Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Protective Effect of the Ethyl Acetate-fraction of Methanol Extract of Ophiophogon japonicus on Amyloid beta Peptide-induced Cytotoxicity in PC12 Cells

소엽맥문동-에틸아세테이트 분획물의 아밀로이드 베타단백질-유발 세포독성에 대한 억제 효능

  • Moon, Ja-Young (Department of Bio-Health Science, College of Natural Sciences, Changwon National University) ;
  • Kim, Eun-Sook (Department of Bio-Health Science, College of Natural Sciences, Changwon National University) ;
  • Choi, Soo-Jin (Department of Bio-Health Science, College of Natural Sciences, Changwon National University) ;
  • Kim, Jin-Ik (Department of Bio-Health Science, College of Natural Sciences, Changwon National University) ;
  • Choi, Nack-Shik (KPROTEC INC.) ;
  • Lee, Kyoung (Department of Bio-Health Science, College of Natural Sciences, Changwon National University) ;
  • Park, Woo-Jin (School of Life Sciences, Kwangju Institute of Science and Technology) ;
  • Choi, Young-Whan (Department of Horticultural Bioscience, College of Natural Resource and Life Sciences, Pusan National University)
  • 문자영 (창원대학교 생명보건학부) ;
  • 김은숙 (창원대학교 생명보건학부) ;
  • 최수진 (창원대학교 생명보건학부) ;
  • 김진익 (창원대학교 생명보건학부) ;
  • 최낙식 (케이프로텍) ;
  • 이경 (창원대학교 생명보건학부) ;
  • 박우진 (광주과학기술원 생명과학부) ;
  • 최영환 (부산대학교 원예생명과학과)
  • Received : 2019.01.03
  • Accepted : 2019.02.12
  • Published : 2019.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

Amyloid ${\beta}$-protein ($A{\beta}$) is the principal component of senile plaques characteristic of Alzheimer's disease (AD) and elicits a toxic effect on neurons in vitro and in vivo. Many environmental factors, including antioxidants and proteoglycans, modify $A{\beta}$ toxicity. It is worthwhile to isolate novel natural compounds that could prove therapeutic for patients with AD without causing detrimental side effects. In this study, we investigated the in vitro neuroprotective effects of the ethyl acetate fraction of methanol extract of Ophiophogon japonicas (OJEA fraction). We used an MTT reduction assay to detect protective effects of the OJEA fraction on $A{\beta}_{25-35}$-induced cytotoxicity to PC12 cells. We also used a cell-based ${\beta}$-secretase assay system to investigate the inhibitory effect of the OJEA fraction on ${\beta}$-secretase activity. In addition, we performed an in vitro lipid peroxidation assay to evaluate the protective effect of the OJEA fraction against oxidative stress induced by $A{\beta}_{25-35}$ in PC12 cells. The OJEA fraction had strong protective effects against $A{\beta}_{25-35}$-induced cytotoxicity to PC12 cells and was strongly inhibitory to ${\beta}$-secretase activity, which resulted in the attenuation of $A{\beta}$ generation. In addition, the OJEA fraction significantly decreased malondialdehyde (MDA) content, which is induced by the exposure of PC12 cells to $A{\beta}_{25-35}$. Our results suggested that the OJEA fraction contained active compounds exhibiting a neuroprotective effect on $A{\beta}$ toxicity.

Amyloid ${\beta}$-단백질($A{\beta}$)은 알츠하이머 질병의 특징인 노인성 반점의 주요 성분이며 in vivo와 in vitro에서 신경세포를 대상으로 독성효과를 유발한다. 항산화물질과 프로테오글리칸을 포함한 많은 환경인자들에는 $A{\beta}$의 독성을 완화하는 물질들이 존재한다. 특히, 천연물질들 중에서 자신은 독성이 없으며, 알츠하이머 환자에게 치료효능을 나타내는 천연화합물들을 순수 분리하는 것은 매우 가치가 있다. 본 연구에서는 소엽맥문동의 메탄올 추출물로부터 에틸아세테이트 유기용매로 분획한 물질(OJEA)을 대상으로 in vitro상에서 신경세포독성 제어효능을 탐색하였다. 본 실험을 위해 PC12 세포주에 $A{\beta}_{25-35}$로 유발한 독성에 대한 OJEA 분획물의 억제효능을 MTT 환원법 분석으로 측정하였으며, ${\beta}$-secretase 활성에 대한 OJEA 분획물의 억제효능을 세포기반 ${\beta}$-secretase assay system으로 측정하였다. 또한 PC12 세포에서 $A{\beta}_{25-35}$에 의해 유도된 산화적 스트레스에 대한 OJEA 분획물의 억제효과를 지질과산화 분석법으로 수행하였다. 본 연구의 결과는 OJEA 분획물이 PC12 세포에서 $A{\beta}_{25-35}$에 의해 유도된 세포독성을 강하게 예방 또는 억제하는 효과가 있음을 확인하였으며, 또한 ${\beta}$-secretase의 활성을 억제함으로써 $A{\beta}$의 생성을 완화하는 효과를 예상할 수 있었다. OJEA 분획물은 또한 PC12 세포에서 $A{\beta}_{25-35}$에의 노출에 의하여 유도되는 malondialdehyde (MDA)의 생성을 강하게 억제하였다. 결론적으로, 본 연구의 결과에 의하면 OJEA 분획물에는 $A{\beta}$ 독성에 대한 신경세포의 보호효능을 함유하는 생리활성물질이 함유되어 있음을 제시한다.

