以Aβ25-35引發PC-12細胞死亡為模式開發抗阿滋海默症食藥材

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder. The decline of memory and cognition in AD is underlied by the progressive deposition of senile plaques, neurofibrillary tangles and neuronal degeneration. The principal component in senile plaques is the β-amyloid peptide (Aβ). Aβ has been shown to lead to oxidative stress and apoptosis of neurons. Many of studies have demonstrated that antioxidants may have the potential to protect Aβ-induced cytotoxicity. The theory of traditional Chinese medicine (TCM) postulated that kidney functions is a key factor in AD development and progression. We screened the dried edible plant materials which have remarkable antioxidation activities and based on the theory of TCM, we chose TCMs which can improve liver and kidney functions to screen for the anti-Alzheimer effect. Scientific preparations of TCMs were extracted with water or methanol at room temperature. The dried edible plant materials were extracted with boiling water or methanol at room temperature. The neuroprotective effect of Ginkgo biloba extract (EGb 761) has been demonstrated by several in vitro and in vivo studies. In vitro, EGb 761 showed dose-dependent effect to protect cultured PC-12 cells against death induced by Aβ. In vivo, daily oral administration of EGb 761 improved cognitive function in patients with Alzheimer’s disease. Therefore, we utilized EGb 761 as the positive control in this study. In this study, we used the “inhibition of aggregated Aβ-induced PC-12 cells death” as the screening model for anti-Alzheimer’s disease activity, and EGb 761 was used as the positive control. PC-12 cells were incubated with samples and 15 μM Aβ25-35 for 48h. The inhibition of Aβ-induced cytotoxicity were observed with methyl thiazolyl tetrazolium (MTT) assay and were represented by cell viability and the percentage of inhibition (inhibition ratio). Among the water extracts, superior inhibition effects were exhibited by Panax ginseng（Pg-W）, Dioscorea opposit（Do-W）, Schisandra chinensis, Polygonum multiflorum（Pm-W）, Lycium barbarum（Lb-W）, Poncirus trifolata（Pt-W）, Psoralea coryhoflia（Pc-W）, green tea（GT-W）, oolong tea（OT-W）, pu-erh tea（PT-W）, GABA tea（GT-W）, Sesamum indicum（Si-W）, nelumbinis folium（Nf-W）, Perilla frutescens（Pf-W）, nelumbinis embryo（Ne-W） and Dimoracarpus longan（Dl-W） at concentration range of 50 ~200 μg/mL. Superior inhibition effects of the ethanol extracts of TCMs were shown by Cornus officinalis（Co-E）, Dioscorea opposite（Do-E）, Acorus tatarinowii（At-E）, Cistanche deserticola（Cd-E）, Lycium barbarum（Lb-E）, Astragalus membranaceus（Am-E）, Angelica sinensis（As-E）, Psoralea coryhoflia（Pc-E） and Polygala Tenuifolia（Pt-E） at concentration range of 50~200 μg/mL. Methanol extract of Sesamum indicum（Si-M）gave the greatest inhibition effects among all the edible plant materials. It also indicated that Si-W, Ne-W and Pt-W were the three most effective extracts, and all of them did not exhibit any cytotoxicity. Microscopic examination of the morphology of PC-12 cells, showed that 200 μg/mL Si-W and Ne-W could induce neurite outgrowth in PC-12 cells even when Aβ25-35 was present. In the cell cycle analysis by flow cytometer, Si-W, Ne-W and Pt-W were demonostrated to significantly inhibit Aβ25-35-induced apoptosis. Si-W, Ne-W and Pt-W were fractionated into ethyl acetate fraction, n-butanol fraction and aqueous fraction. The aqueous fractions of these three extracts were found to have better inhibition effects against Aβ-induced cytotoxicity than the other two fractions. In the cell cycle analysis, the aqueous fractions of Si-W, Ne-W and Pt-W could all significantly inhibit Aβ25-35-induced apoptosis when the concentration was 200 μg/mL. We presumed that Sesamum indicum, nelumbinis embryo and Poncirus trifolata have anti-Alzheimer’s disease capacity could probably be related to compounds having higher polarity.