Total Content Of Kaurene Diterpenes In Annona Vepretorum Stems Via H-1 Qnmr: A Method For Speeding The Identification Of Bioactive Extracts

IntroductionKaurene 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.ObjectiveTo establish a H-1 qNMR method for determining the total KD contents in extracts and fractions obtained from Annona vepretorum stems.MethodologyStems 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-1 qNMR measurements were performed on 400MHz with samples solubilized in deuterated dimethyl sulfoxide. Quantification was carried out using the signals at 4.71and 4.78ppm 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.ResultsUsing the newly developed method, the total KD contents (in g/mg) were 653.8012.15 (HEX-E), 458.9025.94 (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.ConclusionThe developed method offers a fast and simple way of determining total KD contents.In this work a H-1 qNMR method was developed to quantify the total content of kaurene diterpenes (KDs) in extracts and fractions of A. vepretorum stems. The newly developed method was suitable to assess the KDs contents in A. vepretorum samples with good selectivity, precision, robustness, and reproducibility. The quantitative spectra were also submitted to chemometric analysis, which allowed to better evaluate the correlation between the total KD contents in the A. vepretorum samples with their antibacterial activity.