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1 J Korean Soc Food Sci Nutr 한국식품영양과학회지 44(11), 1637~1644(2015) 함양에서재배된여주의추출조건에따른생리활성 문정한 1,5 최동원 1 김성은 1 서문지훈 1 홍수영 1 김현규 1 조계만 2 송진 3 강상수 4 김강화 5 권오경 1 1 콜마비앤에이치 ( 주 ) 신소재식품연구소, 2 경남과학기술대학교식품과학부 3 농촌진흥청기능성식품과, 4 경상대학교해부학교실, 5 전남대학교식품영양과학부 Comparison of Biological Activities of Ethanol Extracts of Unripe Fruit of Bitter Melon (Momordica charantia L.) Cultivated in Hamyang, Korea Jeong-Han Moon 1,5, Dong-Won Choi 1, Seong-Eun Kim 1, Ji-Hoon Seomoon 1, Su-Young Hong 1, Hyun-Kyu Kim 1, Kye-Man Cho 2, Jin Song 3, Sang-Soo Kang 4, Kang-Hwa Kim 5, and Oh-Kyung Kwon 1 1 Nutraceutical Food R&D Center, KolmarBNH Co., Ltd. 2 Department of Food Science, Gyeongnam National University of Science and Technology 3 Functional Food & Nutrition Division, RDA 4 Department of Anatomy, Gyeongsang National University 5 Division of Food and Nutrition, Chonnam National University ABSTRACT To develop health-functional food ingredients for improvement of diabetes, we examined biological activities, including total contents of polyphenols and flavonoids, antioxidant activity, and inhibition of α-glucosidase and pancreatic lipase activities of ethanol extracts from unripe fruit of Momordica charantia L. cultivated in Hamyang, Korea. Ethanol extracts were tested using different ethanol concentrations (0%, 30%, 50%, and 70%) and temperatures (50 C and 70 C). Experimental results showed that total polyphenol and flavonoid contents were achieved concentrations of 7.77 mg/g (tannic acid), 7.66 mg/g (gallic acid), and mg/g (rutin). The hydroxyl radical scavenging, ABTS cation radical scavenging, and FRAP activities were 82.19%, 42.82%, and 0.19 under the same conditions, respectively. The highest inhibitory activities of α-glucosidase and pancreatic lipase were achieved in the 70% ethanol extract at 70 C. These results will be useful for development of health functional foods for anti-diabetes using 70% ethanol extract from unripe fruit of bitter melon Key words: Momordica charantia L., ethanol extract, α-glucosidase, pancreatic lipase, antioxidant 서 우리나라는서구화된식습관과육류섭취의과다로매년비만과당뇨병을비롯한성인병유병률이증가하고있다. 당뇨병 (diabetes mellitus) 은인슐린작용의문제로정상적인혈당조절에이상을초래하는만성소모성질환으로대사증후군의주요한요인이다. 국민건강영양조사 (2013년) 에의하면우리나라의만 30세이상성인의당뇨병유병률은 11.0% 로 2012년과비교해 2.0% 증가하였다. 특히 65세이상에서는 21.7%( 남자 22.3%, 여자 21.3%) 로연령이증가할수록높게나타났다. 당뇨병치료의목적은혈당을일정하게유지하여합병증을예방하는것이며, 실제로당뇨환자들 Received 3 July 2015; Accepted 29 October 2015 Corresponding author: Oh-Kyung Kwon, Nutraceutical Food R&D Center, KolmarBNH Co., Ltd., Sejong 30003, Korea kwonok@kolmarbnh.co.kr, Phone: 론 의 30% 이상이합병증이유발되는것으로보고되고있다 (1). 최근건강에대한관심고조로천연의생리활성물질을이용한다양한식품의활용도가높아짐에따라동남아시아 ( 필리핀, 인도네시아, 말레이시아등 ), 일본, 미국등지에서도오래전부터당뇨개선건강음료를비롯하여차, 환, 분말등의가공제품들이출시되고있으며, 우리나라에서도함양, 정읍, 철원, 제주도등전국각지에서여주를재배 가공 판매하여농가의부가가치를높이고있다 (2,3). 그러나천연물의생리활성을이용한건강기능식품을개발하기위해서는우선적으로천연물의제조표준화가우선적으로확립되어야하고유효성분에대한기능성, 안전성및안정성등의과학적입증이필요하다. 여주 (Momordica charantia L., MC) 는박과 (Cucubitaceae) 의한해살이식물로 Bitter Melon, Balsam Apple, Bitter Gourd, Balsam Pear Bitter 등의이명으로도알려져있다. 열대식물인여주는알칼로이드계열의성분들에의해

