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一种用于同时测定猫须草提取物中橙皮素、泽兰黄酮和3'-羟基-5,6,7,4'-四甲氧基黄酮的简单等度高效液相色谱法。

A simple isocratic HPLC method for the simultaneous determination of sinensetin, eupatorin, and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone in Orthosiphon stamineus extracts.

作者信息

Yam Mun Fei, Mohamed Elsnoussi Ali Hussin, Ang Lee Fung, Pei Li, Darwis Yusrida, Mahmud Roziahanim, Asmawi Mohd Zaini, Basir Rusliza, Ahmad Mariam

机构信息

School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia.

出版信息

J Acupunct Meridian Stud. 2012 Aug;5(4):176-82. doi: 10.1016/j.jams.2012.05.005. Epub 2012 Jun 15.

Abstract

Orthosiphon stamineus extracts contain three flavonoids (3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin) as bioactive substances. Previous reported high performance liquid chromatography- ultraviolet (HPLC-UV) methods for the determination of these flavonoids have several disadvantages, including unsatisfactory separation times and not being well validated according to International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) standard guidelines. A rapid, specific reversed-phase HPLC method with isocratic elution of acetonitrile: isopropyl alcohol: 20mM phosphate buffer (NaH(2)PO(4)) (30:15:55, v/v) (pH 3.5) at a flow-rate of 1ml/minute, a column temperature of 25°C, and ultraviolet (UV) detection at 340 nm was developed. The method was validated and applied for quantification of different types of O stamineus extracts and fractions. The method allowed simultaneous determination of 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin in the concentration range of 0.03052-250 μg/ml. The limits of detection and quantification, respectively, were 0.0076 and 0.061 μg/ml for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, 0.0153 and 0.122 μg/ml for sinensetin and 0.0305 and 0.122 μg/ml for eupatorin. The percent relative standard deviation (% RSD) values for intraday were 0.048-0.368, 0.025-0.135, and 0.05-0.476 for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for intraday precision were 0.333-1.688, 0.722-1.055, and 0.548-1.819, respectively. The accuracy for intraday were 91.25%-103.38%, 94.32%-109.56%, and 92.85%-109.70% for 3'-hydroxy-5,6,7,4'-tetramethoxyflavone, sinensetin, and eupatorin, respectively, and those for interday accuracy were 97.49%-103.92%, 103.58%-104.57%, and 103.9%-105.33%, respectively. The method was found to be simple, accurate and precise and is recommended for routine quality control analysis of O stamineus extract containing the three flavonoids as the principle components in the extract.

摘要

猫须草提取物含有三种黄酮类化合物(3'-羟基-5,6,7,4'-四甲氧基黄酮、华良姜素和泽兰黄酮)作为生物活性物质。先前报道的用于测定这些黄酮类化合物的高效液相色谱-紫外(HPLC-UV)方法存在几个缺点,包括分离时间不理想以及未按照国际人用药品注册技术协调会(ICH)标准指南进行充分验证。开发了一种快速、特异的反相HPLC方法,以乙腈:异丙醇:20mM磷酸盐缓冲液(NaH₂PO₄)(30:15:55,v/v)(pH 3.5)等度洗脱,流速为1ml/分钟,柱温为25°C,并在340nm处进行紫外(UV)检测。该方法经过验证,并应用于不同类型猫须草提取物和馏分的定量分析。该方法能够同时测定浓度范围为0.03052 - 250μg/ml的3'-羟基-5,6,7,4'-四甲氧基黄酮、华良姜素和泽兰黄酮。3'-羟基-5,6,7,4'-四甲氧基黄酮的检测限和定量限分别为0.0076和0.061μg/ml,华良姜素分别为0.0153和0.122μg/ml,泽兰黄酮分别为0.0305和0.122μg/ml。3'-羟基-5,6,7,4'-四甲氧基黄酮、华良姜素和泽兰黄酮日内相对标准偏差(%RSD)值分别为0.048 - 0.368、0.025 - 0.135和0.05 - 0.476,日间精密度的%RSD值分别为0.333 - 1.688、0.722 - 1.055和0.548 - 1.819。3'-羟基-5,6,7,4'-四甲氧基黄酮、华良姜素和泽兰黄酮的日内准确度分别为91.25% - 103.38%、94.32% - 109.56%和92.85% - 109.70%,日间准确度分别为97.49% - 103.92%、103.58% - 104.57%和103.9% - 105.33%。该方法被发现简单、准确且精密,推荐用于以这三种黄酮类化合物为提取物主要成分的猫须草提取物的常规质量控制分析。

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