Allocryptopine, tetrahydropalmatine, and tetrahydroberberine N-oxide alkaloids alleviate cellular stress by modulating calcium homeostasis and the MAPK and akt/GSK-3β/tau signaling pathways.

Introduction: Glaucium grandiflorum Boiss. and A. Huet subsp. refractum (Papaveraceae) is a plant used in traditional medicine for analgesic, anti-inflammatory, sedative, and bronchitis treatment. Benzylisoquinoline derivative alkaloids in its content are responsible for its muscle relaxant, antitussive, and antioxidant effects. It attracts attention as a promising natural source, especially for neurodegenerative diseases (NDs) and conditions associated with oxidative stress, with its neuroprotective, antiproliferative, and calcium homeostasis regulating effects. This study investigates the neuroprotective effects of allocryptopine, tetrahydropalmatine, and tetrahydroberberine N-oxide rich alkaloid extract from Glaucium grandiflorum (GGAE). Methods: The plant was collected, identified, and the GGAE was prepared by macerating dried, pulverized material in chloroform. The GGAE's neuroprotective properties were assessed using the rat pheochromocytoma (PC12) cell line. Intracellular calcium levels were analyzed via flow cytometry; gene expression of L-type voltage-gated calcium channel subtypes was evaluated with qRT-PCR; and the phosphorylation status of key proteins (p-ERK1/2, p-JNK, p-p38, p-Akt, p-GSK-3β, and p-Tau) was determined using Western blotting. The binding energies and contact residues of alkaloids (allocryptopine, tetrahydropalmatine, and tetrahydroberberine N-oxide) found in the GGAE were determined to target proteins (AKT1, CACNA1C, CACNA1D, ERK1/2, GSK3β, JNK, P38, and TAU). Results: The results suggest that the GGAE helps maintain intracellular calcium homeostasis and functions as an L-type Ca2+ channel blocker, crucial for neuronal survival. It modulates key signaling pathways by dephosphorylating stress-related proteins p-ERK1/2, p-JNK, and p-p38, while enhancing cell survival pathways by phosphorylating p-Akt (Ser 473) and p-GSK-3β (Ser 9). Additionally, the GGAE reduces pathological phosphorylation of p-Tau (Ser 396 and Thr 212), proteins associated with NDs. Molecular docking results demonstrated that alkaloids exhibit strong binding energies to target proteins. Discussion: These findings suggest that the GGAE exerts a comprehensive neuroprotective effect, positioning it as a promising therapeutic candidate for the treatment of NDs.