Combined royal jelly 10-hydroxydecanoic acid and aspirin has a synergistic effect against memory deficit and neuroinflammation.

Alzheimer's disease (AD), the most common form of neurodegenerative dementia among the older population, is associated with acute or chronic inflammation. As a nonsteroidal anti-inflammatory drug, aspirin has recently been widely studied in the prevention and treatment of neurodegenerative diseases. However, there is a controversy about the efficacy as well as the adverse effects of aspirin. 10-Hydroxydecanoic acid (10-HDAA) is a characteristic fatty acid found in the honey bee product royal jelly. In this study, we found that 10-HDAA attenuated the activation of the NF-κB pathway, then targeted Ptgs-1/2, the well-known target of aspirin. Hence, combined therapy of 10-HDAA and aspirin was conducted. In vitro assays suggested that this combinatory group alleviated LPS-induced inflammation in BV-2 cells, as assessed by the downregulation of nitric oxide, COX-2, and IL-6 compared to 10-HDAA or aspirin treatment alone. In vivo assays showed that the combined treatment synergistically inhibited the overactivation of glial cells and decreased the levels of pro-inflammatory mediators. Moreover, 10-HDAA alleviated the adverse effects of aspirin on gastrointestinal injuries and microbiota dysbiosis. The Morris water maze test indicated that neither 10-HDAA nor aspirin effectively improved LPS-induced memory dysfunction, but the combined therapy showed synergistic effects. Altogether, our findings support 10-HDAA and aspirin combinatory therapy as the basis for future therapeutics for AD and other neuroinflammation-related diseases with minimal adverse effects.

Prebiotic Membranes and Micelles Do Not Inhibit Peptide Formation During Dehydration.

Cycles of dehydration and rehydration could have enabled formation of peptides and RNA in otherwise unfavorable conditions on the early Earth. Development of the first protocells would have hinged upon colocalization of these biopolymers with fatty acid membranes. Using atomic force microscopy, we find that a prebiotic fatty acid (decanoic acid) forms stacks of membranes after dehydration. Using LC-MS-MS (liquid chromatography-tandem mass spectrometry) with isotope internal standards, we measure the rate of formation of serine dipeptides. We find that dipeptides form during dehydration at moderate temperatures (55 °C) at least as fast in the presence of decanoic acid membranes as in the absence of membranes. Our results are consistent with the hypothesis that protocells could have formed within evaporating environments on the early Earth.

Impact of Intestinal Concentration and Colloidal Structure on the Permeation-Enhancing Efficiency of Sodium Caprate in the Rat.

In this work, we set out to better understand how the permeation enhancer sodium caprate (C10) influences the intestinal absorption of macromolecules. FITC-dextran 4000 (FD4) was selected as a model compound and formulated with 50-300 mM C10. Absorption was studied after bolus instillation of liquid formulation to the duodenum of anesthetized rats and intravenously as a reference, whereafter plasma samples were taken and analyzed for FD4 content. It was found that the AUC and Cmax of FD4 increased with increasing C10 concentration. Higher C10 concentrations were associated with an increased and extended absorption but also increased epithelial damage. Depending on the C10 concentration, the intestinal epithelium showed significant recovery already at 60-120 min after administration. At the highest studied C10 concentrations (100 and 300 mM), the absorption of FD4 was not affected by the colloidal structures of C10, with similar absorption obtained when C10 was administered as micelles (pH 8.5) and as vesicles (pH 6.5). In contrast, the FD4 absorption was lower when C10 was administered at 50 mM formulated as micelles as compared to vesicles. Intestinal dilution of C10 and FD4 revealed a trend of decreasing FD4 absorption with increasing intestinal dilution. However, the effect was smaller than that of altering the total administered C10 dose. Absorption was similar when the formulations were prepared in simulated intestinal fluids containing mixed micelles of bile salts and phospholipids and in simple buffer solution. The findings in this study suggest that in order to optimally enhance the absorption of macromolecules, high (≥100 mM) initial intestinal C10 concentrations are likely needed and that both the concentration and total dose of C10 are important parameters.

Carboxymethyl chitosan coated medium-chain fatty acid nanoliposomes: structure, composition, stability and in vitro release investigation.

