Abstract:

This study aimed to optimize the enzymatic preparation of Cordyceps militaris polypeptides and to elucidate their antioxidant activities and underlying mechanisms. The residual biomass of Cordyceps militaris after cordycepin extraction was ustilized as the raw materials. Cordyceps militaris protein was extracted via alkali extraction-acid precipitation method, followed by the optimization of a multi-enzyme hydrolysis process to produce Cordyceps militaris polypeptides. The scavenging capacities of Cordyceps militaris polypeptides and crude protein against DPPH, ABTS, and ·OH free radicals were determined at different mass concentrations（0.1, 0.2, 0.5, 1.0, 2.0, 4.0 mg/mL）. In addition, HaCaT cells were pretreated with 0.5, 1.0, and 2.0 mg/mL of Cordyceps militaris polypeptides for 24 h, followed by UVB irradiation. At 24 h post-UVB irradiation, cell viability was assessed by the CCK-8 assay; intracellular reactive oxygen species（ROS）levels were measured using the DCFH-DA fluorescent probe method; and the activities of SOD, CAT, and GSH-Px, as well as the cellular content of MDA were detected according to the kit instructions. The activity of the intracellular Nrf2/Keap1 pathway was determined by Western blot. The results show that the degree of hydrolysis of Cordyceps militaris hydrolyzed by the sequential combination of snail enzyme + pepsin + papain + alkaline protease is 84.25%±6.15%, which is identified as the optimal enzyme combination. The IC₅₀ values of Cordyceps militaris polypeptides for DPPH, ABTS, and ·OH free radicals are 1.67, 3.02 and 1.49 mg/mL, respectively. Compared with the model group（M）, pretreatment with low-, medium-, and high-dose Cordyceps militaris polypeptide groups（CMP-L/M/H）and the vitamin C（VC）group significantly increase relative cell viability, decrease relative ROS fluorescence intensity, enhance the activities of SOD, CAT, and GSH-Px, and reduce MDA content, while upregulating nuclear Nrf2 protein levels and downregulating Keap1 protein levels in a dose-dependent manner（P<0.05）. In conclusion, the optimized four-enzyme sequential hydrolysis process developed in this study can efficiently produce Cordyceps militaris polypeptides, which can alleviate UVB-induced oxidative damage in HaCaT cells by directly scavenging free radicals and activating the Nrf2/Keap1 pathway to enhance endogenous antioxidant defenses.

Key words: Cordyceps militaris, polypeptides, enzymatic hydrolysis, antioxidant activity, Nrf2/Keap1 pathway