J Koren Soc Food Sci Nutr 한국식품영양과학회지 43(3), 374~380(2014) http://dx.doi.org/10.3746/jkfn.2014.43.3.374 야관문추출물의창상치유효과 정희경 1 김길수 2 정유석 1 1 ( 재 ) 대구테크노파크바이오헬스융합센터 2 경북대학교수의과대학 Wound Heling Effects of Lespedez cunet Extrct Hee Kyoung Jung 1, Kil-Soo Kim 2, nd Yoo Seok Jeong 1 1 BioHelth Convergence Center, Degu Technoprk, Degu 704-801, Kore 2 College of Veterinry Medicine, Kyungpook Ntionl University, Degu 702-701, Kore ABSTRACT In this study, the nti-inflmmtory effects of Lespedez cunet extrct on mcrophges nd wound-heling in wound-induced niml experiments were investigted. In n nti-inflmmtory test, 0.1 mg/ml of Lespedez cunet extrct did not ffect growth of RAW 264.7 cells, nd Lespedez cunet extrct suppressed nitric oxide (NO) genertion from inflmmtion-induced mcrophges in concentrtion-dependent mnner. Wounds on the skin of rts were treted with vehicle contining Lespedez cunet extrct (SSP), vehicle (SCO), nd commercil ointment (CCO). The wound nd scr sizes in the SSP group were significntly reduced in comprison to the SCO nd CCO groups (P<0.0). The epidermis nd dermis of the SSP group lso recovered fster thn the SCO group sed on Msson's trichrome stining. The gene expression levels of vsculr endothelil growth fctor (VEGF) decresed nd trnsforming growth fctor-et 1 (TGF-β1) incresed in wound tissue from the SSP group compred to tht from the SCO group. These results show tht Lespedez cunet extrct ccelertes wound-heling through nti-inflmmtory ctivity nd induction of collgen regenertion s well s reduces the scr re surrounding wounds. Accordingly, Lespedez cunet extrct could e useful s cosmeceuticl in the cosmetic industry. Key words: Lespedez cunet, cosmeceuticls, nti-inflmmtion, wound heling, wound repir 서 여성의사회진출증가와생활수준향상그리고문화적, 사회적가치관의변화에따라남녀노소를불문하고피부에대한관심이지속적으로증가하고있다. 이에따라화장품의소비도증가하고있으며아름다움만을위한향장용보다효능과효과를중요시하는기능성화장품에대한소비가급증하고있다. 최근기능성화장품은미용기능성에서한단계더나아가치료기능까지포함하는코스메슈티컬 (cosmeceuticls) 이화장품시장에서집중을받고있다 (1,2). 또한화장품에사용되는기능성소재로피부에적합하며부작용이적고안전성이입증된천연물소재의활용과연구가활발하다. 야관문 (Lespedez cunet G. Don) 은콩과의여러해살이식물로비수리, 삼엽초등의여러이름으로불리며우리나라를비롯한일본, 중국, 대만등지에분포하고있는자원으로간과콩팥의기능을보호해주고폐기능강화및혈액순 론 Received 1 Octoer 2013; Accepted 6 Ferury 2014 Corresponding uthor. E-mil: crrot2p@ttp.org, Phone: +82-3-602-188 환에효능이있어관련질병에약재로도쓰이고있다 (3,4). 야관문내알려진유용생리활성물질로는 pinitol, viculrin, trifolin, β-sitosterol, quercetin, kempferol, vitexin 등의플라보노이드가있으며 (), Ding 등 (6) 은야관문내함유되어있는미네랄과아미노산등을분석하여노화방지에유용한 vitmin E 함량이높음을확인하였다. 또한야관문추출물이항균및항산화효과 (3,7), 피부광노화억제효과 (8) 등을나타내는유용한천연물소재임이보고된바있다. 피부는외부자극으로부터몸을보호하는신체방어에중요한역할을하며 (9), 이를구성하고있는외피및진피가손상되어피부구조의연속성이파괴되는데이를창상이라고하고염증반응과신생조직형성및재상피화단계가복합적으로이뤄져창상부위를복구하는과정을창상치유라한다 (10). 일반적으로창상에많이사용하는후시딘산나트륨을함유하는연고가피부에알레르기반응을유도하거나반흔및유착된조직을형성시키는등부작용이보고되고있어 (11,12) 창상치유에있어부작용이적고우수한효과를지닌천연물을약이나화장품소재로개발하려는연구가다양하게진행중이다. Prk 등 (13) 은복합허브추출물의창상치유효과에대해조사하였으며, 참느릅나무 (Ulmus prvifo-