Medium-chain fatty acids (MCFAs) have been proven as an easy energy source and active ingredient to prevent obesity and other metabolic disorders. However, the inherent hydrophobic nature of MCFAs causes poor aqueous solubility and dissolution in the gastrointestinal (GI) tract, thus limiting their applications in aqueous foods. To address these issues, a nutraceutical carrier system was developed by coating nanoliposomes with carboxymethyl chitosan (CMCS) through a series of well-designed processes, including thin-film hydration, dynamic high pressure microfluidization (DHPM) and surface modification. Electron microscopy investigation reveals an obvious morphology evolution from the uncoated nanoliposomes (UC-LPs) to the final CMCS coated nanoliposomes (CMCS-LPs). Together with the FTIR results, it confirms the successful coating of CMCS. More importantly, the resultant CMCS-LPs have a more negatively charged surface with a ζ-potential value of around -18.5 mV, which helps to increase the stability by avoiding severe particle aggregation. Owing to the above benefits, the encapsulated MCFAs can be safely retained in a long storage period of 90 days at 4 °C and the new carrier system also exhibits a more sustained release of MCFAs in the GI fluid.

Ultrasound-assisted dispersive liquid-phase microextraction by solidifying L-menthol-decanoic acid hydrophobic deep eutectic solvents for detection of five fungicides in fruit juices and tea drinks.

An ecofriendly and efficient ultrasound-assisted deep eutectic solvents dispersive liquid-phase microextraction by solidifying the deep eutectic solvents-rich phase was developed to determine azoxystrobin, fludioxonil, epoxiconazole, cyprodinil, and prochloraz in fruit juices and tea drinks by high-performance liquid chromatography. A varieties of environmental hydrophobic deep eutectic solvents serving as extraction agents were prepared using L-menthol and decanoic acid as hydrogen-bond acceptor and hydrogen-bond donor, respectively. The deep eutectic solvents were ultrasonically dispersed in sample solutions, solidified in a freezer and easily harvested. The main variables were optimized by one-factor-at-a-time and response surface test. The new method performs well with relative recovery of 71.75-109.40%, linear range of 2.5-5000 µg/L (r ≥ 0.9968), detection limit of 0.75-8.45 µg/L, quantification limit of 2.5-25 µg/L,, and inter- and intraday relative standard deviations below 13.53 and 14.84%, respectively. As for the extraction mechanism, deep eutectic solvents were disposed into many fine particles in the solution and captured the analytes based on the changes of particle size and quantity in deep eutectic solvents droplets after extraction. The environmental method can successfully detect fungicide residues in real fruit juices and tea drinks.

Formulation of Ocular In Situ Gels with Lithuanian Royal Jelly and Their Biopharmaceutical Evaluation In Vitro.

Royal jelly is a natural substance produced by worker bees that possesses a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, and protective. Although fresh royal jelly is kept at low temperatures, to increase its stability, it needs to be incorporated into pharmaceutical formulations, such as in situ gels. The aim of this study was to formulate in situ ocular gels containing Lithuanian royal jelly for topical corneal use in order to increase the retention time of the formulation on the ocular surface and bioavailability. Gels were evaluated for physicochemical characteristics (pH, rheological properties, refractive index) and in vitro drug release measuring the amount of 10-hydroxy-2-decenoic acid (10-HDA). An ocular irritation test and cell viability tests were performed using the SIRC (Statens Seruminstitut Rabbit Cornea) cell culture line. Results indicated that all the in situ gels were within an acceptable pH and refractive index range close to corneal properties. Rheology studies have shown that the gelation temperature varies between 25 and 32 °C, depending on the amount of poloxamers. The release studies have shown that the release of 10-HDA from in situ gels is more sustained than royal jelly suspension. All gel formulations were non-irritant according to the short-time exposure test (STE) using the SIRC cell culture line, and long-term cell viability studies indicated that the formulations used in small concentrations did not induce cell death. Prepared in situ gels containing royal jelly have potential for ocular drug delivery, and they may improve the bioavailability, stability of royal jelly, and formation of non-irritant ocular formulations.

Intensification of Enzymatic Synthesis of Decyl Oleate Using Ultrasound in Solvent Free System: Kinetic, Thermodynamic and Physicochemical Study.

