Microwave-assisted extraction of bioactive components from date seeds using deep eutectic solvents: Bioactivity and antibiotic adjuvant potential against multidrug-resistant Salmonella Typhimurium DT104

Abstract

Adjuvants offer a promising strategy against antimicrobial resistance. Deep eutectic solvents (DESs) and their subsequent DES-based polyphenol-rich bioactive compounds (PBCs) were assessed for antimicrobial and adjuvant activity against multidrug-resistant Salmonella Typhimurium DT104. PBCs were extracted from date seeds using polyol-based DESs with microwave assistance. FTIR spectra of synthesized DESs confirmed new hydrogen bond formation. Phytocompound profiling revealed that 1,4-Butanediol (1,4-But)-extract showed the highest TPC (30.05 ± 1.33 mg GA g⁻¹) and DPPH activity (35.02 ± 0.34 mmol TE g⁻¹). UHPLC-PDA identified 4-hydroxybenzoic acid and rutin hydrate as the predominant phenolic compounds. Despite limited intrinsic antimicrobial activity, DES extracts and their corresponding DES-enhanced the antibiotic effectiveness. Checkerboard assay showed that the 1,4-But-DES extract exhibited significant synergy with ACSSuT antibiotics, reducing their MICs by 32- to >1024-fold, with FICI values ≤0.5. Time-kill assays confirmed enhanced bactericidal activity of chloramphenicol with both extracts at sub-inhibitory concentrations. Conclusively, 1,4-But-based extracts can enhance antibiotic efficacy against resistant foodborne pathogens.

Graphical abstract

Highlights

• •Polyol-based Deep Eutectic Solvents (DESs) were used for the green extraction of bioactive compounds from date seeds.
• •Choline chloride:1,4- butanediol-based DES exhibited significant synergy with all five antibiotics tested against Salmonella Typhimurium DT104.
• •1,4- butanediol DES-based polyphenolic-rich date seed extracts show potential as novel antibiotic adjuvants against multidrug-resistant Salmonella Typhimurium DT104.

1. Introduction

Antimicrobial resistance (AMR) in pathogenic bacteria is an alarming threat to conventional antimicrobial therapy. AMR is primarily caused by overuse and misuse of antibiotics both in the healthcare and food production sectors (Ramzan et al., 2024). Data from the Global Antimicrobial Resistance and Use Surveillance System shows AMR as one of the leading causes of death globally (Aldulaimi, 2017). In 2021, around 4.7 million deaths were attributed to bacterial AMR (Naghavi et al., 2024). AMR is also slated to overcome cancer as the leading cause of human mortality by the year 2025 (Costanzo & Roviello, 2023). A notable example of the bacterial outbreak is related to multidrug-resistant Salmonella Typhimurium definitive type DT104, which exhibits resistance to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT). Numerous foodborne illness outbreaks worldwide have been caused by Salmonella Typhimurium, underscoring the pressing need for novel approaches to combat AMR (Monte et al., 2019; Zhang et al., 2021). The intrinsic multidrug resistance of bacteria arises from multiple factors, which include a low-permeability outer membrane and the active expression of efflux systems. Efflux pumps (EPs) are protein channels located in the bacterial cell membrane that extrude harmful chemicals, such as antibiotics, heavy metals, detergents, poisons, and disinfectants. They are classified into five families: MFS (major facilitator superfamily), SMR (small multidrug resistance), ABC (ATP-binding cassette), MATE (multidrug and toxic compounds extrusion), and RND (resistance-nodulation-cell division) (Negi et al., 2014). Efflux pumps facilitate the quick development of multidrug-resistant bacteria by driving new resistance mechanisms in addition to expelling drugs. Inhibition is challenging since the same drug might be extruded with multiple pumps, and the reported inhibitors including reserpine, berberine, and quinoline derivatives leads to exhibit severe toxicity (Oyedara et al., 2023).

One anti-AMR intervention strategy is to sensitize multidrug-resistant pathogens to conventional antibiotics by using synergistic antibiotic adjuvants (Costanzo & Roviello, 2023). Many plant-derived compounds, probiotics, antimicrobial proteins, and bacteriophages have demonstrated excellent antimicrobial properties against many pathogens (Łojewska & Sakowicz, 2021). In some cases, even when these bioactive compounds do not exhibit a strong antibacterial effect, they show strong antibiotic adjuvant activity by sensitizing a bacterial strain to an antibiotic against which the strain is originally resistant (Chung and Navaratnam, 2011). Such synergistic interactions lower the MIC of antibiotics and therefore increase the sensitivity of multidrug-resistant bacteria to conventional antibiotics (Ayaz et al., 2019). Consequently, such synergistic interactions could be employed as a promising strategy to mitigate bacterial AMR.

