생명과학회지 (Journal of Life Science)

  • 제29권2호
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  • Pages.173-180
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  • 2019
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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소엽맥문동-에틸아세테이트 분획물의 아밀로이드 베타단백질-유발 세포독성에 대한 억제 효능

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

  • 문자영 (창원대학교 생명보건학부) ;
  • 김은숙 (창원대학교 생명보건학부) ;
  • 최수진 (창원대학교 생명보건학부) ;
  • 김진익 (창원대학교 생명보건학부) ;
  • 최낙식 (케이프로텍) ;
  • 이경 (창원대학교 생명보건학부) ;
  • 박우진 (광주과학기술원 생명과학부) ;
  • 최영환 (부산대학교 원예생명과학과)
  • 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)
  • 투고 : 2019.01.03
  • 심사 : 2019.02.12
  • 발행 : 2019.02.28
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초록

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.

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.

키워드

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.

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