야관문추출물의창상치유효과 37 Tle 1. Composition of formul dded with Lespedez cunet extrct Components D. I. wter EDTA-2N Allntoin 1,2-Hexnediol Glycerin Hydroviton 24 Konjc mnnn Aristolflex AVC Ntrosol 20HHR (1%) Hyluronic cid (1%) Cropol 940 (2%) Ethnol Emlex HC-40 Vitmin E cette L-Arginine Lespedez cunet extrct (3%) 1) Bse smple (vehicle) 6.7 0.03 0.12 3 2. 0.90 0.60 0.10 Rtio (%) Bse smple contining Lespedez cunet extrct 63.7 0.03 0.12 3 2. 0.90 0.60 0.10 2 Totl 100 100 1) ( ): Contents of solid powder. li Jcq.)(14), 꼭두서니 (Cnthium prviflorum)(1), 지근 (Lithospermi rdix) 과치자 (Grdenie fructus) 복합제제 (16) 등도창상치유효과가있다고보고하였다. 또한창상치유과정에서는산화적스트레스원인물질발생으로인해염증관련사이토카인들이발현되어염증반응이일어나는데 (17) 이를개선및치료하기위한다양한천연항염증소재개발도보고되고있다 (18,19). 그러나창상치유가염증반응과조직재생이복합적으로이루어지고있지만상기의연구들은창상치유와관련하여단편적인효과를가지는소재에대해연구를수행한실정이다. 따라서본연구에서는야관문추출물이염증반응억제와함께조직재생에도움을줄수있는코스메슈티컬소재로써적합한지평가하고자야관문추출물의항염증효능과피부결손창을만든동물모델을대상으로야관물추출물이첨가되어진화장품조성물의창상치유효과를조사하여보고하는바이다. 재료및방법시료제조실험에사용되어진야관문에탄올추출물 ( 이하야관문추출물, 고형분 3%) 및야관문추출물함유조성물은소리소 (Soriso Co., Gyeongsn, Kore) 에서제공받아사용하였다. 세포실험시료는야관문추출물을동결건조 (FreeZone plus 12, Lconco, Knss, MO, USA) 하여얻은고형물을 0% 에탄올에녹여사용하였으며, 창상유발마우스모델동물실험에서는 Tle 1과같은야관문추출물첨가 (2%, w/w) 조건이다른화장품조성물을시료로사용하였다. RAW264.7 세포독성측정마우스대식세포 (RAW264.7) 는한국세포주은행 (KCLB, Seoul, Kore) 으로부터분양받아본실험에사용하였다. RAW264.7 세포는 100 unit/ml penicillin-streptomycin (GIBCO BRL Life Technologies, Inc., Githersurg, MD, USA) 과 fetl ovine serum(welgene Inc., Degu, Kore) 을첨가한 Duleco's modified Egle medium (Welgene Inc.) 을사용하여 37 C, % CO 2 조건의배양기 (HERAcell 10, Thermo Fisher Scientific Inc., Wlhm, MA, USA) 에서배양하였다. 야관문추출물에대한세포독성은 3-(4,-dimethylthizole-2-yl)-2,-diphenyl-tetrzolium romide(mtt) 환원법 (20) 을이용하여측정하였다. RAW264.7 세포를 24 well plte에 3 10 4 cells/well 농도로분주하여 24시간배양한후추출물을다양한농도 (0.0, 0.07, 0.1, 0.2, 0. mg/ml) 로처리하였다. 이를 24시간추가배양한후 MTT (Sigm-Aldrich Co., St. Louis, MO, USA) 용액을최종농도 0. mg/ml로첨가하고다시 4시간동안배양한다음배지를제거하고, 형성된 formzn crystl을 1 ml dimethyl sulfoxide(sigm-aldrich Co.) 로용해시켜 microplte reder(infinite M200 PRO, Tecn, Männedorf, Switzerlnd) 를이용하여 70 nm에서흡광도를측정하였다. 시료에대한세포독성은시료를처리하지않은세포의흡광도값을 100% 로하여세포생존율을상대적인값 (%) 으로나타내었다. Nitric oxide 생성량측정 RAW264.7 세포로부터생성되는 nitric oxide(no) 의양은 Griess 법 (21) 으로측정하였다. 24 well plte에 10 4 cells/well의농도로분주하고 24시간동안배양한후야관문추출물을다양한농도 (0.0, 0.07, 0.1, 0.2 mg/ml) 로처리하고 2시간동안반응시킨다음 lipopolyscchride (LPS, Sigm-Aldrich Co.) 1 μg/ml를처리하여 18시간동안배양하였다. RAW264.7 세포로부터생성된 NO의양은배양액과 griess regent(sigm-aldrich Co.) 각 100 μl를혼합하고 1분간상온에서반응시킨다음 microplte reder(tecn) 로 40 nm에서흡광도를측정한후 sodium nitrite(sigm-aldrich Co.) 의표준곡선작성에의해산출 (μm) 하였다. 실험동물및창상유발실험동물은체중 180 210 g의 4주령수컷 Sprgue- Dwley rt을 ( 주 ) 중앙실험동물 (Seoul, Kore) 에서구입하여일정한온도 (21.2±2.0 C), 상대습도 (.0±10.0%) 를유지하고규칙적인명암 (12시간명 / 암 ) 을인공조명으로조절하였으며, 사료와물은충분히공급하면서 1주일동안적