2 1638 문정한 최동원 김성은 서문지훈 홍수영 김현규 조계만 송진 강상수 김강화 권오경 쓴맛을가지고있으며아시아, 동아프리카, 인도, 남아메리카등지에서널리재배되고있고, 건강기능식품과당뇨예방목적의건강식품에도이용되고있다 (4,5). 여주는비타민 A, B 1, B 2, C, E, 엽산뿐만아니라칼슘, 칼륨, 아연, 마그네슘, 인, 철등의미네랄성분이풍부하며, 특히항산화성분인 phenols를비롯하여 carotenoids, flavonoids, isoflavones, terpenes, anthroquinones, essential oil, saponin, triterpenes, alkaloids, momordicine 등과같은생리활성물질들이풍부하다고알려져있다. 항산화성분인 phenolic 화합물은인체의유해한활성산소의발생을억제해체지방을분해시키는효소작용을돕고신진대사를활성화시켜준다. 또한여주는항당뇨, 항암, 항염증등의효과가있으며그중에서도항산화와항당뇨에대한연구가가장많이이루어져있다. 최근에는항바이러스, 인지능력개선효과, 피부의상처치유효과등다양한생리활성기능을가지고있는것으로보고되고있다 (1,5-12). 여주의생리활성성분의추출법에대한연구는대부분메탄올등의유기용매와물을사용하는방법이이용되어왔으며사포닌의최적화된추출방법으로 50~70% 에탄올로추출한보고도있었다 (13). 이러한연구결과들은여주의생리활성물질의효용가치를이용한다양한식품개발과의약품으로의응용가능성을시사한다. 따라서본연구에서는경남함양지역에서재배되고있는여주로부터추출 / 농축제조공정을표준화하고이를이용하여항산화활성과탄수화물및지방흡수억제효과를조사하여혈당개선에최적효능을가지는건강기능식품개발을위한기초자료로활용하고자한다. 재료및방법여주원료확보본실험에사용한여주원료는경남함양에소재한함양영농조합법인천령식품지점으로부터확보하였고, 경남함양군에서 2014년도 4월부터대량재배된 Nakanokoya 품종의여주미숙과열매를 6월중순에수확하여물로세척, 절단가공한후그절편을 40~50 C 조건으로수분함량이 5~7% 가되도록열풍건조 (TJDL-15WA, Joongang Precision Co., Ltd., Daegu, Korea) 하여사용하였다. 여주추출물제조여주의추출은대량생산공정을고려하여다음과같은조건으로수행하였다. 추출용매는정제수 (distilled water), 30, 50, 70% 주정 (ethanol) 을사용하였고추출온도는 50, 70 C 조건에서수행하였다. 추출방법은칭량된여주의 15 배수의용매로 4시간동안 1차추출하고추출물을얻고남은잔여물을다시 10배수의용매로 2시간동안 2차추출하였다. 농축은감압농축기 (Laborata 20 control, Heidolph, Schwabach, Germany) 를사용하여 50~55 C로농축하였 다. 농축된여주추출물을여과한후수분측정기 (MA50C, Satorius Co., Goettingen, Germany) 를사용하여각각의추출물에대한추출수율을측정한다음 -75 C, 5 mtorr 조건으로동결건조 (MCFD8512, Iishinbiobase Co., Dongducheon, Korea) 하였다. 총폴리페놀및총플라보노이드함량측정여주추출시료의총폴리페놀함량은 Folin과 Denis(14) 의방법을변형하여측정하였다. 시료 20 μl에 Folin-Denis' reagent(sigma-aldrich Co., St. Louis, MO, USA) 100 μl를첨가한후 40 C에서 1분간반응시켰다. 반응후 7.5% Na 2CO 3 용액 80 μl를첨가하여 40 C에서 15분간반응시킨다음 765 nm에서흡광도 (Multiskan GO, Thermo Fisher Scientific Inc., Waltham, MA, USA) 를측정하였다. 표준용액은 gallic acid(sigma-aldrich Co.) 와 tannic acid(sigma- Aldrich Co.) 를사용하였으며, 총폴리페놀함량은시료 1 g 중의 mg gallic acid equivalent(gae, dry basis), mg tannic acid equivalent(tae, dry basis) 로나타내었다. 여주추출시료의총플라보노이드함량은 Eum 등 (15) 의 diethylene glycol을이용한방법을변형하여측정하였다. 시료 20 μl에 diethylene glycol(samchun Co., Pyeongtaek, Korea) 200 μl와 1 N NaOH 20 μl를첨가한후, 37 C에서 1시간동안반응시켰다. 반응후 420 nm에서흡광도를측정하였으며표준용액은 rutin(sigma-aldrich Co.) 을사용하였다. 총플라보노이드함량은시료 1 g 중 mg rutin equivalent(re, dry basis) 로나타내었다. ABTS radical 소거활성측정 ABTS cation radical scavenging activity는 Re 등 (16) 의방법을변형하여측정하였다. 이시험법은시료에의해 ABTS cation radical이소거되어시약의색이옅어지는정도를측정하는방법이다. 시료 20 μl에 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma-Aldrich Co.) 시약 180 μl를혼합한후, 실온에서 7분간반응시켰다. 반응후 734 nm에서흡광도를측정하였다. 이때양성대조군으로 ascorbic acid(sigma- Aldrich Co.) 를사용하였다. ABTS cation radical scavenging activity (%) = Blank-Test -100 Blank Blank: blank의흡광도 Test: 시료의흡광도 Hydroxy radical 소거활성측정 Hydroxy radical scavenging activity 측정은 Ji 등 (17) 의방법을변형하여측정하였다. 10 mm FeSO 4 7H 2Oethylenediaminetetra acetic acid disodium salt(edta, Biosesang Co., Seongnam, Korea) 에 10 mm의 2-deox-