Keywords

SMGHBM_2019_v29n2_173_f0001.png 이미지

Fig. 1. Effects of the OJEA fraction (A) and Aβ25-35(B) on viability of PC12 cells. The cells were cultured for 24 hr in DMEM supplemented with 10% FBS, 10% horse serum, 100 units/ml penicillin, and 100 μg/ml streptomycin containing different concentrations of OJEA fraction (A) or Aβ25-35(B). Cell viability in PC12 cells was determined by MTT reduction assay as described in Materials and Method.

SMGHBM_2019_v29n2_173_f0002.png 이미지

Fig. 2. Protective effects of OJEA fraction on Aβ25-35-induced cytotoxicity in PC12 cells. Data are presented as means ± standard deviation from independent experiments performed in triplicate (n=3). *, significantly different from Aβ25-25only treated group, p<0.05.

SMGHBM_2019_v29n2_173_f0003.png 이미지

Fig. 3. Dose-dependent inhibitory potency of the OJEA fraction (A) or three peptidomimetic compounds (B) to BACE. Various amounts of the OJEA fraction were tested for measuring its inhibitory potency. Three peptidomimetic compounds were tested as reference inhibitors for BACE. The assay was performed three times. The curves were fitted using Sigma Plot, yielding IC50 values of the fractions.

SMGHBM_2019_v29n2_173_f0004.png 이미지

Fig. 4. Protective effects of the OJEA fraction against lipid peroxidation induced by Aβ25-35treatment in PC12 cells. Data are presented as means ± standard deviation from independent experiments performed in triplicate (n=3). *, significantly different from Aβ25-25treated group, p<0.05.

