Journal of agriculture & life science (농업생명과학연구)

Institute of Agriculture & Life Science, Gyeongsang National University (경상대학교 농업생명과학연구원)
권호 표지

Preventive Characteristics of Garlic Extracts Using in vitro Model System on Alzheimer's Disease

In vitro model system을 활용한 마늘 추출물의 치매예방 특성

  • Choi, Gwi-Nam (Dept. of A Food Science & Technology(Insti. of Agric. and Life Sci.), Gyeongsang National Univ.) ;
  • Kim, Ji-Hye (Dept. of A Food Science & Technology(Insti. of Agric. and Life Sci.), Gyeongsang National Univ.) ;
  • Kwak, Ji-Hyun (Dept. of A Food Science & Technology(Insti. of Agric. and Life Sci.), Gyeongsang National Univ.) ;
  • Jeong, Chang-Ho (Dept. of A Food Science & Biotechnology, Kyung Hee Univ.) ;
  • Jeong, Hee-Rok (Dept. of A Food Science & Technology(Insti. of Agric. and Life Sci.), Gyeongsang National Univ.) ;
  • Shin, Jung-Hye (Namhae Garlic Research Institue) ;
  • Kang, Min-Jung (Namhae Garlic Research Institue) ;
  • Sung, Nak-Ju (Namhae Garlic Research Institue) ;
  • Heo, Ho-Jin (Dept. of A Food Science & Technology(Insti. of Agric. and Life Sci.), Gyeongsang National Univ.)
  • 최귀남 (경상대학교 식품공학과(농업생명과학연구원)) ;
  • 김지혜 (경상대학교 식품공학과(농업생명과학연구원)) ;
  • 곽지현 (경상대학교 식품공학과(농업생명과학연구원)) ;
  • 정창호 (경희대학교 식품공학과) ;
  • 정희록 (경상대학교 식품공학과(농업생명과학연구원)) ;
  • 신정혜 ((재)남해마늘연구소) ;
  • 강민정 ((재)남해마늘연구소) ;
  • 성낙주 ((재)남해마늘연구소) ;
  • 허호진 (경상대학교 식품공학과(농업생명과학연구원))
  • Received : 2010.05.31
  • Accepted : 2010.08.16
  • Published : 2010.08.31
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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 ($IC_{50}$: $36.1{\mu}g/mL$). 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 ${\beta}$ protein ($A{\beta}$)-induced neurotoxicity. Because $A{\beta}$ 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 $A{\beta}$-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.

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, $IC_{50}$은 $36.1{\mu}g/mL$로 나타났다. MTT reduction assay를 이용해 amyloid ${\beta}$ protein ($A{\beta}$) 유도성 신경세포 독성에 대한 신경세포 보호효과를 측정한 결과, 세 가지 마늘 추출물들은 대부분 40% 미만의 세포생존율을 보였고 이 결과는 $A{\beta}$ 유도성 신경세포 독성보다 상대적으로 더 높은 세포독성을 보여주었다. LDH assay에서는 마늘 물 추출물이 37%의 LDH 방출량을 나타내 $200{\mu}M$의 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.

