Korean J. Plant Res. 31(5):433-452(2018) https://doi.org/10.7732/kjpr.2018.31.5.433 Print ISSN 1226-3591 Online ISSN 2287-8203 Original Research Article 콜린에스테라제저해효과보유식물추출물탐색 박샛별 1, 이정훈 2 김형돈 2, 서경혜 2, 정현수 1, 김동휘 3, 이승은 2 * 1 농촌진흥청국립원예특작과학원인삼특작부, 공무직, 2 농촌진흥청국립원예특작과학원인삼특작부, 농업연구사, 3 농촌진흥청국립원예특작과학원인삼특작부, 농업연구관 Screening of Plant Extracts with Cholinesterase Inhibition Activity Saet-Byul Park 1, Jeong-Hoon Lee 2, Hyung-Don Kim 2, Kyung-Hae Soe 2, Hyeon-Soo Jeong 1, Dong-Hwi Kim 3 and Seung-Eun Lee 2 * 1 Assistant, 2 Researcher and 3 Senior Researcher, Department of Herbal Crop Research, National Institute of Horticultural & Herbal Science, Eumseong 27709, Korea Abstract - This study was conducted to select candidates from plant resources for the purpose of improving or treating Alzheimer s disease, a type of dementia. One hundred and eighty-four plant extracts at a final concentration of 100 μg / ml were screened to determine their capacity to inhibit acetylcholinesterase (AChE) by in vitro assay. From this AChE assay, seven plant extracts - including methanol ext. and water ext. of Phellaodendron amurense Rupr. (bark), methanol ext. of Nelumbo nucifera Gaertn (stamen/ovary), methanol ext. of Persicaria tinctoria H. GROSS (flower), methanol ext. of Coptis chinensis (rhizome), ethanol ext. of Cinnamomum cassia Blume(bark) and ethanol ext. of Carthamus tinctorius L. (fruit) - showed effective inhibition activity ranging from 18.7% to 63.1%. The selected extracts were testified their inhibition activities on AChE and BuChE (butyrylcholinesterase) at concentrations of 25, 50, 100, 200 μg / ml. In the AChE assay, five extracts including methanol ext. of Nelumbo nucifera Gaertn. (stamen/ovary), methanol ext. of Persicaria tinctoria H. GROSS (flower), methanol ext. of Coptis chinensis (rhizome), methanol ext. and water ext. of Phellaodendron amurense Rupr. (bark) showed inhibition activity of 15.0%~73.5%, 19.5%~63.5%, 81.6%~58.5%, 69.9%~80.5%, and 54.8%~78.3%, respectively, at concentrations of 25, 50, 100, 200 μg / ml. In the BuChE assay, the extracts of Nelumbo nucifera Gaertn. (stamen/ovary), Persicaria tinctoria H. GROSS (flower), and Coptis chinensis (rhizome) showed inhibitory capacities of 58.9~81.6%, 45.8%~72.4%, and 33.1%~55.4% at concentrations of 25, 50, 100, 200 μg / ml, respectively. In conclusion, it is suggested that Nelumbo nucifera Gaertn. (stamen/ovary), Persicaria tinctoria H. GROSS, Coptis chinensis (rhizome) and Phellaodendron amurense Rupr. (bark) could be selected as candidate materials for improving or treating Alzheimer s disease on the basis of further study. Key words Alzheimer s disease, Cholinesterase, Cognition, Dementia 서언 고령화현상이사회문제가됨에따라노인성질환에관한연구의중요성이강조되고있으며, 의학발달로높아진평균수명을통해치매는발병률과유병률이점점높아지고있는추세이다 (Evans et al., 1989; Nordberg, 1996). 치매는혈관성치매, 루이소체질환, 알츠하이머병등을포함하는신경퇴행성질환으로서 60세이상의치매환자중 50 * 교신저자 : E-mail herbin3@korea.kr Tel. +82-043-871-5782 60% 가알츠하이머병 (Alzheimer s disease, AD) 을앓고있다. 기본적인치매증상으로기억력감퇴및인지능력저하가있고, 그중알츠하이머치매는인식능력을점차적으로저하시키며기억력감퇴, 인지력감소, 우울증등으로인해일상생활수행에심각한영향을미친다 (Alzheimer s Association, 2006). 알츠하이머병은기억과관련된콜린성신경계에서신경전달물질중하나인아세틸콜린 (Acetylcholine, ACh) 이관여하고있다고알려져있고, 알츠하이머환자의뇌신경절에서콜린성신경계의퇴화는아세틸콜린양의감소에따른결핍현상과기억과연관된해마의구조적이상을만들며, 아세틸콜린농도감 c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons -433- Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Korean J. Plant Res. 31(5) : 433~452(2018) 소로아세틸콜린을생성하는신경세포의손상과아세틸콜린분해효소인 acetylcholinesterase(ache) 활성의증가로인해기억장애는더욱악화된다 (Kim et al., 2005; Kasa et al., 2000). 그러므로, 근래에아세틸콜린의농도감소를늦추기위해 AChE 저해제에대한연구가진행되어왔으며, 알츠하이머증상을완화시키고병의진전속도를늦출수있는치매치료제로타크린 (tacrine:cognex ), 도네페즐 (donepezil hydrochloride: Aricept ), 리바스티그민 (rivastigmine: Exelon ), 갈란타민 (galantamine:reminyl ) 이알려져있다 (Giacobini et al., 2002). 하지만이들치매치료제의사용은메스꺼움, 식욕부진, 구토및설사와같은부작용을유발하여일상생활이크게개선되지않아보다부작용이적고효과가우수한새로운약물또는천연물발견등이요구되고있다 (Lanctôt et al., 2003). 한편, 국내에서천연식물자원의항산화등활성탐색및선발자원에대한연구 (Lee et al., 2013; Lyu et al., 2007; Jang et al., 2016) 는다수이루어져있으나인지기능개선과관련하여서는 Cho et al. (2014) 이한국산양하꽃봉오리가인지기능개선효과를나타낸다고보고하고있을뿐식물자원의인지능개선효과를탐색하고유용자원을선발한연구는미흡하다. 따라서본연구팀은천연식물자원을대상으로치매의일종인알츠하이머병을개선및치료할수있는소재를발굴하기위해콜린성에스테라제 (AChE) 저해활성을중심으로탐색하였으며, 몇가지자원을선발하였기에그결과를보고하고자한다. 재료및방법실험재료실험에사용된어저귀 (Abutilon theophrasti Medicus) 열매의물추출물등 184 개의식물추출물시료는농촌진흥청국립원예특작과학원약용작물과소속식물분류전문가로부터확보된식물과학명을검토받아조제된것을사용하였으며, 각추출물은아래의방법에따라제조되었다 (Table 1). 먼저, 상온에서추출된추출물로서추출조건 A, B 및 C는각각물, 메탄올및에탄올로추출및여과되었다. 또한, 가속용매추출장치 (accelerated solvent system, Dionex ASE-350, Themofischer scientific and its subsidiaries, Sunnyvale, USA) 를이용한추출조건 D와 E는 50 에서메탄올및에탄올로각각추출및여과되었고, 추출조건 F는 85 에서에탄올로추출되었다. 그리고, 환류냉각장치를이용한추출방법으로추출조건 G는 100 에서물로추출된후여과되었으며, 추출조건 H 와 I는 74 에서메탄올및 70% 메탄올로각각추출및여과되었다. 