J Korean Soc Food Sci Nutr 한국식품영양과학회지 38(3), 287~291(9) DOI: 1.3746/jkfn.9.38.3.287 산수유에탄올추출물의생리활성 권승혁 1 양희선 1 김재용 2 박경욱 1 손미예 3 강갑석 4 심기환 5 서권일 1 1 순천대학교식품영양학과, 2 경북대학교식품공학과 3 경상대학교식품영양학과, 4 부산정보대학호텔조리학과 5 경상대학교대학원응용생명과학부 농업생명과학연구원 Biological Activities of Ethanol Extract from Corni fructus Seong-Hyuk Kwon 1, Hee-Sun Yang 1, Jae-Yong Kim 2, Kyoung-Wuk Park 1, Mi-Yae Shon 3, Kap-Suk Kang 4, Ki-Hwan Shim 5, and Kwon-Il Seo 1 1 Dept. of Food and Nutrition, Sunchon National University, Jeonnam 54-742, Korea 2 Dept. of Food Science and Technology, Kyungpook National University, Daegu 72-71, Korea 3 Dept. of Food and Nutrition, Gyeongsang National University, Gyeongnam 66-71, Korea 4 Dept. of Hotel Culinary Arts, Busan College of Information Technology, Busan 616-737, Korea 5 Division of Applied Sciences, Graduae School, Institute of Agricultured & Life Sciences, Gyeongsang National University, Gyeongnam 66-71, Korea Abstract In order to use Corni fructus as functional food materials, we investigated the biological activities of ethanol extracts from Corni fructus (EECF). The hydrogen-donating activity of EECF was increased in a dose dependent manner compared with untreated control, and the activities by EECF were 64 and 74% at 3 and 5 μg/ml concentration, respectively. The NO productions in the RAW264.7 marcrophage cells treated with EECF were increased in dose dependent manners. EECF significantly inhibited the growth of MCF-7 human breast cancer cells in dose and time dependent manners. EECF of 5 μg/ml concentration inhibited the proliferation by over 6% in the MCF-7 cells when treated for 72 hr. Also, the proliferations were increased in the MCF-7 cells cultured in the charcoal-treated FBS (cfbs) medium with environmental hormones such as bisphenol or 17βestradiol of.1 μm whereas the proliferations were decreased in the MCF-7 cells treated with the environmental hormones after treatment of EEFC for 72 hr. The results suggest that Corni fructus would be used as functional food materials. Key words: Corni fructus, antioxidative effect, immuno-activities, NO, environmental hormones, cytotoxic activity 서론최근국민소득증대, 생활패턴의서구화, 핵가족화등으로인해식습관이변하고있으며, 암, 뇌혈관질환및당뇨병등과만성퇴행성질환에의한사망률증가등으로인하여건강에대한관심이증가함에따라식품의 3차기능이강조된기능성식품의요구가증대되고있는실정이다 (1). 최근식품에관한연구는천연식품에서추출한천연소재에대한관심이증대되고있으며, 특히민간요법, 한방에서효능이입증된재료들의생리활성을연구하여기능성식품으로서개발등에응용하고있다 (1). 