J Korean Soc Food Sci Nutr 한국식품영양과학회지 44(5), 681~686(2015) http://dx.doi.org/10.3746/jkfn.2015.44.5.681 약용식물메탄올추출물의항산화및항당뇨활성 이연리 1 윤나라 2 1 대전보건대학교식품영양과 2 충북대학교식품생명공학과 Anti-Oxidative and Anti-Diabetic Effects of Methanol Extracts from Medicinal Plants Youn Ri Lee 1 and Nara Yoon 2 1 Department of Food and Nutrition, Daejeon Health Sciences College 2 Department of Food Science and Biotechnology, Chungbuk National University ABSTRACT The purpose of this study was to measure total phenolic compounds as a measure of antioxidant as well as α-amylase inhibitory and α-glucosidase inhibitory activities as a measure of anti-diabetic efficacy in methanol extracts from 23 kinds of medicinal plants. Extracts of three medicinal plant species showing high total polyphenol contents were selected ( stem, fruit, and leaf). Extracts of six medicinal plant species showing over 60% DPPH radical scavenging were also selected [ barks (80.10%), roots (64.25%), stem (73.59%), (78.20%), Taxus cuspidata fruits (70.52%), and leaf and stem (67.81%)]. Regarding α-glucosidase and α-amylase inhibitory activities acarbose showing approximately 80% inhibitory was selected as a control group, and six species ( heartwood, bark, stem,,, and ) showed greater than 80% α-glucosidase inhibitory. Extracts of nine medicinal plant. species showing over 80% α-amylase inhibitory ( root, bark, leaf, fruits, leaf and stem, stem, whole, leaf and stem, and leaf and stem). Based on these results, medicinal plants showing high antioxidant and antidiabetic activities can be used as fundamental products in developing new medicines, as well as functional foods to prevent adult disease. Key words: total phenolic compounds, DPPH radical scavenging, α-glucosidase inhibition, α-amylase inhibition 서 당뇨병은우리나라의 5대사망원인중하나로비만과운동부족의증가로인해전세계적으로유병률이증가하고있다. 우리나라에서도 2003년전인구의 5.9% 에불과했던당뇨병유병률이 2030년 10.85% 로급격히증가할것으로예상된다 (1). 당뇨병은췌장베타세포의인슐린분비량이부족하거나결핍되어발생하는대사성질환으로열량원의대사를변형시키며, 이러한대사이상은고혈압, 고지혈증, 비만및동맥경화성혈관장애를일으킨다 (2,3). 이러한증상외에도망막증, 신증, 신경장애, 감염증등이당뇨합병증으로나타날수있다. 면역체계에도영향을주게되어세포매개성면역에 Received 8 January 2015; Accepted 1 April 2015 Corresponding author: Youn Ri Lee, Department of Food and Nutrition, Daejeon Health Sciences College, Daejeon 300-711, Korea E-mail: leeyounri@hit.ac.kr, Phone: +82-42-670-9246 론 더큰영향을미치는것으로알려져있다 (4). 당뇨병을근원적으로치료할수있는치료법은아직개발되어있지않는실정이며, 혈당이정상적인수준으로유지되도록하는것이최선의치료법으로알려져있다 (5). 당뇨병의개선을위한방안으로는식이요법, 운동요법을기본으로 (6), 경구혈당강하제와인슐린이주로사용되고있는데 (7), 임상에서사용되고있는경구혈당강하제로는 sulfonylurea 제제및 biguanide 제제가있다. 그러나이약제들은저혈당의위험이높고체증증가, 오심, 구토, 소화불량등의소화기장애와같은부작용을발생시킬수있음이보고되었다 (8). 따라서최근천연물의약품및기능성식품에관한시장이확대됨에따라천연소재에대한연구가활발히진행되고있다 (9). 기능성소재물질에관한탐색연구는주로민간요법이나한방에서이미효능이검증된소재를활용하고있으며, 식이가가능하고부작용이적은천연물을대상으로다양한형태의건강기능성식품과의약품들이개발되는추세이다 (10). 천연항산화제의대부분은식물기원의페놀성항산화