여과된각추출물은감압농축기 (Eyela, N-1200B, Tokyo Rikakikai co. Ltd, Tokyo, Japan) 를이용하여용매를제거하였으며, 남은수분및물추출물은동결건조기 (PVTFD 50R, IlshinBioBase Co., Ltd., Donducheon, Korea) 를사용하여최종추출물을확보하였다. Table 1. Plant part extracts used for the study Sample Scientific name Korean name Used part Resource z Extract No. condition y 1 Abutilon theophrasti Medicus 어저귀 fruit 1 A 2 Abutilon theophrasti Medicus 어저귀 stem 1 A 3 Abutilon theophrasti Medicus 어저귀 root 1 A 4 Abutilon theophrasti Medicus 어저귀 fruit 1 D 5 Abutilon theophrasti Medicus 어저귀 flower/seed 2 E 6 Abutilon theophrasti Medicus 어저귀 leaf 2 E 7 Eleuthero coccus sessiliflorus (Rupr. Maxim.) S.Y.Hu 오갈피나무 leaf 3 A 8 Eleuthero coccus sessiliflorus (Rupr. Maxim.) S.Y.Hu 오갈피나무 root 3 E 9 Eleuthero coccus sessiliflorus (Rupr. Maxim.) S.Y.Hu 오갈피나무 fruit 3 E 10 Eleuthero coccus sessiliflorus (Rupr. Maxim.) S.Y.Hu 오갈피나무 root 3 A 11 Eleuthero coccus sessiliflorus (Rupr. Maxim.) S.Y.Hu 오갈피나무 fruit 3 A 12 Eleuthero coccus sessiliflorus (Rupr. Maxim.) S.Y.Hu 오갈피나무 leaf 3 E 13 Eleuthero coccus sessiliflorus (Rupr. Maxim.) S.Y.Hu 오갈피나무 fruit 4 A 14 Achillea alpinia L. 톱풀 aerial part 1 D 15 Achillea alpina var. discoidea (Regel) Kitam. 산톱풀 root 2 E 16 Aconitum carmichaeli Debeaux 오두 root 14 E -434-
콜린에스테라제저해효과보유식물추출물탐색 Table 1. Continued Sample No. Scientific name Korean name Used part Resource z Extract condition y 17 Aconitum carmichaeli Debeaux 오두 root 14 A 18 Aconitum carmichaeli Debeaux 오두 processed root 14 E 19 Aconitum carmichaeli Debeaux 오두 processed root 14 A 20 Aconitum koreanum R. Raymond 백부자 root 5 A 21 Agrimonia pilosa L. var. japonica Nakai 짚신나물 stem 1 H 22 Agrimonia pilosa L. var. japonica Nakai 짚신나물 leaf 1 H 23 Akebia quinata (Houtt.) Decne 으름 stem 4 A 24 Akebia quinata (Houtt.) Decne 으름 stem 4 E 25 Alpinia oxyphylla Mique 익지 fruit 15 E 26 Alpinia oxyphylla Mique 익지 fruit 15 A 27 Althaea rosea CAV. 접시꽃 flower(red) 1 D 28 Althaea rosea CAV. 접시꽃 root(red) 1 D 29 Amaranthus SP. 으름덩굴 flower 1 A 30 Amaranthus SP. 으름덩굴 root 1 A 31 Amaranthus SP. 으름덩굴 aerial part 1 A 32 Arisaema amurense Maxim. 둥근잎천남성 processed root 14 A 33 Arisaema amurense Maxim. 둥근잎천남성 processed root 14 E 34 Artemisia annua L. 개똥쑥 leaf/stem/ flower 2 D 35 Artemisia argyi H. Lev. & Vaniot 황해쑥 leaf 14 I 36 Aruncus dioicus var. kamtschaticus Hara 눈개승마 aerial part 1 D 37 Aruncus dioicus var. kamtschaticus Hara 눈개승마 aerial part 1 H 38 Asarum sieboldii Mig. 족도리풀 root 14 E 39 Asarum sieboldii Mig. 족도리풀 root 14 A 40 Asparagus cochinchinensis (Lour.) Merr. 천문동 root 14 A 41 Asparagus cochinchinensis (Lour.) Merr. 천문동 root 14 E 42 Aster scaber THUNB. 참취 aerial part 1 D 43 Aster ageratoides Turcz. 까실쑥부쟁이 aerial part 1 D 44 Aster koraiensis NAKAI 벌개미취 aerial part 1 D 45 Astilbe koreana (Kom.) Nakai 숙은노루오줌 aerial part 2 C 46 Astragalus mongholicus Bunge 황기 root 6 D 47 Astragalus mongholicus Bunge 황기 root 6 A 48 Astragalus mongholicus Bunge 황기 root 6 D 49 Bupleurum falcatum L. 시호 root 7 E 50 Bupleurum falcatum L. 시호 root 8 A 51 Calystegia sepium var. japonicum (Choisy) Makino 메꽃 root 1 A 52 Calystegia sepium var. japonicum (Choisy) Makino 메꽃 aerial part 1 A 53 Campanula takesimana Nakai 섬초롱꽃 aerial part 2 C 54 Campsis grandiflora (Thunb.) K.Schum. 능소화 root 1 D 55 Campsis grandiflora (Thunb.) K.Schum. 능소화 aerial part 1 D 56 Caragana sinica (Buchoz) Rehder 골담초 stem 1 H 57 Caragana sinica (Buchoz) Rehder 골담초 leaf 1 H 58 Cardiospermum halicacabum L. 풍선덩굴 seed 1 D 59 Cardiospermum halicacabum L. 풍선덩굴 root 1 D 60 Carthamus tinctorius L. 잇꽃 fruit 9 C 61 Carthamus tinctorius L. 잇꽃 fruit 9 A 62 Chaenomeles sinensis KOEHNE 모과나무 fruit 1 H -435-
Korean J. Plant Res. 31(5) : 433~452(2018) Table 1. Continued Sample Scientific name Korean name Used part Resource z Extract No. condition y 63 Chaenomeles sinensis KOEHNE 모과나무 leaf 1 H 64 Chenopodium ambrosioides L. 양명아주 root 2 E 65 Chionanthus retusus Lindl. & Paxton 이팝나무 branch 2 E 66 Cinnamomum cassia Blume 육계 bark 15 E 67 Cinnamomum cassia Blume 육계 bark 15 A 68 Cinnamomum cassia Presl 육계 stem bark 15 E 69 Cinnamomum cassia Presl 육계 stem bark 15 A 70 Citrus aurantium L. 광귤 unriped fruit 14 D 71 Codonopsis pilosula (FR.) NANNF 만삼 root 1 D 72 Codonopsis pilosula (FR.) NANNF 만삼 aerial part 1 D 73 Coptis chinensis 중국황련 rhizome - D 74 Cornus officinalis Siebold et Zuccarini 산수유 leaf 1 H 75 Crataegus pinnatifida Bunge 산사나무 leaf 1 H 76 Crataegus pinnatifida Bunge 산사나무 leaf 1 D 77 Crataegus pinnatifida Bunge 산사나무 fruit 1 H 78 Cucumis melo L. 메론 fruit stalk 14 E 79 Cucumis melo L. 메론 fruit stalk 14 A 80 Dendranthema boreale (Makino) Ling ex Kitam. 산국 aerial part 1 D 81 Dendranthema zawadskii var. lucidum (Nakai) J.H.Park 울릉국화 aerial part 2 C 82 Dendrobium moniliforme (L.) Sw. 석곡 stem 15 A 83 Dendrobium moniliforme (L.) Sw. 석곡 stem 15 E 84 Aster tartaricus Lf. 개미취 root 1 G 85 Aster tartaricus Lf. 