농업생명과학연구 45(6) pp.213-226 Journal of Agriculture & Life Science 45(6) pp.213-226 천연식물자원으로부터 Acetylcholine esterase 저해활성탐색및인지기능에미치는영향 최장원 1 원무호 2 주한승 3* 대구대학교바이오산업학과 1, 강원대학교신경생물학실 2, 씨앤제이바이오텍 3* Screeening of Natural Plant Resources with Acetylcholine esterase inhibitory activity and Effect on Scopolamine-induced Memory Impairment Jang Won Choi 1 Mu-Ho Won and Han-Seung Joo 3* 초록 1 Dept. of Bioindustry, Daegu University, Gyeongsan 712-714, Korea 2 Dept. of Neurobiology, Kangwon National University, Chuncheon 200-701, Korea 3 C & J Biotech. Bio21 Center, Jinju 660-844, Korea Received: NOV. 14. 2011, Revised: DEC. 16. 2011, Accepted: DEC. 20. 2011 아세틸콜린분해효소 (acetylcholine esterase, AChE) 억제제는아세틸콜린함량을높여콜린성 neuron 을활성화함으로써기억능력의개선및치매개선을가져와현재다양한 AChE 억제제들이개발되어사용되고있다. 본연구에서는 AChE 에대한억제활성을갖는천연물을다양한식물추출물및에센스오일로부터탐색하였으며, 탐색한추출물의 scopolamine 으로기억손상을유발한쥐의기억력개선활성을치매치료제로사용하고있는 donepizil 과비교분석하였다. 그결과자몽 (Citrus paradisi) 유래의에센스오일이 AChE 억제활성이가장높아 20 ug/ml 의농도로처리하였을때 90% 이상의효소억제활성을나타내었다. 수동회피실험결과, 자몽유래의에센스오일 (100 mg/kg, p.o.) 을투여한쥐는치매치료제로사용하고있는 donepizile (0.5 mg/kg) 을투여한쥐와유사한 latency time 을나타내어인지기능이개선되었다. 또한, 수중미로시험결과, 자몽유래에센스오일 (100 mg/kg, p.o.) 을투여한쥐는 donepizile(0.5 mg/kg) 을투여한쥐와유사한 latency time 을나타내어인지기능이개선되었다. 이상의결과로부터자몽유래에센스오일은매우효과적으로기억력을개선하여인지기능을개선해줄수있는안전하고효과적인후보물질이라고사료된다. 검색어 - Acetylcholine esterase, 인지기능개선, 식물추출물, 에센살오일, 저해제 ABSTRACT This study was performed to investigate the effect of essential oils and ethanolic extracts of approximately 650 plant species on acetylcholine esterase (AChE) enzyme activity using Ellman's colorimetric method in 96-well microplates. The results showed that the ethanolic extracts from twig of Sophora subprostrata, twig of Phellodendron amurense, seed of Corylopsis coreana, and essential oil (EO) from Citrus paradisi, Cupressus sempervirens, Ocimum basilicum, Pinus sylvestris and Rosmarinus officinalis inhibited more than 80% of AChE activity. Among these, EO from Pinus sylvestris, C. sempervirens and C paradisi exhibited higher values of AChE inhibitory activity, which were 75, 84 and 99% at a concentration of 50 ug/ml, respectively. Finally, EO from C paradisi (grapefruit, GEO) showed the highest inhibitory activity towards AChE, which showed 91% of inhibition at a concentration of 20 ug/ml. We also examined the anti-dementia effects of GEO in mouse by passive avoidance test and Morris water maze test. The model mouse (male, ICR) of dementia (negative control) was induced by administration of scopolamine (1 mg/kg body weight). The latency time of sample group administrated with GEO (100 mg/kg, p.o.) increased significantly as compared with negative control on passive avoidance test. There were significant recovery from the scopolamine-induced deficits on learning and memory in water maze test through daily administrations with GEO (100 mg/kg, p.o.). From these results, we conclude that GEO treatment might enhance the cognitive function, suggesting that the EO of C. paradis may be a potential candidate for improvement of perceptive ability and dementia. Key words - Acetylcholine esterase, Cognitive function, Essential oil, Plant extract, Inhibitor *Corresponding author: Han-Seung Joo Tel: +82-55-761-7043 Fax: +82-55-761-6043 E-mail: joohs1963@hanmmail.net