산수유 (Corni fructus) 는층층나무과에속하는산수유나무 (Cornus officinalis) 의과육으로, 가을에성숙한붉은색열매의씨를제거한건조한과육을산수유라하며 (2), 예로부 터우리나라를비롯하여중국과일본등에서중요한한약재로많이사용되어왔다 (3). 산수유는그맛이시고성질은따뜻하며, 다뇨증, 요통, 이명, 폐결핵등의치료제로사용되어왔으며, 그과실은자양, 강장, 음위, 이조에약효가있고, 간경화, 신경에좋고, 이뇨작용, 혈압강하작용, 항암및항균작용등의약리작용이있다고한방자료에기록되어있다 (4). 산수유의화학성분에관한연구는 gallic acid, malic acid, tartaric acid, ursolic acid, morroniside, loganin, sweroside 등과같은배당체, tellimagrandin 1,2, 1,2,3-tri-O-galloylβ-D-glucose, 1,2,6-tri-O-galloyl-β-D-glucose, 1,2,3,6- tetra-o-galloyl-β-d-glucose, gemin D, 탄닌성분으로 cornusin A-G, 2,3-di-O-galloyl-D-glucose, 1,7-di-Ogalloyl-D-sedoheptullose 등이분리보고되었다 (5,6). 산수유에대한연구로는산수유열매의렉틴성분 (2), 영양성분 Corresponding author. E-mail: seoki@sunchon.ac.kr Phone: 82-61-75-3655, Fax: 82-61-752-3657
288 권승혁 양희선 김재용 박경욱 손미예 강갑석 심기환 서권일 (7), 산수유종자의항당뇨효과 (8), 물추출물의항히스타민효과 (9), 부종억제효과 (1), 납에의한조직손상억제 (11), 정자운동성증가에미치는효과 (12), 항균효과 (13), 티로시나제저해작용 (14), 항산화 (15,16) 및항암효과 (17) 등이보고되고있으나, 아직은이에대한체계적인연구가미흡한실정으로산수유의유용성분을산업적으로이용하기위해서는더욱많은연구가진행되어야할것으로생각된다. 따라서본연구에서는산수유를기능성식품소재로사용하기위한근거를제시하기위해산수유에탄올추출물에대한항산화, 면역활성, 항암활성및환경호르몬에의한암세포증식억제효과를조사하였다. 재료및방법실험재료본실험에사용된산수유는 7년 1월에전남구례군에서수확하여씨를제거한건조과실을구입하여분쇄시켜 4 o C 냉동고에보관하였다. 산수유에탄올추출물제조및수율산수유 1 g 당 8% 에탄올 ml의비율로첨가하여 65 o C에서 3시간동안 3회열수추출하고여과하였다. 이여과액을회전식감압농축기 (EYELA, Rikakikai Co., Japan) 로감압 농축하여에탄올을완전히제거시킨후동결건조기를이용하여건조한후수율을계산하였다. 수소공여능측정시료에대한수소공여능은 a,a'-diphenyl-β-picrylhydrazine(dpph) 의환원성을이용하여 54 nm에서 UV/Visspectrophotometer로측정하였다 (18). 대조구로사용한 BHT 의농도는.1% 되게조제하였으며, 시료 1 ml과 5 1-4 M DPPH 용액 (DPPH 12 mg 1 ml 에탄올에완전히용해시킨후 1 ml의증류수를가한용액 ) 3 ml를혼합하고암실에서 3분반응한후흡광도를측정하였다. 대조구는시료대신 ethanol을첨가하였으며수소공여능을대조구에대한흡광도의감소비율로나타내었다. 대식세포주 (RAW264.7) 의 NO 생성능 RAW264.7세포를 96 well culture plate에 1 1 5 cells/ well로분주하였다. 세포를안정화시킨후산수유에탄올추출물을농도별로처리하여 48시간더배양하고각조건에따른 NO생성정도를측정하였다. 반응종료후 NO - 2 (nitrite) 의생성량을 Griess 반응을이용하여측정하였다 (19). 세포배양액과 Griess reagent를동량으로혼합하여 1분간반응시킨후 54 nm에서 microplate-elisa reader(titertek Multiscan Plus, Finland) 로흡광도를측정하여 NaNO 2 를 32 μm까지 2배씩희석하여얻은표준곡선과비교하여 NO 생성량을계산하였다. Charcoal treatment FBS 제조 (FBS 내의 steroid hormones 제거 ) Serum의 estrogen 활성을최소화하기위해 FBS에 5% charcoal 을처리하여 55 o C에서 3분동안교반한후 3, rpm, 4 o C, 분원심분리하여상등액을취하였고, 이를 2회반복하여얻은상등액을.45 μl microfilter로여과하여 cfbs(charcoal treatment FBS) 를얻은후 - o C에서보관하며실험에사용하였다 (). 