682 이연리 윤나라 성화합물로나무, 줄기, 잎, 열매, 뿌리, 꽃, 씨앗등의모든부분에존재하며, 이들성분은유리라디칼의생성을지연시키거나활성을저해하는항산화물질로작용한다 (11,12). 이에본연구는약용식물 23종을선정하여총폴리페놀함량을측정한후 DPPH 라디칼소거능을측정하고항당뇨활성으로는 α-amylase 저해활성, α-glucosidase 저해활성을검정하여혈당강하활성을나타낼수있는약용식물을선발하고자한다. 재료및방법 실험재료 23종의소재들은한국식물추출은행 (Daejeon, Korea) 에분양받아시료로사용하였다. 칡 ( 꽃 / 뿌리 ), 겨우살이 ( 잎 / 줄기 / 잎과줄기 ), 두충 ( 줄기심재 / 줄기수피 / 잎 ), 구기자나무 ( 열매 / 잎과줄기 / 뿌리 ), 화살나무 ( 줄기와잎 / 줄기 ), 조릿대 ( 지상부 / 잎 ) 삼백초, 마, 비수리, 주목열매, 율무, 녹두, 둥굴레, 뽕잎나무 ( 잎줄기 ) 를부위별로선정하였다 (Table 1). 총폴리페놀함량측정총폴리페놀함량은 Dewanto 등 (13) 의방법에따라측정하였다. 메탄올추출물 100 μl에 2% Na 2CO 3 용액 2 ml를가한후 50% Folin-Ciocalteu reagent(sigma-aldrich Co., St. Louis, MO, USA) 100 μl를첨가하여실온에서 30분간방치후반응액의흡광도값을 750 nm에서측정하였다. 표준물질로 gallic acid(sigma-aldrich Co.) 를 5, 10, 25 및 50배로희석하여사용하였으며, 검량선작성후총폴리페 Table 1. Medicinal plants and herbs used for experiments 칡겨우살이두충구기자나무화살나무조릿대삼백초마비수리주목율무녹두둥글레뽕잎나무 Viscum album var. coloratum (heart wood) (bark) 놀함량은시료 1 g 중의 mg gallic acid로나타내었다. DPPH radical 소거능측정 DPPH radical 소거능은 Hwang 등 (14) 의방법을변형하여측정하였다. 메탄올추출물 0.2 ml에 0.2 mm DPPH (Sigma-Aldrich Co.) 용액 0.8 ml를가하여실온에서 60 분간방치한후 520 nm에서흡광도를측정하였다. α-glucosidase 저해활성측정 α-glucosidase 저해활성은 Tibbot과 Skadsen(15) 의방법에따라측정하였다. α-glucosidase(0.35 U/mL) 와 ρ- nitrophenyl-α-d-glucopyranoside(1.5 mm, PNPG) 는 0.1 M sodium phosphate buffer(ph 7.0) 에용해하여사용하였으며, 각각의추출물 50 μl를 0.35 unit/ml α-glucosidase 효소액 100 μl와혼합하여 37 C에서 10분간전배양한후 1.5 mm PNPG 50 μl를가하여 37 C에서 20분간반응시켰다. 1 M Na 2CO 3 1 ml를가하여반응을정지시킨후 ELISA (UV-1650PC, Shimadzu, Kyoto, Japan) 를사용하여 405 nm에서흡광도를측정하고저해율 (%) 을계산하였으며, 양성대조군으로 acarbose(sigma-aldrich Co.) 를사용하였다. α-amylase 저해활성 α-amylase 저해활성은 Stirpe과 Corte(16) 의방법을변형하여측정하였다. 추출물 125 μl에 12 unit/ml pancreatin 기원의 α-amylase 효소액 62.5 μl, 200 mm potassium phosphate buffer(ph 6.8) 62.5 μl와혼합하여 37 C에서 10분간전배양한후 1% starch를 125 μl 가하여 37 C에서 5분간반응시켰다. 반응액에 48 mm 3,5-dinitrosalicylic acid(dns, 30% sodium potassium tartrate in 0.5 M NaOH) 발색시약 125 μl를넣고 100 C에서 15분간끓여발색을시킨후충분히냉각시키고, 이반응액에 3배량의증류수를가한후 ELISA(UV-1650PC, Shimadzu) 를사용하여 540 nm에서흡광도를측정하고저해율을계산하였다. 통계처리실험에서얻어진결과의통계적유의성은 SPSS(Statistical Package for Social Sciences, version 12.0, SPSS Inc., Chicago, IL, USA) 를이용하여평균 ± 표준오차로나타내었고, 각농도의평균치의통계적유의성을 P<0.05 수준에서 Duncan's multiple range test에의해검정하였다. 항산화및항당뇨활성간의상관관계는 Pearson's correlation analysis를통하여분석하였다. 결과및고찰약용식물의총폴리페놀함량식물의 2차대사산물로서하나이상의수산기가치환된벤젠환을가지고있는 phenolic acid, flavonoid, antho-