References

  1. Andrea, Z. 2003. The psychopharmacology of huperzine A: an alkaloid with cognitive enhancing and neuroprotective properties of interest in the treatment of Alzheimer's disease. Pharmacol. Biochem. Behav. 75, 675-686. https://doi.org/10.1016/S0091-3057(03)00111-4
  2. Anh, N. T. H., Sung, T., Porzel, A., Franke, K. and Wessjohann, L. A. 2003. Homoisoflavonoids from Ophiopogon japonicus Ker-Gawler. Phytochemistry 62, 1153-1158. https://doi.org/10.1016/S0031-9422(02)00515-0
  3. Bastinetto, S., Ramassamy, C., Dore, S., Christen, Y., Poirier, J. and Quirion, R. 2000. The ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by ${\beta}$-amyloid. European J. Neurosci. 12, 1882-1890. https://doi.org/10.1046/j.1460-9568.2000.00069.x
  4. Braak, H., de Vos, R. A., Jansen, E. N., Bratzke, H. and Braak, E. 1998. Neuropathological hallmarks of Alzheimer's and Parkinson's diseases. Prog. Brain Res. 117, 267-85. https://doi.org/10.1016/S0079-6123(08)64021-2
  5. Chen, X. M., Jin, J., Tang, J., Wang, Z. F., Wang, J. J., Jin, L. Q. 2011. Extraction, purification, characterization and hypoglycemic activity of a polysaccharide isolated from the root of Ophiopogon japonicus. Carbohyd. Polym. 83, 749-754. https://doi.org/10.1016/j.carbpol.2010.08.050
  6. Cole, S. L. and Vassar, R. 2008. The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology. J. Biol. Chem. 283, 29621-29625. https://doi.org/10.1074/jbc.R800015200
  7. Draper, H. H. and Hadley, M. 1990. Malondialdehyde determination as index of lipid peroxidation. Methods in Enzymol. 186, 421-431. https://doi.org/10.1016/0076-6879(90)86135-I
  8. Durand, D., Carniglia, L., Beauquis, J, Caruso, C., Saravia, F. and Lasaga, M. 2014. Astroglial mGlu3 receptors promote alpha-secretase-mediated amyloid precursor protein cleavage. Neuropharmacology 79, 180-189. https://doi.org/10.1016/j.neuropharm.2013.11.015
  9. D'Ursi, A. M., Armenante, M. R., Guerrini, R., Salvadori, S., Sorrentino, G. and Picone, D. 2004. Solution structure of amyloid beta-peptide (25-35) in different media. J. Med. Chem. 47, 4231-4238. https://doi.org/10.1021/jm040773o
  10. Fan, J. and Zhang, X. 2006. Research progress in pharmacology of Ophiopogon japonicus polysaccharides. Chinese Archives of Traditional Chinese Medicine 24, 626-627. https://doi.org/10.3969/j.issn.1673-7717.2006.04.023
  11. Guo, T. and Hobbs, D. W. 2006. Development of BACE1 inhibitors for Alzheimer's disease. Curr. Med. Chem. 13, 1811-1829. https://doi.org/10.2174/092986706777452489
  12. Haass, C. 2004. Take five--BACE and the gamma-secretase quartet conduct Alzheimer's amyloid beta-peptide generation. EMBO J. 23, 483-488. https://doi.org/10.1038/sj.emboj.7600061
  13. Hardy, J. and Selkoe, D. J. 2002. The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353-356. https://doi.org/10.1126/science.1072994
  14. Hoi, C. P, Ho, Y. P., Baum, L. and Chow, A. H. 2010. Neuroprotective effect of honokiol and magnolol, compounds from Magnolia officinalis, on beta-amyloid-induced toxicity in PC12 cells. Phytother. Res. 24, 1538-1542. https://doi.org/10.1002/ptr.3178
  15. Hung, T. M., Thu, C. V., Dat, N. T., Ryoo, S. W., Lee, J. H., Kim, J. C., Na, M., Jung, H. J., Bae, K. and Min, B. S. 2010. Homoisoflavonoid derivatives from the roots of Ophiopogon japonicus and their in vitro anti-inflammation activity. Bioorg. Med. Chem. Lett. 20, 2412-2416. https://doi.org/10.1016/j.bmcl.2010.03.043
  16. Kihara, T., Shimohama, S., Sawada, H., Honda, K., Nakamizo, T., Shibasaki, H., Kume, T. and Akaike, A. 2001. Alpha 7 nicotinic receptor transduces signals to phosphatidylinositol 3-kinase to block A beta-amyloid-induced neurotoxicity. J. Biol. Chem. 276, 13541-13546. https://doi.org/10.1074/jbc.M008035200
  17. Kim, E .S., Choi, S. J., Lee, C. W., Park, K. T. and Moon, J. Y. 2005. Protective effect of Liliaceae root extracts on amyloid ${\beta}$-protein-induced death in neuronal cells. Cancer Prevention Res. 10, 242-250.
  18. Kim, M. H., Kim, S. H. and Yang, W. M. 2014. Mechanisms of action of phytochemicals from medicinal herbs in the treatment of Alzheimerʼs Disease. Planta Med. 80, 1249-1258. https://doi.org/10.1055/s-0034-1383038