214 Journal of Agriculture & Life Science 45(6) Ⅰ. 서론 우리나라의경우고령인구의비중은 9.9% 로다른 OECD 국가에비해아직낮은편이나세계에서가장빠른속도로고령화가진행되고있는국가로 2050년에는세계최고령국가가될전망이다. 치매는노화와밀접하게관련이있는질병으로 65세이상의노인에게있어치매환자의수는매 5년마다약 2배정도씩증가하고있으며 (Johnson & Bosanquet, 2000; Silvestrelli et al., 2006), 그대표적인증상이기억력감퇴, 인식능력및지능퇴화등의다양한인지기능을손상시켜환자의인간적인삶을황폐화시키는질병으로환자자신뿐만아니라보호자의정신적고통과더불어경제적피해등의사회적문제를발생할우려가큰질병으로인식되고있으며병리적인소견은노인반 (senile plaque) 형성및신경섬유덩어리 (neurofibrillary tangle) 생성등이다 (Lee et al., 2008). 서구의경우 85세이상에서는 24~33% 의노인들이고통을받고있으며 (de Lau & Breteler, 2006), 우리나라의경우 65세이상의인구중에서 7~10% 정도가치매를앓고있는것으로보고되어있다 (Suh et al., 2006). 치매중가장많이발병하는알쯔하이머병 (Alzheimer's disease, AD) 이대략 2/3 정도를차지한다고보고되어있다 (Nussbaum & Ellis, 2003). 비록 AD 발병의원인은정확하게밝혀지지않았지만뇌에서세포외 β -amyloid 단백질의침착에의한 amyloid plaques 의생성, tau protein 침착, 산화적손상및아세틸콜린양의감소등과같은여러가지요인들에의하여발병하는것으로알려져있다 (Cummings, 2004; Mattson, 2004; Findeis, 2007; Watanabe et al., 2009). AD는뇌조직중특히기억형성에중요한기관으로알려진 hippocampus( 해마 ) 와기억, 언어및사고에중요한 cerebral cortex( 대뇌피질 ) 에서합성되는중요한신경전달물질인 acetylcholine 계의신경세포감소와그에따른뇌조직부피의감소등, 그리고이로인한뇌활동대사의감소현상을보인다 (Lemiere et al., 1999). 최근의 AD 치료의방법이다양하게개발되고있으며그표적으로는 acetylcholinesterase(ache) 억제제, β-secretase 억제제및 reactive oxygen species scavenger 등 이보고되고있다 (Barnham, 2004; Citron, 2004; Cummings, 2004; Mattson, 2004). 콜린성 neuron 의손상으로대뇌피질에서신경전달물질인아세틸콜린함량을급격하게낮추어주게되며이것이 AD 환자에있어서기억력손상의주요한결과중의하나의원인이되므로, AD 치료법중의하나가 AChE 억제제를사용함으로써뇌에서아세틸콜린의함량을높이는것이다 (Enz et al., 1993; Park et al., 1996; Cummings, 2000; Ingkaninan et al., 2003; Rosengarten et al., 2006). 그러므로아세틸콜린의함량을높이기위하여 AChE 억제제의사용은콜린성 neuron 을활성화하여 AD 증상을개선시키기위한중요한전략이며 (Kalauni et al, 2002; Wenk, 2006), 아세틸콜린의생체내분해를줄이는다양한 AChE 억제제들이개발되고있다. 뿐만아니라, Cottingham et al.(2003) 은 AChE가 β -amyloid 단량체를소중합체로변형시키는작용을하여, β-amyloid 축적을증가시킴으로써, 뇌독성을증진시킨다고보고하고있어, AD의대증적치료제로사용되고있는 AChE 억제제가아세틸콜린양의증가와함께 β-amyloid 축적에의한뇌독성을감소시키는부가적효능이있을것으로추정하고있다 (Wilkinson et al., 2004). 비록많은 AChE 억제제들이 AD를치료하기위한목적으로많이연구되고있지만, tacrine(butini et al., 2008), donepezil (Reale et al., 2006), rivastigmine(grossberg et al., 2004) 및 galanthamine(sharp et al., 2004) 들이미국 FDA에서승인되어판매되고있으며, 아세틸콜린의분해를억제함으로써 synapses 에서아세틸콜린의함량을증가시켜아세틸콜린의작용시간을증가시켜초기및중기 AD 환자의치료에유용하게쓰이고있다. 그러나이러한저해제들은위장애, 간독성, 식욕감퇴와같은부작용이보고되어있으며특히 tarcrine 의경우는심각한부작용이보고되어사용이제한적이어 (Fayuk & Yakel, 2004), 부작용이적으며, 뛰어난효능을갖는인지기능개선및치매치료제를탐색하여효과를입증하는연구가절실히요구되고있다. 이에따라각종식물로부터 AChE 저해활성을갖는연구가지속적으로이루어지고있다. 현재, AChE 억제기능성물질로서식물등으로부터분리추출한천연물에대한연구가활발
Choi et al. : Screeening of Natural Plant Resources with Acetylcholine esterase inhibitory activity and Effect on Scopolamine-induced Memory Impairment 215 하게진행되고있다. 식물유래의천연 AChE 억제제의전형적인예가 Galanthus nivalis L. 로부터유래한 galanthamine, Huperzia serrata 로부터유래한 huperzine A(Wang et al., 2006), Caragana chamlague 로부터유래한 viniferin(sung et al., 2002), Origanum majorana 로부터유래한 ursolic acid(chung et al., 2001) 및 Physostigma venenosum L. 로부터유래한 physostigmine(adams et al., 1984) 등으로이들천연물들은현재 AD 치료를위한의약품개발을위하여임상실험중에있다. 국내에서는, 고추나무, 다래, 두충, 칡, 헛개나무, 치자나무등여러가지천연자원으로부터높은 AChE 저해활성이탐색되었다 (Kim et al., 2005). 또한, 황련, 황박, 오수유, 몰약, 빈랑자, 후추등의생약추출물로부터 AChE 저해활성이탐색되었다 (Kim et al., 2002). 그외, 석곡 (Yoon et al., 2007), 노루궁뎅이버섯배양액 (Jung & Lee, 2007), 해바라기씨 (Park et al., 2007), 초석잠 (Ryu & Kim, 2004), 토사자 (Ryu et al., 2003), 참당귀 (Son et al., 2009), 녹용 (Lee et al., 2009a) 및누에 (Kim et al., 2004) 등에서도 AChE 저해활성이보고되어있다. 본연구에서는국내 외에서서식하고있는약 470여종의식물추출물을대상으로 AChE 저해활성을검색하여독성이낮고효율적인인지기능개선및치매치료후보물질을개발하는기본적인자료를제공하고자하였다. 그결과, 자몽유래에센스오일로부터강력한 AChE 저해활성효과를확인할수있었으며동물모델에서스코폴라민에의해저하된인지기능의개선에효과가있는지를검사하였다. II. 재료및방법 2.1 시험재료및분석기기한방에서쓰이는한약재및여러가지식물체를서울경동시장및한약방에서구입하여사용하였다. 추출에사용된유기용매는시중에서구입한분석용특급시약을사용하였다. 