376 정희경 김길수 정유석 응시킨후실험에사용하였다. 안정화기간이지난후실험동물은 Zoletil 0(Virc, Crros, Frnce) 주사마취하에등부위를제모한다음피혁용펀치 (iopsy punch; 8 mm) 를이용하여좌우대칭의원형결손창을 2개소만들어창상을유발하였다. 동물실험에관련된모든과정은동물보호법의 3R 및 ( 재 ) 대구테크노파크바이오헬스융합센터동물실험윤리위원회 (Institutionl Animl Cre nd Use Committee, IACUC) 의원칙을준수하여수행하였다. 실험군분류및시료처리실험군은창상유발후시료를처리하지않은무처리군 (NO), 창상유발후 vehicle 시료도포대조군 (SCO), 상처치료용시판연고를도포한군 (CCO) 과 2% 야관문추출물이함유된 vehicle 시료도포시험군 (SSP) 총 4개군으로분류하였으며, 각군당 마리씩총 20마리로실험을수행하였다. 야관문추출물의창상치유효능을기존시장제품과성능을비교하기위해상처치료제로써일반인들의인지도가높은 20 mg/g의후시딘산나트륨이함유된시판피부외용제연고를사용하였다. 각시료의도포량은가피가탈각될때까지매일 1회씩창상유발부위가덮일정도로충분하게도포하였다. 육안적변화의관찰창상치유진행과정을관찰하기위해창상유발후 3일간격으로 digitl cmer(camedia TM, Olympus Co., Tokyo, Jpn) 를이용하여각군별로일정한거리에서창상부위를촬영하여상처의변화를육안으로관찰하였으며, 객관적지표로나타내기위하여 digitl cliper(mitutoyo Co., Kwski, Jpn) 로창상의면적을측정하였다. 조직병리학적변화의관찰창상유발 21일째에각실험군의조직병리학적검사를위해실험동물들을 ethyl ether(duksn Pure Chemicl Co., Ltd., Ansn, Kore) 로마취한후창상부위가포함된조직절편을채취하여 10% 중성 formlin 용액에 24시간동안고정하였다. 고정된조직은창상중앙을통과하는절편을취하여탈수하고 prffin 블록에포매한후조직절편기 (RM212, Leic, Wetzlr, Germny) 를이용하여 4 μm 두께로절단한다음, polylysin으로 coting된 slide에부착하였다. 조직절편은 xylene으로 prffin을제거하고알코올과증류수로 10분동안함수시킨후증류수로세척하였다. 이후 Hemtoxylin-Eosin(H&E) 염색을통한피부세포및조직의재생을관찰하였으며, 진피교원섬유변화를관찰하기위하여 Msson's trichrome 염색을실시하였다. 피부조직의유전자발현량측정창상유발후 21일째각군별실험동물들의창상부위조직을채취하여 RNAlter RNA stiliztion regent (Qigen, Hilden, North Rhine-Westphli, Germny) 를사용하여 4 C에보관하면서본실험에이용하였다. 조직으로부터 RNA는 RNesy firous tissue mini kit(qigen) 를이용하여추출한후 RNA 2 μg을 RevertAid First Strnd cdna Synthesis kit(thermo Fisher Scientific Inc.) 를이용하여 cdna 합성을수행하였다. PCR 반응액은 cdna 0. μl, forwrd primer(10 pmole) 1 μl, reverse primer(10 pmole) 1 μl, SYBR Green supermix(bio-rd Lortories Inc., Hercules, CA, USA) 12. μl, 멸균증류수 10 μl를혼합하여준비한다음, icycler iq rel time PCR detection system(bio-rd Lortories Inc.) 을사용하여 RT-PCR(reverse trnscription polymerse chin rection) 수행후 GAPDH(glycerldehyde 3-phosphte dehydrogense) 유전자발현에대한각유전자발현정도를백분율 (%) 로나타내었다. PCR 반응에사용되어진 primer 염기서열은다음과같다 : VEFG(vsculr endothelil growth fctor) forwrd primer '-AGT CTT GCC AAT GTG GAC TC-3', reverse primer '-GGC AGT GGA TTC TCA TCT TG-3'; TGF-β1(trnsforming growth fctor-et 1) forwrd primer '-GCC TCC GCA TCC CAC CTT TG-3', reverse primer '-GCG GGT GAC TTC TTT GGC GT-3'; IL-10(interleukine-10) forwrd primer '-GAA AAC AGA GCT TCA GCA TGC TTG G-3', reverse pimer '-TTT GAG TGT CAC GTA GGC TTC TAT GC-3'; GADPH forwrd primer '-TCC CTC AAG ATT GTC AGC AA-3', reverse primer '-AGA TCC ACA ACG GAT ACA(22). 통계분석본연구의모든실험결과는 3회반복실험에대한평균값 (men) 과평균의표준편차 (SD) 로나타내었으며, 각그룹간의통계적유의성은 SPSS ver 10.0(SPSS Inc., Chicgo, IL, USA) 을이용하여 one-wy ANOVA 검정을적용하여 P<0.0 수준에서유의성검정을실시하였다. 결과및고찰 RAW264.7 세포에대한세포독성측정피부의창상치유과정에서활성화된혈소판이나섬유소에의해상처부위로모이게되는대식구와섬유모세포의역할은매우중요하다 (23). 이중에서대식구는상처부위에파괴된세포와이물질등을제거하고항염증반응과함께교원질의합성을촉진시키며상처치유활성을증가시키기도한다 (24). 본연구에서는대식구에대한독성없이야관문추출물이나타내는항염증반응을조사하고자잘알려진대식세포 RAW264.7에대한야관문추출물농도별세포독성을확인하였으며, 그결과 0. mg/ml 농도조건에서 89% 의생존율을보였으나 0.2 mg/ml 이하의농도에서모두
