kim : 대추열수추출물의피부장벽손상완화효과 355 Table 3. Changes in skin water capacity of SKH-1 hairless mice Days 2 50.34±4.50 35.82±8.02* a) 37.73±6.96* a) 47.93±5.04 45.32±6.87 6 48.62±3.27 25.71±3.35*** a) 26.83±6.12** a) 28.89±2.01*** a) 27.13±4.58*** a) 12 46.01±3.52 17.24±2.98*** a) 23.74±4.57*** a) 25.03±3.59*** a) 29.96±4.03** a), * b) 22 50.27±2.06 17.86±5.14*** a) 19.62±6.05*** a) 23.67±5.80*** a) 25.76±5.35*** a) 28 52.73±6.58 25.41±3.47*** a) 28.46±5.23** a) 31.80±3.23** a) 37.63±4.32* a)c)d), ** b) N: Normal group, C: Control group, VC: Vehicle control group, PC: Positive control group, E: Extract group. Values are mean±sd of 5 mice. Unit: AU (Arbitrary unit) a) Significantly different from the normal group by t-test (*; p 0.05, **; p 0.01, ***; p 0.001); b) Significantly different from the control group by t-test (*; p 0.05, **; p 0.01); c) Significantly different from the vehicle control group by t-test (*; p 0.05); d) Significantly different from the positive control group by t-test (*; p 0.05) N C PC E Fig. 1. Histological observation on SKH-1 hairless mice skin after 4-week experiment. Toluidine blue stain, 200 & 400. Relatively much less number of mast cells (arrows) were found in the extract group and the degree of degranulation was slight compared to the control group. N: Normal group, C: Control group, PC: Positive control group, E: Extract group. 증반응이심할경우, 진피층과피하층내 mast cell 의증가와함께탈과립을일으켜히스타민이방출되어소양증을동반한다 (Chiba et al., 2003). 이러한대추열수추출물의염증을경감시킨조직학적소견은피부의홍반도를감소시킨결과와부합한다. 2) Oil red O 염색관찰표피각질층내 lipid lamella 의형태및지방량을관찰한결과는 Fig. 2 와같다. 정상군은 lipid lamella 가규칙적이며지방량이많은반면, 대조군은 lipid lamella 가많이부서져형태가불규칙적이고지방량
356 J. ENVIRON. TOXICOL. Vol. 24, No. 4 N C PC E Fig. 2. Histological observation on SKH-1 hairless mice skin after 4-week experiment. Oil red O stain, 200 & 400. N: Normal group, C: Control group, PC: Positive control group, E: Extract group. Table 4. Organ weight of SKH-1 hairless mice after 4-week experiment Groups Spleen Thymus 0.10±0.02 1) 0.11±0.03 0.12±0.02 0.18±0.03** a), * b)c) 0.13±0.02* d) 0.43±0.05 2) 0.42±0.15 0.46±0.13 0.60±0.08** a), * b)c) 0.44±0.09* d) 0.03±0.01 0.04±0.01 0.03±0.01 0.03±0.01 0.03±0.01 0.13±0.02 0.17±0.03 0.15±0.03 0.11±0.02* b) 0.14±0.02 N: Normal group, C: Control group, VC: Vehicle control group, PC: Positive control group, E: Extract group. Values are mean±sd of 5 mice. 1) Absolute weight: g; 2) Relative weight: g/100 g body weight a) Significantly different from the normal group by t-test (**; p 0.01); b) Significantly different from the control group by t-test (*; p 0.05); c) Significantly different from the vehicle control group by t-test (*; p 0.05); d) Significantly different from the positive control group by t-test (*; p 0.05) 이적었다. 양성대조군과대추추출물도포군은대조군에비해 lipid lamella 가비교적규칙적이고지방량도많았다. 피부표피의각질세포 (corneocytes) 사이에있는지질은다수의층판으로연결되어있고, 유극층에서의각질세포간지질의전구체를함유한층판소체 (lamellar bodies) 의분비는표피에서지질 합성이증가함에따라촉진되고이러한지질생성은피부장벽의항상성유지에중요한역할을담당한다 (Elias and Menon, 1991). 이러한대추열수추출물의각질층내 lipid lamella 의형태와지방함량을유지시키는효과는피부의유분함량과수분함량을증가시킨일련의결과와부합하는결과이다.
