Antibacterial activities of chemical constituents from the aerial parts of <i>Hedyotis pilulifera</i>

<b>Context:</b><i>Hedyotis pilulifera</i> (Pit.) T.N. Ninh (Rubiaceae) has been used in Vietnamese ethnomedicine; the methanol extract exhibited antibacterial activity in our preliminary screening. <b>Objectives:</b> In this study, compounds from <i>H. pilulifera</i> were isolated and their antibacterial activity <i>in vitro</i> was evaluated. <b>Materials and methods:</b> The aerial parts of <i>H. pilulifera</i> (1.4 kg) were extracted with MeOH, suspended in water and ethyl acetate extract was chromatographed on a silica gel column. The structures of isolated compounds were elucidated by the combination analyses of spectroscopy including 1D-, 2D-NMR, HRMS and in comparison with the reported NMR data in the literature. All isolated compounds were evaluated for inhibitory effect using the microdilution method toward <i>Staphylococcus aureus, Bacillus subtilis</i> and <i>Mycobacterium smegmatis,</i> and MIC values were determined. <b>Results:</b> Twenty compounds were isolated, including five triterpenoids, two steroids, two aromatic compounds, three fatty acids, one quinone derivative, one lignan glycoside, one ceramide and five glycolipids. Among these, oleanolic acid showed significant antibacterial activity against <i>M. smegmatis</i> with the MIC value of 2.5 μg/mL. Remarkably, rotungenic acid showed strong activity against <i>S. aureus, B. subtilis, M. smegmatis</i> with MIC values of 2.5, 2.5 and 1.25 μg/mL, respectively. Rotundic acid exhibited significant antibacterial activity against <i>B. subtilis</i> with the MIC value of 5 μg/mL. To the best of our knowledge, the antibacterial activity of rotungenic acid, stigmast-4-ene-3,6-dione and (2<i>S</i>,3<i>S</i>,4<i>R</i>,2′<i>R</i>)-2-(2′-hydroxytetracosanoylamino) octadecane-1,3,4-triol was reported for the first time. <b>Conclusions:</b> Oleanolic acid, rotungenic acid, and rotundic acid were considered to be useful for developing new antimicrobial therapeutic agents for human.**背景**：球果耳草（*Hedyotis pilulifera* (Pit.) T.N. Ninh，茜草科（Rubiaceae））在越南民族医药中已有应用；我们的预筛选实验显示其甲醇（Methanol，MeOH）提取物具有抗菌活性。 **目的**：本研究旨在分离球果耳草中的化学成分，并体外评价其抗菌活性。 **材料与方法**：取1.4 kg球果耳草地上部分，以甲醇（Methanol，MeOH）进行提取，提取物经水悬浮后获得乙酸乙酯萃取组分，随后通过硅胶柱色谱进行分离纯化。通过一维核磁共振波谱（One-dimensional Nuclear Magnetic Resonance，1D-NMR）、二维核磁共振波谱（Two-dimensional Nuclear Magnetic Resonance，2D-NMR）、高分辨质谱（High Resolution Mass Spectrometry，HRMS）结合已发表文献中的核磁共振数据，解析所有分离得到的化合物的结构。采用微量稀释法，针对金黄色葡萄球菌（*Staphylococcus aureus*）、枯草芽孢杆菌（*Bacillus subtilis*）及耻垢分枝杆菌（*Mycobacterium smegmatis*）测试所有分离化合物的抑菌活性，并测定其最低抑菌浓度（Minimum Inhibitory Concentration，MIC）值。 **结果**：本研究共分离得到20个化合物，包括5个三萜类化合物、2个甾体类化合物、2个芳香族化合物、3个脂肪酸类化合物、1个醌类衍生物、1个木脂素苷、1个神经酰胺类化合物及5个糖脂类化合物。其中，齐墩果酸（oleanolic acid）对耻垢分枝杆菌表现出显著的抗菌活性，最低抑菌浓度为2.5 μg/mL。值得注意的是，罗腾酸（rotungenic acid）对金黄色葡萄球菌、枯草芽孢杆菌及耻垢分枝杆菌均表现出较强的抗菌活性，其最低抑菌浓度分别为2.5、2.5及1.25 μg/mL。土当归酸（rotundic acid）对枯草芽孢杆菌表现出显著的抗菌活性，最低抑菌浓度为5 μg/mL。据我们所知，罗腾酸、豆甾-4-烯-3,6-二酮（stigmast-4-ene-3,6-dione）及(2S,3S,4R,2′R)-2-(2′-羟基二十四烷酰氨基)十八烷-1,3,4-三醇的抗菌活性均为首次报道。 **结论**：齐墩果酸、罗腾酸及土当归酸有望用于开发新型人类抗菌治疗药物。