背景:印度的古代文献,CharakSamhita和SushrutaSamhita,参考了印度和中国的乌龙传统药用,它长期以来一直被用来治疗胃痛,削减,腹泻,和皮肤状况。根茎的这种能力归因于其抗微生物特性。迄今为止的研究已经显示了其抗菌性能。然而,关于其作用方式的科学证据仍然缺乏。
目的:水葫芦提取物及其生物活性成分通过调节膜通透性和脂肪酸组成而发挥抗菌作用。
方法:采用Soxhlet装置在正己烷中提取水杨根茎中的次生代谢产物。提取物抑制多重耐药细菌分离株的能力,即蜡样芽孢杆菌,大肠杆菌,鲍曼不动杆菌,和铜绿假单胞菌使用棋盘法进行评估。Further,使用薄层色谱法纯化提取物,重力柱色谱,和combiflash色谱。使用GC-MS进行活性化合物的结构阐明,FT-IR,和紫外-可见光谱扫描。确定了生物活性级分的作用模式。使用SEM分析细菌膜损伤,使用SYBRGreenI和PI染料测定膜通透性,使用Bradford测定法和Fehling试剂分析细胞质内容物的泄漏。使用EtBr积累测定法确定鲍曼不动杆菌的抑制外排泵的能力,并使用硝基罗芬作为底物分析β-内酰胺酶的抑制作用。此外,使用结晶紫染料测定蜡样芽孢杆菌的生物膜抑制。此外,使用37组分FAME混合物作为标准品,通过GC-FAME分析确定生物活性组分对细菌膜脂肪酸谱的影响。
结果:水龙葵根茎己烷提取物(AC-R-H)对所有测试的分离株都具有广谱抗菌活性。AC-R-H提取物还显著降低了氨苄青霉素对所有测试细菌的MIC,表明其细菌抗性调节特性。分析指导纯化确定了细辛脑作为生物活性级分(S-III-BAF)中存在的主要化合物。发现S-III-BAF可降低氨苄青霉素对大肠杆菌的MIC(100至25mg/mL),铜绿假单胞菌(15至3.25mg/mL),鲍曼不动杆菌(12.5至1.56mg/ml),和蜡样芽孢杆菌(10至1.25mg/mL)。Further,它记录了氨苄青霉素对蜡状芽孢杆菌的协同活性(FICI=0.365),铜绿假单胞菌(FICI=0.456),和鲍曼不动杆菌(FICI=0.245)。S-III-BAF的作用模式可归因于其干扰膜完整性的能力,增强膜的渗透性,减少生物膜的形成,并可能改变细菌细胞膜的脂肪酸组成。
结论:含有细辛脑作为活性化合物的AC-R-H提取物的生物活性部分对所测试的细菌分离株具有抗菌活性和与氨苄青霉素的协同相互作用。这种活性可以归因于细菌膜中存在的脂肪酸的调节,这增强了膜的渗透性并导致膜损坏。
BACKGROUND: India\'s ancient texts, the Charak Samhita and Sushruta Samhita, make reference to the traditional medicinal usage of Acorus calamus L. In India and China, it has long been used to cure stomach aches, cuts, diarrhea, and skin conditions. This ability of the rhizome is attributed to its antimicrobial properties. Research studies to date have shown its antimicrobial properties. However, scientific evidence on its mode of action is still lacking.
OBJECTIVE: Acorus calamus L. rhizome extract and its bioactive fraction exhibits antibacterial effect by modulating membrane permeability and fatty acid composition.
METHODS: The secondary metabolites in the rhizome of A. calamus L. were extracted in hexane using Soxhlet apparatus. The ability of the extract to inhibit multidrug resistant bacterial isolates, namely Bacillus cereus, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa were evaluated using checkerboard assay. Further, the extract was purified using thin layer chromatography, gravity column chromatography, and combiflash chromatography. Structure elucidation of the active compound was done using GC-MS, FT-IR, and UV-Vis spectral scan. The mode of action of the bioactive fraction was determined. Bacterial membrane damage was analyzed using SEM, membrane permeability was determined using SYBR green I and PI dye, leakage of cytoplasmic contents were analyzed using Bradford assay and Fehling\'s reagent. The ability to inhibit efflux pump of A. baumannii was determined using EtBr accumulation assay and β-lactamase inhibition was analyzed using nitrocefin as substrate. Also, the biofilm inhibition of B. cereus was determined using crystal violet dye. Moreover, the effect of the bioactive fraction on the fatty acid profile of the bacterial membrane was determined by GC-FAME analysis using 37 component FAME mix as standard.
RESULTS: Acorus calamus L. rhizome hexane extract (AC-R-H) demonstrated broad-spectrum antibacterial activity against all the isolates tested. AC-R-H extract also significantly reduced the MIC of ampicillin against all tested bacteria, indicating its bacterial resistance modulating properties. The assay guided purification determined Asarone as the major compound present in the bioactive fraction (S-III-BAF). S-III-BAF was found to reduce the MIC of ampicillin against Escherichia coli (100-25 mg/mL), Pseudomonas aeruginosa (15-3.25 mg/mL), Acinetobacter baumannii (12.5-1.56 mg/ml), and Bacillus cereus (10-1.25 mg/mL). Further, it recorded synergistic activity with ampicillin against B. cereus (FICI = 0.365), P. aeruginosa (FICI = 0.456), and A. baumannii (FICI = 0.245). The mode of action of S-III-BAF can be attributed to its ability to disturb the membrane integrity, enhance membrane permeability, reduce biofilm formation, and possibly alter the fatty acid composition of the bacterial cell membranes.
CONCLUSIONS: The bioactive fraction of AC-R-H extract containing Asarone as the active compound showed antibacterial activity and synergistic interactions with ampicillin against the tested bacterial isolates. Such activity can be attributed to the modulation of fatty acids present in bacterial membranes, which enhances membrane permeability and causes membrane damage.