Abstract
Abstract
Background: Isolating amphiphilic natural products and highly polar metabolites from complex microbial extracts remains a significant challenge in natural product chemistry. Conventional liquid-liquid extraction methods suffer from poor selectivity, foam formation, and inefficient recovery of compounds with dual hydrophilic-lipophilic character.
Methods: We developed Sonication-Assisted Vesicle-Mediated Extraction (SAVME), a novel fractionation method that exploits the intrinsic self-assembly behavior of endogenous amphiphilic compounds in crude microbial extracts. The method combines strategic application of bath ultrasonication (20-60 kHz) with sequential solvent partitioning using n-hexane, dichloromethane or chloroform, ethyl acetate, and n-butanol. Dynamic light scattering (DLS) was employed to monitor vesicle size transformations throughout the extraction process.
Results: SAVME converted pre-existing multilamellar vesicles (Z-average ~157 nm, PDI 0.5) into small unilamellar vesicles (~89 nm, PDI 0.48) through controlled bath ultrasonication. The method selectively enriched amphiphilic compound classes (glycerophosphocholines, N-acyl amines, benzoic acid esters, phytosphingosines, lipopeptides) in a single n-butanol fraction, while simultaneously co-recovering vesicle-entrapped polar metabolites, including ectoine (logP ≈ −4.0), via vesicle-mediated co-transport.
Gravimetric analysis of the dried n-butanol fraction showed that SAVME yielded 100 mg/L versus 52 mg/L by conventional extraction, an approximately 2-fold increase. Downstream fractionation enabled isolation of ectoine (via acetone precipitation) and identification of (5E)-3-hydroxydodec-5-enoic acid (Compound N19), a vesicle-associated fatty acyl lipid. The dried n-butanol residue spontaneously reformed vesicular structures (~135 nm, PDI 0.54) upon aqueous redissolution, confirming the intrinsic self-assembly behavior of the enriched amphiphilic fraction.
The SAVME fraction demonstrated 2-fold lower MIC values compared to the conventionally extracted fraction against MDR E. coli (125 vs. 250 µg/mL), carbapenem-resistant K. pneumoniae BAA-1705 (125 vs. 250 µg/mL), and S. aureus NCTC 12493 (250 vs. 500 µg/mL), confirming that selective amphiphile enrichment enhances antimicrobial potency.
Conclusions: SAVME represents a paradigm shift in natural product extraction by utilizing endogenous self-assembled nanostructures as active separation vehicles. The method offers superior selectivity and efficiency compared to conventional approaches, with broad applicability to microbial fermentation systems producing amphiphilic metabolites.
| Has the manuscript been published | not published |
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| Intend to be published in the IJPS ? | NO |