Fruits, seeds, oils, and vegetables are known to be rich in bioactive compounds with numerous health-beneficial attributes (Gil-Martín et al., 2022). Similarly, date fruit is rich in many bioactive phytochemicals, such as polyphenols, flavonoids, anthocyanins, carotenoids, procyanidins, and sterols. Besides antimicrobial activity, date phytochemicals are rich in anti-oxidant, anti-mutagenic, and anti-carcinogenic properties. The date fruit contains a seed known as the date stone or pit, and a fresh pericarp that is eaten in large quantities (Airouyuwa et al., 2023). Date seeds account for 10–15 % of the fruit's overall weight and contribute to an annual post-harvest loss of 852 thousand tons as a byproduct of the date processing industries (Maqsood et al., 2020). Due to the increased global production, use, and industrialization of the date fruit industry, enormous amounts of waste as byproducts, such as date seeds and leaves, are produced. These wastes are frequently dumped in landfills or utilized as animal feed because they have no industrial purpose. In this context, the extraction of polyphenol-rich bioactive compounds from the date fruit waste has been explored for nutraceutical properties, potential functional food additives, or pharmaceutical ingredients (Airouyuwa et al., 2023).

Conventional solvents such as acetone, ethanol, methanol, petroleum ether, dichloromethane, chloroform, and diethyl ether have been used to extract bioactive compounds such as phenolic and flavonoid compounds from date seeds (Airouyuwa et al., 2023). However, issues such as toxicity, flammability, and volatility have limited their use for the extraction of these compounds efficiently (Rebocho et al., 2022). Alternatively, green extraction solvents and technologies have recently gained significant interest as environmentally acceptable alternatives for conventional extraction and techniques (Otero et al., 2021). In recent years, reliable and environmentally friendly techniques for extracting bioactive compounds from lignocellulosic waste have created a demand in the agro-food industries (Shah et al., 2024). Deep eutectic solvents (DESs) have gained popularity as prospective green substitutes for traditional solvents due to their non-toxicity & eco-friendly nature, biodegradability, easy preparation, and efficiency in extracting bioactive compounds from lignocellulosic biowastes (Karpitskiy et al., 2024; Xu et al., 2024). DESs are the preferred option for extraction due to their high polarity and capacity to dissolve in a wide range of organic and inorganic compounds (Ji et al., 2021). DESs are liquid mixtures consisting of two or three different substances, typically a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA), with melting points lower than those of the individual components (Yan et al., 2021).

To the best of our knowledge, this is the first study to investigate the use of polyol-based DESs (TEG and 1,4-But) and their subsequent DES-based date seed extracts as antibiotic adjuvants against a penta-resistant strain of Salmonella Typhimurium exhibiting the ACSSuT (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline) resistance pattern. The presence of major polyphenol-rich bioactive components has been confirmed through UHPLC-PDA (Ultra-High-Performance Liquid Chromatography with Photodiode Array Detector) analysis of the DES-based extracts. Extracts were characterized in terms of total phenolic content (TPC) and antioxidant activity using three different in vitro assays (ABTS radical scavenging activity, DPPH radical scavenging activity, and Ferric reducing antioxidant power (FRAP)) and antimicrobial activity (agar well diffusion, broth dilution method, and dot plating assays) against Salmonella Typhimurium DT104. Additionally, the efficacy of DESs and their subsequent DES-based extracts as adjuvants with the antibiotics was analyzed using a synergistic strategy and time-killing assay.

2. Materials and methods

2.1. Reagents and bacterial strain

Salmonella Typhimurium definitive type DT104, a multidrug-resistant strain, was used in this study. The strain was maintained as glycerol stocks at −80 °C and revived into the appropriate media whenever needed. Mueller Hinton broth (MHB), Brain Heart Infusion (BHI), and Lennox broth (LB) agar were acquired from Sigma-Aldrich, USA. 1,4-Butanediol (1,4 But), tri-ethylene glycol (TEG), lactic acid, urea, fructose, sodium acetate, gallic acid, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) 1,1-diphenyl-1-picrylhydrazyl (DPPH) and Folin-Ciocalteu and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) were procured from Sigma-Aldrich. Choline chloride (ChCl) (> 98 %) was purchased from Alfa Aesar, Karlsruhe, Germany.