3 여주의추출조건에따른생리활성 1639 yribose와농도별로희석한상층액을혼합한후, 10 mm의과산화수소 (H 2O 2) 를첨가하여 Fenton 반응에따라 37 C에서 4시간동안반응시켰다. 그리고 2.8% trichloroacetic acid(tca, Acros Co., Pittsburgh, PA, USA) 1.0 ml와 1.0% thiobarbituric acid(tba) 1.0 ml를반응혼합액에첨가하여끓는물에서 10분간반응시킨후냉각시켜 540 nm에서흡광도를측정하였다. 이때양성대조군으로 ascorbic acid(sigma-aldrich Co.) 를사용하였다. FRAP 활성측정여주추출시료의 ferric reducing antioxidant power (FRAP) 활성은 Benzie와 Strain(18) 의방법을변형하여측정하였다. 실험에사용된 FRAP 용액은 0.3 M sodium acetate buffer(ph 3.6, Daejung Chemical Co., Siheung, Korea) : 0.01 M 2,4,6-tris(2-pyridyl)-s-triazine(TPTZ, Sigma-Aldrich Co.) : 0.02 M FeCl 3 6H 2O(Daejung Chemical Co.) 를 10:1:1 비율로각각혼합하여제조하였다. FRAP 기질용액 950 μl에추출물시료 50 μl를첨가하여 37 C에서 15분간반응시킨후 593 nm에서흡광도를측정하였고양성대조군으로 ascorbic acid(sigma-aldrich Co.) 를사용하였다. α-glucosidase 저해활성측정여주추출시료의 α-glucosidase 저해활성은 Kim 등 (19) 의방법을변형하여수행하였다. 0.1 M phosphate buffer(ph 6.9, Sigma-Aldrich Co.) 50 μl에시료 20 μl, 1 unit/ml α-glucosidase from Saccharomyces cerevisiae(sigma-aldrich Co.) 10 μl를혼합하고 37 C에서 30분간반응시킨후, 1 mm p-nitrophenyl α-glucopyranoside(pnpg, Sigma-Aldrich Co.) 20 μl를첨가하여 37 C에서 15분간반응시켰다. 0.1 M Na 2CO 3(Daejung Chemical Co.) 50 μl를첨가하여반응을정지시킨후 405 nm에서흡광도를측정하였고양성대조군은 α-glucosidase 억제제인 acarbose(sigma-aldrich Co.) 를사용하였다. α-glucosidase inhibition activity (%)= Negative control-(test-blank) 100 Negative control-positive control Test: buffer+enzyme+sample+substrate+stop solution Blank: buffer+enzyme+sample+stop solution Negative control: buffer+enzyme+substrate+stop solution Positive control: buffer+substrate+stop solution Pancreatic lipase 저해활성측정지방흡수억제활성실험은 Kwon 등 (20) 의방법을변형하여수행하였다. 효소원은 enzyme buffer(10 mm MOPS, 1 mm EDTA, ph 6.8) 에 porcine pancreatic lipase (Sigma-Aldrich Co.) 를 0.25 g/100 ml의농도로 4 C에서용해한후 4,000 rpm으로원심분리하여상층액을얻었다. 기질로 p-nitrophenyl butyrate(p-npb, Sigma-Aldrich Co.) 를 10 mm이되게 dimethylformamide에용해하여사용하였다. Tris buffer(100 mm Tris-HCl, 5 mm CaCl 2, ph 7.0) 170 μl에시료 20 μl를첨가한후 enzyme buffer 6 μl와혼합하였다. 효소와시료를먼저 37 C에서 15분동안반응시킨다음기질을 4 μl 첨가하여 37 C에서 30분동안반응시켰다. ELISA reader(multiskan GO, Thermo Fisher Scientific Inc.) 를이용하여 405 nm에서흡광도를측정하였으며 pancreatic lipase 저해활성은시료용액의첨가군과무첨가군의흡광도감소율로나타내었다. 양성대조군은위와췌장의지방분해효소억제제인 orlistat(sigma-aldrich Co.) 을사용하였다. Pancreatic lipase inhibition activity (%)= Negative control-(test-blank) 100 Negative control-positive control Test: buffer+enzyme+sample+substrate+stop solution Blank: buffer+enzyme+sample+stop solution Negative control: buffer+enzyme+substrate+stop solution Positive control: buffer+substrate+stop solution 통계분석각실험결과는 GraphPad Prism version 5.0(GraphPad Software, San Diego, CA, USA) 을이용하여평균 ± 표준편차로나타내었으며, ANOVA의 one way Dunnet's multiple comparison test에의해각실험군간의유의성을검증하였다. 결과및고찰여주주정추출조건별추출수율, 총폴리페놀및총플라보노이드함량여주의주정추출조건별추출수율과각시료들의총폴리페놀및총플라보노이드함량값은 Fig. 1에나타내었다. 추출온도에따른추출수율은 50 C보다 70 C에서높게측정되었고, 주정농도별로비교해보았을때주정농도 30% 에서가장높은수율을나타냈다. 추출온도와주정농도를고려했을때 70 C, 30% 주정농도에서 39.20% 의가장높은수율을나타냈고, 50 C, 70% 주정농도에서 % 의가장낮은추출수율을나타냈다 (Fig. 1A). 식물계에함유되어있는 2차대사산물인폴리페놀과그의일종인플라보노이드는생체내에서항산화, 노화억제, 항암효과, 혈압강하등다양한생리기능을가지는것으로