December 2009 Choi and Kim : 대추열수추출물의피부장벽손상완화효과 357 4. 장기무게변동 적출한비장및흉선무게를측정한결과는 Table 4 와같다. 비장의절대무게와상대무게에있어서양성대조군은다른군에비해유의하게높았다. 흉선의절대무게는모든군에서유의한차이가없었으며상대무게는대조군에비해양성대조군이약 35.3%(p 0.05) 유의하게낮았다. 인체가 retinoic acid 에과량노출시피부건조증, 표피탈락, 소양증과함께간장과비장의비대를초래한다고보고된바있다 (Bollag, 1983). 본연구에서도 retinoic acid 는비장무게의유의한증가를가져와면역학적인측면에서문제점을확인했으며대추열수추출물은정상군과차이를보이지않아상대적으로안전한것으로확인되었다. 결 론 대추열수추출물의염증및피부장벽손상완화효과를알아보기위해 hairless 마우스의피부에 UVB 조사와 SQ-OOH 도포로염증과피부장벽손상을유발시키면서대추열수추출물을병행하여도포한실험에서다음과같은결과를얻었다. 대추열수추출물은피부홍반도를낮추고진피및피하층내비만세포의수와탈과립정도를현저히낮춰염증을경감시키는효과를보였다. 대추열수추출물은피부의유분함량과수분함량을높이고표피각질층내 lipid lamella 의배열과지방함량을유지시키는작용을보임으로써피부장벽손상을완화시키는효과를보였다. 아울러, retinoic acid 는주요면역장기인비장의절대무게와상대무게를유의하게증가시킨반면, 대추열수추출물은유의한차이를보이지않았다. 이상의실험결과를통하여, 대추열수추출물은 hairless 마우스에서피부조직의염증반응과피부장벽손상에개선효과가있음을확인하였다. 참고문헌 남진희. 대추추출물의항산화활성과페놀성화합물의동 정, 계명대학교대학원석사학위논문 2004. 최신욱, 정재형, 김남경, 한상화, 김창수. 오존에의한피부손상확인및이를방어하는피부외용제소재의탐색, 대한화장품학회지 2004; 30(1): 39-51. Blanken R, Van Vilsteren MJT, Tupker RA and Conraads, PJ. Effect of mineral oil and linoleic-acid-containing emulsions on the skin vapour loss of sodium-lauryl-sulphate-induced irritant skin reactions, Cont Dermat 1989; 20(2): 93-97. Bollag W. Vitamin A and retinoids: from nutrition to pharmacotherapy in dermatology and oncology, Lancet 1983; 1(8329): 860-863. Chiba K, Kawakami K, Sone T and Onoue M. Characteristics of skin wrinkling and dermal changes induced by repeated application of squalene monohydroperoxide to hairless mouse skin, Skin Pharmacol Appl Skin Physiol 2003; 16 (4): 242-251. Chiba K, Sone T, Kawakami K and Onoue M. Skin roughness and wrinkle formation induced by repeated application of squalene monohydroperoxide to the hairless mouse, Exp Dermatol 1999; 8(6): 471-479. Choi KS, Suk MS and Chung DS. Studies on the storage of jujube fruit (Sisipus jujuba Miller) var. Bokjo in sealed polyethylene film bag. J Res Develop 1990; 9: 55-61. Elias PM and Menon GK. Structural and lipid biochemical correlates of the epidermal permeability barrier, Adv Lip Res 1991; 24(1): 1-26. Fisher GJ, Esmann J, Griffiths CEM, Talwar HS, Duell EA, Hammerberg C, Elder JT, Karabin GD, Nickoloff BJ, Cooper KD and Voorhees JJ. Cellular, immunologic and biochemical characterization of topical retinoic acid-treated human skin, J Invest Dermatol 1991: 96(5): 699-707. Mukhtar H and Elmets CA. Photocarcinogenesis: mechanisms, models and human health implications, Photochem Photobiol 1996; 63(4): 356-357. Pathak MA. Formation of thymine dimers in mammalian skin by ultraviolet radiation in vivo, Photochem Photobiol 1972; 15(2): 177-185. Schurer NY, Plewig G and Elias PM. Stratum corneum lipid function, J Dermatol 1991; 183(1): 77-94. Soter NA. Acute effects of ultraviolet irradiation the skin, Dermatology 1990; 9(1): 11-15. Weiss JS, Ellis CN, Headington JT and Voorhees JJ. Topical tretinoin in the treatment of aging skin, J Am Acad Dermatol 1988; 19(1): 169-175.