Green Tea Seed Isolated Theasaponin E1 Ameliorates AD Promoting Neurotoxic Pathogenesis by Attenuating Aβ Peptide Levels in SweAPP N2a Cells

Abstract

Alzheimer's disease (AD) is the most frequent type of dementia affecting memory, thinking and behaviour. The major hallmark of the disease is pathological neurodegeneration due to abnormal aggregation of Amyloid beta (Aβ) peptides generated by β- and γ-secretases via amyloidogenic pathway. Purpose of the current study was to evaluate the effects of theasaponin E1 on the inhibition of Aβ producing β-, γ-secretases (BACE1, PS1 and NCT) and acetylcholinesterase and activation of the non-amyloidogenic APP processing α-secretase (ADAM10). Additionally, theasaponin E1 effects on Aβ degrading and clearing proteins neprilysin and insulin degrading enzyme (IDE). The effect of theasaponin E1 on these crucial enzymes was investigated by RT-PCR, ELISA, western blotting and fluorometric assays using mouse neuroblastoma cells (SweAPP N2a). theasaponin E1 was extracted and purified from green tea seed extract via HPLC, and N2a cells were treated with different concentrations for 24 h. Gene and protein expression in the cells were measured to determine the effects of activation and/or inhibition of theasaponin E1 on β- and γ-secretases, neprilysin and IDE. Results demonstrated that theasaponin E1 significantly reduced Aβ concentration by activation of the α-secretase and neprilysin. The activities of β- and γ-secretase were reduced in a dose-dependent manner due to downregulation of BACE1, presenilin, and nicastrin. Similarly, theasaponin E1 significantly reduced the activity of acetylcholinesterase. Overall, from the results it is concluded that green tea seed extracted saponin E1 possess therapeutic significance as a neuroprotective natural product recommended for the treatment of Alzheimer's disease.

Keywords: Alzheimer’s disease; SweAPP N2a cells; amyloid precursor proteins; amyloidogenic pathway; theasaponin E1; α-secretase.

Figure 1

LC/TOF/MS analysis of Camellia sinensis seed extracted saponin mixture and the isolated pure saponins fraction (Base peak intensity chromatogram at m/z 1050–1300). (a) LC-MS chromatogram of the saponin mixture showed the presence of theasaponin E1, theasaponin C, assamsaponin A (C₅₇H₈₈O₂₅), theasaponin E3 (C₅₇H₈₈O₂₆), theasaponin A1 (C₅₇H₉₀O₂₆), assamsaponin B (C₆₁H₉₂O₂₈) and theasaponin A3, (C₆₁H₉₄O₂₈). (b) LC-MS chromatogram of the isolated theasaponin E1 reached fraction. (c) LC-MS/MS spectra of the isolated saponin fraction showed theasaponin E1.

Figure 2

Thin layer chromatography (TLC) of the green tea seed extracted saponin mixture and the purified isolated theasaponin E1 in comparison with the commercial grade standard theasaponin E1.

Figure 3

Toxicity determination of theasaponin E1 on mouse neuroblastoma cells by MTT assay. Percentage cell viability was determined for control and various concentrations of theasaponin E1. Experiments were performed in triplicate. Data are the mean ± SEM. Data are statistically significant at p < 0.05. (* represents significance at p < 0.05, ** represents significance at p < 0.01 to control group).

Figure 4

Acetylcholinesterase inhibitory activity of theasaponin E1. Percent inhibition was measured for each concentration and control according to the manufacturer’s protocol. Data are expressed as mean ± SEM. Data were analysed with one-way ANOVA (n =  3). Data are statistically significant at p < 0.05. (* represents significance at p < 0.05, ** represents significance at p < 0.01).

Figure 5

Effects of theasaponin E1 on the activities of APP cleaving proteases and Aβ degrading enzymes in vitro determined by fluorometric assays. Percent activity was measured for control and treatment groups for each enzyme. (a) Effects of theasaponin E1 on β-secretase (BACE1) activities. (b) Effects of theasaponin E1 on γ-secretase (PS1) activities. (c) Effects of theasaponin E1 on γ-secretase (NCT). (d) Effects of Theasaponin E1 on α-secretase (ADAM10) activities. (e) Effects of theasaponin E1 on neprilysin activities. (f) Effects of theasaponin E1 on insulin degrading enzyme (IDE) activities. Data are expressed as mean ± SEM. Data were analysed with one-way ANOVA (n =  3). (*p < 0.05; ** p < 0.01).

Figure 6

Effects of theasaponin E1 on mRNA expression levels of APP cleaving proteases and Aβ degrading enzymes in vitro determined by RT-PCR. Relative mRNA expression levels of genes of various enzymes under the effects of theasaponin E1. (a) Relative mRNA expression level of α-secretase (ADAM10). (b) Relative mRNA expression level of neprilysin. (c) Relative mRNA expression level of insulin degrading enzyme (IDE). (d) Relative mRNA expression level of β-secretase (BACE1). (e) Relative mRNA expression level of γ-secretase (PS1). (f) Relative mRNA expression level of γ-secretase NCT. (g) Expression level of ADAM10, BACE1, PS1, NCT, neprilysin and IDE genes. Visualized the PCR products on agarose gel. Data are expressed as mean ± SEM. Data were analysed with one-way ANOVA (n = 3). Data are statistically significant at p < 0.05. (* p < 0.05; ** p < 0.01 to control group).

Figure 7

Effect of theasaponin E1 on the level of Aβ peptides and APP level quantified by ELISA (due to enhancement or reduction in the activities of APP processing and Aβ amyloid producing or degrading enzymes). (A) Aβ quantification by ELISA after treatment of the cells with various concentrations of E1 theasaponin. (B) APP quantification by ELISA after treatment of the cells with various concentration of E1 theasaponin. Data are expressed as mean ± SEM. Data were analysed with one-way ANOVA (n  =  3). Data are statistically significant at p < 0.05. (* p < 0.05; ** p < 0.01 to control group).

Figure 8

Effects of theasaponin E1 on the protein expression level of ADAM10, BACE1, PS1, NCT, neprlysin, IDE and APP determined by western blotting. Relative proteins expression levels of various proteins normalized (fold change) to control (β-actin). (a) Relative proteins expression level of α-secretase (ADAM10). (b) Relative proteins expression level of neprilysin. (c) Relative proteins expression level of insulin degrading enzyme (IDE). (d) Relative proteins expression level of β-secretase (BACE1). (e) Relative proteins expression level of γ-secretase presenilin 1 (PS1). (f) Relative proteins expression level of γ-secretase nicastrin NCT. (g) Relative expression level of APP. (h) Expression of APP, ADAM10, BACE1, PS1, NCT, neprilysin and IDE proteins, visualized bands on membrane. Data are expressed as mean ± SEM. Data were analysed with one-way ANOVA (n = 3). Data are statistically significant at p < 0.05. (*p < 0.05; ** p < 0.01). Data are expressed as mean ± SEM.