1640 문정한 최동원 김성은 서문지훈 홍수영 김현규 조계만 송진 강상수 김강화 권오경 A B C D Fig. 1.

Levels of significant between each water extracts and ethanolic extracts were determined using one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparison test.

a, water extract on 50 C; b, 30% ethanol extract on 50 C; c, 50% ethanol extract on 50 C; d, 70% ethanol extract on 50 C; e, water extract on 70 C; f, 30% ethanol extract on 70 C; g, 50% ethanol

4 1640 문정한 최동원 김성은 서문지훈 홍수영 김현규 조계만 송진 강상수 김강화 권오경 A B C D Fig. 1. Extraction yield (A) and content of total polyphenol (B and C) and flavonoid (D) according to ethanol extraction conditions of Momordica charantia L. Values are mean±sd. Levels of significant between each water extracts and ethanolic extracts were determined using one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparison test. For significant differences: *** implies P 0.001, ** implies P 0.01, * implies P 0.05, ns implies no significant difference. a, water extract on 50 C; b, 30% ethanol extract on 50 C; c, 50% ethanol extract on 50 C; d, 70% ethanol extract on 50 C; e, water extract on 70 C; f, 30% ethanol extract on 70 C; g, 50% ethanol extract on 70 C; h, 70% ethanol extract on 70 C. 보고되고있으며천연생리활성물질로서의활용도가높아지고있다 (21). 총폴리페놀함량은시료 1 g 중의 mg gallic acid equivalent(gae, dry basis), mg tannic acid equivalent(tae, dry basis) 로나타내었다. 그결과총폴리페놀함량은여주추출조건중 50 C의추출조건에서는주정농도영향을크게받지않았다. 하지만 70 C 추출조건에서는주정농도가증가할수록총폴리페놀함량이높게측정되었고 70% 주정에서 gallic acid는 7.66 mg/g extract, tannic acid는 7.77 mg/g extract로가장높게측정되었다 (Fig. 1B, 1C). 총플라보노이드함량은 50 C, 70 C 추출조건모두주정농도가높아질수록함량이농도의존적으로증가되었다. 총플라보노이드함량은 70 C, 70% 주정추출조건에서 rutin을표준물질로하여 mg/g extract로가장높게나타났다 (Fig. 1D). Boo 등 (22) 은 80% 주정, 40 C 온도, 6시간 2회반복추출조건에서여주일본품종인 Nikko, Peacock, Dragon, Erabu의경우총폴리페놀함량이 13.20~16.39 μg/ml로 Nakanokoya 품종보다낮은폴리페놀함량을나타냈으며, 총플라보노이드의경우도 2.39~ 4.38 μg/ml의수준으로 Nakanokoya 품종보다낮게나타났다. 본연구에사용된여주는현재경남함양지역에서대대적으로재배를하고있으며국내함양에서생육조건이우수하고비타민 C 함량이높은 Nakanokoya 단일품종을 사용하였는데본실험결과에서폴리페놀함량이타품종보다높게나타났다. 또한 Lee 등 (23) 과 Ahn 등 (21) 에따르면건조여주의총폴리페놀함량은 mg GAE/g extract, 총플라보노이드함량이 9.79 mg rutin/g extract로측정되었다고보고되어총폴리페놀함량차이가있지만플라보노이드함량은본연구와유사한결과를나타냈다. 이는추출조건에따른폴리페놀및플라보노이드함량의차이인것으로판단된다. 또한 Kim 등 (24) 의연구보고에따르면사과 pomace의용매추출시식물의총페놀성화합물함량은물이나단일용매보다 70% 주정으로추출시더우수한효율을보이고추출온도가높아질수록총폴리페놀함량이증가한결과는본연구에서추출온도와주정농도가높아질수록총폴리페놀, 총플라보노이드함량도증가한결과와도일치하였다. 여주주정추출조건별항산화활성여주주정추출조건별항산화활성은 ABTS cation radical 소거활성, hydroxy radical 소거활성, FRAP 활성을측정한결과모두 70 C, 70% 주정추출조건에서가장높게나타났다. 양성대조군인 ascorbic acid의활성은시료의농도보다낮은농도 (100 μg/ml) 에서 100% 이상의활성을나타냈으며 ABTS cation radical 소거활성은 1 mg/ml의

여주의추출조건에따른생리활성 1641 Fig. 2. ABTS cation radical scavenging activities according to ethanol extraction conditions of Momordica charantia L. Values are mean±sd.

