College of Plant Protection, Hunan Agricultural University, Changsha 410128, China.
College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha 410128, China.
Int J Mol Sci. 2021 Dec 28;23(1):312. doi: 10.3390/ijms23010312.
The aim of the present investigation was to determine the active ingredients in L. leaves and develop a biological pesticide. Organic solvent extraction, column chromatography, liquid chromatography, ODS-C18 reverse elution, Sephadex LH-20 gel filtration, H spectrum, and C spectrum were used to isolate the pure product for an assessment of the agricultural activity and bacteriostatic mechanisms. The results showed that the activity of the crude extract following carbon powder filtration was 1.63-fold that of the non-filtered extract. Further isolation was performed to obtain two pure products, namely, hydroxybenzoic acid (HBA) and benzo[b]furan-2-carboxaldehyde (BFC), and their molecular formulas and molecular weights were CHO and 138.12, and CHO and 146.12, respectively. Our study is the first to determine that HBA has bacteriostatic activity (MIC 125 μg/mL) and is also the first to isolate BFC from . The ultrastructure observation results showed that HBA caused the bacteria to become shriveled, distorted, and deformed, as well as exhibit uneven surfaces. After HBA treatment, 70 differentially expressed metabolites were detected in the bacteria, of which 9 were downregulated and 61 were upregulated. The differentially expressed metabolites were mainly strigolactones, organic acids and derivatives, fatty acids, benzene and substituted benzene derivatives, amino acids and associated metabolites, and alcohols and amines. Among all of the downregulated differentially expressed metabolites, MEDP1280 was the most critical, as it participates in many physiological and biochemical processes. The enrichment analysis showed that the differentially expressed metabolites mainly participate in tyrosine metabolism, biosynthesis of amino acids, cysteine and methionine metabolism, and arginine and proline metabolism. Additionally, HBA was found to disrupt cell membrane permeability and integrity, causing the leakage of substances and apoptosis. The physiological and biochemical test results showed that HBA could increase the pyruvate levels in bacteria but could decrease the activities of respiratory enzymes (malate dehydrogenase (MDH) and NADH oxidase) and antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX)). Inverse molecular docking was used to study the binding between HBA and respiratory and antioxidant enzymes. The results showed that HBA could bind to MDH, NADH oxidase, SOD, and GSH-PX, suggesting that these enzymes may be the effector targets of HBA. Conclusion: The optimal active ingredient in that can inhibit subsp. was identified as HBA. HBA mainly disrupts the cell membrane, damages the metabolic system, and inhibits respiration and antioxidant enzyme activity to control bacterial growth. These results provide a reference for the further development of biological pesticides.
本研究旨在确定 叶片中的活性成分，并开发一种生物农药。采用有机溶剂萃取、柱层析、液相色谱、ODS-C18 反相洗脱、Sephadex LH-20 凝胶过滤、氢谱和碳谱对纯产物进行分离，以评估其农业活性和抑菌机制。结果表明，经碳粉过滤后的粗提物的活性是未过滤提取物的 1.63 倍。进一步分离得到两种纯产物，即羟基苯甲酸（HBA）和苯并[b]呋喃-2-甲醛（BFC），其分子式和分子量分别为 CHO 和 138.12、CHO 和 146.12。本研究首次确定 HBA 具有抑菌活性（MIC 为 125μg/mL），也是首次从 中分离出 BFC。超微结构观察结果表明，HBA 导致细菌皱缩、变形、表面不均匀。经 HBA 处理后，在细菌中检测到 70 种差异表达代谢物，其中 9 种下调，61 种上调。差异表达的代谢物主要是独脚金内酯、有机酸及其衍生物、脂肪酸、苯及其取代苯衍生物、氨基酸及其相关代谢物、醇和胺。在所有下调的差异表达代谢物中，MEDP1280 最为关键，因为它参与了许多生理和生化过程。富集分析表明，差异表达的代谢物主要参与酪氨酸代谢、氨基酸生物合成、半胱氨酸和蛋氨酸代谢以及精氨酸和脯氨酸代谢。此外，HBA 被发现破坏细胞膜通透性和完整性，导致物质泄漏和细胞凋亡。生理生化试验结果表明，HBA 可增加细菌中丙酮酸的水平，但可降低呼吸酶（苹果酸脱氢酶（MDH）和 NADH 氧化酶）和抗氧化酶（超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-PX））的活性。采用反向分子对接研究 HBA 与呼吸和抗氧化酶的结合。结果表明，HBA 可与 MDH、NADH 氧化酶、SOD 和 GSH-PX 结合，提示这些酶可能是 HBA 的效应靶标。结论：鉴定出能抑制 亚种的 最佳活性成分是 HBA。HBA 主要通过破坏细胞膜、破坏代谢系统、抑制呼吸和抗氧化酶活性来控制细菌生长。这些结果为进一步开发生物农药提供了参考。