야관문추출물의창상치유효과 377 9% 이상의생존율을나타내었다 (dt not shown). 이는 Eun(2) 이야관문의면역능에미치는영향을알아보고자수행한마우스의흉선세포, 비장세포및대식세포에대한야관문추출물의세포독성을평가한결과와유사하였다. RAW264.7 세포로부터 NO 생성에미치는효과 NO는신경전달계및혈관이완, 혈소판응집억제, 염증반응매개등여러생물학적반응에관여하는활성산소중의하나로대식구에의해활성화되어염증유발에중요한역할을한다 (26,27). RAW264.7 세포에염증유발후 NO 생성억제정도를통해야관문추출물의염증반응저해효과를확인한결과, LPS를처리하지않은정상군에서 NO 생성량은 1.04±0.60 μm인반면 LPS를처리한대조군에서는 31.02 ±4.30 μm로정상군에비해약 30배정도증가하였다. 야관문추출물 0.0, 0.07, 0.1, 0.2 mg/ml 처리후 LPS로활성화된 RAW264.7 세포에서 NO의생성량은각각 18.1 ±0.47,.92±1.66, 2.44±0.32 및 1.91±0.23 μm로야관문추출물에농도의존적으로감소하는경향을보였다 (Fig. 1). Yoon과 Moon(28) 은 LPS 처리를통해활성화된염증반응이창상치유를지연시키고대식구가창상치유에관여함 NO concentrtion (μm). 0 40 30 20 10 0 LPS (μg/ml) Lespedez cunet extrct (mg/ml) d - + + + + + - - 0.0 0.07 0.1 0.2 Fig. 1. Effect of Lespedez cunet extrct on NO (nitric oxide) production in LPS-stimulted RAW264.7 cells. The levels of nitrite were determined y the Griess method s descried in Mterils nd Methods. The results re expressed s mens (r) with SD (error r) of triplicte determintions. Differences were considered sttisticlly different t P<0.0. c d d 을확인하였다. 또한 Schffer 등 (29) 은마우스모델에서창상부위의콜라겐섬유의침착정도와물리적인강도가 NO의농도와반비례함을보고하였다. 따라서염증반응이유도되어진대식세포에대한야관문추출물의 NO 생성저해활성은야관문추출물이염증반응억제를통해창상치유에긍정적효과를가져올수있을것으로기대된다. 상처면적및피부표면변화창상발생후상처부위에는염증기단계와함께혈관수축및혈소판응고반응이일어나고그후교원질의합성, 신생혈관의형성등복잡한병리학적과정이일어나며육아조직이상처부위를채우는재상피화후상처부위가수축되게된다 (12,30). Tle 2와같이 8 mm 창상유발후시료처리 3일째상처면적을확인한결과, NO군과 SCO군그리고 CCO군은약 % 정도감소하였으나 SSP군에서는약 17% 의상처치유율을보였다. 시료처리 12일째에는 NO군, SCO 군과 CCO군은각각 4.33±1.09, 3.86±0.28, 3.6±0.11 mm의상처면적을나타낸반면, SSP군에서는 1.92±0.19 mm로유의적으로크게상처면적감소를보였으며 (P< 0.0), NO군과비교해약 30% 정도빠른회복을나타내었다. 창상치유시발생되는일반적인부작용으로미용적관심대상인반흔 (scr) 형성을시료처리 21일째비교해본결과, NO군, SCO군, CCO군및 SSP군에서반흔크기는각각.6±0.1,.88±0.70,.64±0.37과 4.66±0.29 mm로 SSP군이유의적으로가장작게형성되었다 (P<0.0). 본연구에서는상처치유에대해일반소비자들의인지도가높은후시딘산나트륨이함유된시판피부외용제를양성대조군으로사용하였으며상처면적은 NO군및 CCO군과의유의적차이는관찰되지않았다. 사용되어진시판피부외용제는세균의단백질합성을저해하는항생물질인 fusidic cid를주성분으로한다. 창상치유는염증반응억제후재상피화가완료되고상처부위수축을시작하므로 (31) NO군과비교시 CCO군에의한상처면적감소경향은세균감염방지에의한것이며, 일반시판상처치유용피부외용제는재상피화에대한유효성은없는것으로생각되어진다. 그러나야관문추출물은항염증효과와함께재상피화가속화를통해상처 Tle 2. Comprisons of wound size nd scr size chnges NO 1) SCO 1) CCO 1) SSP 1) Wound size (mm) Scr size (mm) 0 dy 3 dys 6 dys 9 dys 12 dys 18 dys 21 dys 8.00±0.00 2) 7.8±0.19 3).63±0.10 4.10±0.64 4.33±1.09 8.00±0.00 7.1±0.46 6.3±0.44 3.90±0.17 3.86±0.28 8.00±0.00 7.7±0.3 6.6±0.26 4.67±0.24 3.6±0.11 8.00±0.00 6.62±0.38 ±0.21 c 2.84±0.06 c 1.92±0.19 6.38±0.47 7.42±0.39 7.48±0.04 3.4±0.32 c.6±0.1.88±0.70.64±0.37 4.66±0.29 1) NO: wound of non-treted rt, SCO: wound of rt treted with vehicle, CCO: wound of rt treted with commercil ointment (fucidin ointment), SSP: wound of rt treted with vehicle contining Lespedez cunet extrct. 2) Vlues re men±sd of dorsl 2 hole in rts (n=). 3) Vlues within row with different superscripts letters re significntly different ech other groups (n=) t P<0.0.