효소활성저해실험에사용한효소원으로뱀장어유래의 acetylcholine esterase, acetylthiocholine, DTNB(5,5'-dithio bis -2-nitrobenzoic acid) 는 Sigma(Saint Louis, USA) 에서구입하였다. 분석을위한기기로 ELISA reader 는 Multimode Microplate Reader(Molecular Device, USA) 를사용하였다. 2.2 AChE 의활성억제측정 AChE 의효소활성은 Ellman 방법 (Ellman et al., 1961) 에따라 acetylthiocholine 을기질로사용하였으며 AChE에의해생성되는 thiocholine 을 DTNB와반응시켜그결과생성되는 5-thio-2-nitrobenzoate 를 405nm 에서흡광도변화를측정하여수행하였다. 간단하게, 96 well microplate 에 100 μl의 AChE assay buffer(0.1 M Tris-HCl, ph 8.2), 10 μl의 0.5 U/ml AChE(dissolved in assay buffer containing 10% glycerol) 및 10 μl의적당하게희석된시료를가한후 shaking 하면서상온에서 10분동안 incubation 한후, 10 μl의 10 mm DTNB와 5 μl의 100 mm acetylthiocholine 을가한후 2분동안반응시킨후 microplate reader(spectramax PLUS 384, Molecular Device, 미국 ) 를사용하여 405 nm에서흡광도를측정하였다. 이때효소대신동량의 assay buffer 를가한것을 control 로하였다. AChE inhibitor 에의한억제활성은다음의수식을이용하여계산하였다. 억제율 (%) = 100-(As/Ac) 100 이때, Ac는대조군의흡광도, As는시료군의흡광도이다. 2.3 AChE 억제활성을갖는천연물탐색천연물로부터 AChE 억제활성을갖는물질을추출하기위하여에탄올을이용하여추출하였다. 추출에사용한천연물은믹서기로잘분쇄한후사용하였다. 경동시장에서구입한건조천연물 5 g을 50 ml의 50% 에탄올을가한후상온에서 6시간동안추출한후, 원심분리 (12,000 rpm, 30 min, 4 ) 하여불용성물질을제거하였으며, 얻어진상층액을 AChE 억제활성측정에사용하였다. 에센스오일 (Essence oil, EO) 은뉴디렉션스코리아로부터구입하여사용하였다. 2.4 열및 ph 안정성열에대한안정성을시험하기위하여추출한시료
216 Journal of Agriculture & Life Science 45(6) 를 40~121 범위에서 1시간동안 incubation 한시료의 AChE 억제활성을측정하였다. 이때, 대조군으로는 25 에서처리한시료를 100% 로하였다. 또한, ph에대한안정성을시험하기위하여시료를 ph 3~11 범위로조정하고 1시간동안 incubation 한후 AChE 억제활성을측정하였다. 이때, 대조군으로는 ph 7에서처리한시료를 100% 로하였다. 2.5 인지기능개선활성측정 1) 실험동물및시료투여체중이약 35±5 g의 ICR 생쥐 (8주령, 웅성, 중앙실험동물 ) 49마리를사용하였다. 인지기능검사는수동회피시험 (Passive avoidance test) 및수중미로시험 (Morris water maze test) 으로습득검사를실시하였다. 훈련에대한스트레스를감소시키기위해 1주일간환경적응기간을거치게하였다. 사육실의온도는 22±2, 습도 55±5% 를유지하였으며, 명암은 12시간주기로하였다. 실험동물의관리는 NIH 가이드라인을준수하였다. 실험동물은대조군, 스코폴라민 (scopolamine, Sigma, USA) 단독투여군, 스코폴라민과여러농도의시료투여군으로하여각군당 7마리씩사용하였다. 인지기능향상검사에사용한시료는 0.5 ml을 2회 / 일로 32일간구강투여하였으며, 무스카리닉 (Muscarinic) 수용체의길항제인스코폴라민은 1 mg/kg 을수중미로시험을진행하기 30분전에복강투여하였다. 2) 수동회피시험쥐는야행성동물이라밝은곳에서어두운곳으로가려는습성이있기때문에. 어두운곳에전기적충격이가하는장치를이용하였다 (50 x 15 x 40 cm, electrofiable frid floor). 이상자는칸막이를이용하여두구획으로구분하는데동물이어두운구획으로넘어가는즉시 0.25 ma의전류를바닥의 stainless grid를통해 1초동안흘려전기충격을주는장치이다. 조명이켜진구획에실험동물을넣은후부터이동물이어두운구획으로넘어가입구가닫힐때까지의시간 (latency) 을타이머로측정하였다. 따라서시험동물은어두운방과전기충격과의관계를기억하게되며, 24시간후다시셔틀박스에위치시켰을때밝은구획에서어두운구획으로들어 가기를망설이게되며, 이때어두운구획으로들어가기까지의시간을측정하였다. 기억력이좋은쥐는이러한전기적인충격을기억하고있어어두운곳으로들어가는시간이지체되어밝은곳에머무르는시간 (latency time) 이길어지고이 latency time을측정하여인지기능개선효능을측정하였다. 3) 수중미로시험수중미로측정장치 (Ethovision 사, 네덜란드 ) 는공간학습및기억력을측정하는장비로널리이용되고있다. 지름이 150 cm, 높이가 50 cm인원형수조안에지름이 10 cm, 높이가 35 cm인원형플랫폼을넣고수면아래 0.5 cm가되도록물높이를조절한다. 물의온도는 21±5 가되도록한다. 미로주위를어둡게하고 60 W 빛을비추고 video camera 로 monitor하였다. 수중미로는 1일 4회씩 5일간연속적으로실시하였다. 만약실험동물이 120 초내에플랫폼을찾지못하면꺼내어플랫폼에올려놓고 10초동안방치하여플랫폼의위치를학습하도록하였다. 실험동물은부동한 3면에서입수하여플랫폼에도달하는시간 (escape latency) 을측정하였으며, 플랫폼을찾지못하면 120초로기록하였다. 시료군은수중미로시험을측정하기 1시간전에구강투여하였다. III. 결과및고찰 3.1 AChE 억제활성을갖는천연물 screening 경동시장에서구입한천연물로부터 50% 에탄올로추출한시료 ( 약 400 가지 ) 및에센스오일 ( 약 70 가지 ) 들을대상시료로하여전기뱀장어유래의 AChE에대한 in vitro 억제효능을 Ellman et al. (1961) 의방법으로조사한결과는 Table 1과같다. 산두근 (S. subprostrata), 히어리 (C. coreana) 및황백 (P. amurense) 은 80% 이상의 AChE 억제활성을나타내었으며, 소엽 (P. frutescens), 노회 (A. ferox), 해바라기씨 (H. annuus), 계혈등 (S. suberectus), 산철쭉 (R. yedoense), 대황 (R. undulatum), 고추나물 (H. erectum), 쇄양 (C. songaricum), 빈랑 (A. catechu) 및측백나무 (T. orientalis) 등의 10종은 70 79% 정도의억제활성을나타내어 AChE 효소억제활성이비교적우수한