암세포증식억제효과본실험에사용한인체유방암세포주인 MCF-7(breast carcinoma) 를한국세포주은행 (KCLB) 에서분양받아 1% FBS(fetal bovine serum) 를첨가한 RPMI164 배지를첨가하여 37 o C, 5% CO 2 incubator에서계대배양하면서실험에사용하였다. Monolayer로자란유방암세포를.25% trypsin-edta 를처리하여 single 세포로만든후최종세포농도가 1 1 5 cells/ml로되도록희석하여 24 well plate에각 well 당분주한다음 37 o C, 5% CO 2 incubator에서 24시간동안배양한후, 산수유에탄올추출물을농도별로첨가하여 24, 48 및 72 시간동안배양한후세포증식을 SRB(sulforhodamine) 방법 (21) 및세포를 single로만들어혈구계산반을이용하여세포를 counting 하였다. 한편환경호르몬에의한암세포증식억제효과는 MCF-7 암세포주를최종농도가 1 1 4 cell/ml 되도록희석하여 96 well plate에분주하고 37 o C, 5% CO 2 incubator에서 24시간배양하였다. 상층을제거한후 1% cfbs phenol free RPMI164 배지, 환경호르몬과산수유에탄올추출물을농도별처리하여일정시간동안배양한후 SRB 방법으로그억제능을측정하였다. 암세포형태학적관찰대조구와실험군의세포모양변화를관찰하기위해암세포증식억제능을측정하기전에광학현미경으로세포의형태학적변화를관찰하였다. 통계처리실험결과는평균과표준편차로표시하였으며, 각실험군을대조군에대한백분율로나타내었다. 대조군과실험군간의통계적유의성에대한검증은 Student's t-test를이용하여, p-value가.5 미만일때통계적으로유의성이있다고판단하였다. 결과및고찰산수유에탄올추출물의수소공여능산수유 (1 g) 을 8% 에탄올로추출및동결건조한후고형분의함량은측정한결과 61.8% 으로나타났으며, 에탄올추출물의항산화활성을측정한결과는 Fig. 1과같다. 산수
산수유에탄올추출물의생리활성 289 1 Hydrogen donating activity (%) 8 6 4 NO 2- concentration (μm). 15 1 5.1% BHT 1 3 5 Fig. 1. Hydrogen donating activity of 8% ethanol extracts from Corni fructus. Data values were expressed as mean±sd of triplicate determinations. 유에탄올추출물을 1, 3 및 5 μg/ml의농도로수소공여능을측정한결과농도의존적으로항산화효과를나타냈으며, 5 μg/ml 농도의경우합성항산화제보다는효과가낮았지만, 73% 이상의항산화활성을나타내어그효과를확인할수있었다. Seo 등 (4) 은산수유에탄올추출물 1 μl 첨가시 68.7%, μl 첨가시 92.3% 의높은수소공여능이있음을보고하였으며, Kim(16) 의연구에서는산수유에탄올추출물 1 mg/kg 투여로지질의불포화지방산들이산화적분해를일으키는 lipid peroxide 반응에서 MDA와세포내의자유라디칼을제거하는 glutathione 함량이유의적인감소와증가를나타내었다. 따라서, 본연구결과에서도산수유에탄올추출물이항산화활성을나타내는것으로미루어볼때, 항산화작용을일으키는물질들이이추출물에함유되어있음을확인할수있었다. 산수유에탄올추출물의 NO 생성능 대식세포는박테리와같은항원이침입하였을때, 일차적인면역반응을담당하는세포로서, 이러한박테리아를사멸시키기위해대식세포는다양한물질을분비하며, 이중에서일산화질소 (mitric oxide) 가대표적인물질로알려져있다 (22). 따라서산수유에탄올추출물이대식세포의일산화질소생산에어떤영향을미치는지알아보기위하여대식세포주인 RAW264.7에산수유에탄올추출물을농도별로처리하여 48시간배양한후, 배양액중의대식세포가생산한일산화질소로부터산화된 NO - 2 를농도를측정한결과는 Fig. 2 와같다. 즉산수유에탄올추출물을 1, 1 및 5 μg/ml 농도로처리한실험군에서는 NO 생성량이각각 9.65, 11.44 및 12.34 μm로나타났다. Lee 등 (23) 은산수유에탄올추출물이비장및흉선림프구의증식을촉진시켰으며, 복강대식세포의탐식능및 NO 생성을유발하여면역조절작용이있다고보고하고있다. 따라서산수유에는면역능을증가시킬수있는성분들이함유되어있다는것을추측할수있으나, 앞으로산수유에 1 1 5 Fig. 2. Effects of 8% ethanol extracts from Corni fructus on the production of nitric oxide (NO) in macrophage cells (RAW264.7). Data values were expressed as mean±sd of triplicate determinations. Significant differences were compared with control at p<.5 by Student t-test. 탄올추출물에함유되어있는물질을분리하여보다구체적인실험을수행해야할것으로사료된다. 