The inhibition mechanism of polyphenols from Phyllanthus emblica Linn. fruit on acetylcholinesterase: A interaction, kinetic, spectroscopic, and molecular simulation study.

The present study aimed to investigate the inhibition mechanism of polyphenols from Phyllanthus emblica Linn. fruit (PEF, family Euphorbiaceous) on acetylcholinesterase (AChE). Interaction assay, enzyme kinetics, spectroscopic methods, and molecular simulations were performed. Results showed that myricetin, quercetin, fisetin, and gallic acid were the most active components in PEF, because of their low docking scores and strong inhibition ability on AChE with IC50 values of 0.1974 ± 0.0047, 0.2589 ± 0.0131, 1.0905 ± 0.0598 and 1.503 ± 0.0728 mM, respectively. Among them, the results of kinetic study showed that myricetin, quercetin, and fisetin reversibly inhibited AChE in a competitive manner, while gallic acid inhibited it through a noncompetition type. The interaction assay implied that a combination of the four polyphenols at the selected concentrations manifested a synergistic inhibition effect on AChE in a mixed inhibition type. Fluorescence and UV-vis spectrophotometry revealed that the active PEF polyphenols could strongly quench the intrinsic fluorescence of AChE via a static quenching mechanism. Circular dichroism spectroscopy analysis indicated that the active PEF polyphenols gave rise to the secondary structure changes of AChE by increasing the content of α-helix and reducing ß-sheet and random coil conformation. The molecular dynamics simulation results validated that all the four docked polyphenol-AChE complexes were relatively stable according to their root-mean-square distance, root-mean-square fluctuations, solvent accessible surface area, radius of gyration values and hydrogen bonds evaluations during the whole simulation process. Overall, our study provides a creative insight into the further utilization of PEF polyphenols as functional components in exploring natural AChE inhibitors.

The Composition and Anti-Aging Activities of Polyphenol Extract from Phyllanthus emblica L. Fruit.

Phyllanthus emblica L. (PE) is commonly known as a medicine and food homologous plant, which is abundant in natural products polyphenols. In the present study, polyphenols were extracted from PE fruit by response surface method, and the anti-aging ability was determined. PE fruit polyphenols exhibited strong antioxidant capacities in scavenging free radicals, and anti-cholinesterase ability by inhibition of AChE (IC50 0.2186 ± 0.0416 mg/mL) and BuChE (IC50 0.0542 ± 0.0054 mg/mL) in vitro. Moreover, PE fruit polyphenols showed strong protective effect against the aging process in Caenorhabditis elegans model, including increased thermal resistance, extended lifespan by 18.53% (p < 0.05), reduced activity of AChE by 34.71% and BuChE by 45.38% (p < 0.01). This was accompanied by the enhancement in antioxidant enzymes activity of SOD by 30.74% (p < 0.05) and CAT by 8.42% (p > 0.05), while decrease in MDA level by 36.25% (p < 0.05). These properties might be interrelated with the presence of abundant flavonols and phenolic acids identified by UPLC-ESI-QTOF-MS, such as quercetin, myricetin, ellagic, gallic, and chlorogenic acids, together with their glycosides. The remarkable antioxidant and anti-aging potential of PE fruit polyphenols could be implemented in the food and pharmaceutical industry.

Neuroprotection Effect of Astragaloside IV from 2-DG-Induced Endoplasmic Reticulum Stress.

