不同产地、不同龄期蚕沙中多类型资源性化学成分的分析与评价

目的：对不同产地、不同龄期蚕沙中多类型资源性化学成分进行分析评价，为蚕沙的资源化利用提供科学依据。方法：采用超高效液相色谱串联三重四极杆质谱法（UPLC-TQ/MS法）分析蚕沙中黄酮类、生物碱类、核苷及氨基酸类等资源性化学成分组成及含量。分析条件：黄酮类成分，采用Acquity UPLC BEH C₁₈色谱柱（100 mm×2.1 mm，1.7 μm），以0.1%甲酸水（A）-乙腈（B）为流动相，梯度洗脱（0~8 min，90% A→40% A；8~8.5 min，40% A→20% A），流速0.4 mL·min^-1，检测方式为多反应监测（MRM）；生物碱类、核苷及氨基酸类成分，采用ACQUITY UPLC BEH Amide色谱柱（100 mm×2.1 mm，1.7 μm），流动相为含有5 mmol·L^-1甲酸铵、5 mmol·L^-1乙酸铵和0.2%甲酸的水溶液（A）-含有1 mmol·L^-1甲酸铵、1 mmol·L^-1乙酸铵和0.2%甲酸的乙腈（B），梯度洗脱（0~3 min，10% A；3~9 min，10% A→18% A；9~15 min，18% A→20% A；15~16 min，20% A→46% A；16~18 min，46% A），流速0.4 mL·min^-1，检测方式为多反应监测（MRM）。采用紫外-可见分光光度法分析蚕沙中可溶性多糖及叶绿素含量：采用葡萄糖和葡萄糖醛酸作为中性多糖和酸性多糖的对照品，检测波长分别是490 nm和512 nm；通过在645和663 nm检测波长下的吸收度，根据Amon公式计算叶绿素含量。结果：可溶性多糖类成分在蚕沙中含量最高，其中收集于云南大理的蚕沙中可溶性多糖含量最高，达11.9%；黄酮类成分主要为异槲皮苷、芦丁、紫云英苷，不同产地含量差别显著，其中收集于云南大理的蚕沙中黄酮类成分含量约为河南蚕沙的25倍；苏州产蚕沙中生物碱类成分含量最高，为1.63 mg·g^-1；共检出5个核苷类成分，14个氨基酸类成分，其中苏州产蚕沙核苷类和氨基酸类成分含量最高，达0.26%；叶绿素含量以镇江三龄蚕沙为最高，达2.62 mg·g^-1。结论：除产地外，蚕沙中资源性化学成分的含量与龄期关系密切，其中黄酮类、可溶性多糖类、叶绿素类成分含量均随龄期的增加而降低，生物碱类成分含量随龄期的增加而升高。研究结果为蚕沙的资源化利用与产业化开发提供了科学依据。

Objective:To analyze the multiple types of chemical constituents in silkworm excrement from different habitats and at different various instars, and to provide the basis for the resource utilization of silkworm excrement. Methods:The flavonoids, alkaloids, nucleosides and amino acids in the silkworm excrement were determined by UPLC-TQ/MS. Flavonoids were separated on an Acquity UPLC BEH C₁₈ column (100 mm×2.1 mm, 1.7 μm) and the mobile phase was composed of water (containing 0.1%formic acid) (A) and acetonitrile (B) with gradient elution (0-8 min, 90%A→40%A;8-8.5 min, 40%A→20%A) at a flow rate of 0.4 mL·min^-1. Alkaloids, nucleosides and amino acids were separated on an ACQUITY UPLC BEH Amide column (100 mm×2.1 mm, 1.7 μm) and the mobile phase was composed of water (containing 5 mmol·L^-1 ammonium formate, 5 mmol·L^-1 ammonium acetate and 0.2%formic acid) (A) and acetonitrile (containing 1 mmol·L^-1 ammonium formate, 1 mmol·L^-1 ammonium acetate and 0.2%formic acid) (B) with gradient elution (0-3 min, 10%A;3-9 min, 10%A→18%A;9-15 min, 18%A→20%A;15-16 min, 20%A→46%A;16-18 min, 46%A) at a flow rate of 0.4 mL·min^-1. The above components were detected by MS with multiple reaction monitoring (MRM). The soluble polysaccharides and chlorophyll were determined by UV-Vis spectrophotometry. Glucose and glucuronic acid were used as the control substances of neutral polysaccharide and acid polysaccharide with detection wavelengths of 490 and 512 nm, respectively. The content of chlorophyll was calculated by Amon formula based on the absorbance values of detection wavelengths at 645 and 663 nm. Results:The results showed that the contents of soluble polysaccharides were the highest among the measured chemical constituents, and the silkworm excrement from Dali had the highest content (11.9%). The main constituents of flavonoids were isoquercitrin, rutin and astragalin and the contents in different habitats were significantly different. Flavonoids in silkworm excrement from Dali were approximately 25 times of than those in sample from Henan. The contents of alkaloids in silkworm excrement from Suzhou were the highest (1.63 mg·g^-1). 5 nucleosides and 14 amino acids were detected, which were most abundant (0.26%) in sample Suzhou. The silkworm excrement at third instar from Zhenjiang had the highest content of chlorophyll (2.62 mg·g^-1) among all measured samples. Conclusion:In addition to the habitats, the contents of the chemical components in the silkworm excrement are closely related to their instars. The contents of flavonoids, soluble polysaccharides and chlorophyll all decreased with the increase of the instar, and the contents of alkaloids increased with the increase of the instar. The results of this study provide scientific basis for the precise utilization and industrialization development of silkworm excrement.

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