  19. Kou, J., Tian, Y., Tang, Y., Yan, J. and Yu, B. 2006. Antithrombotic activities of aqueous extract from Radix Ophiopogon japonicus and its two constituents. Biol. Pharm. Bull. 29, 1267-1270. https://doi.org/10.1248/bpb.29.1267
  20. Le Bars, P. L., Katz, M. M., Berman, N., Itil, T. M., Freedman, A. M. and Schatzberg, A. F. 1997. A placebo-controlled, double-blind, randomized trial of an extract of Ginko biloba for dementia, North American Egb Study Group. J. Am. Med. Assoc. 278, 1327-1332. https://doi.org/10.1001/jama.1997.03550160047037
  21. Lin, X., Koelsch, G., Wu, S., Downs, D., Dashti, A. and Tang, J. 2000. Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc. Natl. Acad. Sci. USA. 97, 1456-1460. https://doi.org/10.1073/pnas.97.4.1456
  22. Li, Y. N., Zhu, D. N., Qi, J., Qin, M. J. and Yu, B. Y. 2010. Characterization of homoisoflavonoids in different cultivation regions of Ophiopogon japonicus and related antioxidant activity. J. Pharm. Biomed. Anal. 52, 757-762. https://doi.org/10.1016/j.jpba.2010.02.016
  23. Masters, C. L., Multhaup, G., Simms, G., Pottgiesser, J., Martins, R. N. and Beyreuther, K. 1985. Neuronal origin of a cerebral amyloid: neurofibriliary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels. EMBO J. 4, 2757-2763. https://doi.org/10.1002/j.1460-2075.1985.tb04000.x
  24. Muthaiyah, B., Essa, M. M., Chauhan, V. and Chauhan, A. 2011. Protective effects of walnut extract against amyloid beta peptide-induced cell death and oxidative stress in PC12 cells. J. Neurosci. 36, 2096-2103.
  25. Oh, M. S., Kim, S. Y., Oh, Y. S., Choi, D. Y., Sin, H. J., Jung, I. M. and Park, W. J. 2003. Cell-based assay for ${\beta}$-secretase activity. Anal. Biochem. 323, 7-11. https://doi.org/10.1016/j.ab.2003.08.036
  26. Oken, B. S., Storzbach, D. M. and Kaye, J. A. 1998. The efficacy of Ginkgo biloba on cognitive function in Alzheimer disease. Arch. Neurol. 55, 1409-1415. https://doi.org/10.1001/archneur.55.11.1409
  27. Park, S. Y. and Kim, D. S. 2002. J. Nat. Prod. 65, 1227-1231. https://doi.org/10.1021/np010039x
  28. Pike, C. J., Walencewicz-Wasserman, A. J., Kosmoski, J., Cribbs, D. H., Glabe, C. G. and Cotman, C. W. 1995. Structure-activity analyses of beta-amyloid peptides: contributions of the beta 25-35 region to aggregation and neurotoxicity. J. Neurochem. 64, 253-265. https://doi.org/10.1046/j.1471-4159.1995.64010253.x
  29. Qian, X., Cao, H., Ma, Q., Wang, Q., He, W., Qin, P., Ji, B., Yuan, K., Yang, F. and Liu, X. 2015. Allopregnanolone attenuates $Ab_{25-35}$-induced neurotoxicity in PC12 cells by reducing oxidative stress. Int. J. Clin. Exp. Med. 8, 13610-13615.
  30. Salloway, S., Mintzer, J., Weiner, M. F. and Cummings, J. L. 2008. Disease-modifying therapies in Alzheimer's disease. Alzheimers Dement 4, 65-79. https://doi.org/10.1016/j.jalz.2007.10.001
  31. Varadarajan, S., Kanski, J., Aksenova, M., Lauderback, C. and Butterfield, D. A. 2001. Different mechanisms of oxidative stress and neurotoxicity for Alzheimer's A beta (1-42) and A beta (25-35). J. Am. Chem. Soc. 123, 5625-5631. https://doi.org/10.1021/ja010452r
  32. Vassar, R. 2004. BACE1: the beta-secretase enzyme in Alzheimer's disease. J. Mol. Neurosci. 23, 105-114. https://doi.org/10.1385/JMN:23:1-2:105
  33. Yankner, B. A., Duffy, L. K. and Kirschner, D. A. 1990. Neurotrophic and neurotoxic effects of amyloid ${\beta}$ protein: reversal by tachykinin neuropeptides. Science 250, 279-282. https://doi.org/10.1126/science.2218531
  34. Yu, X. W., Du, H. Z., Sun, L. and Yuan, S. T. 2014. Research progress on the pharmacological effects of Ophiopogonins. Prog. Pharm. Sci. 38, 279-284.
  35. Zheng, Q., Feng, Y., Xu, D. S., Lin, X. and Chen, Y. Z. 2009. Influence of sulfation on antimyocardial ischemic activity of Ophiopogon japonicus polysaccharide. J. Asian Nat. Prod. Res. 11, 306-321. https://doi.org/10.1080/10286020902727363
  36. Zhou, Y. H., Xu, D. S., Feng, Y., Fang, J. N., Xia, H. L. and Liu, J. 2003. Effects on nutrition blood flow of cardiac muscle in mice by different extracts in radix Ophiopogonis. Chin. J. Exp. Tradit. Med. Form. 9, 22-23.