산수유에탄올추출물의유방암세포증식억제효과 산수유에탄올추출물을다양한농도로유방암세포주 (MCF-7) 에처리하여 24, 48, 72시간동안배양한후암세포증식억제능을측정한결과는 Fig. 3, 4와같다. 즉산수유에탄올추출물은대조구에비하여시간및농도에의존적으로암세포의증식을억제하였으며, SRB 방법으로암세포성장억제효과를측정한결과산수유에탄올추출물을처리후 24, 48 및 72시간배양시 5 μg/ml 농도에서유방암세포의성장억제율이각각 44, 52 및 63% 으로나타났다 (Fig. 3). 또한 trypan blue 염색을통한결과에서도시간및농도에의존하여세포의증식을억제하는것을확인할수있었다 (Fig. 4). Kim 등 (17) 의연구에서산수유에탄올추출물은폐암세포주 (A549) 와유방암세포주 (MCF-7) 의증식을유의하게억제시켰으며, 암세포의세포주기를변화시키는등항암에효과가있다고보고하였다. 또한 Chung 등 (2) 의연구에서도산수유의헥산추출물이다양한인체암세포주 (MDA- Survival rate (%). 1 1 8 6 4 1 3 5 h 24 h 48 h 72 h Time Fig. 3. Cell growth inhibition effects in the MCF-7 cells treated with 8% ethanol extracts from Corni fructus for 24, 48 and 72 h by SRB assay. Data values were expressed as mean±sd of triplicate determinations.
29 권승혁 양희선 김재용 박경욱 손미예 강갑석 심기환 서권일 Cell density ( 1 4 ). 15 1 5 24 h 48 h 72 h 24 h 48 h 72 h 1 3 5 Fig. 4. Cell growth inhibition effects by trypan blue dye in the MCF-7 cells treated with 8% ethanol extracts from Corni fructus for 24, 48, and 72 h. Results were expressed as the percentage of control. The bars and line are means of the cell densities and cell deaths, respectively. Data values were expressed as mean±sd of triplicate determinations. Significant differences were compared with the control at p<.5 by Student t-test. MB-231, B16, A549) 에서암세포성장을억제한다고보고하였다. 따라서, 본연구결과에서도산수유에탄올추출물이유방암세포의성장을억제함을확인할수있었으며, 이는산수유내에함유된인체암세포억제효능을가진 ursolic acid와같은항암물질등에의해기인되는것으로생각된다 (17). 1 8 6 4 Dead cells (%). 암세포의형태학적관찰부착형의세포들은다양한종류의외부자극에의해세포사멸이유도되어지면세포가축소되어지고크기와수가줄면서배양 plate 바닥으로부터분리되어부유하게된다. 산수유에탄올추출물이 MCF-7 세포의증식을억제하는지를알아보기위하여산수유에탄올추출물을 3 및 5 μg/ml 농도로처리하고 24시간배양한후세포의형태학적변화를광학현미경으로대조군과비교하여관찰한결과는 Fig. 5와같다. 대조구세포는세포막을유지하며 plate의기벽에부착되어있었으나산수유에탄올추출물을처리한실험군은사멸한세포가배양 plat에서떨어져배지에부유하는것을관찰할수있었고, 대조구에비하여세포의밀도감소현상과세포모양변화가관찰되어산수유에탄올추출물이 MCF-7 세포의증식을저해함을확인할수있었다. 유방암세포에서산수유에탄올추출물의환경호르몬방어효과산수유에탄올추출물이환경호르몬에의해자극되어진에스트로겐수용체를가지는유방암세포주 (MCF-7) 의증식을억제할수있는지의여부를확인한결과는 Fig. 6과같다. 즉 bisphenol A와 17β-estradiol을산수유에탄올추출물을처리하기전에.1 μm의농도로처리하여암세포의증식을유도한후산수유에탄올추출물을농도별로처리하 Fig. 5. Photomicrographs ( ) of the MCF-7 treated with 8% ethanol extracts from Corni fructus (EECF) for 24 h. A:, B: Treated with 3 μg/ml EECF, C: Treated with 5 μg/ml EECF. Absorbance (54 nm). 2. 1.5 1..5 (A) Absorbance (54 nm). 2. 1.5 1..5 (B). Bisphenol A 1 3 5 Bisphenol A + extracts. 