Choi et al. : Screeening of Natural Plant Resources with Acetylcholine esterase inhibitory activity and Effect on Scopolamine-induced Memory Impairment 217 Table 1. Inhibitory activity of acetylcholinesterase of the extracts of plants in vitro Sample Scientific name Inhibition(%) Mistletoe Ramulus visci 28.7 Cinnamon Cinnamomum cassia 46.6 Erect St. Johnswort Hypericum erectum 70.9 Madder color Rubia akane 34.2 Seed of radish Raphanus sativus 61.8 Alloe Aloe ferox 76.8 Seed of Evening Primrose Oenothera odorata 1.9 False Chinese Swertia Swertia dilut 38.3 Rhubarb Rheum undulatum 72.5 Rodger's Bronze Leaf Rodgersia podophylla 27.9 Cocklebur Xanthium strumarium 47.4 Stonecro Sedum sarmentosum 30.9 Garlic Allium sativum 47.8 Ephedra Ephedra sinica 53.6 Korean berchemia Berchemia berchemiaefolia 40.2 Quince Chaenomeles sinensis 58.8 Moutan Radicis Cortex Paeonia suffruticosa 63.8 Mongolian dandelion Taraxacum mongolicum 35.3 White mustard Sinapis alba 41.2 Amur adonis Adonis amurensis 36.0 Common cattail Typha orientalis 68.6 Loquat Eriobotrya japonica 28.5 Betel nut Areca catechu 69.9 Sophora Sophora subprostrata 89.8 Japanese Cornelian Cherry Cornus officinalis 26.2 Royal Azalea Rhododendron yedoense 72.5 Ginger Zingiber officinale 27.2 Pine Pinus pinaster 59.8 Perilla color Perilla frutescens 79.4 Cynomorium Herb Cynomorium songaricum 70.5 Black Cohosh Cimicifuga heracleifolia 43.5 Nanking Cherry Prunus tomentosa 68.1 Chinese Lespedeza Lespedeza cuneata 52.8 Yacón Polymnia sonchifolia 49.1 Galangal Alpinia officinarum 45.7 Phyllanthus Phyllanthus ussuriensis 25.0 Japanese Mallotus Mallotus japonicus 38.0
218 Journal of Agriculture & Life Science 45(6) Sample Scientific name Inhibition(%) Oregano Origanum vulgare 38.0 Mume fruit Prunus mume 47.4 Gingko leaf Ginkgo biloba 28.8 Tree of heaven Ailanthus altissima 57.1 Fortune Loosestrife Arthraxon hispidus 34.5 Chinese matrimony vine Lycium chinense 31.8 Burnet bloodwort Sanguisorba longifolia 34.5 Baby brier Rosa multiflora 50.7 Shortstalk Bushclover Lespedeza cyrtobotrya 60.1 Sasa borealis Phyllostachys bambusoides 51.9 Coptis Coptis japonica 66.3 Greenbrier Smilax china 42.1 Cardamom Amomum tsao-ko 38.0 Alpiniae Katsumadaii Semen Aplinia katsumadai 62.5 Chinese arbor vitae Thuja orientalis 69.6 Clove Syzygium aromaticum 37.9 White Clover Trifolium repens 29.3 Chinese Fir Tetrapanax papyriferus 32.2 Chinese Star Anise Illicium verum 31.5 Seed of grape Vitis vinifera 41.9 Peony flower Magnolia sieboldii 29.6 Seed of Sun flower Helianthus annuus 74.5 Thunbergii Geranium nepalense 63.4 Beautiful Corydalis Corydalis turtschaninovii 41.0 Phellodendro Phellodendron amurense 80.2 Korean winter Hazel Corylopsis coreana 84.0 천연물로확인되었다. 에센스오일들중에서자몽 (C. paradisi) 의경우 95% 이상의억제효능을나타내었으며, 사이프러스 (C. sempervirens), 바질 (O. basilicum), 파인스카치 (P. sylvestris) 및로즈마리 (R. officinalis) 는 80% 이상의억제효능을나타내었으며, 유칼립투스 (E. globulus), 티트리 (M. alternifolia) 및편백피톤치드 (C. obtusa) 는 70% 이상의억제활성을나타내어 AChE 억제활성이비교적우수한에센스오일 (EO) 로확인되었다 (Table 1, 2). 이들중에서 80% 이상의억제활성을갖는 8 종류의천연물 ( 히어리, 황백, 산두근, 바질 EO, 파인스카치 EO, 로즈마리 EO, 사이프러스 EO, 자몽 EO) 을농도별 (50 및 20 μg/ml) 로처리하여 AChE 효소억 제활성을비교측정한결과, 두가지에센스오일 ( 사이프러스 EO, 자몽 EO) 에서높은 AChE 억제활성을나타내었다. 사이프러스 EO 및자몽 EO는 50 μg/ml 의농도로처리하였을때각각 84% 와 99%, 20 μg/ml 의농도로처리하였을때각각 43% 와 91% 의활성억제를나타내어낮은농도에서도강한활성억제효과를나타냄이확인되었으며, 자몽 EO 가가장높은 AChE 억제활성을나타내었다 (Fig. 1). 이상의결과로부터, 자몽유래의에센스오일의열안정성및수동회피시험및수중미로측정방법으로인지기능개선효능을검색하여인지기능개선효능을갖는기능성식품첨가제로서유용성을더검증하였다.