약용식물메탄올추출물의항산화및항당뇨활성 683 cyanin, condensed tannin과같은다양한구조의화합물을폴리페놀화합물이라고하며 (17,18), 이러한페놀성분자들은체내에서항산화, 항비만및항염증등과같은생리활성을가지고있는것으로알려져있다 (19,20). 특히페놀성화합물에존재하는 hydroxyl group은 ROS를제거하는역할과동시에 ROS의생성에기여하는금속이온을흡착하는특별한구조를가지기때문에높은항산화활성을가지는것으로알려져있다 (21,22). 약용식물 23종의총폴리페놀함량은 Table 2와같다. 총폴리페놀함량은 gallic acid를표준용액으로하여작성한검정곡선으로부터약용추출물의폴리페놀함량을분석한결과 1.50~15.25 mg gallic acid equivalent(gae)/g의함량범위로나타났다. 10~15 mg GAE/g 함량범위로는두충잎, 화살나무줄기, 마, 비수리, 주목열매, 뽕잎나무잎줄기부위로나타났다. Shin 등 (23) 은한약재열수추출물의총폴리페놀함량측정결과황금추출액 (2.25 mg GAE/ ml) 과금은화추출액 (2.21 mg GAE/mL) 이가장높은함량을보였으며, 의이인추출액 (0.01 mg GAE/mL), 백복령추출액 (0.01 mg GAE/mL), 길경추출액 (0.02 mg GAE/mL), 총백추출액 (0.02 mg GAE/mL) 이낮은함량의연구결과를나타냈다. 약용식물류를이용한항산화물질의검정결과시료중의총폴리페놀함량이약용식물의종류에따라매우상이한것으로나타났다. 본연구에서총폴리페놀함량이높은약용식물부위로는화살나무줄기, 마, 주목열매, 뽕잎나무잎 줄기로기능성소재로써의이용가능성이높을것으로예상된다. 약용식물의 DPPH radical 소거능측정특유의자색을나타내는 DPPH는분자내에 free radical 을가지고있어항산화작용을나타내는 ascorbate, tocopherol, BHA, Maillard형갈변생성물질등에의해환원되어탈색이되는특성을가지고있다 (17). DPPH 라디칼소거법은 DPPH의짙은자색이탈색되는정도에따라그물질의항산화능을나타내는방법으로일반적으로많이사용되고있다 (24). Ascorbic acid를양성대조군으로하여 23종약용식물메탄올추출물 (4 mg/ml) 의 DPPH radical 소거능에대한결과는 Table 3과같다. 60% 이상의 DPPH 라디칼소거능을보인약용식물로는두충줄기수피 (80.10%), 구기자나무뿌리 (64.25%), 화살나무줄기 (73.59%), 비수리 (78.20%), 주목열매 (70.52%), 뽕잎나무잎줄기 (67.81%) 부위로나타났다. Nam과 Kang(25) 은 130여종의식물성한약재열수추출물의항산화활성을검증한결과 25종이대조군에비해 80% 이상의전자공여능을나타내었으며항변이원성효과를동시에가지는한약재도 12종이라고보고하였다. Jung 등 (26) 은한약재의식물체가그사용부위에따라전자공여능의차이를나타내기도한다고보고하였다. Kang 등 (27) 의보고에의하면전자공여능이 phenolic acids와 flavonoids 및 Table 2. Total phenolic contents of the methanol extracts from medicinal plants 칡 겨우살이 두충 구기자나무 화살나무 조릿대 삼백초마비수리주목율무녹두둥글레뽕잎나무 Viscum album var. coloratum (heart wood) (bark) Total polyphenol (mg GAE 1) /extract g) 6.05±0.05 h2)3) 5.59±0.01 i 5.35±0.03 j 5.32±0.03 j 5.10±0.06 K 4.02±0.01 o 3.57±0.03 q 14.24±0.08 b 3.25±0.02 s 7.51±0.02 g 7.83±0.03 f 4.92±0.03 l 15.12±0.03 a 4.68±0.09 m 3.72±0.01 p 3.19±0.02 s 13.47±0.02 d 13.69±0.06 c 15.25±0.08 a 3.45±0.01 r 4.31±0.02 n 1.50±0.08 t 12.50±0.12 e 1) GAE gallic acid equivalents. 2) Mean±SD (n=3). 3) Different superscripts (a-t) in a column indicate significant differences at P<0.05 by Duncan's multiple range test.