17β-estradiol 17β-estradiol + extracts 1 3 5 17β-estradiol+extracts Fig. 6. Anti-proliferation effects of 8% ethanol extracts from Corni fructus in the MCF-7 cells treated with bisphenol A and 17β-estradiol (.1 μm) for 72 h. Data values were expressed as mean±sd of triplicate determinations. Significant differences were compared with control at p<.5 by Student t-test. (A) Bisphenol A, (B) 17β-estradiol.
산수유에탄올추출물의생리활성 291 여 72시간후에그활성을측정하였다. Bisphenol A와 17βestradiol에의해유방암세포는대조구에비하여 2배정도의증식을보였으며, 산수유에탄올추출물이첨가된실험군에서는앞서유방암세포주에대한산수유추출물억제실험의결과와마찬가지로농도에의존적으로 bisphenol A와 17β -estradiol에의해유도되어진암세포의증식을억제하였다. Han 등 (11) 은산수유에탄올추출물을투여한실험군이대조군에비하여납과같은중금속을해독하는효과가있다고보고한바도있다. 요 산수유를기능성식품소재로서이용하기위하여산수유에탄올추출물의생리활성을조사하였다. 산수유에탄올추출물의수소공여능은농도의존적으로증가되었으며, 3 및 5 μg/ml 농도에서각각 64, 73% 의활성을나타내었다. 대식세포 RAW264.7에산수유에탄올추출물을 1, 3 및 5 μg/ml의농도로처리하였을때산수유추출물은농도의존적으로 NO(nitric oxide) 의생성을유도하였다. 또한산수유에탄올추출물을유방암세포에다양한농도로처리하여 24, 48, 72시간반응시킨결과농도및시간에의존적으로유방암세포의증식을억제하였으며, 5 μg/ml 농도로 72 시간처리시 6% 이상의높은증식억제율을보여주었다. 또한, MCF-7 유방암세포에 bisphenol과 17β-estradiol과같은환경호르몬을.1 μm의농도로처리하였을때세포의증식이유도되었으며, 이세포에산수유에탄올추출물을농도별로 72시간처리하였을때유방암세포의증식이억제되었다. 따라서본연구결과는산수유의기능성식품소재로서의활용가능성을시사한다. 약 감사의글 본논문은 6년도순천대학교친환경바이오산업전문인력양성사업단산학공동연구과제연구비지원으로수행된연구결과의일부이며, 그지원에감사드립니다. 문 1. Oh HS, Kim JH. 6. Development of functional soy-based stew sauce including hot water extracts of Cornus officinalis Sieb. et Z. Korean J Food Culture 21: 55-558. 2. Chung SR, Jeune KH, Park SY, Jang SJ. 1993. Toxicity lectins constituents from the seed of Cornus officinalis. Korean J Pharmacogn 24: 177-182. 3. Lee JY. 1981. Iridoid glycosides of Cornus officinalis. MS Thesis. Seoul National University. 4. Seo KI, Lee SW, Yang KH. 1999. Antimicrobial and antioxidative activities of Corni fructus extracts. Korean J 헌 Postharvest Sci Technol 6: 99-13. 5. Toheu E, Chiro TH. 1973. Constituents of Cornus officinalis. Yakugaku Zasshi 93: 3-36. 6. Guilian T, Zhang T, Yang F, Ito Y.. Separation of gallic acid from Cornus officinalis Sieb. et Zucc by high-speed counter-current chromatography. J Chromatogr A 886: 39-312. 7. Kim YD, Kim HK, Kim KJ. 3. Analysis of nutritional components of Corni fructus. J Korean Soc Food Sci Nutr 32: 785-789. 8. Park YK, Whang WK, Kim HI. 1995. The antidiabetic effects of extracts from Cornus officinalis seed. Chung-Ang J Pharm Sci 9: 5-11. 