The present study evaluates the potential use of ultrasound irradiation to synthesize decyl oleate using Fermase CALBTM10000 under the solvent-free system (SFS). The optimal condition to achieve a maximum yield of 97.14% was found to be 1:2 oleic acid:decanol ratio, 1.8% (w/w) enzyme loading, 45°C temperature, 200 rpm agitation speed, 50 W power input, 50% duty cycle, 22 kHz frequency and reaction time of 25 minutes. The thermodynamic study was done to determine the change in entropy, Gibb's free energy, and change in enthalpy at various temperatures. The experimental results and kinetic study showed that the reaction followed ordered bi-bi model with kinetic parameters as rate of reaction (V max ) = 35.02 M/min/g catalyst, Michaelis constant for acid (K A ) = 34.47 M, Michaelis constant for alcohol (K B ) = 3.31 M, Inhibition constant (Ki) = 4542.4 M and sum of square error (SSE) = 0.000334. The application of ultrasound irradiation combined with biocatalyst and the absence of solvent intensified the process compared to the traditional stirring method using hexane as solvent.

Phase-Transition Thermal Charging of a Channel-Shape Thermal Energy Storage Unit: Taguchi Optimization Approach and Copper Foam Inserts.

Thermal energy storage is a technique that has the potential to contribute to future energy grids to reduce fluctuations in supply from renewable energy sources. The principle of energy storage is to drive an endothermic phase change when excess energy is available and to allow the phase change to reverse and release heat when energy demand exceeds supply. Unwanted charge leakage and low heat transfer rates can limit the effectiveness of the units, but both of these problems can be mitigated by incorporating a metal foam into the design of the storage unit. This study demonstrates the benefits of adding copper foam into a thermal energy storage unit based on capric acid enhanced by copper nanoparticles. The volume fraction of nanoparticles and the location and porosity of the foam were optimized using the Taguchi approach to minimize the charge leakage expected from simulations. Placing the foam layer at the bottom of the unit with the maximum possible height and minimum porosity led to the lowest charge time. The optimum concentration of nanoparticles was found to be 4 vol.%, while the maximu possible concentration was 6 vol.%. The use of an optimized design of the enclosure and the optimum fraction of nanoparticles led to a predicted charging time for the unit that was approximately 58% shorter than that of the worst design. A sensitivity analysis shows that the height of the foam layer and its porosity are the dominant variables, and the location of the porous layer and volume fraction of nanoparticles are of secondary importance. Therefore, a well-designed location and size of a metal foam layer could be used to improve the charging speed of thermal energy storage units significantly. In such designs, the porosity and the placement-location of the foam should be considered more strongly than other factors.

Melting phase change heat transfer in a quasi-petal tube thermal energy storage unit.

In the present study, the thermal energy storage of a hot petal tube inside a shell-tube type Thermal Energy Storage (TES) unit was addressed. The shell is filled with the capric acid Phase Change Material (PCM) and absorbs the heat from a hot U-tube petal. The governing equations for the natural convection flow of molten PCM and phase change heat transfer were introduced by using the enthalpy-porosity approach. An automatic adaptive mesh scheme was used to track the melting interface. The accuracy and convergence of numerical computations were also controlled by a free step Backward Differentiation Formula. The modeling results were compared with previous experimental data. It was found that the present adaptive mesh approach can adequately the melting heat transfer, and an excellent agreement was found with available literature. The effect of geometrical designs of the petal tube was investigated on the melting response of the thermal energy storage unit. The phase change behavior was analyzed by using temperature distribution contours. The results showed that petal tubes could notably increase the melting rate in the TES unit compared to a typical circular tube. Besides, the more the petal numbers, the better the heat transfer. Using a petal tube could increase the charging power by 44% compared to a circular tube. The placement angle of the tubes is another important design factor which should be selected carefully. For instance, vertical placement of tubes could improve the charging power by 300% compared to a case with the tubes' horizontal placement.

Prebiotic Peptide Synthesis and Spontaneous Amyloid Formation Inside a Proto-Cellular Compartment.

Cellular life requires a high degree of molecular complexity and self-organization, some of which must have originated in a prebiotic context. Here, we demonstrate how both of these features can emerge in a plausibly prebiotic system. We found that chemical gradients in simple mixtures of activated amino acids and fatty acids can lead to the formation of amyloid-like peptide fibrils that are localized inside of a proto-cellular compartment. In this process, the fatty acid or lipid vesicles act both as a filter, allowing the selective passage of activated amino acids, and as a barrier, blocking the diffusion of the amyloidogenic peptides that form spontaneously inside the vesicles. This synergy between two distinct building blocks of life induces a significant increase in molecular complexity and spatial order thereby providing a route for the early molecular evolution that could give rise to a living cell.

Chelation Crosslinking of Biodegradable Elastomers.

