Post-Graduate Program in Biotechnology, State University of Feira de Santana (UEFS), Feira de Santana, BA, Brazil.
Federal University of the San Francisco Valley (UNIVASF), Petrolina, PE, Brazil.
Phytochem Anal. 2019 Jan;30(1):83-88. doi: 10.1002/pca.2792. Epub 2018 Aug 29.
Kaurene diterpenes (KDs) constitute a chemical class often found in the genus Annona with interesting biological activities. To date, chromatographic tools have been mostly used to determine KDs. Quantitative nuclear magnetic resonance (qNMR) has distinguished itself in quantitative estimation of natural products and is an interesting choice to assess total KD contents.
To establish a H qNMR method for determining the total KD contents in extracts and fractions obtained from Annona vepretorum stems.
Stems were extracted with hexane and methanol, resulting in the hexane extract (HEX-E) and the methanol extract (MeOH-E). The former was partitioned with the acid-base method to obtain the total alkaloid fraction (TA-F) and the neutral dichloromethane fraction (NDM-F). H qNMR measurements were performed on 400 MHz with samples solubilized in deuterated dimethyl sulfoxide. Quantification was carried out using the signals at 4.71 and 4.78 ppm related to hydrogens of the exocyclic double bond of the basic skeleton of KDs and gallic acid as the standard reference. The selectivity, intra- and inter-day precision, reproducibility, limit of detection, limit of quantification, and robustness of the methodology were evaluated.
Using the newly developed method, the total KD contents (in μg/mg) were 653.80 ± 12.15 (HEX-E), 458.90 ± 25.94 (NDM-F), 375.60 ± 27.52 (TA-F), and 315.10 ± 19.20 (MeOH-E). For determining the most promising bioactive sample, the KD contents and the sample discriminations obtained by principal component analysis were correlated to the antibacterial activity. Such approach pointed out HEX-E as a potential source of KDs.
The developed method offers a fast and simple way of determining total KD contents.
卡瑞二萜（KDs）构成了一个在番荔枝属中经常发现的化学类别，具有有趣的生物活性。迄今为止，色谱工具主要用于测定 KDs。定量核磁共振（qNMR）在天然产物的定量估计方面表现出色，是评估总 KD 含量的一个有趣选择。
建立一种 H qNMR 方法，用于测定从番荔枝茎中提取和分离得到的提取物和馏分中的总 KD 含量。
用正己烷和甲醇提取茎，得到正己烷提取物（HEX-E）和甲醇提取物（MeOH-E）。前者用酸碱法进行分配，得到总生物碱馏分（TA-F）和中性二氯甲烷馏分（NDM-F）。在 400 MHz 下进行 H qNMR 测量，样品溶解在氘代二甲亚砜中。使用与 KDs 基本骨架中环外双键氢相关的 4.71 和 4.78 ppm 的信号以及没食子酸作为标准参考进行定量。评估了方法的选择性、日内和日间精密度、重现性、检测限、定量限和稳健性。
使用新开发的方法，总 KD 含量（以μg/mg 计）分别为 653.80±12.15（HEX-E）、458.90±25.94（NDM-F）、375.60±27.52（TA-F）和 315.10±19.20（MeOH-E）。为了确定最有前途的生物活性样品，通过主成分分析得到的 KD 含量和样品判别与抗菌活性相关。这种方法指出 HEX-E 是 KDs 的潜在来源。
所开发的方法提供了一种快速简单的测定总 KD 含量的方法。