378 정희경 김길수 정유석 크기와창상치유기간을감소시킬뿐만아니라반흔형성을최소화할수있는소재로기대된다. 조직병리학적변화창상유발후 21일째에실험동물들로부터채취한창상부위의피부조직을 H&E 염색과 Msson's trichrome 염색을실시하여광학현미경을통해관찰되어진결과를 Fig. 2와 Fig. 3에나타내었다. 조직염색소견으로 NO군, SCO군, CCO군및 SSP군의모든실험군에서섬유아세포의증식및교원질 (collgen fiers, CF) 의합성이관찰되었으며 SSP 군의경우 NO군에비해상피층 (epidermis, ED) 의두께가얇고일정하고부드럽게형성된것을확인할수있었다. 또한 SSP군이진피층 (dermis, DM) 에서의모낭 (hir follicle, HF) 을비롯한기타피부부속기관과구성조직의재형성이 NO군에비해더욱치밀하고활발하게일어난것을확인하였다. SSP군을제외한다른실험군은육아조직을보이는반면 SSP군은시험되어진다른군에비해교원질이일정하게가장빨리재형성된것을관찰할수있었다. 복잡한다단계과정을통해일어나는창상치유는염증기이후상피화기를통해창상조직으로세포들이이동증식하여창상표피재생에관여하게되고, 섬유아세포가교원질을형성하는활성으로창상내부구조재생에의관여는섬유증식기가일어나게되고, 창상부위내외부적구성성분이수축활동을통해결합및구성조직들이재배치되게되는수축기를마지막으로완료되게된다 (32). 동일실험기간내 SSP군이가장빠른조직재생을나타냄으로써야관문추출물은염증반응은물 론교원질과같은피부조직재생수준까지의창상치유속도를가속화시킬수있음을확인할수있었다. 반흔형성은염증반응 (33) 과신생조직의과형성및교원질의불균형적형성 (34) 으로인해발생되어진다고알려져있다. 다른실험군대비야관문추출물처리군에서감소된반흔형성은조직병리학적결과로미루어볼때, 야관문추출물이균형적인교원질형성을유도함으로써나타난결과로사료된다. 피부조직의유전자발현량변화창상치유과정은 PDGF(plttele-derived growth fctor), VEGF, EGF(epiderml growth fctor), TGF-β, IL-10 등의 growth fctor와 cytokine이세포및조직들에서복합적으로활성을나타내어이뤄진다 (3,36). 본연구에서는 VEGF, TGF-β1, IL-10 유전자를대상으로시료가유전자발현에미치는영향을확인하였다 (Fig. 4). 창상유발후 21일째실험군들의조직에서 VEGF 유전자는 NO군 66.6%, SCO군 66.1%, CCO군 6.%, SSP군 61.4% 로 SSP군이유의적으로낮은발현정도를나타내었으며, TGFβ1 유전자는 NO군 7.2%, SCO군 70.33%, CCO군 70.3%, SSP군 79.8% 로 SSP군이유의적으로높은발현정도를나타내었다 (P<0.0). VEGF는창상유발후초기단계에발현되어져염증성사이토카인및저산소상태등의자극에의해신생혈관형성에관여하며 (34,37), TGF-β1은섬유아세포의분화와증식으로인한육아조직형성과재상피화단계에관여하여창상치유에중요한역할을하는것으로알려져있다 (34,38). 본연구에서 SSP군이다른실험군에비해낮은 Fig. 2. Histologicl ppernce of wounded skin from experimentl groups y H&E (Hemtoxylen nd Eosin) stining on 21 dys fter initil wounding. A: NO group (wounded skin of nontreted rts), B: SCO group (wounded skin of rts treted with vehicle), C: CCO group (wounded skin of rts treted with commercil ointment, fucidin ointment), D: SSP group (wounded skin of rts treted with vehicle contining Lespedez cunet extrct). ED: epidermis, DM: dermis, HF: hir follicle. Fig. 3. Histologicl ppernce of wounded skin from experimentl groups y Msson trichrome stining on 21 dys fter initil wounding. A: NO group (wounded skin of non-treted rts), B: SCO group (wounded skin of rts treted with vehicle), C: CCO group (wounded skin of rts treted with commercil ointment, fucidin ointment), D: SSP group (wounded skin of rts treted with vehicle contining Lespedez cunet extrct). ED: epidermis, CF: collgen fiers.
