美洲大蠊抗肝纤维化活性部位HPLC指纹图谱的建立及13种成分测定
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摘要
目的:建立美洲大蠊抗肝纤维化活性部位(ML-HB)氨基酸类指纹图谱,评价不同批次ML-HB的质量,并测定氨基酸的含量。方法:采用盐酸水解法制备水解氨基酸样品液,领苯二甲醛柱前衍生化后进行HPLC分析。色谱柱为Agilent ZORBAX Eclipse XDB-C18(4.6 mm×250 mm,5μm);柱温35℃;检测波长338 nm;流动相为甲醇-17.6 mmol/L的磷酸二氢钠溶液(pH 7.80),进行梯度洗脱。依据相似度评价、聚类分析和主成分分析结果进行不同批次ML-HB质量评价。结果:建立了10批ML-HB指纹图谱,标定共有峰15个,指认出13个峰,10批样品相似度均大于0.980;测定了10批样品中13种氨基酸的含量。结论:所建立的ML-HB氨基酸类指纹图谱及含量测定方法稳定性、重复性好,可为ML-HB的质量控制和评价提供参考。
Objective: To establish an amino acid fingerprint of the active site of anti-hepatic fibrosis of Periplaneta americana(MLHB), evaluate the quality of different batches of ML-HB, and determine the amino acid content. Methods: Hydrolyzed amino acid sample solution was prepared by hydrochloric acid hydrolysis method, and HPLC analysis was performed after precolumn derivatization of o-phthalaldehyde. The column was Agilent ZORBAX Eclipse XDB-C18(4.6 mm × 250 mm, 5 μm); column temperature was 35 °C;detection wavelength was 338 nm; mobile phase was methanol-17.6 mmol/L NaH2 PO4 solution(pH 7.80), and gradient elution was applied. According to the similarity evaluation, cluster analysis and principal component analysis results, the quality of different batches of ML-HB was evaluated. Results: Ten batches of ML-HB fingerprints were established, 15 common peaks were identified, 13 peaks were identified, and the similarity of 10 batches was greater than 0.980. The content of 13 amino acids in 10 batches was determined. Conclusion: The established ML-HB amino acid fingerprints and content determination methods have good stability and repeatability, which can provide reference for quality control and evaluation of ML-HB.
Objective: To establish an amino acid fingerprint of the active site of anti-hepatic fibrosis of Periplaneta americana(MLHB), evaluate the quality of different batches of ML-HB, and determine the amino acid content. Methods: Hydrolyzed amino acid sample solution was prepared by hydrochloric acid hydrolysis method, and HPLC analysis was performed after precolumn derivatization of o-phthalaldehyde. The column was Agilent ZORBAX Eclipse XDB-C18(4.6 mm × 250 mm, 5 μm); column temperature was 35 °C;detection wavelength was 338 nm; mobile phase was methanol-17.6 mmol/L NaH2 PO4 solution(pH 7.80), and gradient elution was applied. According to the similarity evaluation, cluster analysis and principal component analysis results, the quality of different batches of ML-HB was evaluated. Results: Ten batches of ML-HB fingerprints were established, 15 common peaks were identified, 13 peaks were identified, and the similarity of 10 batches was greater than 0.980. The content of 13 amino acids in 10 batches was determined. Conclusion: The established ML-HB amino acid fingerprints and content determination methods have good stability and repeatability, which can provide reference for quality control and evaluation of ML-HB.
引文
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[3]李夸巧,肖培云,黄丽,等.美洲大蠊不同溶剂提取物对肝星状细胞增殖的影响[J].时珍国医国药,2014,25(4):780-781.
[4]郝芳芳,杨永寿,肖培云.美洲大蠊抗肝纤维化活性部位的筛选[J].中国临床药理学杂志,2019,35(3):241-244.
[5]肖培云,杨永寿,施贵荣,等.美洲大蠊药材中氨基酸含量的柱前衍生化高效液相色谱法测定[J].时珍国医国药,2012,23(3):525-526.
[6]谢志茹,程敏,谭丽容,等.OPA-FMOC柱前衍生化HPLC法测定两种常用食用菌中蛋白氨基酸和非蛋白氨基酸的含量[J].现代食品科技,2018,34(10):240-246.
[7]肖培云,杨永寿,施贵荣,等.蜚蠊抗肝纤维化活性提取物的制备及检测方法:CN104352529B[P].2018-07-10.
[8]杨华蕊,何正春,杨永寿,等.美洲大蠊抗肝纤维化活性部位纯化工艺研究[J].世界最新医学信息文摘,2019,19(8):1-4.
[9]黄晶,梅明,许润春,等.美洲大蠊提取物抗肝损伤药效比较及工艺评价[J].中国实验方剂学杂志,2013,19(19):46-49.
[10]王心龙,向斌,李亚美,等.虫药美洲大蠊水溶性成分及其促血管生成活性[J].天然产物研究与开发,2017,29(12):2004-2009.
[11]王璐萱,巩荣艳,曾陈娟,等.药用美洲大蠊氨基酸和维生素含量分析[J].四川动物,2019,38(1):87-91.
[12]杨芳,邵金良,汪庆平,等.美洲大蠊水解氨基酸含量测定及营养评价[J].氨基酸和生物资源,2010,32(1):9-11.
[13]四川省食品药品监督管理局.四川省中药材标准(2010年版)[M].成都:四川科学技术出版社,2010.
[14]李海波,蒋学云,赵超仁,等.美洲大蠊药材质量控制方法的研究[J].大理学院学报,2007,6(8):3-5.
[15]马殿飞.美洲大蠊药材质量标准研究[D].昆明:云南中医学院,2017.
[16]张臻,宋芹,胡慧玲,等.美洲大蠊提取物的质量标准研究[J].中药与临床,2013,4(5):20-21.