Widely present in nature and in manufactured goods, elastomers are network polymers typically crosslinked by strong covalent bonds. Elastomers crosslinked by weak bonds usually exhibit more plastic deformation. Here, chelation as a mechanism to produce biodegradable elastomers is reported. Polycondensation of sebacic acid, 1,3-propanediol, and a Schiff-base (2-[[(2-hydroxyphenyl) methylene]amino]-1,3-propanediol) forms a block copolymer that binds several biologically relevant metal ions. Chelation offers a unique advantage unseen in conventional elastomer design because one ligand binds multiple metal ions, yielding bonds of different strengths. Therefore, one polymeric ligand coordinated with different metal ions produces elastomers with vastly different characteristics. Mixing different metal ions in one polymer offers another degree of control on material properties. The density of the ligands in the block copolymer further regulates the mechanical properties. Moreover, a murine model reveals that Fe3+ crosslinked foam displays higher compatibility with subcutaneous tissues than the widely used biomaterial-polycaprolactone. The implantation sites restore to their normal architecture with little fibrosis upon degradation of the implants. The versatility of chelation-based design has already shown promise in hydrogels and highly stretchy nondegradable polymers. The biodegradable elastomers reported here would enable new materials and new possibilities in biomedicine and beyond.

A modified quick, easy, cheap, effective, rugged, and safe method with hydrophobic natural deep eutectic solvent as extractant and analyte protectant for analyzing pyrethroid residues in tomatoes.

In this work, a novel quick, easy, cheap, effective, rugged, and safe technique with hydrophobic natural deep eutectic solvent as both extractant and analyte protectant was developed and combined with gas chromatography-tandem mass spectrometry to analyze pyrethroid residues in tomatoes. Eight hydrophobic natural deep eutectic solvents were first evaluated as analyte protectants and those with decanoic acid or lactic acid as hydrogen bond donor were demonstrated to be effective in compensating for the matrix effects of pyrethroids in the gas chromatography system. Hence, they were added to solvent standards for correcting the quantitation errors instead of matrix-matched calibration standards. Then the abilities of these acid-based deep eutectic solvents to extract pyrethriods from tomatoes were evaluated. Results showed the recoveries of all pyrethroids reached to over 80% with only 5 mL menthol:decanoic acid (1:1) used, and good phase separation was easily achieved without the addition of inorganic salt in the extraction step, indicating hydrophobic natural deep eutectic solvent could be a green substitute for acetonitrile in the quick, easy, cheap, effective, rugged, and safe extraction. Compared with the conventional method, the proposed protocol improved the recoveries, reduced the matrix effects, and simplified the extraction step, demonstrating to be an effective, fast, and green method.

Hydrophobic deep eutectic solvent as a green extractant for high-performance liquid chromatographic determination of tetracyclines in water samples.

A green extractant, hydrophobic deep eutectic solvent was first introduced for extraction of tetracycline, oxytetracycline, and chlortetracycline from environmental water samples prior to high-performance liquid chromatography determination. Deep eutectic solvents consist of methyltrioctylammonium chloride and various medium-chain alcohols/acids, and are easy in preparation, low cost and toxicity, desirably biodegradable, and biocompatible. The overall time required for sample preparation was 6 min and the volume of organic solvent used for extraction was only 400 µL. Under the optimized extraction condition, the present method yielded low limit of detection (0.5-2.0 ng/mL), acceptable precision (relative standard deviations < 9.7%), good linearity from 2.0 to 500 ng/mL (r2  ≥ 0.9991). This optimized procedure was applied for determination of tetracyclines in different water samples with desirable spiked recovery ranged from 77.5 to 87.6%. There is, therefore, a great potential to further expand application of the method for investigation of other ultra-trace analyte(s) in environmental matrixes.

Insulin-Sensitizing Activity of Sub-Nanoscaled Polyalkoxyvanadate Clusters.

Sub-nanoscaled polyalkoxyvanadates (PAOVs) functionalized with various aliphatic acids are evaluated for their insulin-sensitizing activity in lowering the blood glucose levels of diabetic mice in typical glucose tolerance tests. All the PAOVs can restore the blood glucose to normal levels after a single oral administration of PAOVs. Among them, the myristic acid-modified PAOVs enable the response of insulin to the repeated glucose challenges, lasting for up to 13 h. The combined administration of PAOVs exerts better glucose control over insulin alone, while the capric acid- and myristic acid-modified ones can enhance the responsiveness of insulin to glucose challenge and is comparable to a clinical-used derivative of insulin. Interestingly, continuous glucose monitoring shows that myristic acid-modified PAOV derivatives sensitize the responsiveness of insulin, almost matching with that of a healthy pancreas. These discoveries open up new opportunities for the application of PAOVs to promote glucose-responsive and long-lasting activity of insulin, which are expected to aid the accurate blood glucose control in insulin therapy while reducing the number of insulin administrations.