5 여주의추출조건에따른생리활성 1641 Fig. 2. ABTS cation radical scavenging activities according to ethanol extraction conditions of Momordica charantia L. Values are mean±sd. Levels of significant between each water extracts and ethanolic extracts were determined using one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparison test. For significant differences: *** implies P 0.001, ** implies P a, water extract on 50 C; b, 30% ethanol extract on 50 C; c, 50% ethanol extract on 50 C; d, 70% ethanol extract on 50 C; e, water extract on 70 C; f, 30% ethanol extract on 70 C; g, 50% ethanol extract on 70 C; h, 70% ethanol extract on 70 C. Fig. 4. FRAP activities according to ethanol extraction conditions of Momordica charantia L. Values are mean±sd. Levels of significant between each water extracts and ethanolic extracts were determined using one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparison test. For significant differences: *** implies P 0.001, ** implies P 0.01, * implies P 0.05, ns implies no significant difference. a, water extract on 50 C; b, 30% ethanol extract on 50 C; c, 50% ethanol extract on 50 C; d, 70% ethanol extract on 50 C; e, water extract on 70 C; f, 30% ethanol extract on 70 C; g, 50% ethanol extract on 70 C; h, 70% ethanol extract on 70 C. Fig. 3. Hydroxy radical scavenging activities according to ethanol extraction conditions of Momordica charantia L. Values are mean±sd. Levels of significant between each water extracts and ethanolic extracts were determined using one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparison test. For significant differences: *** implies P a, water extract on 50 C; b, 30% ethanol extract on 50 C; c, 50% ethanol extract on 50 C; d, 70% ethanol extract on 50 C; e, water extract on 70 C; f, 30% ethanol extract on 70 C; g, 50% ethanol extract on 70 C; h, 70% ethanol extract on 70 C. 산화활성에대해입증한바있고, Boo 등 (22) 은여주의총폴리페놀물질과총플라보노이드물질이 ABTS cation radical 소거활성에영향을미친다는연구결과를보고한바있다. 또한 Lee 등 (26) 은차가버섯의온도단계별물추출물의폴리페놀함량과항산화효과를비교하였는데, 동일온도의 1차, 2차, 3차추출물의총페놀화합물의농도는 1 차 > 2차 > 3차순으로높았고그에따른 ABTS cation radical 소거활성도페놀화합물의농도에비례하게증가하였다고보고하였다. 본연구에서도총폴리페놀및플라보노이드함량이추출온도나주정농도가증가할수록높게측정되었고이에따른항산화활성도같은경향을나타내었다. 이결과로보았을때총폴리페놀및플라보노이드물질은항산화활성과상관관계가있음을더욱입증하였다. 또한 ABTS cation radical 소거활성뿐아니라다른항산화측정지표인 hydroxy radical 소거활성및환원력도폴리페놀물질과플라보노이드물질이같은영향을미치고있음을추측할수있었다. 이러한여주의항산화능력은항산화체계의불안정성으로유발되는질병중당뇨병개선에큰도움이될수있을것으로기대된다. 농도에서 42.82%, hydroxy radical 소거활성은동일농도에서 82.19%, FRAP 활성은흡광도 593 nm에서측정한결과동일농도에서 0.19의흡광도가측정되어 3종류의항산화활성모두 70 C, 70% 주정추출조건에서가장높게측정되었다. 항산화활성은물추출조건에비해주정추출조건에서유의성있는효과를나타냈다 (Fig. 2~4). Park 등 (25) 은여주의항산화활성을부위별로측정하여여주의항 여주주정추출조건별탄수화물및지방흡수억제활성여주추출물의혈당개선건강기능식품원료개발을위해추출조건별탄수화물흡수억제활성을 α-glucosidase 저해활성측정을통해평가하였다. α-glucosidase는소장상피세포에존재하는효소로입으로섭취된음식의탄수화물을포도당등의단당으로분해하여소장에서흡수시킨다 (27). α-glucosidase 저해제는소장점막의미세융모막에