개미취 root 1 H 86 Syringa patula var. Kamibayashii (Nakai) M.Y.Kim 정향나무 flower 14 3 87 Euphorbia lathyris L. 속수자 aerial part 12 A 88 Fagopyrum dibotrys (D.DON) HARA 금교맥 aerial part 1 A 89 Fagopyrum dibotrys (D.DON) HARA 금교맥 root 1 A 90 Forsythia viridissima Lindley 의성개나리 fruit 9 A 91 Forsythia viridissima Lindley 의성개나리 fruit 9 E 92 Forsythia viridissima Lindley 의성개나리 root 1 D 93 Forsythia viridissima Lindley 의성개나리 aerial part 1 A 94 Gentiana macrophylla Pallas 진교 root 14 A 95 Gentiana macrophylla Pallas 진교 root 14 E 96 Geranium thunbergii Siebold & Zucc. 이질풀 aerial part 1 D 97 Geum aleppicum Jacq. 큰뱀무 aerial part 2 E 98 Geum japonicum Thunb. 뱀무 leaf/stem 1 D 99 Gleditsia japonica Mig. 주엽나무 thorn 1 D 100 Gleditsia japonica Mig. 주엽나무 thorn 1 A 101 Glycine max Merr. 콩 sprouted seed 14 E 102 Glycine max Merr. 콩 sprouted seed 14 A 103 Glycyrrhiza uralensis Fischer 감초 root 14 E 104 Glycyrrhiza uralensis Fischer 감초 root 14 A 105 Gossyium hirsutum Lam. 목화 root 1 A 106 Gossyium hirsutum Lam. 목화 aerial part 1 A 107 Gossyium hirsutum Lam. 목화 seed 1 A -436-
콜린에스테라제저해효과보유식물추출물탐색 Table 1. Continued Sample No. Scientific name Korean name Used part Resource z Extract condition y 108 Hibiscus manihot L. 닥풀 root 1 A 109 Hibiscus manihot L. 닥풀 aerial part 1 A 110 Hibiscus manihot L. 닥풀 flower 1 A 111 Iris germariea L. 독일붓꽃 aerial part 1 A 112 Iris germariea L. 독일붓꽃 root 1 D 113 Iris koreana Nakai 노랑붓꽃 root 1 A 114 Iris koreana Nakai 노랑붓꽃 aerial part 1 A 115 Ledebouriella seseloides (Hoffm.) H.Wolff 방풍 root 1 A 116 Ledebouriella seseloides (Hoffm.) H.Wolff 방풍 root 1 D 117 Ligularia fischeri (Ledeb.) Turcz. 곰취 aerial part 2 C 118 Linum usitatissimum L. 아마 aerial part 1 A 119 Linum usitatissimum L. 아마 fruit 1 A 120 Euphorbia lathyris L. 속수자 fruit 1 A 121 Lycium chinense Mill 구기자나무 fruit 1 D 122 Lycium chinense Mill 구기자나무 fruit 1 A 123 Lythrum salicaria L. 털부처꽃 aerial part 1 A 124 Lythrum salicaria L. 털부처꽃 aerial part 1 D 125 Lythrum salicaria L. 털부처꽃 flower 1 A 126 Magnolia obovata Thumb. 일본목련 bark 4 E 127 Magnolia obovata Thumb. 일본목련 bark 4 A 128 Melissa officinalis L. 레몬밤 aerial part 1 D 129 Nelumbo nucifera Gaertn. 연꽃 stamen/ovary 2 D 130 Oenothera odorata 달맞이꽃 seed 1 D 131 Onoclea sensibilis var. interrupta Maxim. 야산고비 aerial part 1 H 132 Patrinia scabiosaefolia Fisch. ex. Trevir. 마타리 root 1 A 133 Patrinia scabiosaefolia Fisch. ex. Trevir. 마타리 aerial part 1 A 134 Paulownia coreana Uyeki 오동나무 leaf 1 H 135 Paulownia coreana Uyeki 오동나무 fruit 1 H 136 Penthorum chinense Pursh 낙지다리 aerial part 1 A 137 Penthorum chinense Pursh 낙지다리 root 1 A 138 Persicaria orientalis (L.) Spach 털여뀌 leaf 2 E 139 Persicaria orientalis (L.) Spach 털여뀌 stem 1 H 140 Persicaria tinctoria (Aiton) H. Gross 쪽 flower 1 H 141 Peucedanum japonicum Thunberg 갯기름나물 root 1 D 142 Peucedanum japonicum Thunberg 갯기름나물 root 1 A 143 Peucedanum japonicum Thunberg 갯기름나물 aerial part 1 D 144 Phellaodendron amurense Rupr. 황벽나무 bark 13 H 145 Phellaodendron amurense Rupr. 황벽나무 bark 13 G 146 Physalis angulata L. 땅꽈리 aerial part 1 D 147 Physalis angulata L. 땅꽈리 fruit 1 F 148 Physalis angulata L. 땅꽈리 root 1 D 149 Phytolacca americana L. 미국자리공 flower 1 A 150 Phytolacca americana L. 미국자리공 aerial part 1 A 151 Pinellia ternata (Thunb.) Breitenb. 반하 whole plant 1 H 152 Plantago lanceolata L. 창질경이 root 1 D 153 Plantago lanceolata L. 창질경이 aerial part 1 D -437-
Korean J. Plant Res. 31(5) : 433~452(2018) Table 1. Continued Sample No. Scientific name Korean name Used part Resource z Extract condition y 154 Plantago lanceolata L. 창질경이 flower 1 D 155 Plantago lanceolata L. 창질경이 aerial part 1 H 156 Platycodon grandiflorum (Jacg.) A. DC. 도라지 root 14 I 157 Polygonatum sibiricum F. Delaroche 층층갈고리둥글레 root 11 B 158 Potentilla chinensis SER 딱지꽃 aerial part 1 D 159 Potentilla chinensis SER 딱지꽃 root 1 D 160 Potentilla chinensis SER 딱지꽃 stem 1 D 161 Prunus aremeniaca Linne 살구나무 seed 14 I 162 Prunus persica (L.) Batsch 복사나무 seed 16 E 163 Prunus persica (L.) Batsch 복사나무 seed 16 A 164 Rosa rugosa Thunb. 해당화 fruit 1 I 165 Rosa rugosa Thunb. 해당화 seed 1 H 166 Rosa rugsa Thunb. 해당화 leaf 1 H 167 Rumex acetocella L. 애기수영 whole plant 1 A 168 Rumex acetocella L. 애기수영 aerial part 1 D 169 Rumex acetosa L. 수영 root 1 A 170 Rumex acetosa L. 수영 aerial part 1 A 171 Rumex obtusifolius L. 돌소리쟁이 flower 1 A 172 Salvia plebeia 배암차즈기 whole plant 1 D 173 Schisandra chinensis Baill 오미자 fruit 12 E 174 Schisandra chinensis Baill 오미자 fruit 13 A 175 Scrophularia buergeriana Miq. 현삼 aerial part 1 B 176 Scrophularia buergeriana Miq. 현삼 aerial part 1 D 177 Silene armeria L. 끈끈이대나물 flower 1 D 178 Silybum marianum 흰무늬엉겅퀴 aerial part 1 D 179 Synurus deltoides (Aiton) Nakai 수리취 aerial part 1 D 180 Thalictrum aquilegifolium var. sibiricum Regel & Tiling 뀡의다리 stem 1 D 181 Trichosanthes kirilowii Maxim. 