야관문추출물의창상치유효과 379 Gene expression (%, reltive rtio. of GAPDH mrna expression). 90 80 70 60 0 40 30 20 10 0 c VEGF TGFβ1 IL-10 NO SCO CCO SSP Fig. 4. Gene expression of experimentl groups on 21 dys fter wounding. Comprison of gene expression y RT-PCR. The results re expressed s mens (r) with SD (error r) of triplicte determintions (n=). Differences were considered sttisticlly different t P<0.0. VEGF 유전자발현과높은 TGF-β1 유전자발현정도를결과하는것은야관문추출물이창상치유가속화에기여하여나타난것으로사료된다. IL-10은대식세포활성과 TNF-α, IL-1β, IL-6와같은전염증 cytokine 생산을저해함으로써염증반응을완화시키는항염증인자로창상치유과정에관여한다 (39,40). 유의적이진않으나 CCO군과 SSP 군에서 IL-10 유전자발현이낮은경향을보이는것은창상치유단계에서의염증반응이완료되어나타난결과로써사료된다. Kpoor 등 (41) 은창상회복기간중 VEGF 발현이낮아지며, Shh 등 (38) 과 Rolfe 등 (42) 은 TGF-β1의과발현이반흔을감소시켜주며콜라겐합성에기여한다고보고한바있다. 상기의결과들을종합적으로보았을때화장품조성물내첨가된야관문추출물은창상치유속도를가속화하는동시에창상과반흔면적감소에기여함으로써피부창상치유와관련한코스메슈티컬소재로써유효성을나타내며이는산업적활용에유용할것으로사료된다. 요 본연구에서는야관문추출물의마우스대식세포에대한항염증활성과창상유발동물실험모델을통한창상치유효과를조사하였다. RAW264.7 세포에서야관문추출물은 0.2 mg/ml 이하농도에서세포생존에영향을주지않았으며, 염증반응이활성화된대식세포에대해농도의존적으로유의적인 NO 생성감소를나타내었다. 창상유발동물실험모델에서야관문추출물을함유한화장품조성물의창상치유효과에대해육안적으로관찰한결과, SCO군과 CCO군보다야관문추출물을함유한 SSP군에서약 20~30% 빠른상처면적감소효과를나타내었으며, 반흔크기역시약 12% 작게형성되었다. 또한 SSP군조직의외피와진피재생회복속도가빨라진것을 Msson's trichrome 염색을통해확인할수있었으며, VEGF 및 TGF-β1 유전자발현이 SCO군과비교시각각감소및증가하였다. 이러한결과는야관문 약 추출물이항염증및교원질생성유도를통한조직재생활성에기여하여창상치유속도를가속화하고반흔면적을감소시킬수있는피부창상치유와관련한코스메슈티컬소재로써산업적활용이가능함을보여준다. 감사의글 본연구는지식경제부의지역산업기술개발사업 ( 과제번호 : A000200004) 의지원에의해수행되었으며이에감사드립니다. REFERENCES 1. Prk JM, Lee SP, Son ES. 2002. An nlysis on the regultions, technology, nd mrket of cosmeceuticls. Kor J Technol Innovt Soc : 293-306. 2. Lee HH. 2007. A prospect on development of functionl foods in the cosmetics industry. Kor J Aesthet Cosmetol : 83-90. 3. Lee HJ, Lim GN, Prk MA, Prk SN. 2011. Anticteril nd ntioxidtive ctivity of Lespedez cunet G. Don. Koren J Microiol Biotechnol 39: 63-69. 4. Kwon DJ, Kim JK, Hm YH, Be YS. 2007. Flvone glycosides from the eril prts of Lespedez cunet G. Don. J Koren Soc Appl Biol Chem 0: 344-347.. Numt A, Hokimoto K, Ymguchi H. 1980. C-Glycosylflvones in Lespedez cunet. Chem Phrm Bull 23: 964-96. 6. Ding JL, Lim IJ, Lee HD, Ch WS. 2006. Anlysis of minerls, mino cids nd vitmin of Lespedez cunet. Koren J Biotechnol Bioeng 21: 414-417. 7. Kim SJ, Kim DW. 2007. Antioxidtive ctivity of hot wter nd ethnol extrcts of Lespedez cunet seeds. Koren J Food Preserv 14: 332-33. 8. Kim HJ, Kim KS, Kim DI. 2012. Inhiitory effects of Lespedez cunet ethnol extrct on ultrviolet-induced photo ging. J Koren Soc Food Sci Nutr 41: 140-14. 9. Choi KM, Lee CW, Lee MY. 2008. Effect of ore minerls in the heling of full-thickness skin injury model of rt. J Environ Sci 17: 809-816. 10. Wldron DR, Zimmermn-Pope N. 2003. Superficil skin wounds. In Textook of Smll Animl Surgery. Elsevier Helth Science, Phildelphi, PA, USA. p 29-273. 