Purification And Characterization Of Bioactive Peptides From Skin Secretion Of Chinese Tiger Frog (Hoplobatrachus Rugulosus)

Bioactive peptides with physiological function are widespread in nature. In order to survive in different ecological conditions, amphibians can secrete a variety of bioactive peptides of different structure and function. The types, function and quantities of the bioactive peptides depend on the amphibian species and their geographical distributions. Accumulating evidence indicated that the isolated peptides possess a variety of physiological activities, such as antimicrobial, antioxidant, anticancer, anti-aging, neuromodulatory and immunomodulatory activities.Much research has been focused on the skin secretions of Anura amphibian, including frogs and toads. The main bioactive substances are antimicrobial and antioxidant peptides. Peptides of 15-30 residues with strong antioxidant activities were isolated from the skin secretions of Rana pleuraden by Chinese scholars and were classified as the third antioxidant system.Tiger frogs (Hoplobatrachus rugulosus, also known as Rana rugulosa) belong to the Ranidae, Rana tigrina genus. They are widely distributed from the southeast coast to the southwest inland in China, and can also be found in South and Southeast Asia. To this date, only one class of antimicrobial peptides obtained from the Indian tiger frog skin secretions and an insulin-releasing peptide extracted from the Vietnam tiger frog skin have been reported. There is no published research on the antioxidant peptides from tiger frog skin secretions.In this research, Chinese tiger frogs were collected from the paddy fields of Fuqing, Fujian Province. Skin secretions were obtained by 9V electrical stimulation of the dorsal skin glands of young adult frogs. Bioactive peptides with antimicrobial and antioxidant activities were isolated and purified using a two-step procedure. Finally, a dipeptide with antioxidant activity was purified and characterized. The antioxidant mechanism was discussed.Sephadex G-50 gel filtration chromatography and reverse phase high performance liquid chromatography (RP-HPLC) were adopted for the separation of Chinese tiger frog skin secretions. The antimicrobial and antioxidant activities were tracked by disk diffusion and DPPH (2,2-diphenyl-l-picrylhydrazyl) radical scavenging assay respectively. The antibacterial activity weakened after separation, which might be attributed to the synergistic antimicrobial action in the skin secretions or the loss of effective fractions in the purification process. A novel antioxidant peptide was discovered. Its molecular weight is 277.10 Da by HPLC-ESI-MS analysis. The amino acid is identified as Lys-Met by ESI-MS/MS method.Antioxidant activities of the dipeptide were evaluated by ABTS radical scavenging activity, DPPH radical scavenging activity, hydroxyl radical scavenging activity, superoxide anion radical scavenging activity as well as ferrous ion-chelating and reducing power. The dipeptide Lys-Met showed high antioxidant activities. The antioxidant activities of Lys-Met, Met-Lys and free amino acids of lysine and methionine were compared. The results indicated that the antioxidant activity was associated with not only the amino acid composition but also the sequence. Besides, preliminary research on the antioxidant mechanism was carried out using solution NMR.