6 1642 문정한 최동원 김성은 서문지훈 홍수영 김현규 조계만 송진 강상수 김강화 권오경 존재하고있는이당류의분해효소를가역적으로억제함으로써탄수화물의흡수를지연시키는역할을하고, 식후혈당상승을완만하게한다 (28). 현재시판되고있는 α-glucosidase 저해제로는 acarbose, voglibods 및 miglitol이있으나이들제제들은장기복용시복부팽만감, 구토, 설사등의부작용이나타날수있어사용이제한되고있다 (19). 그러므로이러한부작용을줄이고장기복용이가능한 α-glucosidase 저해활성물질을함유하고있는천연식품기능성소재로서여주추출물의항당뇨활성을측정하고자하였다. 본연구에서는 α-glucosidase 저해활성실험의양성대조군으로 acarbose를사용하여활성을비교한결과를 Fig. 5 에나타내었다. 여주추출조건별로비교해보았을때주정농도 70%> 50%> 30%> 0% 의순서대로저해활성이높게측정되었고 50 C보다 70 C 온도추출조건에서높은저해활성을나타냈으며, 물추출조건보다주정추출조건에서유의성있는효과를나타내었다. 또한추출분말시료를 0.25, 0.5, 1 mg/ml의농도별로각각처리했을때주정농도 70%, 추출온도 70 C에서농도의존적으로 24.71, 41.53, % 의저해활성을나타내었다. 양성대조군인 acarbose는 0.25 mg/ml 농도에서 80.20%, 0.50 mg/ml 농도에서 93.39%, 1.00 mg/ml 농도에서는 100% 이상의활성을나타내었다. 동일농도에서양성대조군인 acarbose보다저해활성이높지는않지만천연물인점을감안했을때 1 mg/ml 의농도에서 81.03% 로비교적높은저해활성을나타내었다. 여주에서알려져있는대표적인항당뇨활성에관련된성분들은 charantin, momordicin 등으로이물질들의특성은물보다에탄올, 메탄올등과같은유기용매에용해가더 잘된다. 그래서여주의활성물질들은대부분열수추출물보다주정추출물에존재할가능성이클것으로예상되며향후국내에서재배된여주로부터항당뇨활성을나타내는기능성분탐색도필요할것으로사료된다. Pancreatic lipase는지방가수분해효소로체내에서지방을 monoglyceride와 fatty acid로분해하고담즙산염과미셀을형성하여흡수된다. 흡수된지방산은소장상피세포에서 triglyceride로재합성되어혈액속에들어가지방조직, 근육등으로사용된다. 그리고남은 triglyceride는각조직에축적되는데이축적이과도하면비만을초래할수있게된다 (20). 현재의약품으로판매되고있는 lipase 저해활성제로 lipstatin의유도체인 orlistat(tetrahydrolipstatin) 은 Streptomyces toxitricini로부터유래된것으로서음식으로섭취된지방의약 30% 를흡수시키지않고배설해낼정도로우수한효능을가진다 (4). 하지만 orlistat 부작용으로지방변, 대변실금등의소화기증상과불안, 우울, 요통, 근육통, 관절통등의신경학적증상이보고된바있어 (29) 본연구에서는여주추출물을이용하여 pancreatic lipase의저해활성을추출조건별로알아보고자하였다. 양성대조군으로는위와췌장의지방분해효소억제제인 orlistat을사용하여저해활성을비교하였으며이물질은 0.1 mg/ml 농도에서 100% 이상의저해활성을나타냈다. 여주추출조건중주정농도별로 0.25, 0.5, 1 mg/ml를각각처리했을때주정농도 70%, 추출온도 50 C에서 35.74, 40.44, 59.34% 의저해활성을나타냈으며추출온도 70 C에서는 33.42, 39.80, 59.72% 로농도의존적으로저해활성이높게나타 Fig. 5. α-glucosidase inhibition activities according to ethanol extraction conditions of Momordica charantia L. Values are mean±sd. Levels of significant between each water extracts and ethanolic extracts were determined using one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparison test. For significant differences: *** implies P 0.001, ** implies P a, water extract on 50 C; b, 30% ethanol extract on 50 C; c, 50% ethanol extract on 50 C; d, 70% ethanol extract on 50 C; e, water extract on 70 C; f, 30% ethanol extract on 70 C; g, 50% ethanol extract on 70 C; h, 70% ethanol extract on 70 C. Fig. 6. Pancreatic lipase inhibition activities according to ethanol extraction conditions of Momordica charantia L. Values are mean±sd. Levels of significant between each water extracts and ethanolic extracts were determined using one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparison test. For significant differences: *** implies P 0.001, ** implies P 0.01, * implies P 0.05, ns implies no significant difference. a, water extract on 50 C; b, 30% ethanol extract on 50 C; c, 50% ethanol extract on 50 C; d, 70% ethanol extract on 50 C; e, water extract on 70 C; f, 30% ethanol extract on 70 C; g, 50% ethanol extract on 70 C; h, 70% ethanol extract on 70 C.

