Environmental Biotechnology Lab, Centre for Rural Development and Technology, Indian Institute of Technology, Delhi, India.
Environmental Biotechnology Lab, Centre for Rural Development and Technology, Indian Institute of Technology, Delhi, India.
J Ethnopharmacol. 2024 Sep 15;331:118323. doi: 10.1016/j.jep.2024.118323. Epub 2024 May 9.
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.
Acorus calamus L. rhizome extract and its bioactive fraction exhibits antibacterial effect by modulating membrane permeability and fatty acid composition.
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.
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.
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.
印度古代文献《恰拉克萨姆希塔》和《苏斯拉塔萨姆希塔》提到了菖蒲根茎的传统药用用途。在印度和中国，菖蒲根茎长期以来一直被用于治疗胃痛、割伤、腹泻和皮肤病。这种根茎的能力归因于其抗菌特性。迄今为止的研究表明，其具有抗菌特性。然而，其作用机制的科学证据仍然缺乏。
菖蒲根茎提取物及其生物活性部分通过调节膜通透性和脂肪酸组成表现出抗菌作用。
使用索氏提取器从菖蒲根茎中提取次级代谢产物。使用棋盘法评估提取物对多药耐药菌分离株，即蜡状芽孢杆菌、大肠杆菌、鲍曼不动杆菌和铜绿假单胞菌的抑制作用。进一步使用薄层层析、重力柱层析和 combiflash 层析对提取物进行纯化。使用 GC-MS、FT-IR 和 UV-Vis 光谱扫描对活性化合物的结构进行了阐明。测定了生物活性部分的作用方式。使用 SEM 分析细菌膜损伤，使用 SYBR 绿色 I 和 PI 染料测定膜通透性，使用 Bradford 测定法和菲林试剂分析细胞质内容物泄漏。使用 EtBr 积累测定法测定对鲍曼不动杆菌外排泵的抑制作用，并用硝基头孢菌素作为底物分析β-内酰胺酶抑制作用。此外，使用结晶紫染料测定枯草芽孢杆菌生物膜的抑制作用。使用 37 种成分 FAME 混合物作为标准，通过 GC-FAME 分析确定菖蒲根茎正己烷提取物（AC-R-H）中生物活性部分对细菌膜脂肪酸组成的影响。
菖蒲根茎正己烷提取物（AC-R-H）对所有测试的分离株均表现出广谱的抗菌活性。AC-R-H 提取物还显著降低了氨苄西林对所有测试细菌的 MIC，表明其具有细菌耐药性调节特性。基于测定的纯化结果表明，烯丙基苯甲醚是生物活性部分（S-III-BAF）中的主要化合物。发现 S-III-BAF 降低了氨苄西林对大肠杆菌（100-25mg/ml）、铜绿假单胞菌（15-3.25mg/ml）、鲍曼不动杆菌（12.5-1.56mg/ml）和蜡状芽孢杆菌（10-1.25mg/ml）的 MIC。此外，它对枯草芽孢杆菌（FICI=0.365）、铜绿假单胞菌（FICI=0.456）和鲍曼不动杆菌（FICI=0.245）与氨苄西林具有协同作用。S-III-BAF 的作用方式可归因于其干扰膜完整性、增强膜通透性、减少生物膜形成以及可能改变细菌细胞膜脂肪酸组成的能力。
AC-R-H 提取物的生物活性部分含有烯丙基苯甲醚作为活性化合物，对测试的细菌分离株表现出抗菌活性和与氨苄西林的协同作用。这种活性可归因于对存在于细菌膜中的脂肪酸的调节，这增强了膜通透性并导致膜损伤。