2.2. Sample preparation

The date seeds (Khalas variety) were supplied by Al-Foah Date Company, located in Abu Dhabi, United Arab Emirates. The date seeds (DS) were finely ground using an industrial seed grinder (Retsch ZM 200 Ultra-Centrifugal Mill, Retsch GmbH, Germany). The date seed powder (DSP) was obtained by sieving through a 250 μm mesh and stored at 4 °C for all analyses.

2.3. Preparation and physicochemical characterization of DESs

Various DESs were prepared using the heat and stirring technique described by Airouyuwa et al., (2023). Choline chloride as a hydrogen bond acceptor (HBA) was used to prepare DESs in combination with four categories of hydrogen bond donors (HBD): polyols (1,4 butanediol, triethylene glycol), sugar (fructose), acid (lactic acid), and alkaline (urea). After measuring the required components according to the molar ratios (Table 1), the mixtures were heated to 80 °C on a magnetic stirrer until transparent and homogenous liquids were formed. For the extraction process, 70 % DESs were used, which were prepared by adding 30 % water content. Polyphenol-rich bioactive components (PBCs) were extracted from date seeds using freshly made DES.

Table 1.

DESs used in this work and information on their synthesis.

Codes	HBA⁎	HBD⁎	Water content (%)	Molar Ratio	Viscosity at 45 ± 1 °C (mPa. S)	pH	Appearance	Assisted Techniques	Zone of Inhibition NADES only	Zone of Inhibition NADES based Extract	Total Phenolic Content (mg GA/g powder)	Antioxidant Activity (mmol TE/g powder)
												DPPH	ABTS	FRAP
ChCl: LA	Choline Chloride	Lactic Acid	30 %	1:2	45	0.44	Transparent and viscous	Heating and stirring	∼14.5	∼15.6 mm	43.64 ± 1.16	31.24 ± 0.28	202.36 ± 1.6	21.19 ± 0.12
ChCl: UR	Choline Chloride	Urea	30 %	1:2	30⁎⁎	9.34	Transparent and viscous	Heating and stirring	Nil	∼ 3 mm	41.74 ± 1.71	29.58 ± 0.52	183.81 ± 0.49	16.59 ± 0.311
ChCl: TEG	Choline Chloride	Tri-ethylene Glycol	30 %	1:2	45	4.56	Transparent and viscous	Heating and stirring	Nil	∼ 4 mm	27.28 ± 0.66	33.46 ± 0.33	206.51 ± 0.66	18.49 ± 0.375
ChCl:1,4-But	Choline Chloride	1,4-Butanediol	30 %	1:2	55	4.54	Transparent and viscous	Heating & stirring	Nil	∼ 3.5 mm	30.05 ± 1.33	35.02 ± 0.34	188.27 ± 3.54	15.54 ± 0.873
ChCl: FR	Choline Chloride	Fructose	30 %	1:2	2290⁎⁎	5.7	Pale yellow and highly viscous	Heating and stirring	Nil	∼ 3.2 mm	44.59 ± 1.65	34.35 ± 0.64	200.95 ± 1.3	24.34 ± 1.84

Nil: No Inhibition, DPPH: 1,1-diphenyl-1-picrylhydrazyl radical scavenging activity, ABTS: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid radical scavenging, and FRAP: ferric reducing antioxidant power.

^⁎HBA: Hydrogen Bond Acceptor, HBD: Hydrogen Bond Donor, LA: Lactic Acid, UR: Urea, TEG: Tri-ethylene glycol, 1,4-Butanediole, FR: Fructose.

^⁎⁎Viscosity was measured at 60 ± 5 °C due to high crystallization of ChCl:UR and high viscosity of ChCl:FR respectively.

The pH values were measured with a digital pH meter in the range of 0–14. The viscosities of the DES were measured using a Brookfield digital viscometer (AMETEX, USA) with a spindle number of 6 at temperatures of 45 ± 1 °C at a speed of 200 rpm (Airouyuwa et al., 2024).

2.4. Microwave-assisted extraction of DSP

The extraction of polyphenol-rich bioactive compounds (PBCs) from the DSP was executed using a microwave-assisted extraction method (Airouyuwa et al., 2023). The pretreatment of 6.0 % (w/v) DSP was performed using a microwave (LABOTRON 12 T, Lyon, France) on previously optimized conditions, such as 70 % of DESs, at 80 °C for 5 min at 600 W (Airouyuwa et al., 2023). After the treatment, all the samples were centrifuged at 10,000 ×g, at 5 °C for 15 min. The supernatants were collected after filtration and maintained at 4 °C for further study.