7 여주의추출조건에따른생리활성 1643 났다 (Fig. 6). Pancreatic lipase 저해활성또한물추출조건에비해주정추출조건에서유의성있는효과를나타내었다. 추출온도조건별로비교했을때는추출시온도조건이 pancreatic lipase 저해활성에크게영향을미치지않음을확인하였다. 요 본연구에서는항당뇨소재로잘알려져있는여주를이용한혈당개선건강기능성원료개발의기초연구로주정추출조건에따른항산화, 항당뇨, 항비만효능을검증하였다. 여주주정추출물의총폴리페놀과총플라보노이드함량, 항산화능, 탄수화물과지방의흡수지연능을평가하는 α-glucosidase 및 pancreatic lipase 저해활성은온도 70 C, 주정농도 70% 추출조건에서가장높게나타났다. 본연구결과를바탕으로향후여주의항산화, 항당뇨, 항비만효능등대사성질환에대한예방및개선효과를가지는건강기능식품을개발하는데기초자료로활용될것으로기대된다. 약 감사의글 본연구는농촌진흥청에서수행한농업연구개발사업의지원 ( 과제번호 : PJ010003) 에의한연구결과의일부로이에감사드립니다. REFERENCES 1. Leung L, Birtwhistle R, Kotecha J, Hannah S, Cuthbertson S Anti-diabetic and hypoglycaemic effects of Momordica charantia (bitter melon): a mini review. Br J Nutr 102: Lee HJ, Moon JH, Lee WM, Lee SG, Kim AK, Woo YH, Park DK Charantin contents and fruit characteristics of bitter gourd (Momordica charantia L.) accessions. J Bio- Environ Control 21: Yuk HJ, Noh GM, Choi JS, Kwon OK, Hong SY, Kang SS, Cho KM, Park DS α-glucosidase inhibitory effect of vicine and α-eleostearic acid from the seeds of Momordica charantia. Korean J Environ Agric 34: Hadváry P, Lengsfeld H, Wolfer H Inhibition of pancreatic lipase in vitro by covalent inhibitor tetrahydrolipstatin. Biochem J 256: Joseph B, Jini D Antidiabetic effects of Momordica charantia (bitter melon) and its medicinal potency. Asian Pac J Trop Dis 3: Liu JQ, Chen JC, Wang CF, Qiu MH New cucurbitane triterpenoids and steroidal glycoside from Momordica charantia. Molecules 14: Singh R, Kumar A, Giri DD, Bhuvaneshwari K, Pandey KD Gas chromatography-mass spectrometry analysis and phytochemical screening of methanolic fruit extract of Momordica charantia. J Rec Adv Agri 1: Kenny O, Smyth TJ, Hewage CM, Brunton NP Antioxidant properties and quantitative UPLC-MS analysis of phenolic compounds from extracts of fenugreek (Trigonella foenum-graecum) seeds and bitter melon (Momordica charantia) fruit. Food Chem 141: Asiamah D, Verghese M, Boateng JO, Kanda B, Shackelfor L, Walker LT Chemopreventive portential of bitter melon (Momordica charantia) against precancerous lesions in the colon of fisher 344 male rats. Int J Cancer Res 7: Choi JS, Kim HY, Seo WT, Lee JH, Cho KM Roasting enhances antioxidant effect of bitter melon (Momordica charantia L.) increasing in flavan-3-ol and phenolic acid contents. Food Sci Biotechnol 21: Kwatra D, Subramaniam D, Ramamoorthy P, Standing D, Moran E, Velayutham R, Mitra A, Umar S, Anant S Methanolic extracts of bitter melon inhibit colon cancer stem cells by affecting energy homeostasis and autophagy. J Evidence-Based Complementary Altern Med 2013: Article ID Pişkin A, Altunkaynak BZ, Tümentemur G, Kaplan S, Yazıcı OB, Hökelek M The beneficial effects of Momordica charantia (bitter gourd) on wound healing of rabbit skin. J Dermatolog Treat 25: Tan SP, Vuong QV, Stathopoulos CE, Parks SE, Roach PD Optimized aqueous extraction of saponins from bitter melon for