Choi et al. : Screeening of Natural Plant Resources with Acetylcholine esterase inhibitory activity and Effect on Scopolamine-induced Memory Impairment 219 Table 2. Inhibitory activity of acetylcholinesterase of essential oils in vitro. Sample Scientific name Inhibition(%) Aniseed Illicium verum 5.9 Basil Ocimum basilicum 83.0 Benzoin Styrax benzoin 22.8 Bergamot Citrus bergamia 2.5 Cedarwood Cedrus atlantica 7.2 Cinnamon Cinnamomum zeylanicum 33.0 Citronella Cymbopogon nardus 2.7 Cypress Cupressus sempervirens 88.7 Eucalyptus Eucalyptus globulus 77.8 Frankincense Boswellia carteri 8.0 Geranium Pelargonium roseum asperum 16.1 Ginger Nicolaia elatior 7.5 Grapefruit Citrus paradisi 97.9 Juniper berry Juniperus communis 14.0 Lavender Lavendula officinalis 30.4 Lemon Citrus limonum 1.1 Lemongrass Cymbopogon citratus 6.7 Lilac Syringa vulgaris 12.1 Lime Citrus aurantifolia 32.7 Marjoram Oringanum marjorana 53.3 Myrrh Myrtus ommunis 14.4 Neroli Citrus aurantium 24.6 Peppermint Mentha piperita 11.1 Pine scotch Pinus sylvestris 85.3 Phytoncide Chamaecyparis obtusa 70.3 Rosemary Rosmarinus officinalis 81.3 Sage Salvia officinalis 36.3 Orange Sweet Citrus sinensis 25.0 Tea Tree Melaleuca alternifolia 76.2 Thyme Thymus serpyllium 57.3 Winter Green Gaultheria procumbens 2.5 3.2 자몽 EO의안정성자몽 EO의열에대한안정성을시험하기위하여 40~100 범위에서 1시간동안처리한시료및 autoclave(121, 20 min) 한시료의 AChE 억제활성을측정하였을때, 자몽유래 EO의 AChE 억제활성은 40~100 에서 1시간동안열처리를하여도활 성에변함이없었으며 121 에서 20분간 autoclave 하여도활성을잃지않아열에매우안정한물질임을확인하였다 (Fig. 2A). 또한, 자몽유래 EO의 ph 에대한안정성을시험하기위하여시료를 ph 3~12 범위로조정하고 1시간동안처리한후 AChE 억제활성을측정한결과, 자몽유래 EO의 AChE 억제
220 Journal of Agriculture & Life Science 45(6) Fig. 1. The inhibitory activity of acetylcholinesterase by different concentrations of some natural extracts and essential oils. 활성은 ph 3~12 범위에서효소억제활성에큰변함이없는광범위한 ph에매우안정한물질임을확인하였다 (Fig. 2B). 이상의결과로부터, 자몽유래의에센스오일은열안정성및 ph 안정성이매우우수한천연물임을확인하였다. 3.3 자몽 EO의인지기능개선효능측정자몽유래 EO를농도별로쥐에투여하여수동회피시험및수중미로시험으로인지기능개선효능을측정하였다. 이때, 각시료는 1주일마다신선하게제조하여투여하였으며, 투여동안각시료는냉장에서보관하였으며투여직전 vortexing 하여잘혼합한후투여하였다. 각시료군은 6마리씩의 male ICR mouse 를사용하였다. 시료투여량은각 mouse 당제조한시료 0.5 ml 씩투여하였으며, 투여회수는하루에 2회씩투여하였다. 총 4주간투여한후인지기능개선효능을측정하였다. 이때, 사용한 positive control 로는 donepezil 을, negative control로는학습과단기기억에특별한영향을줌으로써치매를유발하는 scopolamine 을사용하였다. 이때, scopolamine 은 1 mg/kg body weight, 자몽유래 EO는 50, 100 및 400 mg/kg body weight, positive control 로사용한 donepezil 은 0.5 mg/kg body weight 로투여하였다. 1) 수동회피시험 Scopolamine(1 mg/kg body weight) 을투여하여기억력을감퇴시킨쥐를이용하여자몽유래 EO가기억력손상을억제하는효과가있는지의여부를동물의기억력측정장치인수동회피측정장치를이용하여측정하였다. 그결과, 정상쥐 (normal, positive control) 의 latency time은 128초이었으며, 인지기능에손상을유발하는 scopolamine(1 mg/kg body weight) 으로처리한시험군 (negative control) 의 latency time은약 11초로정상쥐와비교하였을때인지기능 ( 또는기억력 ) 이손상되었음알수있었다. 그러나, 자몽유래 EO의투여에의하여인지기능개선에효능을보였으며, 100 mg/kg body weight의자몽유래 EO를투여하였을때 latency t i me ( 3 1 초 ) 은치매치료제로사용하고있는 donepezil(0.5 mg/kg body weight) 을투여하였을때의 latency time(34 초 ) 수준으로회복되는것을확인하였다. 특히 400 mg/kg body weight 의자몽유래 EO를투여하였을때의 latency time(118 초 ) 은거의정상상태로회복되는것을확인하였다 (Fig. 3). Latency time이길어진다는것은 scopolamine 으로유도된인