11. Lee AY. 200. Allergic contct dermtitis due to sodium fusidte, n ingredient of fucidin ointment. Medicl Postgrdutes : 261-263. 12. Hn DO, Kim GH, Choi YB, Shim IS, Lee HJ, Lee YG, Kim JH, Chng GT, Hhm DH. 200. Heling effects of Astrgli rdix extrcts on experimentl open wounds in rts. Koren J Orientl Physiology & Pthology 19: 92-97. 13. Prk ST, Kim JW, Jeong SH, Seo YM. 2012. The effect of extrct from severl hers grown nturlly in nmwon province wound tretment. J Koren Biol Nurs Sci 14: 122-128. 14. Song JH. 2006. Simple evlution of the wound heling ctivity of crude extrct of root rk of Ulmus prvifoli. J Wonkwng Medi Sci 20: 313-318. 1. Mohideen S, Ilvrsn R, Hemlth S, Anith N, Ssikl E. 2003. Wound heling nd diuretic ctivities of Cnthium prviflorum lm. Nt Prod Sci 9: 102-104.
380 정희경 김길수 정유석 16. Min DH, Kim DK, Lim JP, Yng JH. 200. Trnsderml drug delivery & therpeutic effect of the preprtions of Lithospermi rdix nd Grdenie fructus extrcts on the urn & wound heling. J Kor Phrm Sci 3: 2-263. 17. Kder KN, Coyle CH. 2007. Rective oxygen nd nitrogen species: implictions for crdiovsculr device engineering. J Biomed Mter Res Prt B 83B: 138-144. 18. Kim HH, Prk GH, Prk KS, Lee JY, An BJ. 2010. Anti-oxidnt nd nti-inflmmtion ctivity of frctions from Aster glehni Fr. Schm. Kor J Microiol Biotechnol 38: 434-441. 19. Kim JM, Kim DJ, Kim TH, Kim HS, Choe M. 2010. Antioxidtive nd nti-inflmmtory effect of wter extrct from Perille semen in RBL-2H3 cells. Koren J Nutr 43: 367-373. 20. Sevnn BC, Sreekumr C, Bnsl GC, Ry D, Ro JR, Mishr AK. 2003. A rpid MTT colorimetric ssy to ssess the prolifertive index of two Indin strins of Theileri nnult. Vet Prsitol 113: 211-216. 21. Kim DH, Yi HS, Yun HJ, Ch CM, Prk SD. 2010. Anti-inflmmtory effect of methnol extrct of Keum-Ryung-J- Sn in mouse mcrophges. Kor J Herology 2: 89-98. 22. Hyshi Y, Tsuji S, Tsuji M, Nishid T, Ishii S, Iilim H, Nkmur T, Eguchi H, Miyoshi E, Hyshi N, Kwno S. 2008. Topicl implnttion of mesenchyml stem cells hs eneficil effects on heling of experimentl colitis in rts. J Phrmcol Exp Ther 326: 23-31. 23. Knighton DR, Hunt TK, Thkrl KK, Goodcon WH. 1982. Role of pltelets nd firin in the heling sequence. Ann Surg 196: 379-387. 24. Hunt TK, Knighton DR, Thkrl KK, Goodson WH 3rd, Andrews WS. 1984. Studies on inflmmtion nd wound heling: ngiogenesis nd collgen synthesis stimulted in vivo y resident nd ctivted wound mcrophges. Surgery 96: 48-4. 2. Eun JS. 2011. Effect of Lespedez cunet G. Don on the ctivity of murine immune cells. Koren J Orientl Physiology & Pthology 2: 837-842. 26. Thippeswmy T, Mcky JS, Quinn JP, Morris R. 2006. Nitric oxide, iologicl doule-fced jnus-is this good or d. Histol Histopthol 21: 44-48. 