production of a saponin-enriched bitter melon powder. J Food Sci 79: Folin O, Denis W A colorimetric method for the determination of phenols (and phenol derivatives) in urine. J Biol Chem 22: Eum MA, Kang YH, Kwon DJ, Jo KS The nitrite scavenging and electron donating ability of potato extracts. Korean J Food & Nutr 12: Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26: Ji N, Song JL, Kil JH, Park KY Protective effect of Perilla frutescens Britt var. japonica extracts oxidative stress in human HaCaT keratinocytes. J Korean Soc Food Sci Nutr 42: Benzie IF, Strain JJ The ferric reducing ability of plasma (FRAP) as a measure of antioxidant power the FRAP assay. Anal Biochem 239: Kim KK, Kang YH, Kim DJ, Kim TW, Choe M Comparison of antioxidant, α-glucosidase inhibition and anti-inflammatory activities of the leaf and root extracts of Smilax china L. J Nutr Health 46: Kwon OJ, Lee HY, Kim TH, Kim SG Antioxidant and pancreatic lipase inhibitory activities of Anemarrhena asphodeloides. Korean J Food Preserv 21: Ahn MJ, Yuk HJ, Lee HY, Hwang CE, Jeong YS, Hong SY, Kwon OK, Kang SS, Kim HR, Park DS, Cho KM Effect of the enhanced biological activities and reduced bitter taste of bitter melon (Momordica charantia L.) by roasting. J Agric Life Sci 49: Boo HO, Lee HH, Lee JW, Hwang SJ, Park SU Different of total phenolics and flavonoids, radical scavenging activities and nitrite scavenging effects of Momordica charantia L. according to cultivars. Korean J Medicinal Crop Sci 17: Lee HY, Park KS, Joo OS, Hwang CE, Ahn MJ, Jeong YS, Hong SY, Kwon OK, Kang SS, Yuk HJ, Kim HR, Park DS, Cho KM Changes in quality characteristics and antioxidant activity of bitter melon (Momordica charantia L.) pickle during ageing. J Korean Soc Food Sci Nutr 44:

8 1644 문정한 최동원 김성은 서문지훈 홍수영 김현규 조계만 송진 강상수 김강화 권오경 24. Kim YS, Kim R, Moon JH, Ji JR, Choi HD, Park YK Optimization of extraction conditions of polyphenolic compounds from apple pomace by response surface methodology. Korean J Food Sci Technol 41: Park Y, Boo HO, Park YR, Cho DH, Lee HH Antioxidant activity of Momordica charantia L. extracts. Korean J Medicinal Crop Sci 15: Lee SO, Kim MJ, Kim DG, Choi HJ Antioxidative activities of temperature-stepwise water extracts from Inonotus obliquus. J Korean Soc Food Sci Nutr 34: Lee BB, Park SR, Han CS, Han D, Park EJ, Park HR, Lee SC Antioxidant activity and inhibition activity against α-amylase and α-glucosidase of Viola mandshurica extracts. J Korean Soc Food Sci Nutr 37: Kim HY, Lim SH, Park YH, Ham HJ, Lee KJ, Park DS, Kim KH, Kim S Screening of α-amylase, α-glucosidase and lipase inhibitory activity with Gangwon-do wild plants extracts. J Korean Soc Food Sci Nutr 40: Kim DH, Lee EH, Hwang JC, Jeung JH, Kim DH, Cheong JY, Cho SW, Kim YB A case of acute cholestatic hepatitis associated with orlistat. Korean J Hepatol 8:

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,