Choi et al. : Screeening of Natural Plant Resources with Acetylcholine esterase inhibitory activity and Effect on Scopolamine-induced Memory Impairment 221 Fig. 2. Heat and ph stability of essential oil from grapefruit(c. paradisi). Panel A, Heat stability. The grapefruit essential oil was incubated at various temperature ranging from 25 to 121 for 1 h, and then measured the residual inhibitory activity against acetylcholinesterase. The activity at 25 was defined as 100%. Panel B, ph stability. The grapefruit essential oil was incubated at various ph ranging from 3 to 11 for 1 h, and then assayed the residual inhibitory activity against acetylcholinesterase. The activity at ph 7 was defined as 100%. 지기능손상으로부터회복, 즉학습및기억력이회복되었다는것을의미한다. 이는, 자몽유래 EO가실제로뇌로들어가서뇌의 acetylcholinesterase 의활성을억제하여, 결과적으로신경전달물질인 acetylcholine 의작용을증진시켜주어학습과기억력향상효과를나타내고있음을나타낸다. 이상의결과로부터, 자몽유래 EO는인지기능개선효능이있음을확인하였으며, 공간학습및기억력을측정하는방법으로널리 이용되고있는수중미로시험으로인지기능개선효능을더검증하였다. 2) 수중미로시험수동회피시험을통하여확인된 scopolamine 에의하여손상된기억력개선효과를더수준이높은기억력 ( 공간기억력 ) 을검증할수있는장치인수중미로시험을이용하여자몽유래 EO의인지기능개선에
222 Journal of Agriculture & Life Science 45(6) 어떤영향을미치는지를검사하였다. Learning trial 을 1일 2회씩각시험군 ( 군당 6마리이상 ) 에대하여 5일째까지실시를하면반복된학습을통하여습득된기억력으로대부분의흰쥐들이빠른시간내에안전지대인플랫폼에도달하게되며, 이는인지기능이정상적으로작동하고있다는것을의미한다. 정상쥐 (positive control) 의 5일째의 latency time은약 40 초이었으며, 인지기능에손상을유발하는 scopolamine(1 mg/kg body weight) 으로처리한시험군 (negative control) 의 latency time은약 80초로정상쥐와비교하였을때인지기능 ( 또는기억력 ) 이손상되었음알수있었다. 그러나, 자몽유래 EO의투여에의하여인지기능의개선에효능을보였으며, 50~100 mg/kg body weight로자몽유래 EO를투여하였을때 latency time은약 60초로감소하여, 치매치료제인 donepezil(0.5 mg/kg body weight) 을투여하였을때의 latency time( 약 58초 ) 수준으로회복되는것을확인하였으며 (Fig. 4A), 수영거리의패턴 (Fig. 4B) 도 latency time과유사한결과를나타내었다. 수중미로시험에서 latency time이짧아진다는것 (Fig. 4A) 과이동거리의감소 (Fig. 4B) 는인지기능 ( 또는기억력 ) 이개선되었다는것을의미하며, 자 몽유래 EO의투여는인지기능개선효능이우수함을알수있었다. Kim et al. (2004) 은누에 (Bombyx mori) 로부터추출한 BF-7(10 mg/kg body weight) 을경구투여하여수동회피시험및수중미로시험을하였을때거의정상상태의 latency time으로회복되었음을보고하였다. Lee et al. (2008) 은홍삼추출물 (200 mg/kg body weight) 이수동회피시험및수중미로시험을하였을때인지기능개선효능을보고하였다. 또한, 녹용추출물 (200 mg/kg body weight) 은수중미로시험에서인지기능개선효능을나타내는것으로보고되어있다 (Lee et al., 2009b). 이상의결과로부터, 자몽유래 EO가실제로뇌혈관장벽 (blood brain barrier) 을통과하여뇌의콜린성신경계기능을높여주는것으로추측되어, 천연물유래의우수한 AChE inhibitor 후보물질임을알수있었다 (Fig. 2 및 3). 이러한측면에서, 자몽유래 EO는인지기능개선효능을갖는기능성첨가제로서가치가매우클것으로사료되며, 자몽유래 EO의인지기능개선효과를분자적수준에서의 mechanism 에대한심도있는연구가더진행되면효과적인천연물유래의부작용이적은인지기능개선, 치매치료및예방의후보물질로개발할만한가치가충분하다고사료된다. Fig. 3. The effect of grapefruit essential oil on escape latency time during the training trial in scopolamine-induced memory deficits mice by passive avoidance test. Memory impairment was induced by treated with scopolamine(1 mg/kg body weight; i.p.), and the task was performed on days 3~4 after injection of scopolamine. Donepezil(0.5 mg/kg body weight; p.o.), saline(normal; same volume) and various amount of grapefruit essential oil(50, 100 and 400 mg/kg body weight; p.o) were administered to mice, and then measured the latency time.
Choi et al. : Screeening of Natural Plant Resources with Acetylcholine esterase inhibitory activity and Effect on Scopolamine-induced Memory Impairment 223 Fig. 4. The effect of grapefruit essential oil on the performance of scopolamine-treated mice in the memory trial by Morris water maze test. The training trials were carried out for 5 days(four per day) and the working memory trials were carried out for 5 days after administration of scopolamine(1 mg/kg body weight). At 60 min before Morris water maze test, donepezil(0.5 mg/kg body weight), saline (normal) and various amount of grapefruit essential oil(50 and 100 mg/kg body weight) were administered to mice, and then measured the latency time. Panel A, Change of latency time; Panel B, Change of moving distance. Ⅳ. 감사의글 본연구는중소기업기술개발지원사업의연구비지원으로수행된연구결과의일부로이루어졌으며이에감사드립니다. Literature Cited Adams R. L., P. L. Craig, and O. A. Parsons. 1984. Neuropsychology of dementia. Neurolog. Clin. 4: 387-405. Barnham K. J., C. L. Masters, and A. I. Bush. 2004. Neurodegenerative diseases and oxidative stress. Nature. Rev. Drug Discovery. 3: 205-214. Borjesson-Hanson A., E. Edin, T. Gislason, and I.
