In vitro model system을 활용한 마늘 추출물의 치매예방 특성
Preventive Characteristics of Garlic Extracts Using in vitro Model System on Alzheimer's Disease
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초록
본 연구에서는 대표적인 퇴행성 신경질환인 알츠하이머성 치매에 대한 마늘 물, 100% 메탄올, 디클로로메탄 추출물들의 acetylcholinesterase (AChE) 저해 및 신경세포 보호효과를 조사하였다. 마늘 디클로로메탄 추출물은 농도 의존적으로 AChE를 저해하는 것으로 나타났으며, IC50은 36.1 μg/mL로 나타났다. MTT reduction assay를 이용해 amyloid β protein (Aβ) 유도성 신경세포 독성에 대한 신경세포 보호효과를 측정한 결과, 세 가지 마늘 추출물들은 대부분 40% 미만의 세포생존율을 보였고 이 결과는 Aβ 유도성 신경세포 독성보다 상대적으로 더 높은 세포독성을 보여주었다. LDH assay에서는 마늘 물 추출물이 37%의 LDH 방출량을 나타내 200 μM의 vitamin C과 유사한 세포막손상 보호효과를 보였다. 마지막으로 neutral red uptake assay를 실시한 결과, MTT reduction assay와 마찬가지로 모든 마늘 추출물들에서 세포생존율의 감소를 확인하였으며 특히 디클로로메탄 추출물의 경우 현저하게 낮은 세포생존율을 나타내었다. AChE 저해활성을 갖는 마늘 디클로로메탄 추출물로부터 얻은 column fractionations에 함유된 생리활성물질을 탐색하기 위해 HPLC 분석을 실시하였으며, 마늘 98:2 fraction의 LC-MS 분석을 통하여 allyl methyl disulfide, diallyl monosulfide, diallyl disulfide로 추정되는 물질군이 확인되었다.
Abstract
In this study, the acetylcholinesterase (AChE) inhibition and neuronal cell protective effects of water, 100% methanol and dichlromethane extracts from garlic were investigated. We found that dichloromethane extract of garlic resulted in a dose-dependent manner on AChE inhibition (IC50: 36.1 μg/mL). In cell viability assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT), cell viabilities of water, 100% methanol and dichlromethane extracts were lower (almost under 40%) than amyloid β protein (Aβ)-induced neurotoxicity. Because Aβ is also known to increase neuronal cell membrane breakdown, neuronal apoptosis was further confirmed by lactate dehydrogenase (LDH) and neutral red uptake (NRU) assay. Water extract presented relative protection against Aβ-induced membrane damage in LDH assay. However all garlic extracts showed significant problem with decrease of cell viability in NRU assay, especially at dichloromethan extract. To determine active compounds in column fractions (98:2 fraction) from dichloromethane extract which showed significant AChE inhibitory effect, we performed HPLC and LC-MS analysis. It was supposed that garlic may contain allyl methyl disulfide, diallyl monosulfide, and diallyl disulfide as active compounds.
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