하늘타리 seed - D 182 Trichosanthes kirilowii Maxim. 하늘타리 seed - A 183 Veronicastrum sibiricum (L.) Pennell 냉초 leaf 1 H 184 Veronicastrum sibiricum (L.) Pennell 냉초 stem 1 H z 1, Suwon (RDA); 2. Eumsung (RDA); 3, Bongwha; 4, Boryeong; 5, Cheongwon; 6, Teabeaksan; 7, Imsil; 8, Bosung; 9, Uiseong; 10, Odaesan; 11, Yeongju; 12, Jecheon; 13, Korea; 14, China; 15, Vietnam; 16, Iran y Solvents and conditions for extraction ; A, water at room temperature; B, methanol at room temperature; C, ethanol at room temperature; D, methanol at 50 by accelerated solvent system; E, ethanol at 50 by accelerated solvent system; F, ethanol at 85 by accelerated solvent system, G, water at 100 ; H, methanol at 74 ; I, 70% methanol at 74. 시약실험에사용된 sodium phosphate dibasic, sodium phosphate monobasic, acetylcholinesterase, acetylthiocholine iodide, 5,5'-dithiobis-(2-nitrobenzoic acid), sodium bicarbonate, butyrylcholinesterase, butyrylthiocholine iodide 및 tacrine hydrochloride 등의시약은 Sigma-Aldrich Co. 에서구매하였다. 단일농도에서의아세틸콜린에스테라제 (AChE) 저해활성측정확보된 184개의식물추출물의아세틸콜린에스테라제저해능을 100 μg / ml의단일농도에서평가하기위해 Ellman 방법을 -438-
콜린에스테라제저해효과보유식물추출물탐색 변형한 Kim and Chung (2011) 의방법에따라실험하였다. 먼저 100 mm sodium phosphate buffer (ph 8.0) 는 sodium phosphate dibasic, sodium phosphate monobasic 을각각 100 mm로만들어 ph meter을이용하여 ph 8.0 으로맞춰사용하였으며, acetylcholinesterase 는 10 unit 농도로 50 μl씩나누어냉동보관하면서 100 mm sodium phosphate buffer (ph 8.0) 950 μl를넣어최종농도 0.5 U/ ml의농도가되게하여사용하였다. 또한, 기질인 75 mm acetylthiocholine iodide 는 buffer (ph 8.0) 에녹여서사용하였고, 양성대조물질인 tacrine 은 0.1 μg / ml농도로녹여사용하였다. DTNB 시약을제조하기위해 10 mm 5,5'-dithiobis-(2-nitrobenzoic acid), DTNB) 와 15 mm sodium bicarbonate 는각각 100 mm sodium phosphate buffer (ph 8.0) 에녹여 1:1 비율로혼합하였다. 최종농도를 100 μg / ml이되도록준비한추출물시료와 positive control인 tacrine, control인 buffer을각각 75 μl씩 96 well microplate 에분주한후 buffer 130 μl, 10 mm DNTB 시약 5 μl, 75 mm acetylthiocholine iodide 1 μl를차례대로분주하여상온에서 10분간반응시키고 AChE 효소 2.5 μl를분주하여 1시간반응시킨후 microplate reader (BIO-TEK, USA) 를이용하여 410 nm에서흡광도를측정하였고결과는 control 군대비저해율 (%) 로표시하였다. Inhibition (%) = 1-(Abs/Abc) 100 Abs = Absorbance of tested sample after 60 min of incubation Abc = Absorbance of control after 60 min of incubation 선발자원의아세틸콜린에스테라제 (AChE) 저해활성측정단일농도실험결과로부터선발한 7개의추출물의처리농도별아세틸콜린에스테라제 (AChE) 저해활성을확인하기위한실험을수행하였다. 선발시료추출물의최종농도가 25, 50, 100, 200 μg / ml가되도록조제한후앞서언급한 AChE 저해활성측정방법에준해실험하였다. 선발자원의부티릴콜린에스테라제 (BuChE) 저해활성측정선발자원 7종에대한농도별부티릴콜린에스테라제 (BuChE) 에대한저해활성을확인하기위해앞에서서술한 AChE 저해활성측정방법에준해실험하였다. Butyrylcholinesterase (BuChE) 는 10 unit 농도로 50 μl씩나누어냉동보관하면서 100 mm sodium phosphate buffer (ph 8.0) 950 μl를넣어최종농도 0.5 U/ ml로사용하였고 buffer 로 75 mm의 butyrylthiocholine iodide 와 0.1 μg / ml의 tacrine (positive control) 을만들어사용하였다. 최종농도가 25, 50, 100, 200 μg / ml이되게조제한각선발시료와 tacrine 를각각 75 μl를 96 well microplate 에분주한후 buffer (control) 130 μl, DNTB 시약 5 μl및 75 mm butyrylthiocholine iodide 1 μl를차례대로분주하여상온에서 10분간반응시킨후 BuChE 효소 2.5 μl를분주하여 1시간반응시키고, ELISA reader 를이용하여 410 nm에흡광도를측정하였다. 실험결과는 AChE 저해활성분석법과같은방식으로 control 대비저해율 (%) 로표시하였다. 선발자원의 UPLC-UV-MS 프로파일분석선발자원의성분프로파일을확인하기위해선발시료를 UPLC (Waters Acquity Ultra Performance LC system, Waters Corp., Milford, USA) 와 Waters Xevo G2 Q-TOF (Waters Corp., Milford, USA) 를사용하고컬럼 (ACQUITY UPLC BEH C18 column (2.1 mm 150 mm, 1.7 μm, Waters Corp., Milford, USA) 을이용하여각식물별로 Table 2와 Table 3에나타낸조건으로분석하였다. 통계처리모든실험결과는평균 ± 표준편차 (means±sd, n=3 4) 로나타내었으며, Microsoft excel 의 Student s t-test 에의해유의성을검정하였다. 결과및고찰단일농도에서의 AChE 저해활성콜린기능의결손은인지, 행동및일상생활활동에영향을미치는알츠하이머병 (Alzheimer s disease, AD) 의발병에관련되며, 이러한결손에대한약물적중재는아세틸콜린에스테라제 (Acetylcholinesterase, AChE) 저해에기초하므로확보된식물추출물 184개를 100 μg / ml의단일농도에서 AChE 저해활성을분석하였다 (Giacobin, 2003). 실험결과, 52개의추출물이 0% 이상의 AChE 저해활성을나타내었으며, 나머지 132개의시료는음의값을나타내 AChE 효소에대한저해효과가없었다 (Table 4). 0% 이상의 AChE 저해활성을보였던 52개의시료중에서는시료번호 144 ( 황벽나무, Phellaodendron amurense Rupr., 수피, 메탄올추출물 ), 시료번호 145 ( 황벽나무, Phellaodendron amurense Rupr., 수피, 물추출물 ), 시료번호 129 ( 연꽃, Nelumbo nucifera Gaertn., 수술 / 자방, 메탄올추출물 ), 시료번호 140 ( 쪽, Persicaria tinctoria H. GROSS, 꽃, 메탄올추출 -439-
Korean J. Plant Res. 31(5) : 433~452(2018) Table 2. UPLC analysis conditions of the selected plant extracts of sample no. 60 (Carthamus tinctorius L. fruit), sample no. 66 (Cinnamomum cassia Blume, bark), sample no. 73 (Coptis chinensis, rhizome), sample no. 129 (Nelumbo nucifera Gaertn., stamen/ovary), sample no. 140 (Persicaria tinctoria H.GROSS, flower), sample no. 144 (Phellaodendron amurense Rupr., bark, methanol ext.), and sample no. 145 (Phellaodendron amurense Rupr., bark, water ext.) Sample No. 60 66 73 129 140 144 145 Solvent for dissolving sample 10 mg / ml in 50% MeOH 1 mg / ml in MeOH 0.25 mg / ml in MeOH 10 mg / ml in MeOH 10 mg / ml in MeOH 10 mg / ml in MeOH 10 mg / ml in MeOH Injection volume ( μl ) 2 4 3 2 2 2 2 Eluent solvent A : 0.1% formic acid in water