9. Seo YB, Kil GJ, Lee YK, Lee YC. 2. Study on the effects of Corni fructus about the anti-allergic action. Korean J Herboogy 1: 1-17. 1. Won DH, Cho JH, Kim HS, Ko JH, Lee J, Park SA, Lee HJ, Yook CS, Kim IH, Won BP. 1996. Studies on the analysis of Corni fructus and its preparation. The Annual Report KFDA 1: 197-1. 11. Han SH, Shin MK, Lee SB. 3. Effects of extracts of shanshuyu (Cornus officinalis Sieb). Korean J Food Culture 18: 544-55. 12. Jeng H, Wu CM, Su SJ, Chang WC. 1997. A substance isolated from Cornus officinalis enhances the motility of human sperm. Am J Chinese Med 25: 31-36. 13. Kim YD, Kim HK, Kim KJ. 3. Antimicrobial activity of solvent fraction from Cornus officinalis. J Korean Soc Food Sci Nutr 32: 829-832. 14. Fukushima M, Kimura S. 1989. Studies on cosmetic ingredients from crude drugs (I). Shoyakugaku Zasshi 43: 142-147. 15. Kim YE, Lee YC, Kim HK, Kim CJ. 1997. Antioxidative effect of ethanol fraction for several Korean medicinal plant hot water extracts. Korean J Food Nutr 2: 141-144. 16. Kim OK. 5. Antidiabetic and antioxidative effects of Corni fructus in streptozotocin-induced diabetic rats. Korean J Oil Chem Soc 2: 157-167. 17. Kim BH, Park KU, Kim JY, Jeong IY, Yang GH, Cho YS, Lee ST, Seo KI. 4. Purification and characterization of anticarcinogenic compound from Corni fructus. Korean J Food Sci Thechnol 36: 11-17. 18. Biois MS. 1958. Antioxidant determination by the use of a stable free radical. Nature 181: 1199-1. 19. Kondo Y, Takano F. 1994. Nitric oxide production in mouse peritoneal macrophage enhanced with glycyrrhizin. Biol Pharm Bull 17: 759-761.. Yamada K, Chujo H, Yamasaki M, Nou S, Koyanagi N. 3. Effect of conjugated linoleic acid isomers on growth factor-induced proliferation of human breast cancer cells. Cancer Letters 2: 81-87. 21. Skehan P, Storeng S, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Keeney S, Boyd MR. 199. New colorimetric cytotoxicity assay for anticancerdrug screening. J Nat Cancer Inst 82: 117-1112. 22. Kang HI, Kim JY, Moon KD, Seo KI, Cho YS, Lee SD, Yee ST. 4. Effect of the crude polysaccharide of Pleurotus eryngii on the activation of immune cells. J Korean Soc Food Sci Nutr 33: 192-197. 23. Lee WB, Jung HS, Kwon J, Oh CH, Lee KG. 2. Immunoregulatory action of Cornus officinalis Sieb. et Zucc. Korean J Orient Physiol Pathol 16: 267-271. (8 년 12 월 29 일접수 ; 9 년 1 월 29 일채택 )