Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso (UFMT), Av. Fernando Correa da Costa, no. 2367, Coxipó, Cuiabá, Mato Grosso 78060-900, Brazil.
Research Laboratory of Natural Products, Federal University of Tocantins, Faculty of Medicine, Palmas, Tocantins 77020-210, Brazil.
J Ethnopharmacol. 2016 May 26;184:128-37. doi: 10.1016/j.jep.2016.03.005. Epub 2016 Mar 3.
Gallesia integrifolia (Phytolaccaceae) is commonly known as "pau-d'alho" in Brazil or "garlic plant" due to the strong scent of garlic peculiar to all parts of the plant. The bark decoction is used for the treatment of microbial infections among other diseases by different ethnic groups in Brazil, Peruvian Amazonians, Bolivia and Mosetene Indians. This study aimed to advance in the antibacterial activity and characterize the mode of action of the hydroethanolic extract of the inner stem bark of G. integrifolia (HEGi) using in vivo and in vitro experimental models.
The qualitative and quantitative phytochemical analyzes of HEGi were carried out using colorimetric and HPLC technique. The cytotoxic potential of HEGi was evaluated against CHO-K1 cells by Alamar blue assay and its acute toxicity was assessed by the Hippocratic screening test using Swiss-Webster mice. The antibacterial activity was evaluated by micro- dilution method against ten strains of Gram-positive and Gram-negative bacteria. The mode of action of HEGi was investigated by outer membrane permeability, nucleotide leakage and potassium efflux assays. In vivo infection model was established by using Staphylococcus aureus infection model Wistar rats.
Qualitative phytochemical analysis of HEGi revealed the presence of saponins, alkaloids, phenolic compounds and flavonoids. Phytochemical quantification of HEGi showed that higher total phenolic (80.10±0.62mg GAE/g) and flavonoid (16.10±0.03mg RE/g) contents. HPLC fingerprint analysis revealed the presence of gallic acid, rutin, and morin. In the Alamar blue assay no cytotoxic effect of HEGi in CHO-K1 cells was observed up to 200µg/mL, and no signs or symptoms of acute toxicity were observed in mice of both sexes at higher doses of up to 2000mg/kg, p.o. HEGi demonstrated bacteriostatic effect against selected Gram positive and Gram negative bacterial pathogens. Its mode of action is associated, at least partly, with changes in the permeability of bacterial membranes, evidenced by the increased entry of hydrophobic antibiotic in Pseudomonas aeruginosa, intense K(+) efflux and nucleotides leakage in Shigella flexneri, Streptococcus pyogenes and S. aureus. HEGi attenuated the experimental blood borne S. aureus infection in rats at all the tested doses levels (10, 50 and 250mg/kg).
HEGi is safe at the dose tested when used acutely, and it presented broad antibacterial effect, which support its traditional use in the treatment of bacterial infections. It contains well known important phytochemicals, recognized to be active against bacterial pathogens in vitro and might be collectively responsible for the antibacterial activity of HEGi. It is bacteriostatic in nature, with membrane perturbation being one of it mode of action. HEGi represent a potential phytotherapic antibacterial agent.
Gallesia integrifolia（商陆科）在巴西通常被称为“pau-d'alho”或“大蒜植物”，因为植物的所有部分都散发出强烈的大蒜气味。巴西、秘鲁亚马逊地区、玻利维亚和莫塞泰内印第安人的不同族群使用树皮煎剂治疗微生物感染等疾病。本研究旨在通过体内和体外实验模型，深入研究 Gallesia integrifolia 内皮茎水醇提取物（HEGi）的抗菌活性和作用方式。
使用比色法和 HPLC 技术对 HEGi 进行定性和定量的植物化学分析。通过 Alamar blue 测定法评估 HEGi 对 CHO-K1 细胞的细胞毒性，通过 Hippocratic 筛选试验用瑞士-韦伯斯特小鼠评估其急性毒性。采用微量稀释法测定 HEgi 对十种革兰氏阳性和革兰氏阴性细菌的抗菌活性。通过外膜通透性、核苷酸泄漏和钾流出测定研究 HEGi 的作用方式。使用金黄色葡萄球菌感染 Wistar 大鼠建立体内感染模型。
HEGi 的定性植物化学分析显示存在皂甙、生物碱、酚类化合物和类黄酮。HEGi 的植物化学定量分析显示，总酚（80.10±0.62mg GAE/g）和类黄酮（16.10±0.03mg RE/g）含量较高。HPLC 指纹图谱分析显示存在没食子酸、芦丁和桑色素。在 Alamar blue 测定中，在 200µg/mL 及以下浓度时，HEGi 对 CHO-K1 细胞没有细胞毒性作用，在雌雄小鼠中，即使在高达 2000mg/kg 的更高剂量下，也没有观察到急性毒性的迹象或症状。HEGi 对所选革兰氏阳性和革兰氏阴性细菌病原体具有抑菌作用。其作用方式至少部分与细菌膜通透性的变化有关，这表现在绿脓假单胞菌中疏水性抗生素的进入增加、福氏志贺菌、化脓链球菌和金黄色葡萄球菌中强烈的 K(+)流出和核苷酸泄漏。HEGi 在所有测试剂量水平（10、50 和 250mg/kg）下均能减轻大鼠实验性血源性金黄色葡萄球菌感染。
在急性使用时，HEGi 在测试剂量下是安全的，它具有广泛的抗菌作用，这支持了其在治疗细菌感染方面的传统用途。它含有众所周知的重要植物化学物质，体外对细菌病原体具有活性，可能共同负责 HEGi 的抗菌活性。它的性质是抑菌的，其作用方式之一是膜扰动。HEGi 代表一种有潜力的植物抗菌剂。