B : 0.1% formic acid in acetonitrile A : 0.1% formic acid in water B : 0.1% formic acid in acetonitrile A : 0.1% formic acid in water B : 0.1% formic acid in acetonitrile A : 0.1% formic acid in water B : 0.1% formic acid in acetonitrile A : 0.1% formic acid in water B : 0.1% formic acid in acetonitrile A : 0.1% formic acid in water B : 0.1% formic acid in acetonitrile A : 0.1% formic acid in water B : 0.1% formic acid in acetonitrile Eluent condition 0-5 min; 10% (B) 5-10 min; 10-15% (B) 10-20 min; 15-25% (B) 20-25 min; 25-100% (B) 25-27 min; 100% (B) 27-27.5 min; 100-10% (B) 27.5-30 min; 10% (B) 0-1 min; 15% (B) 1-20 min; 15-40% (B) 20-26 min; 40-100% (B) 26-28 min; 100% (B) 28-28.5 min; 100-15% (B) 28.5-30 min; 15% (B) 0-5 min; 10% (B) 5-10 min; 10-15% (B) 10-20 min; 15% (B) 20-28 min; 15-100% (B) 28-28.5 min; 100-10% (B) 28.5-30 min; 10% (B) 0-20 min; 10% (B) 20-30 min; 10-100% (B) 30-33 min; 100% (B) 33-33.5 min; 100-10% (B) 33.5-35 min; 10% (B) 0-1 min; 10% (B) 1-20 min; 10-20% (B) 20-30 min; 20-100% (B) 30-33 min; 100% (B) 33-33.5 min; 100-10% (B) 33.5-35 min; 10% (B) 0-5 min; 5% (B) 5-10 min; 5-10% (B) 10-20 min; 10-20% (B) 20-25 min; 20-100% (B) 25-27 min; 100% 27-27.5 min; 100-5% (B) 27.5-30 min; 5% (B) 0-5 min; 5% (B) 5-10 min; 5-10% (B) 10-20 min; 10-20% (B) 20-25 min; 20-100% (B) 25-27 min; 100% (B) 27.5-30 min; 5% (B) Flow ( ml /min) UV wavelength ( nm ) 0.3 290 0.30 270 0.3 320 0.3 340 0.3 340 0.3 280 0.3 280-440-
콜린에스테라제저해효과보유식물추출물탐색 Table 3. The method of operation at mass spectrometer Voltage Temperature ( ) Gas Flow (L/h) Mode Range (m/z) Capillary (kv) Cone (V) Source Desolvation Cone Desolvation Positive 2.5 15 50 350 30 500 50-1200 Table 4. Inhibition effect of 184 plant extracts (at 100 μg / ml ) on the acetylcholinesterase activity Sample No. Inhibition (%) at 100 μg / ml Sample No. Inhibition (%) at 100 μg / ml 144 63.1 ± 1.3 *** y 20 3.4 ± 1.7 * 145 61.5 ± 1.7 *** 38 3.2 ± 5.7 129 59.5 ± 3.5 *** 103 3.2 ± 0.6 *** 140 45.6 ± 4.3 *** 53 3.1 ± 1.9 73 38.3 ± 0.7 *** 108 3.1 ± 1.1 *** 66 22.1 ± 3.1 *** 141 3.0 ± 2.6 60 18.7 ± 7.8 * 142 3.0 ± 1.4 * 96 9.7 ± 1.3 *** 65 2.3 ± 6.3 157 9.4 ± 3.7 * 91 2.3 ± 1.4 *** 123 9.3 ± 6.9 109 2.3 ± 0.8 *** 68 8.2 ± 1.4 *** 98 2.0 ± 5.4 71 7.5 ± 1.4 *** 45 1.9 ± 4.6 171 6.7 ± 1.2 *** 72 1.8 ± 4.0 *** 46 6.6 ± 2.2 ** 126 1.6 ± 0.8 * 67 6.6 ± 1.7 ** 47 1.5 ± 2.9 162 6.6 ± 0.8 *** 88 1.4 ± 1.2 *** 152 6.4 ± 2.1 *** 124 1.4 ± 6.8 58 6.3 ± 1.7 *** 25 1.3 ± 1.1 16 6.1 ± 1.1 *** 18 1.2 ± 1.0 *** 62 5.7 ± 7.4 59 1.2 ± 11.9 153 5.3 ± 1.1 ** 48 0.6 ± 6.3 101 5.1 ± 5.3 113 0.5 ± 2.7 78 4.6 ± 1.5 *** 69 0.4 ± 1.7 *** 181 3.6 ± 0.9 ** 146 0.3 ± 6.7 105 0.1 ± 2.3 *** 159-2.8 ± 2.4 7 0.0 ± 1.4 *** 19-2.9 ± 0.4 *** 21 0.0 ± 10.1 137-2.9 ± 3.7 *** 83 0.0 ± 1.0-175 -2.9 ± 3.9 180-0.2 ± 2.2 29-3.1 ± 1.8 * 173-0.4 ± 2.0-13 -3.2 ± 1.0 *** 1-0.4 ± 0.4 63-3.2 ± 4.4 132-0.6 ± 1.6 104-3.2 ± 1.0 *** 80-0.7 ± 3.3 166-3.2 ± 5.7 155-0.7 ± 1.4 147-3.2 ± 4.2 120-0.8 ± 1.6 *** 139-3.3 ± 2.0 136-0.8 ± 0.3 *** 70-3.4 ± 5.3 164-0.9 ± 2.8 118-3.4 ± 1.3 ** 77-1.2 ± 1.5 84-3.5 ± 3.1 79-1.3 ± 0.6 *** 9-3.7 ± 1.4 *** 8-1.4 ± 0.6 *** 51-3.7 ± 3.9 27-1.5 ± 0.3 *** 174-3.7 ± 1.3 ** -441-
Korean J. Plant Res. 31(5) : 433~452(2018) Table 4. Continued Sample No. Inhibition (%) at 100 μg / ml Sample No. Inhibition (%) at 100 μg / ml 158-1.6 ± 0.2 *** 75-3.8 ± 1.3 *** 15-1.7 ± 0.7 * 143-3.8 ± 8.3 106-1.9 ± 1.0 *** 169-3.8 ± 3.0 *** 154-1.9 ± 1.3 *** 34-3.9 ± 3.3 127-2.1 ± 1.1 * 165-3.9 ± 2.0 * 167-2.4 ± 5.6 148-4.0 ± 0.4 110-2.6 ± 2.4 52-4.0 ± 2.8 * 28-2.7 ± 2.8 111-4.0 ± 2.5 2-2.8 ± 0.9 *** 112-4.2 ± 1.5 ** 128-4.4 ± 4.5 10-7.4 ± 3.0 *** 130-4.4 ± 10.1 90-7.4 ± 0.3 *** 32-4.5 ± 1.5 *** 100-7.4 ± 3.6 * 56-4.5 ± 1.7 *** 119-7.5 ± 3.2 *** 17-4.6 ± 1.2 *** 87-8.3 ± 1.5 *** 89-4.8 ± 1.2 ** 55-8.4 ± 2.3 ** 156-4.8 ± 6.6 122-8.6 ± 1.6 *** 115-5.0 ± 6.2 4-8.6 ± 1.8 * 30-5.0 ± 0.9 *** 33-8.7 ± 1.6 *** 170-5.2 ± 1.1 ** 151-8.7 ± 2.8 ** 97-5.4 ± 1.3 ** 183-8.7 ± 1.2 *** 26-5.5 ± 2.0 * 82-9.1 ± 2.1 ** 57-5.9 ± 3.8 *** 179-9.2 ± 5.8 3-6.1 ± 0.9 *** 172-9.3 ± 1.7 *** 23-6.4 ± 2.7 * 92-9.5 ± 3.9 *** 54-6.5 ± 0.7 *** 138-10.6 ± 3.3 * 76-6.6 ± 1.4 *** 133-10.8 ± 2.1 *** 178-6.6 ± 6.8 39-11.3 ± 1.9 *** 116-6.9 ± 4.8 149-11.4 ± 0.7 *** 81-6.9 ± 7.1 11-11.5 ± 1.4 *** 40-7.1 ± 1.2 *** 176-11.6 ± 3.6 ** 49-7.1 ± 6.1 74-11.9 ± 3.6 ** 99-7.2 ± 4.2 * 37-12.1 ± 0.6 *** 121-7.2 ± 1.8 ** 36-12.1 ± 3.6 ** 50-7.3 ± 3.4 * 86-12.3 ± 7.6 * 114-7.3 ± 1.9 ** 85-19.6 ± 3.7 *** 41-13.4 ± 1.6 *** 160-20.1 ± 8.0 * 31-13.7 ± 0.4 *** 94-20.6 ± 6.3 ** 161-14.7 ± 7.8 * 150-20.8 ± 2.3 *** 134-14.8 ± 0.8 *** 93-21.1 ± 1.2 *** 107-15.7 ± 1.9 *** 22-21.9 ± 5.6 ** 125-15.7 ± 9.7 * 95-23.9 ± 1.2 *** 182-15.7 ± 2.3 *** 44-26.2 ± 4.8 ** 117-16.1 ± 5.5 ** 61-27.2 ± 6.8 ** 43-16.8 ± 10.0 * 5-13.0 ± 5.6 * 135-17.1 ± 0.5 *** 102-30.2 ± 0.9 *** 184-17.3 ± 1.6 *** 163-31.5 ± 0.7 *** 64-17.9 ± 4.0 * 6-32.2 ± 5.2 ** -442-