OBJECTIVE: Astragaloside IV shows neuroprotective activity, but its mechanism remains unclear. To investigate whether astragaloside IV protects from endoplasmic reticulum stress (ERS), we focus on the regulation of glycogen synthase kinase-3ß (GSK-3ß) and mitochondrial permeability transition pore (mPTP) by astragaloside IV in neuronal cell PC12. METHODS AND RESULTS: PC12 cells treated with different concentrations of ERS inductor 2-deoxyglucose (2-DG) (25-500 µM) showed a significant increase of glucose-regulated protein 78 (GRP 78) and GRP 94 expressions and a decrease of tetramethylrhodamine ethyl ester (TMRE) fluorescence intensity and mitochondrial membrane potential (∆Ψm), with the peak effect seen at 50 µM, indicating that 2-DG induces ERS and the mPTP opening. Similarly, 50 µM of astragaloside IV increased the GSK-3ß phosphorylation at Ser9 most significantly. Next, we examined the neuroprotection of astragaloside IV by dividing the PC12 cells into control group, 2-DG treatment group, astragaloside IV plus 2-DG treatment group, and astragaloside IV only group. PC12 cells treated with 50 µM 2-DG for different time courses (0-36 hr) showed a significant increase of Cleaved-Caspase-3 with the peak at 6 hr. 2-DG significantly induced cell apoptosis and increased the green fluorescence intensity of Annexin V-FITC, and these effects were reversed by astragaloside IV. Such a result indicates that astragaloside IV protected neural cell survival from ERS. 2-DG treatment significantly increased the expressions of inositol-requiring ER-to-nucleus signal kinase 1 (IRE1), phosphor-protein kinase R-like ER kinase (p-PERK), but not affect the transcription factor 6 (ATF6) expression. 2-DG treatment significantly decreased the phosphorylation of GSK-3ß and significantly reduced the TMRE fluorescence intensity and ∆Ψm, following mPTP open. Astragaloside IV significantly inhibited the above effects caused by 2-DG, except the upregulation of ATF6 protein. Taken together, astragaloside IV significantly inhibited the ERS caused by 2-DG. CONCLUSION: Our data suggested that astragaloside IV protects PC12 cells from ERS by inactivation of GSK-3ß and preventing the mPTP opening. The GRP 78, GRP 94, IRE1, and PERK signaling pathways but not ATF6 are responsible for GSK-3ß inactivation and neuroprotection by astragaloside IV.

Tauroursodeoxycholic acid inhibits endoplasmic reticulum stress, blocks mitochondrial permeability transition pore opening, and suppresses reperfusion injury through GSK-3ß in cardiac H9c2 cells.

This study investigates whether inhibition of endoplasmic reticulum (ER) stress prevents opening of the mitochondrial permeability transition pore (mPTP) and evaluates the corresponding signaling pathways involved in this process. Exposure of cardiac H9c2 cells to 800 µM H2O2 for 20 min opened mPTP in response to oxidative stress, as demonstrated by quenching of tetramethylrhodamine ethyl ester (TMRE) fluorescence. Oxidative stress-induced mPTP opening was rescued by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) in a dose-dependent manner at low concentrations. The PI3K and PKG inhibitors LY294002 and KT5823 inhibited the effect of TUDCA on mPTP opening, suggesting the involvement of PI3K/Akt and PKG signaling pathways. TUDCA significantly increased glycogen synthase kinase 3 (GSK-3ß) phosphorylation at Ser-9, with peak effect at 30 µM TUDCA. The level of GRP78 (ER chaperone) expression was significantly upregulated by 30 µM TUDCA. TUDCA-induced increases in Akt and GSK-3ß phosphorylation were inhibited by LY294002, whereas KT5823 suppressed TUDCA-induced increases in VASP and GSK-3ß phosphorylation. Oxidative stress severely affected cell morphology and ultrastructure. TUDCA prevented H2O2-induced ER swelling and mitochondrial damage. TUDCA boosted the viability of cells disrupted by ischemia/reperfusion (I/R), indicating that TUDCA eased reperfusion injury. However, TUDCA did not improve the viability of cells expressing the constitutively active GSK-3ß mutant (GSK-3ß-S9A-HA) that were subjected to I/R, suggesting an essential role of GSK-3ß inactivation in TUDCA-mediated cardioprotection against reperfusion damage. These data indicate that ER stress inhibition prevents mPTP opening and attenuates reperfusion injury through GSK-3ß inactivation. The PI3K/Akt and PKG pathways may mediate GSK-3ß inactivation.