de Paula Joelma Abadia Marciano, Brito Lucas Ferreira, Caetano Karen Lorena Ferreira Neves, de Morais Rodrigues Mariana Cristina, Borges Leonardo Luiz, da Conceição Edemilson Cardoso
Unidade Universitária de Ciências Exatas e Tecnológicas, Universidade Estadual de Goiás, Anápolis, Goiás, Brazil.
Curso de Farmácia, Instituto de Ciências da Saúde, Universidade Paulista, Goiânia, Goiás, Brazil.
Talanta. 2016;149:77-84. doi: 10.1016/j.talanta.2015.11.005. Epub 2015 Nov 3.
Azadirachta indica A. Juss., also known as neem, is a Meliaceae family tree from India. It is globally known for the insecticidal properties of its limonoid tetranortriterpenoid derivatives, such as azadirachtin. This work aimed to optimize the azadirachtin ultrasound-assisted extraction (UAE) and validate the HPLC-PDA analytical method for the measurement of this marker in neem dried fruit extracts. Box-Behnken design and response surface methodology (RSM) were used to investigate the effect of process variables on the UAE. Three independent variables, including ethanol concentration (%, w/w), temperature (°C), and material-to-solvent ratio (gmL(-1)), were studied. The azadirachtin content (µgmL(-1)), i.e., dependent variable, was quantified by the HPLC-PDA analytical method. Isocratic reversed-phase chromatography was performed using acetonitrile/water (40:60), a flow of 1.0mLmin(-1), detection at 214nm, and C18 column (250×4.6mm(2), 5µm). The primary validation parameters were determined according to ICH guidelines and Brazilian legislation. The results demonstrated that the optimal UAE condition was obtained with ethanol concentration range of 75-80% (w/w), temperature of 30°C, and material-to-solvent ratio of 0.55gmL(-1). The HPLC-PDA analytical method proved to be simple, selective, linear, precise, accurate and robust. The experimental values of azadirachtin content under optimal UAE conditions were in good agreement with the RSM predicted values and were superior to the azadirachtin content of percolated extract. Such findings suggest that UAE is a more efficient extractive process in addition to being simple, fast, and inexpensive.
印楝（Azadirachta indica A. Juss.），也被称为印度楝，是一种来自印度的楝科树木。它因其柠檬苦素四环三萜衍生物（如印楝素）的杀虫特性而闻名于世。这项工作旨在优化印楝素的超声辅助提取（UAE）方法，并验证用于测定印楝干果提取物中该标志物的高效液相色谱-光电二极管阵列检测（HPLC-PDA）分析方法。采用Box-Behnken设计和响应面方法（RSM）来研究工艺变量对超声辅助提取的影响。研究了三个独立变量，包括乙醇浓度（%，w/w）、温度（℃）和料液比（g/mL）。印楝素含量（μg/mL），即因变量，通过HPLC-PDA分析方法进行定量。采用乙腈/水（40:60）进行等度反相色谱分析，流速为1.0 mL/min，检测波长为214 nm，使用C18柱（250×4.6 mm²，5 µm）。根据国际人用药品注册技术协调会（ICH）指南和巴西法规确定主要验证参数。结果表明，在乙醇浓度范围为75 - 80%（w/w）、温度为30℃、料液比为0.55 g/mL的条件下获得了最佳超声辅助提取条件。HPLC-PDA分析方法被证明是简单、选择性好、线性、精密、准确且稳健的。在最佳超声辅助提取条件下印楝素含量的实验值与响应面方法预测值吻合良好，且优于渗漉提取物中印楝素的含量。这些发现表明，超声辅助提取除了简单、快速和廉价外，还是一种更高效的提取工艺。
