Abstract
Anti-biofilm represents an urge to face drug resistance. Nymphaea alba L. flowers and rhizomes have been traditionally used in Ayurvedic medicine for dyspepsia, enteritis, diarrhea and as an antiseptic. This study was designed to identify the main constituents of Nymphaea alba L. rhizomes and their anti-biofilm activity. 70% aqueous ethanolic extract (AEE) of N. alba rhizomes was analyzed by liquid chromatography, high resolution, mass spectrometry (LC-HRMS) for its phytoconstituents in the positive and negative modes in addition to column chromatographic separation. Sixty-four phenolic compounds were identified for the first time in N. alba rhizomes. Hydrolysable tannins represent the majority with identification of galloyl hexoside derivative, hexahydroxydiphenic (HHDP) derivatives, glycosylated phenolic acids and glycosylated flavonoids. Five phenolics have been isolated and identified as gallic acid and its methyl and ethyl ester in addition to ellagic acid and pentagalloyl glucose. Minimum inhibitory concentrations (MIC) and anti-biofilm activity for the extract and the major isolated compounds were determined. Radical scavenging activity using 2.2Di (4-tert-octylphenyl)-1-picryl-hydrazyl (DPPH) assay as well as cytotoxic activity using 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay have also been evaluated. MIC of N. alba rhizomes against Staphylococcus aureus was 0.25 mg/mL compared with 0.1 mg/mL for methyl gallate. The best reduction in biofilm formation (84.9%) as well as the best radical scavenging (IC50 3 µg/mL) and cytotoxic (IC50 9.61 ± 0.3 µg/mL) activities were observed with methyl gallate. This is the first study for in-depth characterization of phenolic compounds in N. alba rhizomes revealing it as a valuable source of phenolic compounds and promising anti-biofilm forming agent of natural origin. Key words: Hydrolysable tannins, Nymphaeaceae, LC-HRMS.
Highlights
The total phenolic content (TPC) of N. alba aqueous ethanolic extract (AEE) was estimated as 32.96 ± 0.86 mg/g gallic acid equivalent (GAE) while the Flavonoid content (FC) was evaluated as 0.43 ± 0.59 mg/g quercetin equivalent (QE)
The considerable high phenolic and moderate flavonoid contents have a great impact on biological activities
HPLC-MS-MS provides a powerful tool for phytochemical analysis in crude plant extracts
Summary
Bacterial illnesses are caused by many virulence factors. Biofilm forming capacity is an additional virulence factor. Cells in biofilms grow as communities, surrounded by a selfproduced thick layer of extracellular polymeric substances (EPS, known as matrix or slime) (Sauer and Camper, 2001). The extracellular matrix of biofilmembedded microorganisms is capable of sequestering and concentrating environmental nutrients such as carbon, nitrogen and phosphate. They can evade multiple clearance mechanisms produced by host and synthetic sources such as antimicrobial and antifouling agents, shear stress, host phagocytic elimination and host radical and protease defenses (Archer et al, 2011).
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