콜린에스테라제저해효과보유식물추출물탐색 Table 4. Continued Sample No. Inhibition (%) at 100 μg / ml Sample No. Inhibition (%) at 100 μg / ml 12-18.0 ± 1.5 *** 24-33.2 ± 4.9 *** 177-18.3 ± 7.3 * 14-36.5 ± 5.0 *** 131-18.4 ± 3.2 *** 168-39.3 ± 12.0 ** 35-19.1 ± 9.5 * 42-44.6 ± 12.5 ** x Symbol indicates significance of the data, *, p < 0.05; **, p < 0.01; ***, p < 0.001. y Final concentration of sample for the assay of acetylcholinesterase activity inhibition was 100 μg / ml. 물 ), 시료번호 73 ( 중국황련, Coptis chinensis, 뿌리줄기, 메탄올추출물 ), 시료번호 66 ( 육계, Cinnamomum cassia Blume, 수피, 에탄올추출물 ) 및시료번호 60 ( 잇꽃, Carthamus tinctorius L., 열매, 에탄올추출물 ) 등 7개의시료는 18.7±7.8%~63.1± 1.3% 의비교적우수한저해활성을보인것을확인하였다. 이러한결과에따라단일농도에서 AChE 효소에대해우수한저해활성을보였던 7개의시료에대해서는처리농도별로추가실험을추진하였다. 선발자원의 AChE 저해활성앞에서언급한바와같이 100 μg / ml의단일농도에서우수한 AChE 저해활성을나타내었던 7개시료를대상으로최종처리농도가 25, 50, 100, 200 μg / ml이되도록처리하여 AChE 저해활성을분석하였으며그결과는 Fig. 1에나타내었다. 실험결과를살펴보면, 잇꽃 (Carthamus tinctorius L.) 열매의에탄올추출물 (A), 육계 (Cinnamomum cassia Blume) 수피의에탄올추출물 (B) 은처리농도에따른 AChE 저해효과가각각 2.6±5.2~12.8± 4.3% 및 9.1±1.3%~15.2±3.2% 를보여전반적으로낮은수치를보였다. 또한연꽃 (Nelumbo nucifera Gaertn.) 수술 / 자방의메탄올추출물 (D) 은 25, 50, 100, 200 μg / ml의처리농도에서 15.0±3.4%~73.5±3.6%, 쪽 (Persicaria tinctoria H. GROSS) 꽃의메탄올추출물 (E) 는 19.5±9.1%~63.5±5.4% 의 AChE 저해활성을보여처리농도가증가함에따라 AChE 저해활성이유의하게증가하는결과를보였다. 그리고, 중국황련 (Coptis chinensis) 뿌리줄기의메탄올추출물 (C), 황벽나무 (Phellaodendron amurense Rupr.) 수피의메탄올추출물 (F) 및물추출물 (G) 은처리농도에따라각각 81.6±0.4%~58.5±2.4%, 69.9± 1.8%~80.5±0.9% 및 54.8±0.6%~78.3±2.6% 의값을보여처리농도에따라고르게매우높은저해활성을나타내는것을알수있었다. Tabet (2006) 에의하면 AChE 저해제는약 ~ 중정도의 AD 치료에유용하게이용될수있으므로처리농도별 AChE 저해능이 우수하였던연꽃 (Nelumbo nucifera Gaertn.) 수술 / 자방의메탄올추출물 (D), 쪽 (Persicaria tinctoria H. GROSS) 꽃의메탄올추출물 (E), 중국황련 (Coptis chinensis) 뿌리줄기의메탄올추출물 (C), 황벽나무 (Phellaodendron amurense Rupr.) 수피의메탄올추출물 (F) 및물추출물 (G) 은경증내지중증의 AD 치료용소재로의활용가능성이높을것으로사료되었다. 선발자원의 BuChE 저해활성아세틸콜린에스테라제 (AChE) 와마찬가지로아세틸콜린의가수분해에관여하고 AD에서중요한역할을담당하는또다른콜린에스테라제 (ChE) 인부티릴콜린에스테라제 (BuChE) 에대한저해활성을확인하기위해단일농도에서우수한 AChE 저해활성을나타내었던 7개의시료를 25, 50, 100, 200 μg / ml의최종처리농도로실험하였다 (Giobini et al., 2002). 그결과, Fig. 2 에나타낸바와같이 7개의시료중에서연꽃 (Nelumbo nucifera Gaertn.) 수술 / 자방의메탄올추출물 (D), 쪽 (Persicaria tinctoria H.GROSS) 꽃의메탄올추출물 (E) 및중국황련 (Coptis chinensis) 뿌리줄기의메탄올추출물 (C) 이각각 58.9±7.8~81.6±1.9% 및 45.8±9.8%~72.4±2.5% 및 33.1±9.9%~55.4±5.4% 의순으로높은 BuChE 저해활성을보이는것을알수있었다. 한편, 잇꽃 (Carthamus tinctorius L.) 열매의에탄올추출물 (A), 육계 (Cinnamomum cassia Blume) 수피의에탄올추출물 (B), 황벽나무 (Phellaodendron amurense Rupr.) 수피의메탄올추출물 (F) 및물추출물 (G) 은 19.4±5.6%~12.7±12.7% 범위의비교적낮은수치를보여 BuChE 에대한저해활성이낮은것으로확인되었다. 한편, Giacobini (2003) 에의하면심각한 AD에서는 AChE 와 choline acetyltransferase (ChAT) 의 90% 정도가감소되는반면, 부티릴콜린에스테라제 (butyrylcholinesterase, BuChE) 의수준은증가하므로본실험결과, 우수한 BuChE 저해효과가확인된연꽃 (Nelumbo nucifera Gaertn.) 수술 / 자방의메탄올추출물 (D), 쪽 (Persicaria tinctoria H. GROSS) 꽃의메탄올추출 -443-
Korean J. Plant Res. 31(5) : 433~452(2018) Fig. 1. Inhibition effect of the selected plant extracts of (A) Carthamus tinctorius L. (fruit, sample no. 60), (B) Cinnamomum cassia Blume (bark, sample no. 66), (C) Coptis chinensis (rhizome, sample no. 73), (D) Nelumbo nucifera Gaertn. (stamen/ovary, sample no. 129), (E) Persicaria tinctoria H.GROSS (flower, sample no. 140), (F) Phellaodendron amurense Rupr. (bark, sample no. 144, methanol ext.), and (G) Phellaodendron amurense Rupr. (bark, sample no. 145, water ext.) on acetylcholinesterase (AChE) activity. Each extracts was experimented at the final concentration of 25, 50, 100, and 200 μg / ml. Cont means control which treated with 100 mm sodium phosphate buffer (ph 8.0) and tacrine as positive control was used at the concentration of 0.1 μg / ml. -444-
콜린에스테라제저해효과보유식물추출물탐색 Fig. 2. Inhibition effect of the selected plant extracts of (A) Carthamus tinctorius L. (fruit, sample no. 60), (B) Cinnamomum cassia Blume (bark, sample no. 66), (C) Coptis chinensis (rhizome, sample no. 73), (D) Nelumbo nucifera Gaertn. (stamen/ovary, sample no. 129), (E) Persicaria tinctoria H.GROSS (flower, sample no. 140), (F) Phellaodendron amurense Rupr. (bark, sample no. 144, methanol ext.), and (G) Phellaodendron amurense Rupr. (bark, sample no. 145, water ext.) on butyrylcholinesterase (BuChE) activity. Each extracts was experimented at the final concentration of 25, 50, 100, and 200 μg / ml. Cont means control which treated with 100 mm sodium phosphate buffer (ph 8.0) and tacrine as positive control was used at the concentration of 0.1 μg / ml. -445-