2.5. FTIR (Fourier-transform infrared) spectroscopy

The FTIR spectra of various DESs and DES components were analyzed with a Fourier transform infrared spectrophotometer (103,146, PerkinElmer, UK) operated in attenuated total reflectance (ATR) mode, at a resolution of 4 cm⁻¹, and accumulation of 30 scans, and in the range of 400 to 4000 cm⁻¹ (Fakhlaei et al., 2025).

2.6. Determination of Total phenolic content (TPC)

The TPC of all the samples was determined by following the standardized protocols (Airouyuwa et al., 2023). The TPC was expressed as milligrams of gallic acid equivalents (mg GAE/g powder) per gram of DSP with gallic acid as the reference standard.

2.7. Identification and quantification of the individual phenolic compounds

Quantification and identification of the major phenolic compounds from date seed extract were performed using a UHPLC-PDA chromatography system (Thermo Scientific Dionex Ultimate 3000 UHPLC system) equipped with a quaternary Series RS pump, and PDA detector controlled by Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, USA and Dionex Softron GmbH subsidiary of Thermo Fisher Scientific, Bremen, Germany). The UHPLC unit was coupled with an autosampler and column compartment (Thermo Scientific Dionex Ultimate 3000 Series TCC-3000RS) (Airouyuwa et al., 2023). All Samples were filtered using a 0.45 μm micropore membrane (PTFE, Waters, Milford, MA, USA). The compound separation was executed on a UHPLC C₁₈ column (Suplecosil LC-18-DD 150 × 4.6 mm). The mobile phase consisted of (eluent A) 0.1 % formic acid in water and (eluent B) 0.1 % formic acid in methanol. The gradient program was set as follows: 0–0.1 min, B (15.0 %); 0.1–7.1 min, B (65.0 %); 7.1–7.9 min, B (95.0 %); 7.9–8.0 min, B (95.0 %); 8.0–10.0 min, B (15.0 %). The flow rate in the mobile phase was constant at 1.0 mL/min, the column temperature was set at 30 °C, and the effluents were measured at 280 nm, where the UHPLC-PDA method typically achieves an LOD of ∼0.33–4 ng and an LOQ of ∼0.5–10 ng per injection. Pure phenolic standards were run to calibrate a standard curve with a R² = 0.95.

2.8. Determination of antioxidant assay

The antioxidant activity of the DES-based extracts was evaluated by estimating the radical scavenging ability using the DPPH and ABTS and the ferric-reducing antioxidant power (FRAP) assay. Each DES-based extract, 20× dilution, was exposed to all three mentioned antioxidant assays, and the absorbance was recorded at 734 nm, 517 nm, and 593 nm for ABTS, DPPH, and FRAP, respectively, using a spectrophotometer microplate reader (Multiskan Sky, Thermo Fisher Scientific, Cambridge, MA, USA) (Airouyuwa et al., 2023).

2.9. Determination of antimicrobial activity

2.9.1. Agar diffusion assay

A 10 μL aliquot of 24 h-grown Salmonella Typhimurium DT104 bacterial culture (1–5 × 10⁵ CFU/mL) was spread plated on the MHB agar plates. Further, wells (∼ 6 mm in diameter) were punctured in each plate using a sterile borer. Then, 50 μL of each sample was loaded into the well. The samples were allowed to diffuse for 1 h at room temperature in a sterile environment. Subsequently, the plates were incubated for 24 h at 37 °C. After 24 h, inhibition zones (mm) were measured (Martí et al., 2018). The diameter of the inhibition zone (z) and the well diameter (d) were measured with a digital caliper. The width of the zone of inhibition was measured by using Equation (i).

$$\mathit{Zone\; of\; Inhibition}=\frac{z-d}{2}$$ (i)