224 Journal of Agriculture & Life Science 45(6) Skoog. 2004. The prevalence of dementia in 95 year olds. Neurol. 63: 2436-2438. Butini S., E. Guarino, G. Campiani, M. Brindisi, S. S. Coccone, I. Fiorini, E. Novellino, T. Belinskaya, A. Saxena, and S. Gemmaa. 2008. Tacrine based human cholinesterase inhibitors: Synthesis of peptidic-tethered derivatives and their effect on potency and selectivity. Bioorganic Med. Chem. Lett. 18: 5213-5216. Chung Y. K., H. J. Heo, E. K. Kim, H. K. Kim, T. L. Huh, Y. Lim, S. K. Kim, and D. H. Shin. 2001. Inhibitory effect of ursolic acid purified from Origanum majorana L on the acetylcholinesterase. Mol. Cells. 11: 137-143. Citron M. 2004. Strategies for disease modification in Alzheimer's disease. Nature Rev. Neurosci. 5: 677-685. Cottingham M. G., J. L. Voskuil, and D. J. Vaux. 2003. The intact human acetylcholinesterase c-terminal oligomerization domain is alpha-helical in situ and in isolation, but a shorter fragment forms beta-sheet-rich amyloid fibrils and protofibrillar oligomers. Biochem. 42: 10863-10873. Cummings J. L. 2000. Cholinesterase inhibitors: a new class of psychotropic compounds. Am. J. Psychiatry. 157: 4-15. Cummings J. L. 2004. Alzheimer's Disease. Nwe. Engl. J. Med. 351: 56-67. de Lau, L. M. and M. M. Breteler. 2006. Epidemiology of Parkinson's disease. Lancet Neurol. 5: 525-535. Ellman G. L., D. K. Courtney, V. Andres, and R. M. Featherstone. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88-95. Enz A., R. Amstutz, H. Boddeke, G. Gmelin, and J. Malonowski. 1993. Brain selective inhibition of acetylcholinesterase: a novel approach to therapy for Alzheimer's disease. Prog. Brain Res. 98: 431-445. Fayuk D. and J. L. Yakel. 2004. Regulation of nicotinic acetylcholine receptor channel function by acetylcholinesterase inhibitors in rat hippocampal CA1 interneurons. Mol. Pharmacol. 66: 658-666. Findeis M. A. 2007. The role of amyloid β peptide 42 in Alzheimer's disease. Pharmacol. Therapeut. 116: 266-286. Grossberg G., P. Irwin, and A. Satlin. 2004. Rivastigmine in Alzheimer's disease: efficacy over two years. Am. J. Geriatr. Psychiatry. 12: 420-431. Ingkaninan K., P. Temkitthawon, K. Chuenchom, T. Yuyaem, and W. Thongnoi. 2003. Screening for acetylcholin esterase inhibitory activity in plants used in Thai traditional rejuvenating and neurotonic remedies. J. Ethnopharmacol. 89: 261-264. Johnson N. T. D. and N. Bosanquet. 2000. The epidermic of Alzheimer's disease. Pharmacoeconomics. 18: 215-223. Jung J. H. and S. Y. Lee. 2007. AChE Inhibitory Effect and Antioxidative Activity of Submerged Cultured Products from Hericium erinaceum. Kor. J. Biotechnol. Bioeng. 22: 30-36. Kalauni S. K., M. I. Choudhary, A. Khalid, M. D. Manandhar, F. Shaheen, F. Atta-ur-Rahman, and M. B. Gewali. 2002. New cholinesterase inhibiting steroidal alkaloids from the leaves of Sarcococca coriacea of Nepalese origin. Chem. Pharm. Bull. 50: 1423-1426. 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. Kor. Soc. Food Sci. Nutr. 34: 427-432. Kim D. K., J. Y. Lee, J. J. Sung, E. T. Kim, Y. S. Kim, O. S. Kwon, Y. C. Yun, T. J. Lee, Y. K. Kang, Y. H. Chung, S. S. Kim, K. Y. Kim, and W. B. Lee. 2004. The Role of BF-7 on Enhancement of Memory and Cognitive Function. The Korean J. Anat. 37: 519-527. Kim, J. S., Y. S. Kim, S. K. Kim, J. H. Heor, B. H. Lee, B. W. Choi, G. S. Ryu, E. K. Park, O. P Zee, and S. Y. Ryu. 2002. Inhibitory Effects of Some Herbal Extracts on the Acetylcholinesterase (AChE) In Vitro. Kor. J. Pharmacogn. 33: 211-218. Lee E. H., Y. C. Youn, K. Y. Park,J. H. Min, O. S. Kwon, H. O. Lee, and H. J. Hong. 2009a. The
