Optimization of hydrolysis conditions, isolation, and identification of neuroprotective peptides derived from seahorse Hippocampus trimaculatus

Ratih Pangestuti, BoMi Ryu, SWA. Himaya, Se-Kwon Kim

Hippocampus trimaculatus is one of the most heavily traded seahorse species for traditional medicine purposes in many countries. In the present study, we showed neuroprotective effects of peptide derived from H. trimaculatus against amyloid-β42 (Aβ42) toxicity which are central to the pathogenesis of Alzheimer's diseases (AD). Firstly, H. trimaculatus was separately hydrolyzed by four different enzymes and tested for their protective effect on Aβ42-induced neurotoxicity in differentiated PC12 cells. Pronase E hydrolysate exerted highest protection with cell viability value of 88.33 ± 3.33 %. Furthermore, we used response surface methodology to optimize pronase E hydrolysis conditions and found that temperature at 36.69 °C with the hydrolysis time 20.01 h, enzyme to substrate (E/S) ratio of 2.02 % and pH 7.34 were the most optimum conditions. Following several purification steps, H. trimaculatus-derived neuroprotective peptides (HTP-1) sequence was identified as Gly-Thr-Glu-Asp-Glu-Leu-Asp-Lys (906.4 Da). HTP-1 protected PC12 cells from Aβ42-induced neuronal death with the cell viability value of 85.52 ± 2.22 % and up-regulated pro-survival gene (Bcl-2) expressions. These results suggest that HTP-1 has the potential to be used in treatment of neurodegenerative diseases, particularly AD. Identification, characterization, and synthesis of bioactive components derived from H. trimaculatus have the potential to replace or at least complement the use of seahorse as traditional medicine, which further may become an approach to minimize seahorse exploitation in traditional medicine.