27. McMicking J, Xie QW, Nthn C. 1997. Nitric oxide nd mcrophge function. Annu Rev Immunol 1: 323-30. 28. Yoon JH, Moon HB. 1994. The effect of mcrophges on the wound heling delyed y immunosuppression. J Koren Surg Soc 46: 10-22. 29. Schffer MR, Tntry U, Gross SS, Wsserkrug HL, Brul A. 1996. Nitric oxide regultes wound heling. J Surg Res 63: 237-240. 30. Hn SM, Lee KG, Yeo JH, Kweon HY, Woo SO, Bek HJ, Prk KK. 2006. The effects of Bomyx mori silk protein for the expression of epithelil growth fctor in the wound heling process of the hirless mouse. Koren J Seric Sci 48: 6-60. 31. Kim JS, Hn SH, Jung BK, Prk SJ, Kim HW, Che H, Kwon YK, Kim BJ. 2010. Effects of Koren herl thing cndidtes on llergic contct or wound induced dermtitis mice model. Koren J Orientl Physiology & Pthology 24: 481-489. 32. Mrtin P. 1997. Wound Heling-iming for perfect skin regenertion. Science 276: 7-81. 33. Muller MJ, Hollyok MA, Moveni Z, Brown TLH, Herndon DN, Heggers JP. 2003. Retrdtion of wound heling y silver sulfdizine is reversed y Aloe ver nd nysttin. Burns 29: 834-836. 34. Diegelmnn RF, Evns MC. 2004. Wound heling: An overview of cute, firotic nd delyed heling. Front Biosci 9: 283-289. 3. Mcri L, Silverstein D, Clrk RAF. 2007. Growth fctor inding to the pericellulr mtrix nd its importnce in tissue engineering. Adv Drug Delivery Rev 9: 1366-1381. 36. Cornelissen LH. 2004. Which molecules of the initil phse of wound heling my e used s mrkers for erly detection of skin dmge. http://www.mte.tue.nl/mte/pdfs/4703.pdf. 37. Hong JH, Kim HS, Kim HR, Prk MK, Yoon CH, Lee SH, Kim HY, Prk SH. 200. Elevted serum levels of vsculr endothelil growth fctor in Behcet's syndrome. J Koren Rheum Assoc 12: 189-196. 38. Shh M, Revis D, Herrick S, Billie R, Thorgenirson S, Ferguson M, Roerts A. 1999. Role of elevted plsm trnsforming growth fctor-β1 levels in wound heling. Am J Pthol 14: 111-1124. 39. Crouvezier S, Powell B, Keir D, Yqoo P. 2001. The effects of phenolic components of te on the production of pro- nd nti-inflmmtory cytokines y humn leukocytes in vitro. Cytokine 13: 280-286. 40. Lee JH, Byun JA, Yeo SW, Kim JK, Prk YS. 2011. Role of interleukin-10 on mouse otitis medi model using lipopolyscchride. Koren J Otorhinolryngol-Hed Neck Surg 43-47. 41. Kpoor M, Howrd R, Hll I, Appleton I. 2004. Effects of epictechin gllte on wound heling nd scr formtion in full thickness incisionl wound heling model in rts. Am J Pthol 16: 299-307. 42. Rolfe KJ, Richrdson J, Vigor C, Irvine LM, Groelr AO, Linge C. 2007. A role for TGF-β1-induced cellulr responses during wound heling of the non-scrring erly humn fetus. J Invest Dermtol 127: 266-2667.