Study on extraction technology and antioxidant activity of "Sibai" extracts based on orthogonal design and Caenorhabditis elegans

This study examined the antioxidant, whitening, anti-aging, and safety properties of "Sibai" extract obtained from Bombyx Batryticatus , Ampelopsis japonica , Radix Paeoniae Alba , and Atractylodes macrocephala . The "Sibai" extract was optimized by orthogonal design and evaluated for in vitro antioxidant activity through hydroxyl radical scavenging, DPPH scavenging, and total reducing capacity measurements. Tyrosinase inhibition and hemolysis assays assessed its whitening potential and biosafety. Anti-aging effects were examined using Caenorhabditis elegans (C. elegans ) lifespan and fecundity assays, while antioxidant capacity under H 2 O 2 , juglone, and heat stress was evaluated. Motor function was analyzed via head thrashing, body bending, and spontaneous locomotion tests. Finally, aging-related changes in lipofuscin, reactive oxygen species (ROS), malondialdehyde (MDA), and catalase (CAT) levels were measured. The optimal ratio of Bombyx Batryticatus , Ampelopsis japonica , Radix Paeoniae Alba , and Atractylodes macrocephala rhizome was determined to be 3:3:1:1. At specific concentrations, the extract demonstrated significant biological activity: the scavenging rate for hydroxyl radicals was 61.70% (at 48 ​mg/mL), the scavenging rate for DPPH radicals was 93.53% (at 4 ​mg/mL), and its total reducing power at 48 ​mg/mL was comparable to that of Vitamin C (Vc). The tyrosinase inhibition rate was 74.90% (at 24 ​mg/mL), and the hemolysis rate remained below 15%, indicating good in vitro safety. In lifespan experiments using C. elegans as a model, treatment with 48 ​mg/mL extract reduced fecundity and extended the normal lifespan by 38.5%. Under oxidative stress induced by H 2 O 2 and juglone, as well as under heat stress, the survival time of C. elegans in the 48 ​mg/mL treatment group increased by 37.9%, 69.5%, and 49.2% respectively. In terms of motor ability, the 48 ​mg/mL treatment group showed increases in head thrashing and body bending frequencies by 61.4% and 220.7% respectively, along with excellent autonomous movement capacity. At the cellular level, levels of lipofuscin, ROS, and MDA were reduced by 65.8%, 41.5%, and 51.3% respectively, while CAT activity increased by 329.1%. This study employed an orthogonal experimental design to determine the optimal formulation. Subsequent validation confirmed that the optimized extract possessed remarkable antioxidant and whitening properties. Furthermore, it was found to significantly enhance stress resistance in C. elegans and improve age-related phenotypes. These results indicated that this formulation may effectively address issues related to aging and oxidative stress. • The results indicate that the optimized “Sibai” extract formulation (3:3:1:1) exhibits potent antioxidant activity. • A significant tyrosinase inhibition rate confirms “Sibai” extract’s strong skin-whitening potential. • In vivo ​studies show Sibai extract extends ​ C. elegans ​lifespan and notably boosts resistance to oxidative/thermal stress. • “Sibai” extract reduces oxidative stress (lower ROS/MDA levels) and significantly enhances CAT activity.