684 이연리 윤나라 Table 3. DPPH radical scavenging of medicinal plant methanol extracts 칡 겨우살이 두충 구기자나무 화살나무 조릿대 삼백초마비수리주목율무녹두둥글레뽕잎나무 Viscum album (heart wood) (bark) DPPH radical scavenging (%) 13.73±0.78 p1)2) 35.60±0.34 j 24.48±0.49 l 20.49±0.00 m 20.07±0.06 m 27.61±0.82 k 80.10±0.10 a 10.95±0.88 q 15.65±0.10 o 39.42±0.10 h 64.25±0.40 f 38.59±0.48 i 73.59±0.17 c 1.76±0.60 s 14.26±0.49 p 16.16±0.28 o 45.80±0.55 g 78.20±0.15 b 70.52±0.15 d 15.58±0.20 o 18.55±0.26 n 8.69±0.25 r 67.81±0.06 e 1) Mean±SD (n=3). 2) Different superscripts (a-s) in a column indicate significant differences at P<0.05 by Duncan's multiple range test. 기타 phenol성물질에대한항산화작용의지표라고하였으며, 이러한물질은환원력이큰것일수록전자공여능이높다고하였다. 본연구에서도약용식물추출물들의폴리페놀함량의결과와일치하는경향으로 DPPH 라디칼소거능을가지는것을확인하였다. 약용식물의 α-glucosidase 및 α-amylase 저해활성측정당뇨병환자의경우급격히상승하는과도한혈당및고혈당증이지속됨에따라발생하는활성산소 (reactive oxygen species; ROS) 들로인해당뇨병의복합증세인신경장애, 신장해, 그리고망막증등과같은질병이발생하게된다 (28). 따라서효율적인당뇨병의관리를위해서는 α-glucosidase 및 α-amylase 저해활성과활성산소를제거할수있는항산화활성을갖는소재가필요하다. α-glucosidase 저해제는소장점막의미세융모막에존재하는이당류의분해효소를가역적으로억제하여탄수화물의흡수를지연시키는역할을하며, 소장전체에포도당이흡수되어식후혈당상승을완만하게한다 (29). 본연구에서는약용식물 23종을대상으로 α-glucosidase 저해활성을측정하여향후혈당상승억제제로활용될수있는소재를선발및탐색하였다. 실험결과 (Table 4) 대조구인 acarbose는 80% 정도의 α-glucosidase 저해활성을나타냈으며, 이에약용식물 23종을대상으로제조한메탄올추출물 (5 mg/ml) 의소재도역시 80% 이상의 α-glucosidase 저해활성을나타내는소재들로선발하게되었다. 약 용식물메탄올추출물에서두충줄기심재, 두충줄기수피, 화살나무줄기, 마, 율무, 녹두 6종이 80% 이상의우수한 α-glucosidase 저해활성을나타내었다. 두충은 astragalin, isoquercitrin, qeurcetin 3-O-β-D-xylopyranosyl(1-2)- β-d-glucopyranoside 3종의 flavonoid 화합물을가지고있으며, lignan과 iridoid 등의생리활성물질을지니는것으로보고되고있다 (30). α-amylase는탄수화물의 α-d-(1,4)-glucan 결합을분해하는효소이고, α-glucosidase 소장상피세포의 brushborder membrane에존재하는효소로소장에서음식물중의전분을포도당과같은단당으로분해하여흡수시킨다 (31). 따라서소장의 α-amylase와 α-glucosidase를저해함으로써포도당의흡수를지연시킬수있어 α-amylase와 α-glucosidase의저해활성은혈당수치상승억제의지표로사용된다 (31,32). 23종의메탄올추출물 5 mg/ml의농도로대조구인 acarbose의저해활성과같은 α-amylase를 80% 이상저해한약용식물로는칡뿌리, 두충줄기수피, 두충잎, 구기자열매, 화살나무잎줄기, 화살나무줄기, 조릿대지상부, 마, 뽕잎나무잎줄기부위추출물 9종이선발되었다 (Table 4). 특히 α-amylase 90% 이상저해활성을나타내는약용식물로는두충잎추출물, 뽕잎나무잎줄기추출물, 화살나무줄기추출물로나타났다. 이와같이약용식물에는탄수화물의소화효소를억제하는성분들이풍부하게분포되어 α-glucosidase, α-amylase