Keywords

Acknowledgement

Supported by : 재단법인 남해마늘연구소

References

  1. Agarwal, K. C. 1996. Therapeutic actions of garlic constituents. Med. Res. Rev. 16: 111-124. https://doi.org/10.1002/(SICI)1098-1128(199601)16:1<111::AID-MED4>3.0.CO;2-5
  2. Agusti, K. T. 1996. Therapeutic values of onion (Allium cepa L.) and garlic (Allium sativum L.). Indian. J. Exp. Biol. 34: 634-640.
  3. Alkam, T., A. Nitta, H. Mizoguchi, K. Saito, M. Seshima, A. Itoh, K. Yamada, and T, Nabeshima. 2008. Restraining tumor necrosis factor-alpha by thalidomide prevents the amyloid ${\beta}$-induced impairment of recognition memory in mice. Behav. Brain. Res. 189: 100-106. https://doi.org/10.1016/j.bbr.2007.12.014
  4. Alkreathy, H., Z. A. Damanhouri, N. Ahmed, M. Slevin, SS. Ali, and M. Osman Abdel-Moneim. 2010. Aged garlic extract protects against doxorubicin-induced cardiotoxicity in rats. Food Chem. Toxicol. doi:10.1016/j.fct.2010.01.005.
  5. Banerjee, S. K., P. K. Mukherjee, and S. K. Maulik. 2003. Garlic as an antioxidant: the good, the bad and the ugly. Phytother. Res. 17: 97-106. https://doi.org/10.1002/ptr.1281
  6. Brodnitz, M. H., J. V. Pascale, and L. van Derslice. 1971. Flavor components of garlic extract. J. Agric. Food Chem. 19: 273-275. https://doi.org/10.1021/jf60174a007
  7. Chung, Y. K, H. J. Heo, E. K. Kim, H. K. Kim, T. L. Huh, Y. H. 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.
  8. Das, A., N. L. Banik, and S. K. Ray. 2007. Garlic compounds generate reactive oxygen species leading to activation of stress kinases and cysteine proteases for apoptosis in human glioblastoma T98G and U87MG cells. Cancer 110: 1083-1095. https://doi.org/10.1002/cncr.22888
  9. Ellmans, G. L., K. D. Courtney, V. J. Andress, and R. M. Featherstone. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88-95. https://doi.org/10.1016/0006-2952(61)90145-9
  10. Filomeni, G., K. Aquilano, G. Rotilio, and M. R. Ciriolo. 2003. Reactive oxygen species- dependent c-Jun NH2-terminal kinase/c-Jun signaling cascade mediates neuroblastoma cell death induced by diallyl disulfide. Cancer Res. 63: 5940-5949.
  11. Fimognari, C., M. Lenzi, and P. Hrelia. 2009. Apoptosis induction by sulfur-containing compounds in malignant and nonmalignant human cells. Environ. Mol. Mutagen. 50: 171-189. https://doi.org/10.1002/em.20447
  12. Heo, H. J., S. J. Choi, S. G. Choi, D. H. Shin, J. M. Lee, and C. Y. Lee. 2008. Effect of banana, orange, and apple on oxidative stress-induced neurotoxicity in PC12 cells. J. Food Sci. 73: H28-H32. https://doi.org/10.1111/j.1750-3841.2007.00632.x
  13. Heo, H. J., D. O. Kim, S. J. Choi, D. H. Shin, and C. Y. Lee.. 2004. Potent inhibitory effect of flavonoids in Scutellaria baicalensis on amyloid ${\beta}$ protein-induced neurotoxicity. J. Agric. Food Chem. 52: 4128-4132. https://doi.org/10.1021/jf049953x
  14. Heo, H. J. and C. Y. Lee. 2006. Phenolic phytochemicals in cabbage inhibit amyloid ${\beta}$ protein-induced neurotoxicity. Lebensm. Wiss. Technol. 39: 330-336.
  15. Hugo, L. H., D. M. Ricardo, and C. L. Nibaldo. 1996. Tetrameric(G4) acetylcholinesterase: structure, localization, and physiological regulation. J. Neurochem. 66: 1335-1346.
  16. Iciek, M., I. Kwiecien, and L. Wlodek. 2009. Biological properties of garlic and garlic-derived organosulfur compounds. Environ. Mol. Mutagen. 50: 247-265. https://doi.org/10.1002/em.20474
  17. Ide, N., and B. H. Lau. 2001. Garlic compounds minimize intracellular oxidative stress and inhibit nuclear factor-${\kappa}B$ activation J. Nutr. 131: 1020S-1026S. https://doi.org/10.1093/jn/131.3.1020S
  18. Jo, J. S. 1990. Food materials. pp. 154-155. Gijeunyumgusa, Seoul, Korea.
  19. Kim, E. H., S. S., Kang, D. H. Kang, and K. H. Kyung. 2009. Antimicrobial activity of garlic heated under different conditions, time of heating, and pH. Food Sci. Biotechnol. 18: 771-775.
  20. Kim, J. H., C. H. Jeong, G. N. Choi, J. H. Kwak, S. G. Choi, and H. J. Heo. 2009. Antioxidant and neuronal cell protective effects of methanol extract from Schizandra chinensis using an in vitro system. Korean J. Food Sci. Technol. 41: 712-716.
  21. Lu, P., T. Mamiya, L. L. Lu, A. Mouri, L. Zou, T. Nagai, M. Hiramatsu, T. Ikejima, and N. Nabeshima, 2009. Silibinin prevents amyloid beta peptide-induced memory impairment and oxidative stress in mice. Br. J. Pharmacol. 157: 1270-1277. https://doi.org/10.1111/j.1476-5381.2009.00295.x
  22. Papandreou, M. A., A. Dimakopoulou, Z. I. Linardaki, P. Cordopatis, D. Klimis-Zacas, M. Margarity, and F. N. Lamari. 2009. Effect of a polyphenol-rich wild blueberry extract on cognitive performance of mice, brain antioxidant markers and acetylcholinesterase activity. Behav. Brain Res. 198: 352-358. https://doi.org/10.1016/j.bbr.2008.11.013
  23. Trubetskaya, V. V., M. Y. Stepanichev, M. V. Onufriev, N. A. Lazareva, V. A. Markevich, and N. V. Gulyaeva. 2003. Administration of aggregated beta-amyloid peptide 25-35 induces changes in long-term potentiation in the hippocampus in vivo. Neurosci. Behav. Physiol. 33: 95-98. https://doi.org/10.1023/A:1021761310435