2.9.2. Broth dilution method

The broth dilution method was used to calculate the Minimum Inhibitory Concentration (MIC) for Salmonella Typhimurium DT104. Initially, stock solutions of DESs and DES-based extracts were prepared, and 100 μL of the prepared solution was loaded into 96-well microplates containing 100 μL of MHB broth, and then serially diluted throughout the 96-well plates. To define positive and negative control, 100 μL of the MHB broth was transferred to both wells. 10 μl of the Salmonella Typhimurium DT104 culture (1–5 × 10⁵ CFU/mL) was added to all the wells except the negative control well. The microplates were then incubated at 37 °C for 24 h, and bacterial growth was visually recorded (Phasaludeen et al., 2025). Since the MIC assay relies on visual observation of bacterial growth turbidity, the inherent color of the date seed extract may interfere with the assay. So, a resazurin dye-based assay was additionally performed to indicate bacterial growth. Resazurin powder (0.015 g) was dissolved in 100 ml of DW and filter sterilized with 0.22 μm and stored at 4 °C for further use. The resazurin dye assay was performed after 24 h on the MIC plates. Briefly, 30 μL of dye solution was added to each well and incubated for 2–3 h at 37 °C. For the analysis of the dye assay, the wells with a blue color indicated negative results for bacterial growth; however, a pink color depicted positive results with bacterial growth (Adelakun et al., 2024). The color change from blue to pink confirmed the induction of resazurin by bacteria, and the MIC value was specified by the minimum sample concentration that prevents this color change.

2.9.3. Dot plating method

The Minimum Bactericidal Concentration (MBC) was determined using the Dot Plate Screening Assay, following the method described by Al-Akayleh et al., (2022), with slight modifications. From the 96-well broth microdilution assay used for MIC determination, a 5 μL aliquot was taken from the well corresponding to the MIC concentration and from at least two higher concentrations. These aliquots were spotted onto BHI agar plates in triplicate. The plates were then incubated at 37 °C for 18 h under aerobic conditions. The lowest concentration at which no bacterial growth was observed in any of the spotted dilutions was recorded as the MBC.

2.10. Evaluation of synergism between DESs (TEG and 1,4-but) and their subsequent DES-based extracts with antibiotics

The synergistic effect of DESs and DES-based extract with antibiotics was investigated by the checkerboard method (Requena et al., (2019). In the microreader plate (96 wells), antibiotics and DES-based extracts were loaded horizontally and vertically using the double-fold dilution techniques, respectively, in such a manner that each column and row denoted various ratios of the two samples. The MIC values of both the DES-based extracts and antibiotics were used as the initial concentrations for the experiment. After the serial dilution, each well of the 96-well plates contained 50 μL of antibiotic and 50 μL of DES-based extract in MHB culture broth medium. The bacterial inoculum (10⁵ CFU/mL), equivalent to 0.5 McFarland standard, was inoculated in all the wells, including the positive control of the plates. Furthermore, the resazurin dye assay, as described in Section 2.9, was performed to assess bacterial growth. The particular combination of antibiotics and DES-based extract concentration in the 96-well plate, where the well remained blue (indicating no bacterial growth in the resazurin dye assay), was considered MIC. Similarly, the same procedure was followed for testing the combination of DES and antibiotics.

The results from the checkerboard assay were analyzed, and the degree of synergy or antagonism was quantitatively evaluated using the fractional inhibitory concentration index (FICI). The FICI is calculated as follows (equation ii):

$$\mathit{FICI}=\frac{{\mathit{MIC}}_{\mathit{AB}}}{{\mathit{MIC}}_{\mathit{A}}}+\frac{{\mathit{MIC}}_{\mathit{BA}}}{{\mathit{MIC}}_{\mathit{CB}}}$$ (ii)

Where MIC_AB is the MIC of drug A when combined with drug B, MIC_A is the MIC of drug A alone, MIC_BA is the MIC of drug B when combined with drug A, and MIC_B is the MIC of drug B alone. A FICI ≤0.5 indicates synergy, a FICI between >0.5 and 4 suggests no interaction or indifference, and a FICI >4 is interpreted as antagonism (Phasaludeen et al., 2025).

2.11. Time kills kinetic studies

A time-kill kinetic study was conducted using 1× MIC, 0.5× MIC, and 0.25× MIC of DESs and DES-based extracts on a Salmonella Typhimurium DT104 culture growing in MHB supplemented with 0.25× MIC of chloramphenicol. Briefly, a single colony of Salmonella Typhimurium DT104 was added to 5 mL of LB broth and cultured overnight at 37 °C. The Salmonella Typhimurium DT104 cells were then washed twice with sterile 1× phosphate-buffered saline (PBS) and inoculated into MHB with chloramphenicol (0.25× MIC) and incubated at 37 °C. At the mid-logarithmic phase, DESs or DES-extracts were added at different combinations of either 1× MIC, 0.5× MIC, and 0.25× MIC, and incubated at 37 °C. At every 2-h interval, bacterial growth was evaluated by serial dilution and plating. The bacterial colonies were counted by scan 1200 automatic colony counter (Interscience, UK), and the growth curve was plotted.