Antifungal Activity of Capric Acid, Nystatin, and Fluconazole and Their In Vitro Interactions Against Candida Isolates from Neonatal Oral Thrush.

Due to the increasing resistance of various Candida species to azole drugs, particularly fluconazole, it would be of significant importance to look for alternative therapies. The aim of this study was to investigate the antifungal activity of capric acid and its in vitro interactions with nystatin and fluconazole against Candida isolates. A total of 40 Candida isolates (C. albicans, 36; C. kefyr, 2; C. tropicalis, 1; C. glabrata, 1) collected from the oral cavity of neonates with oropharyngeal candidiasis and a reference strain of C. albicans (ATCC 10231) were used in this study. Antifungal activity of capric acid and two comparator antifungal drugs, namely fluconazole and nystatin, was tested according to CLSI M27-A3/M60 method. The in vitro interaction between capric acid with fluconazole and nystatin was determined following a checkerboard method and results were interpreted using fractional inhibitory concentration index. Nystatin had the lowest minimum inhibitory concentrations (range, 0.125-8 µg/mL; geometric mean [GM], 0.6229 µg/mL) followed by fluconazole (range, 0.5-16 µg/mL; GM, 1.9011 µg/mL) and capric acid (range, 128-2,048 µg/mL; GM, 835.9756 µg/mL). When tested in combination, capric acid with fluconazole demonstrated synergistic, indifferent, and antagonistic interactions in 3 (7.317%), 24 (58.536%), and 14 (34.146%) cases, respectively. For combination of capric acid with nystatin, synergistic, indifferent, and antagonistic interactions were observed in 1 (2.439%), 19 (46.341%), and 21 (51.219%) cases, respectively. All cases of synergistic interactions were against resistant or susceptible dose-dependent isolates. Fluconazole, nystatin, and capric acid seem to be more effective when they are used alone compared with their combination. However, their combination might be effective on resistant isolates.

A head-to-head Caco-2 assay comparison of the mechanisms of action of the intestinal permeation enhancers: SNAC and sodium caprate (C10).

Salcaprozate sodium (SNAC) and sodium caprate (C10) are the two leading intestinal permeation enhancers (PEs) in oral peptide formulations in clinical trials. There is debate over their mechanism of action on intestinal epithelia. The aims were: (i) to compare their effects on the barrier function by measuring transepithelial electrical resistance (TEER), permeability of FITC-4000 (FD4) across Caco-2 monolayers, and on immunohistochemistry of tight junction (TJ)-associated proteins; and (ii) to compare cellular parameters using conventional end-point cytotoxicity assays and quantitative high content analysis (HCA) of multiple sub-lethal parameters in Caco-2 cells. C10 (8.5 mM) reversibly reduced TEER and increased FD4 permeability across monolayers, whereas SNAC had no effects on either parameter except at cytotoxic concentrations. C10 exposure induced reorganization of three TJ proteins, whereas SNAC only affected claudin-5 localization. High concentrations of C10 and SNAC were required to cause end-point toxicology changes in vitro. SNAC was less potent than C10 at inducing lysosomal and nuclear changes and plasma membrane perturbation. In parallel, HCA revealed that both agents displayed detergent-like features that reflect initial membrane fluidization followed by changes in intracellular parameters. In conclusion, FD4 permeability increases in monolayers in response to C10 were in the range of concentrations that altered end-point cytotoxicity and HCA parameters. For SNAC, while HCA parameters were also altered in a similar overall pattern as C10, they did not lead to increased paracellular flux. These assays show that both agents are primarily surfactants, but C10 has additional TJ-opening effects. While these in vitro assays illucidate their epithelial mechanism of action, clinical experience suggests that they over-estimate their toxicology in the dynamic intestinal environment.

A Step toward Molecular Evolution of RNA: Ribose Binds to Prebiotic Fatty Acid Membranes, and Nucleosides Bind Better than Individual Bases Do.