약용식물메탄올추출물의항산화및항당뇨활성 685 Table 4. α-glucosidase and α-amylase inhibition of medicinal plant methanol extracts 칡 겨우살이 두충 구기자나무 화살나무 조릿대 삼백초마비수리주목율무녹두둥글레뽕잎나무 Viscum album Acarbose (5 mg/ml) (heart wood) (bark) α-glucosidase inhibition (%) 41.50±0.23 1)n2) 61.84±0.16 i 2.49±0.09 p 78.45±0.23 d 58.28±0.09 j 82.13±0.28 c 83.50±0.23 b 43.65±0.58 m 77.43±0.33 e 77.14±0.44 e 44.90±0.53 l 73.70±0.47 g 82.06±0.19 c 74.72±0.55 f 57.88±0.38 j 40.94±0.30 n 88.71±0.51 a 48.45±0.32 k 57.88±0.23 j 83.22±0.69 b 83.67±0.29 b 57.94±0.26 i 62.78±0.51 h 80.28±0.02 α-amylase inhibition (%) 42.20±0.23 o 83.02±0.36 f 3.49±0.28 r 74.56±0.65 g 57.63±0.65 k 72.07±0.36 i 89.92±0.70 c 92.52±0.40 b 87.97±0.73 d 61.17±0.44 j 73.24±0.47 h 84.63±0.43 e 92.26±0.43 b 82.65±0.43 f 6.84±0.28 q 40.94±0.30 p 89.91±0.76 c 48.45±0.32 m 51.76±0.73 l 41.54±0.57 op 57.15±0.57 k 44.96±0.49 n 96.66±0.37 a 80.88±0.02 1) Values are mean±se (n=3). 2) Different superscripts (a-r) in a column indicate significant differences at P<0.05 by Duncan's multiple range test. 효소저해물질을찾아낼필요성이있으며본실험결과활성이우수했던약용식물들은추가적으로세포실험및동물실험을통해항당뇨관련효능검증을실시하고활성이검증된약용식물들은향후새로운의약품개발및성인병예방의건강기능식품으로경쟁력을확보할수있을것으로기대된다. 상관관계약용식물메탄올추출물의항산화활성및항당뇨활성간의상관관계를분석한결과는 Table 5와같다. 총폴리페놀함량은 DPPH 라디칼소거능및 α-amylase 저해활성과양의상관관계를보였으며, 상관계수는각각 0.618과 0.334 Table 5. Correlation coefficients among total polyphenol contents, DPPH radical scavenging, α-glucosidase and α- amylase inhibition of medicinal plant methanol extracts Total polyphenol content DPPH radical scavenging α-glucosidase inhibition ** P<0.01. DPPH radical scavenging α-glucosidase inhibition α-amylase inhibition 0.618 ** 0.333 0.334 ** 0.165 0.326 ** 0.710 ** 로나타났다 (P<0.01). DPPH 라디칼소거활성역시 α- amylase 저해활성과양의상관관계를보였다 (0.326, P< 0.01). α-glucosidase 저해활성과 α-amylase 저해활성은 0.710으로높은양의상관관계를나타내었다 (P<0.01). 요약약용식물 23종메탄올추출물의폴리페놀함량을측정한후항산화활성을측정하고, 항당뇨활성으로는 α-amylase 저해활성, α-glucosidase 저해활성을검정하여혈당강하활성을나타낼수있는약용식물을선발하고자한다. 약용식물의폴리페놀함량을분석한결과 1.50~15.25 mg GAE/g 의다양한함량범위로나타났으며가장높은함량은주목열매 (15.25 mg GAE/g), 화살나무줄기 (15.12 mg GAE/g), 두충잎 (14.24 mg GAE/g) 으로 3종이나타났다. 60% 이상 DPPH 라디칼소거능을본약용식물로는두충줄기수피 (80.10%), 구기자나무뿌리 (64.25%), 화살나무줄기 (73.59 %), 비수리 (78.20%), 주목열매 (70.52%), 뽕잎나무잎줄기 (67.81%) 부위추출물로 6종이선발되었다. α-glucosidase를 80% 이상저해한약용소재로는두충줄기심재, 두충줄기수피, 화살나무줄기, 마, 율무, 녹두 6종이선발되었다. α-amylase를 80% 이상저해한약용식물로는칡뿌리, 두충줄기수피, 두충잎, 구기자열매, 화살나무잎줄기, 화살나무줄기, 조릿대지상부, 마, 뽕잎나무잎줄기추출물등
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