关键词: 家蚕, 全蚕体, 抗氧化能力, 酚类化合物, 非靶向代谢组, 解毒酶基因

Abstract: 【Aim】 To analyze the types, effects and monomer composition of the main natural antioxidant components in the larval body of Bombyx mori, so as to provide a reference for their development and utilization as well as the study of their pharmacodynamic effects.【Methods】 The antioxidant capacity (AC) of the extracts from the leaves of three varieties of mulberry trees (Heyebai, Jisang2 and Boluo), as well as the extracts from excrement and whole larval bodies of the 5th instar of four varieties of B. mori (Jingsong×Haoyue, Dongfei, Cai3 and Cai4) fed on Jisang2 leaves, were tested by the scabenging abilities to free radicals of 2, 2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS) and 2, 2-diphenyl-1-picrylhydrazyl (DPPH). Meanwhile, the contents of total flavonoids, total phenols, polysaccharids and 1-deoxynojirimycin (1-DNJ) in the above extracts were determined by spectrophotometry and reverse-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) to analyze the main antioxidant active substances from the whole larval body of B. mori. The daily contents of total phenol and the AC for scavenging ABTS radicals (AC_ABTS) in the hemolymph of the last instar larvae (the 4th instar of 3-molter silkworms, the 5th instar of 4-molter silkworms) of six varieties of B. mori (Dongfei, Ansiwuhua, Caigan, 7903, Cai0 and Cai3) were detected. Furthermore, liquid chromatograph-mass spectrometer (LC-MS) was used for the non-targeted qualitative and quantitative analysis of antioxidant active components in the whole bodies of four varieties of larval B. mori (Jingsong×Haoyue, Dongfei, Cai3 and Cai4) on the last day of the 5th instar and in the leaves of Jisang2. 【Results】The contents of total flavonoids, total phenols and polysaccharids of the extracts from mulberry leaves showed highly significant positive correlations with the AC of extracts from mulberry leaves, and the contents of total flavonoids and total phenols showed highly significantly positive correlations with the AC of extracts from excrement and whole bodies of the 5th instar larvae of B. mori. The AC_ABTS and AC_DPPH of extracts from whole bodies of the 5th instar larvae at the last day of four varieties of B. mori were ［(22.61±1.37)-(36.70±2.12) mg VC/g］ and ［(16.77±0.47)-(23.43±2.34) mg VC/g］, respectively, which were 1.46-2.38 and 1.81-2.54-fold of those of Jisang2 leaf extracts, and also 0.96-2.27 and 1.23-7.73-fold of those of excrement extracts from the 5th instar larve at the last day of B. mori, respectively. The content of total phenols in the extracts from whole body of the 5th instar larvae at the last day of B. mori ［(9-14±0.13)-(13.28±0.78) mg/g］ was close to those of Jisang2 leaf ［(19.74±0.58) mg/g］ and excrement of the 5th instar larvae at the last day of B. mori ［(10.19±0.19)-(16.74±0.24) mg/g］, but the contents of total flavonoids and 1-DNJ in the extracts from whole body of the 5th instar larvae at the last day of B. mori were significantly lower than those of Jisang2 leaf and excrement  of the 5th instar larvae at the last day of B. mori. The above data indicated that total phenols were the main antioxidant components in the extracts from whole body of the 5th instar larvae at the last day of B. mori. The daily AC_ABTS value and daily content of total phenols in larval hemolymph from six varieties of B. mori at the last instar were significantly positively correlated, which further confirmed that total phenols are the important antioxidant components of B. mori. A total of 436 metabolites were identified in the whole bodies of the 5th instar larvae at the last day of four varieties of B. mori and in the Jisang2 leaves. Correlation analysis and principal component analysis (PCA) revealed that the differences in the contents of the 436 metabolites could be used to effectively distinguish mulberry leaves from whole bodies of B. mori, and whole bodies of Cai4 from those of the other three varieties of B. mori. The clustering analysis result of 375 differential metabolites showed that the monomers with obviously higher content in the extracts from whole bodies of the 5th instar larvae at the last day of B. mori than those of the Jisang2 leaves, mainly belonged to benzene and its derivatives, organic acids, organic oxygen and organic nitrogen compounds. The specific monomer composition of phenols and amino acids was the main reason that the AC of extract from whole bodies of the 5th instar larvae at the last day of B. mori was higher than those of Jisang2 leaf extracts.【Conclusion】 The present study confirmed that the AC of whole bodies of the 5th instar larvae at the last day of B. mori was higher than that of Jisang2 leaves and B. mori excrement. Phenols are the main natural antioxidants of B. mori. B. mori can selectively absorb and transform the substances of ingested mulberry leaves to form their unique antioxidant components. More importantly, it was discovered that the sequence polymorphism and expression pattern changes of detoxification enzyme genes are the important reasons for causing the differences in the phenolic composition among B. mori varieties. The above results obtained provide a scientific basis for the application and development of natural antioxidant substances of B. mori.

Key words: Bombyx mori, whole silkworm body, antioxidant capacity, phenolic compounds, non-targeted metabolome, detoxification enzyme genes