Guizhou Provincial Engineering Technology Research Center for Chemical Drug R & D, School of Pharmacy, Guizhou Medical University, Guiyang, 550025, Guizhou, China.
Engineering Research Center of Medical Biotechnology, Key Laboratory of Biology and Medical Engineering, Key Laboratory of Infectious Immune and Antibody Engineering in Guizhou Province, School of Biology and Engineering, Guizhou Medical University, Guiyang, 550025, Guizhou, China.
J Ethnopharmacol. 2022 Nov 15;298:115602. doi: 10.1016/j.jep.2022.115602. Epub 2022 Aug 24.
The fruit of Ginkgo biloba L. (Ginkgo nuts) has been used for a long time as a critical Chinese medicine material to treat cough and asthma, as well as a disinfectant. Similar records were written in the Compendium of Materia Medica (Ben Cao Gang Mu, pinyin in Chinese) and Sheng Nong's herbal classic (Shen Nong Ben Cao Jing, pinyin in Chinese). Recent research has shown that Ginkgo biloba exocarp extract (GBEE) has the functions of unblocking blood vessels and improving brain function, as well as antitumour activity and antibacterial activity. GBEE was shown to inhibit methicillin-resistant Staphylococcus aureus (MRSA) biofilm formation as a traditional Chinese herb in our previous report in this journal. AIM OF THE STUD: yThe antibiotic resistance of clinical bacteria has recently become increasingly serious. Thus, this study aimed to investigate the Ginkgo biloba exocarp extract (GBEE) antibacterial lineage, as well as its effect and mechanism on S. haemolyticus biofilms. This study will provide a new perspective on clinical multidrug resistant (MDR) treatment with ethnopharmacology herbs.
The microbroth dilution assay was carried out to measure the antibacterial effect of GBEE on 13 types of clinical bacteria. Bacterial growth curves with or without GBEE treatment were drawn at different time points. The potential targets of GBEE against S. haemolyticus were screened by transcriptome sequencing. The effects of GBEE on bacterial biofilm formation and mature biofilm disruption were determined by crystal violet staining and scanning electron microscopy. The metabolic activity of bacteria inside the biofilm was assessed by colony-forming unit (CFU) counting and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2HY-tetrazolium bromide (MTT) assay. Quantitative polymerase chain reaction (qPCR) was used to measure the gene expression profile of GBEE on S. haemolyticus biofilm-related factors.
The results showed that GBEE has bacteriostatic effects on 3 g-positive (G) and 2 g-negative (G) bacteria among 13 species of clinical bacteria. The antibacterial effect of GBEE supernatant liquid was stronger than the antibacterial effect of GBEE supernviaould-like liquid. GBEE supernatant liquid inhibited the growth of S. epidermidis, S. haemolyticus, and E. faecium at shallow concentrations with minimum inhibitory concentrations (MICs) of 2 μg/ml, 4 μg/ml and 8 μg/ml, respectively. Genes involved in quorum sensing, two-component systems, folate biosynthesis, and ATP-binding cassette (ABC) transporters were differentially expressed in GBEE-treated groups compared with controls. Crystal violet, scanning electron microscopy (SEM) and MTT assays showed that GBEE suppressed S. haemolyticus biofilm formation in a dose-dependent manner. Moreover, GBEE supernatant liquid downregulated cidA, cidB and atl, which are involved in cell lysis and extracellular DNA (eDNA) release, as well as downregulated the cbp, ebp and fbp participation in encoding cell-surface binding proteins.
GBEE has an excellent antibacterial effect on gram-positive bacteria and also inhibits the growth of gram-negative bacteria, such as A. baumannii (carbapenem-resistant Acinetobacter baumannii) CRABA and S. maltophilia. GBEE inhibits the biofilm formation of S. haemolyticus by altering the regulation and biofilm material-related genes, including the release of eDNA and cell-surface binding proteins.
银杏的果实（银杏坚果）长期以来一直被用作中药的重要材料，用于治疗咳嗽和哮喘，以及消毒剂。类似的记录写在《本草纲目》（本草纲目，拼音在中文）和《神农本草经》（神农本草经，拼音在中文）中。最近的研究表明，银杏外种皮提取物（GBEE）具有血管疏通和改善大脑功能、抗肿瘤活性和抗菌活性的功能。在我们之前在本杂志上的报告中，GBEE 被证明可以抑制耐甲氧西林金黄色葡萄球菌（MRSA）生物膜的形成，作为一种传统中药。
临床细菌的抗生素耐药性最近变得越来越严重。因此，本研究旨在探讨银杏外种皮提取物（GBEE）的抗菌谱系，以及其对溶血葡萄球菌生物膜的作用和机制。这将为临床多药耐药（MDR）治疗提供新的视角。
采用微量肉汤稀释法测定 GBEE 对 13 种临床细菌的抗菌作用。在不同时间点用或不用 GBEE 处理绘制细菌生长曲线。通过转录组测序筛选 GBEE 对溶血葡萄球菌的潜在靶标。通过结晶紫染色和扫描电子显微镜观察测定 GBEE 对细菌生物膜形成和成熟生物膜破坏的影响。通过平板计数法（CFU）和（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基-2HY-四唑溴化物（MTT）测定评估生物膜内细菌的代谢活性。定量聚合酶链反应（qPCR）用于测定 GBEE 对溶血葡萄球菌生物膜相关因子的基因表达谱。
结果表明，GBEE 对 13 种临床细菌中的 3 种革兰氏阳性（G）和 2 种革兰氏阴性（G）细菌具有抑菌作用。GBEE 上清液的抑菌效果强于 GBEE 超滤液。GBEE 上清液在浅浓度下抑制表皮葡萄球菌、溶血葡萄球菌和粪肠球菌的生长，最小抑菌浓度（MIC）分别为 2μg/ml、4μg/ml和 8μg/ml。与对照组相比，GBEE 处理组中与群体感应、双组分系统、叶酸生物合成和 ABC 转运体相关的基因表达差异。结晶紫、扫描电子显微镜（SEM）和 MTT 检测结果表明，GBEE 以剂量依赖的方式抑制溶血葡萄球菌生物膜的形成。此外，GBEE 上清液下调了 cidA、cidB 和 atl，这三个基因参与细胞裂解和细胞外 DNA（eDNA）释放，下调了 cbp、ebp 和 fbp 参与编码细胞表面结合蛋白的表达。
GBEE 对革兰氏阳性菌具有极好的抗菌作用，对革兰氏阴性菌如耐碳青霉烯类鲍曼不动杆菌（CRABA）和嗜麦芽窄食单胞菌也有抑制作用。GBEE 通过改变调节和生物膜物质相关基因，包括 eDNA 和细胞表面结合蛋白的释放，抑制溶血葡萄球菌的生物膜形成。