Choi et al. : Screeening of Natural Plant Resources with Acetylcholine esterase inhibitory activity and Effect on Scopolamine-induced Memory Impairment 225 Effect of Acetylcholine Esterase Inhibitor on Cerebrospinal Fluid β-amyloid 1-42 and Phosphorylated Tau Protein in Korean Alzheimer's Disease Patients: Preliminary Study. J. Kor. Neurol. Assoc. 26: 224-230. Lee M. R., B. S. Sun, L. J. Gu, C. Y. Wang, E. K. Mo, S. A. Yang, S. Y. Ly, and C. K. Sung. 2008. Effect of white ginseng extract and red ginseng extract on learning performance and acetylcholinesterase activity inhibition. J. Ginseng Res. 32: 341-346. Lee M. R., B. S. Sun, L. J. Gu, C. Y. Wang, Z. M. Fang, Z. Wang, E. K. Mo, S. Y. Ly, and C. K. Sung. 2009b. Effect of the Deer Antler Extract on Scopolamine-Induced Memory Impairment and Its Related Enzyme Activities. J. Korean Soc. Food Sci. Nutr.38: 409-414. Lemiere J., J. D. Van, and R. Dorn. 1999. Treatment of Alzheimer's disease: an evaluation of cholinergic approach. Acta Neurol. Belg. 99: 96-106. Mattson M. P. 2004. Pathways towards and away from Alzheimer's disease. Nature 430: 631-639. Nussbaum R. L. and C. E. Ellis. 2003. Alzheimer's disease and Parkinson's disease. New Engl. J. Med, 348: 1356-1364. Park C. H., S. H. Kim, W. Choi, Y. J. Lee, J. S. Kim, S. S. Kang, and Y. H. Suh. 1996. Novel anticholinesterase and antiamnesic activities of dehydroevodiamine, a constituent of Evodia rutaecarpa. Planta Med. 62: 405-409. Park J. Y., S. U Woo, J. C. Heo, and S. H. Lee. 2007. Protective Effects of Helianthus annuus Seed Extract against Chemical-Induced Neuronal Cell Death. Kor. J. Food Preserv. 14: 213-219. Reale M., C. Iarlori, F. Gambi, C. Feliciani C., L. Isabella, and D. Gambi. 2006. The acetylcholinesterase inhibitor, Donepezil, regulates a Th2 bias in Alzheimer's disease patients. Neuropharmacol. 50: 606-613. Rosengarten B., S. Paulsen, S. Molnar, R. Kaschel, B. Gallhofer, and M. Kaps. 2006. Acetylcholine esterase inhibitor donepezil improves dynamic cerebrovascular regulation in Alzheimer patients. J. Neurol. 253: 58-64. Ryu B. H. and S. O. Kim. 2004. Effects of Methanol Extract of Stachys sieboldii MIQ on Acetylcholine Esterase and Monoamine Oxidase in Rat. Kor. J. Food Nutr. 17: 347-355. Ryu S. Y., Park Y. K. and Kang B. S. 2003. Studies on the Acetylcholinesterase inhibitors Effects of Cuscutae Semen. Kor. J. Herbiol. 18: 289-299. Sabbagh M. N. 2009. Drug Development for Alzheimer's Disease: Where Are We Now and Where Are We Headed? Am. J. Geriatric Pharmacother. 7: 167-185. Sharp B. M., M. Yatsula, and V. Fu. 2004. Effects of Galantamine, a Nicotinic Allosteric Potentiating Ligand, on Nicotine-Induced Catecholamine Release in Hippocampus and Nucleus Accumbens of Rats. J. Pharmacol. Experimental Therapeut. 309: 1116 1123. Silvestrelli G., A. Lanari, L. Parnetti. D. Tomassoni, and F. Amenta. 2006. Treatment of Alzheimer's disease: from pharmacology to a better understanding of disease pathophysiology. Mech. Ageing Dev. 127: 148-157. Son C. Y., I. H. Baek, G. Y. Song, J. S. Kang, and K. I. Kwon. 2009. Pharmacological Effect of Decursin and Decursinol Angelate from Angelica gigas Nakai. Yakhak Hoeji. 53: 303-313. Suh, G. H., M. Knapp, and C. J. Kang, 2006. The economic costs of dementia in Korea, Int. J. Geriatr Psychiatry, 21: 722-728. Sung S. H., S. Y. Kang, K. Y. Lee, M. J. Park, J. H. Kim., J. H. Park, Y. C. Kim, J. Kim, and Y. C. Kim. 2002. (+)-α-viniferin, a Stilbene Trimer from Caragana chamlague, Inhibits Acetylcholinesterase. Biol. Pharm. Bull. 25: 125-127. Wang P., H. Yan, and X. Tang. 2006. Progress in studies of huperzine A, a natural cholinesterase inhibitor from Chinese herbal medicine. Acta Pharmacologica Sinica. 27: 1-26. Watanabe T., N. Yamagata, T. Takasaki, K. Sano, K. Hayakawa, S. Katsurabayashi, N. Egashira, K.
226 Journal of Agriculture & Life Science 45(6) Mishima, K. Iwasaki. and M. Fujiwara. 2009. Decreased acetylcholine release is correlated to memory impairment in the Tg2576 transgenic mouse model of Alzheimer's disease. Brain Res. 1249: 222-228. Wenk G. L. 2006. Neuropathologic changes in Alzheimer's disease: potential targets for treatment. J. Clin. Psychiatry. 67: 3-7. Wilkinson D. G., P. T. Francis, E. Schwam, and J. Payne-Parrish. 2004. Cholinesterase inhibitors used in the treatment of Alzheimer's disease: the relationship between pharmacological effects and clinical efficacy. Drugs Aging. 21: 453-478. Yoon M. Y., J. Y. Kim, J. H. Hwang, M. R. Cha, M. R. Lee, K. J. Jo, and H. R. Park. 2007. Protective Effect of Methanolic Extracts from Dendrobium nobile Lindl. on H 2O 2-induced Neurotoxicity in PC12 cells. J. Kor. Soc. Appl. Biol. Chem. 50: 63-67. Zotova E., J. A. R. Nicoll, K. Ralaria, C. Holmes, and D. Boche. 2010. Inflammation in Alzheimer's disease: relevance to pathogenesis and therapy. Alzheimer's Res. Therapy. 2: 1-9.