Korean J. Plant Res. 31(5) : 433~452(2018) 물 (E) 및중국황련 (Coptis chinensis) 뿌리줄기의메탄올추출물 (C) 은심각한수준의 AD 치료에이용될수있는후보소재라고사료되었다. 이상과같이본실험에서는치매와관련하여아세틸콜린에스테라제 (AChE) 및부티릴콜린에스테라제 (BuChE) 등콜린성에스테라제에대해식물추출물들의저해활성을분석하였으므로이와관련된연구보고를살펴보고자한다. 치매의한종류인알츠하이머병 (AD) 은전뇌의콜린성뉴런손실과점진적아세틸콜린 (ACh) 의감소가특징이며, 아세틸콜린의활성은콜린에스테라제의가수분해작용에의해종결된다. 따라서, 이들효소에대한저해물질은 AD를가진환자에서콜린성뉴런의활성을증폭시키며, 뇌의 BuChE를저해시키면 AD를치료하여인지및신경병리학적인마커를조절할수있다 (Greig et al., 2005). 콜린계는신경전달계로서대뇌피질을조절하며도파민, 노르에피네프린및글루타메이트계에영향을미치고, 콜린계기능의결손은인지, 행동및일상생활활동에영향을미치는알츠하이머병 (Alzheimer s disease, AD) 의발병에관련된다. 심각한 AD 에서는 AChE 와 choline acetyltransferase (ChAT) 의 90% 가감소되지만, 부티릴콜린에스테라제 (BuChE) 의수준은증가한다. 그러나베타- 아밀로이드를함유한노인성프라크에는 AChE 와 BuChE 가함께있고로위체를가진치매에서심각한콜린계손상이발견되므로, 파키슨성치매에대해 AChE 저해요법이적절할것으로보고되고있다 (Giacobini, 2003). 한편, AChE 는콜린성신경말단에존재하는반면, BuChE 는신경교세포나신경단위와관련되는데, AChE 는정상뇌의측두골피질에있는 total ChE 의 90% 를구성하면서대부분의시냅스성아세틸콜린의불활성화를매개하고있으나, BuChE 도아세틸콜린의가수분해에관여하고 AD에서중요한역할을담당하므로 AChE 와 BuChE 에대한저해는 AD에서인지저하를치료하는데유익할것으로보고되었다 (Giaobini et al., 2002). 또한, AD 의발병에서중요한역할을하는 Aβ의독성은 H 2 O 2 등의활성산소 (ROS) 증가와관련되며, 활성산소와 H 2 O 2 는미토콘드리아기능에직접영향을미치는것으로알려져있다. 또한, 항산화제나유리기소거제는 Aβ에의해유도되는독성에대해세포보호를하며 AD 환자에서인지결함의정도는 ChAT 활성의감소와콜린성뉴런의손실과유의하게관련된다. 그러므로활성산소생성을감소시키고 AChE 활성저해를통한 ACh 의생성을증가시키며 ChAT 활성을증가시키는것은 AD 치료를위한중요한전략이될수있다 (Ruan et al., 2010). AChE 저해제는경중정도의 AD 치료에유용하게이용될수있으므로대부분의환자에게잘통용 되며, 주요용도는인지저하의안정화에있지만치매에서의행동학적정신적증상을개선한다. 또한, 유리기와아밀로이드독성에대한작용과뇌와혈액에서활성화된소교세포 (microglia) 로부터사이토카인의분비감소를통해서 AChE inhibitor 의항염작용이가능하다 (Tabet, 2006). 이러한보고들을고려할때, 본연구에서확인되었던 AChE 에대한저해활성이우수하였던연꽃 (Nelumbo nucifera Gaertn.) 수술 / 자방의메탄올추출물, 쪽 (Persicaria tinctoria H. GROSS) 꽃의메탄올추출물, 중국황련 (Coptis chinensis) 뿌리줄기의메탄올추출물, 황벽나무 (Phellaodendron amurense Rupr.) 수피의메탄올추출물및물추출물등의 5가지추출물시료는경도부터중간정도의 AD에서효과를발휘할수있을것으로예상된다. 그리고, BuChE 에대해서도저해효과가우수하였던연꽃 (Nelumbo nucifera Gaertn.) 수술 / 자방의메탄올추출물, 쪽 (Persicaria tinctoria H.GROSS) 꽃의메탄올추출물및중국황련 (Coptis chinensis) 뿌리줄기의메탄올추출물등 3개의식물추출물은심각한 AD에서도개선효과를나타낼수있는소재로확인되었으므로활용성검토를위해추가연구가필요하다고사료되었다. 선발자원의 UPLC-UV-MS 화학성분프로파일분석결과한편, 선발된추출물 7개에대한 UPLC-UV-MS 분석을통한화학성분프로파일예측을진행한결과를 Fig. 3 및 Fig. 4 에나타내었다. 본실험은기존문헌에보고된개별식물에함유된화학성분의분자량정보를활용하여동일분자량을가지는피크들을임의적으로동일화학성분으로추정하는방식으로진행되었다. 시료번호 60 ( 잇꽃열매의에탄올추출물 ) 에대해분석한 UPLC-UV 크로마토그램 (A) 을분석하였을때 arachidonoylserotonin (MW 462), n-serotonin (MW 322) 및 n-feruroylserotonin (MW 352) 로추정되는화학성분이확인되었으며, 시료번호 66 ( 육계수피의에탄올추출물 ) 의경우 (B) 에는 cinnamic acid (MW 148) 및 cinnamaldehyde (MW 132) 가확인되었다. 시료번호 73 ( 중국황련뿌리줄기의메탄올추출물 ) 의경우 (C) 는 thalifendine (MW 322), jateorrhizine (MW 338), columnbammine (MW 338), coptisine (MW 320), berberine (MW 336), palmitine (MW 352) 로추정되는화학성분이확인되었으며, 시료번호 129 ( 연꽃수술 / 자방의메탄올추출물 ) 의 UPLC- UV 크로마토그램 (D) 에서는 kaempferol-6-methoxy-3-oglucoside (MW 478), kaempferol-3-o-glucoside (MW 448), kaempferol-3-o-glucuronide (MW 462) 및 kaempferol-3- -446-
콜린에스테라제저해효과보유식물추출물탐색 (A) (B) (C) (D) (E) (F) (G) Fig. 3. UPLC-UV chromatograms and tentative identification of metabolites obtained by UPLC-UV-MS analysis. (A) Carthamus tinctorius L. (fruit, sample no. 60), (B) Cinnamomum cassia Blume (bark, sample no. 66), (C) Coptis chinensis (rhizome, sample no. 73), (D) Nelumbo nucifera Gaertn. (stamen/ovary, sample no. 129), (E) Persicaria tinctoria H.GROSS (flower, sample no. 140), (F) Phellaodendron amurense Rupr. (bark, sample no. 144, methanol ext.), and (G) Phellaodendron amurense Rupr. (bark, sample no. 145, water ext.). -447-
Korean J. Plant Res. 31(5) : 433~452(2018) (A-1) (A-2) (A-3) (A-4) (B-1) (B-2) (C-1) (C-2) (C-3) Fig. 4. Mass spectrums of selected plant extracts. (A) Carthamus tinctorius L. (fruit, sample no. 60), (B) Cinnamomum cassia Blume (bark, sample no. 66), (C) Coptis chinensis (rhizome, sample no. 73), (D) Nelumbo nucifera Gaertn. (stamen/ovary, sample no. 129), (E) Persicaria tinctoria H.GROSS (flower, sample no. 140), (F) Phellaodendron amurense Rupr. (bark, sample no. 144, methanol ext.), and (G) Phellaodendron amurense Rupr. (bark, sample no. 145, water ext.). -448-
콜린에스테라제저해효과보유식물추출물탐색 (D-1) (D-2) (D-3) (D-4) (E-1) (E-2) (E-3) (E-4) (E-5) Fig. 4. Continued. -449-
Korean J. Plant Res. 31(5) : 433~452(2018) (F-1) (F-2) (F-3) (F-4) (F-5) (G-1) (G-2) (G-3) Fig. 4. Continued. (G-4) -450-
콜린에스테라제저해효과보유식물추출물탐색 (G-5) Fig. 4. Continued. (6" malonylglucoside)-glucoside (MW 696) 으로추정되는화학성분이확인되었다. 또한, 시료번호 140 ( 쪽꽃의메탄올추출물 ) 의 UPLC-UV 크로마토그램 (E) 에서는 quercetin-3-orhamnoside (MW 448), kaempferol-3-o-rhamnoside (MW 432), quercetin-3-o-(4" malonyl) rhamnoside (MW 534), quercetin-3-o-(4" acetyl) rhamnoside (MW 490) 및 kaempferol- 3-O-(3" or 4" acetyl) rhamnoside (MW 471) 로추정되는화학성분확인되었고, 시료번호 144 및 145 ( 황벽나무수피의메탄올추출물과물추출물의크로마토그램인 (F) 와 (G) 에서는 phellodendrine (MW 342), 3-O-feruloyl quinate (MW 368), menispermine (MW 356), plamtine (MW 336) 및 berberine (MW 352) 이검출되었다. 따라서, 이러한 UPLC-UV-MS 분석을통해각선발식물별로기존문헌에서보고된특징적인화학성분이존재하는것으로추정되었다. 적요본연구는천연식물자원을대상으로치매의일종인알츠하이머병을개선및치료할수있는소재를발굴하기위해식물추출물에대해콜린성에스테라제 (AChE) 저해활성을탐색하였다. 