Identification of prenylated phenolics in mulberry leaf and their neuroprotective activity

Background: Neurodegenerative diseases are becoming increasingly prevalent over the world. Therefore, drug development in this field is urgently required. Neuron impairment leads to the pathogenesis of neurodegenerative diseases, while amelioration of oxidative stress can inhibit the impairment. As a traditional Chinese medicine, mulberry leaf exhibits various pharmacological properties, including neuroprotective activity. But the major components responsible for the neuroprotective activity of mulberry leaf remained unknown. Phytochemicals were potent candidates of neuroprotective drug. Prenylated phenolics are the leading phytochemicals present in mulberry leaf. Purpose: The aim of this study was to investigate the neuroprotective activities and mechanisms of prenylated phenolics. Methods: The chemical structure of isolated compounds were elucidated by MS and NMR. UPLC-MS/MS was used to determine the contents of prenylated phenolics in fresh mulberry leaf. Neurotoxicity was induced by erastin in HT22 cells. CCK-8 assay was performed to assess cell viability. ROS production, GSH level and iron release were monitored by using DCFH-DA, monobromobimane, and FeRhoNox T-1, respectively. qRT-PCR and Western blotting assays were performed to assess gene and protein expression, respectively. Results: Four prenylated phenolics, including isobavachalcone, morachalcone B, moracin N and morachalcone A were isolated and identified from mulberry leaf. Their levels in fresh mulberry leaf were in a decreasing order, moracin N > morachalcone A > morachalcone B > isobavachalcone. Moreover, moracin N showed a good neuroprotective activity with an EC50 < 0.50 mu M. The neuroprotective mechanisms of moracin N included inhibition of glutathione depletion, glutathione peroxidase 4 (GPx4) inactivation, reactive oxygen species (ROS) overproduction and iron accumulation, as well as improvement of intracellular antioxidant enzyme activities. Moracin N augmented the transcriptional levels of genes involved in antioxidant defense and glutathione biosynthesis in the early state of ferroptosis induction, and downregulated expression of genes related to iron accumulation and lipid peroxidation. Conclusion: The results confirmed that moracin N was a good ferroptosis inhibitor, which exerted neuroprotective activity through preventing from oxidative stress.

Automated summary

The study identified four prenylated phenolics in mulberry leaf, with moracin N demonstrating good neuroprotective activity by inhibiting oxidative stress through multiple mechanisms and enhancing antioxidant defense and regulation of iron death.