A major challenge in understanding how biological cells arose on the early Earth is explaining how RNA and membranes originally colocalized. We propose that the building blocks of RNA (nucleobases and ribose) bound to self-assembled prebiotic membranes. We have previously demonstrated that the bases bind to membranes composed of a prebiotic fatty acid, but evidence for the binding of sugars has remained a technical challenge. Here, we used pulsed-field gradient NMR spectroscopy to demonstrate that ribose and other sugars bind to membranes of decanoic acid. Moreover, the binding of some bases is strongly enhanced when they are linked to ribose to form a nucleoside or - with the addition of phosphate - a nucleotide. This enhanced binding could have played a role in the molecular evolution leading to the production of RNA.

Ionophore-based pH independent detection of ions utilizing aggregation-induced effects.

Ionophores have been integrated into various electrochemical and optical sensing platforms for the selective detection of ions. Previous ionophore-based optical sensors rely on a H+ chromoionophore as the signal transducer and consequently, suffered from a pH cross-response. pH independent methods were proposed very recently by utilizing the solvatochromic dyes or the exhaustive mode. Here, we report a pH independent sensing principle based on nanospheres containing ionophores. As the ion-exchange occurs, the signal transducer undergoes aggregation-induced emission (AIE) or aggregation-caused quenching (ACQ), leading to a dramatic change in fluorescence intensity. The principle was evaluated on different ionophores including those selective for K+, Na+, Ca2+, and Pb2+. The nanospheres were also introduced into microfluidic chips and successfully applied for the determination of sodium and potassium ion concentrations in diluted blood serum and urine samples.

Molecular hybridization of grape seed extract: Synthesis, structural characterization and anti-proliferative activity in vitro.

The grape seed extract (GSE) hybridized with medium-chain saturated fatty acids (decanoic acid) exhibited higher lipophilicity, antioxidant activity, and anti-proliferative activity than its parents. The chemical structures of individual hybridized GSE derivatives were identified as 3'-O-decanoyl catechin, 3'-O-decanoyl epicatechin, 3', 5'-2-O-decanoyl epigallocatechin, and 3', 4', 3″, 5″-4-O-decanoyl epicatechin gallate by HPLC-MS2 and 1H and 13C NMR. For growth inhibitory effect on HepG2 cells, hybridized GSE derivatives (EC50 = 44.38 µg/mL) were significantly (p < 0.01) stronger than natural GSE (EC50 = 60.83 µg/mL) due to increased lipophilicity. The effects of GSE derivatives on apoptosis and cell cycle in HepG2 cells were further evaluated by flow cytometry. The results showed that the percentage of apoptotic cells increased markedly in the presence of hybridized GSE derivatives. Moreover, hybridized GSE derivatives were capable of inducing cell cycle arrest in G1 phase. This research suggests that hybridized GSE derivatives are effective lipophilic antioxidants and show the potential as adjuvant therapy for cancer.

Hydrophobic borneol-based natural deep eutectic solvents as a green extraction media for air-assisted liquid-liquid micro-extraction of warfarin in biological samples.

In the present study, a new generation of water-immiscible natural deep eutectic solvents (DESs) was synthesized using borneol as a hydrogen-bonding acceptor and decanoic acid, oleic acid, and thymol as a hydrogen-bonding donor in different molar ratios. These green hydrophobic solvents which are chemically stable in aqueous solutions were used as extraction solvents for isolation and pre-concentration of warfarin in biological samples. In this method, fine droplets of DESs were dispersed into the sample solution by using the air-assisted liquid-liquid micro-extraction method to accelerate the cloudy emulsion system formation and increase the mass transfer of the analyte to the DES-rich phase. The borneol based deep eutectic solvent is a worthy generation of the extraction solvents in the ALLME method due to low-cost and less toxicity. A Plackett-Burman design was utilized for screening the experimental parameters. The effective parameters were then optimized by Box-Behnken design (BBD). Optimized extraction conditions were pH of sample solution of 3.9, number of aspiration/dispersion cycles of 15, the volume of DES of 60 µL, and rate and time of centrifuge of 6000 rpm and 10 min, respectively. Under the optimized conditions, the developed NADES-ALLME method exhibited a wide linear range of 5-500 µg L - 1 for plasma and urine samples with satisfactory recoveries above 88.80%. Limit of detections (LODs) and Limit of quantifications (LOQs) of warfarin were in the ranges of 0.5-2.7 and 1.65-8.91, respectively. The enrichment factors were obtained in the range of 148-164 and precisions were lower than 5.87%. Finally, the proposed method was successfully employed for the analysis of warfarin in human urine and plasma samples.