184 개의식물추출물을 100 μg / ml의단일농도에서아세틸콜린에스테라제 (AChE) 을실험한결과, 10% 이상의 AChE 저해활성을보였던추출물은황벽나무 (Phellaodendron amurense Rupr., 수피, 메탄올추출물 ), 황벽나무 (Phellaodendron amurense Rupr., 수피, 물추출물 ), 연꽃 (Nelumbo nucifera Gaertn., 수술 / 자방, 메탄올추출물 ), 쪽 (Persicaria tinctoria H. GROSS, 꽃, 메탄올추출물 ), 중국황련 (Coptis chinensis, 뿌리줄기, 메탄올추출물 ), 육계 (Cinnamomum cassia Blume, 수피, 에탄올추출물 ) 및잇꽃 (Carthamus tinctorius L., 열매, 에탄올추출물 ) 등 7개가확인되었다. 선발된 7개의추출물에대해서는 25, 20, 100 및 200 μg / ml의최종농도에서 AChE 및 BuChE 에대한저해활성을검정하였다. 그결과, AChE 저해활성에서는연꽃 (Nelumbo nucifera Gaertn.) 수술 / 자방의메탄올추출물이 15.0±3.4%~73.5±3.6%, 쪽 (Persicaria tinctoria H. GROSS) 꽃의메탄올추출물이 19.5±9.1%~63.5±5.4%, 중국황련 (Coptis chinensis) 뿌리줄기의메탄올추출물이 81.6±0.4%~58.5± 2.4%, 황벽나무 (Phellaodendron amurense Rupr.) 수피의메탄올추출물이 69.9±1.8%~80.5±0.9%, 그리고황벽나무 (Phellaodendron amurense Rupr.) 수피의물추출물이 54.8± 0.6%~78.3±2.6% 의값을보였다. BuChE 저해활성에서는연꽃 (Nelumbo nucifera Gaertn.) 수술 / 자방의메탄올추출물, 쪽 (Persicaria tinctoria H.GROSS) 꽃의메탄올추출물및중국황련 (Coptis chinensis) 뿌리줄기의메탄올추출물이각각 58.9± 7.8~81.6±1.9% 및 45.8±9.8%~72.4±2.5% 및 33.1±9.9%~ 55.4±5.4% 의순으로높은 BuChE 저해활성을나타내었다. 이러한실험결과를종합하여볼때, 연꽃 (Nelumbo nucifera Gaertn.) 의수술 / 자방, 쪽 (Persicaria tinctoria H. GROSS) 의꽃, 중국황련 (Coptis chinensis) 의뿌리줄기, 황벽나무 (Phellaodendron amurense Rupr.) 의수피는알츠하이머병 (AD) 의개선효과를나타낼수있는후보소재로사료되었다. 사사본연구는농촌진흥청시험연구사업 (PJ01193201) 의연구비지원으로수행된결과이며, 이에깊은감사를드립니다. References Alzheimer s Association. 2006. An Overview of Alzheimer s Disease & Related Dementias. Los Angeles, CA (USA). pp. 4-10 (in Korean). -451-
Korean J. Plant Res. 31(5) : 433~452(2018) Cho, K.H., M.S. Oh, H.G. Kim, S.H. Lee, K.S. Chung and A.J. Kim. 2014. Effects of Korean Zingiber mioga R. (flower buds and rhizome) extract on memory. J. Korean Soc. Food Sci. Nutr. 43:1519-1526. Evans, D.A., H.H. Funkenstein, M.S. Albert, P.A. Scherr, N.R. Cook, M.J. Chown, L.E. Hebert, C.H. Hennekens and J.O. Taylor. 1989. Prevalence of Alzheimer s disease in community population of older persons. Higher than previously reported. J. Amer. Med. Assoc. 262:2551-2556. Giacobini, E., 2003. Cholinergic function and Alzheimer s disease. Int. J. Geriatr. Psych. 18:S1-S5.., R. Spiegel, A. Enz, A.E. Veroff and N.R. Cutler. 2002. Inhibition of acetyl- and butyryl-cholinesterase in the cerebrospinal fluid of patients with Alzheimer s disease by rivastigmine: correlation with cognitive benefit. J. Neural. Transm. 109:1053-1065. Greig, N.H., T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.S. Yu, J. Mamczarz, H.W. Holloway, T. Giordano, D. Chen, K. Furukawa, K. Sambamurti, A. Brossi and D. K. Lahiri. 2005. Selective butyrylcholinesterase inhibition elevates brain acetylcholine, augments learning and lowers Alzheimer β-amyloid peptide in rodent. PNASU. 102:17213-17218. Jang, T.W., S.H. Nam and J.H. Park. 2016. Antioxidant activity and inhibitory effect on oxidative DNA damage of ethyl acetate fractions extracted from cone of red pine (Pinus densiflora). Korean J. Plant Res. 29:163-170. Kasa, P., H. Papp and K.P. Torok. 2000. Donepezil dosedependently inhibits acetylcholinesterase activity in various areas and in the presynaptic cholinergic and the postsynaptic cholinoceptive enzyme-positive structures in the human and rat brain. Neuroscience 101:89-100. Kim, D.I., S.H. Lee, E.Y. Hur, S.M. Cho and H.J. Park. 2005. Screening of natural plant resources with acetylcholinesterase inhibition and antioxidant activity. J. Korean Soc. Food Sci. Nutr. 34:427-432. Kim, S.H. and D.K. Chung. 2011. Nootropic and anti-amnestic effect of PPA on scopolamine-induced cognitive impairment in mice. J. Orient. Neuropsych. 22:185-199. Lanctôt, K.L., N. Herrmann, K.K. Yau, L.R. Khan, B.A. Liu, M.M. LouLou and T.R. Einarson. 2003. Efficacy and safety of cholinesterase inhibitors in Alzheimer s disease: a meta analysis. Can. Med. Assoc. J. 169:557-564. Lee, S.E., J. Choi, J.H. Lee, H.J. Noh, G.S. Kim, J. Kim, H.Y. Chung and S.Y. Kim. 2013. Screening of useful plants with anti-inflammatory and antioxidant activity. Korean J. Plant Res. 26:441-449. Lyu, H.N., M.H. Park, S.G. Hong, D.Y. Lee, K.M. Han, J.S. Yoo, S.Y. Kim, Y.D. Rho and N.I. Baek. 2007. Development of biologically active compounds from edible plant sources- ⅩⅩⅤ. Immunostimulating effect of edible plant extracts. Korean J. Food Sci. Technol. 39:708-714. Nordberg, A. 1996. Pharmacological treatment of cognitive dysfunctionin in dementia disorders. Acta. Neurol. Scand., Suppl. 168:87-92. Ruan, C.J., L. Zhang, D.H. Chen, Z. Li, G.H. Du and L. Sun. 2010. Effects of trans-2,4-dimethoxystibene against the neurotoxicity induced by Aβ 25-35 both in vitro and in vivo. Neurosci. Res. 67:209-214. Tabet, N. 2006. Acetylcholinesterase inhibitors for Alzheimer s disease: anti-inflammatories in acetylcholine clothing! Age. Ageing 35:336-338. (Received 5 May 2018 ; Revised 14 August 2018 ; Accepted 14 September 2018) -452-