2.12. Statistical analysis

Statistical data analyses were carried out using the GraphPad Prism software (version 10.2.0). All data are presented as mean ± standard error of the mean (SE) and analyzed by two-way ANOVA. Differences were considered significant at P < 0.05 and non-significant at P > 0.05.

3. Results and discussion

3.1. Screening and characterization of deep eutectic solvents (DESs)

Deep eutectic solvents (DESs) are frequently marketed as environmentally friendly substitutes for traditional solvents due to their low volatility, nonflammability, chemical and thermal stability, and application-tunable nature. Moreover, DESs, in contrast to conventional solvents such as ethanol and methanol, which often require complex and energy-demanding manufacturing and purification processes, are made from naturally derived, biodegradable substances that need considerably less energy for their production and recycling thereby substantially lowering both energy use and carbon emissions (Dai et al., 2013). Their low volatility and non-flammable nature contribute to reduced environmental release and safety risks (Huang et al., 2023). Studies show that processes using NADES benefit from greater energy efficiency, leading to a smaller carbon footprint per unit of solvent compared to conventional options (Paiva et al., 2014). This makes NADES a promising choice for advancing sustainable and eco-conscious industrial applications.

However, a quantitative evaluation is necessary to determine their actual sustainability (Zaib et al., 2022). In this context, Zaib et al. (2022) carried out a cradle-to-gate life-cycle assessment of a representative ChCl:Urea, DES, and compared its environmental profile to that of methanol, ethanol, dichloromethane (DCM), and ethyl acetate for the same functional use (solvent in the oxidation of an alcohol to acetophenone). However, solvent reuse was not considered in this study. The effects of DES are generally lower than those of DCM and ethyl acetate, but higher than those of methanol and ethanol in several categories, including freshwater eutrophication (FEP), terrestrial acidification (TAP), mineral depletion (MDP), water depletion (WDP), freshwater ecotoxicity (FETP), human toxicity (HTP), and global warming potential (GWP). Xia et al. (2021), on the other hand, recycled a ChCl/oxalic acid DES nine times during the bioplastic synthesis process and found that recycling more than nine cycles could further mitigate the environmental effects of producing lignocellulose bioplastics

In the present study, five choline chloride-based DESs were employed to extract bioactive compounds from DSP, and the reaction was assessed for antimicrobial activity and physiochemical properties. The antimicrobial activity of the DES-based DSP extracts will depend on the types of DESs and the quantity of the polyphenol-rich bioactive compounds (PBCs) that are extracted by the DESs. The selection and screening of DESs for antimicrobial properties analysis depend on bacterial sensitivity to pH. Generally, bacteria have an optimal growth pH range of 6.5–7.5 (Zhao et al., 2015). Our preliminary pH analysis suggested using polyol-based DESs with a pH of around 4.5 instead of ChCl: Urea and ChCl: Lactic acid-based DES, due to their basic and acidic nature, respectively (Table 1). The HBDs used for DESs were sugar-based (fructose), carboxylic acid-based (lactic acid), alkaline (urea), and poly alcohol-based (1,4-butanediol and triethylene glycol) compounds. Pure DESs hindered the extraction process because of their viscous nature (Gajardo-Parra et al., 2020). The viscosities of the studied polyol-based DESs were higher for 1,4-But (55 mPa·S) than for TEG (45 mPa.S), as shown in Table 1. The viscosity of pure DESs results from the intense hydrogen-bonding network between HBA and HBD, which decreases the mass transfer and diffusion rate. To resolve this, dilution by the addition of water has been suggested to reduce the viscosity of DESs and to improve extraction efficiency (Wojeicchowski et al., 2021). In this study, the water percentage was optimized following previously established protocols (Airouyuwa et al., 2023). A lower water content of 30 % enhances the PBCs extraction efficiency and enhances the DESs system's overall performance by reducing solvent viscosity. Therefore, here, 70 % DESs were used for the extraction of PBCs, which were prepared by mixing 30 % of distilled water. The extraction of PBCs was carried out utilizing a microwave-assisted approach with a temperature of 80 °C, a duration of 5 min, and a power level of 600 W, as optimized earlier (Airouyuwa et al., 2023). All the DESs used in this study (Table 1) showed transparency in appearance after the synthesis.

3.1.1. Structural characteristics

FTIR-ATR spectroscopy was employed to study the structural changes during the intermolecular interaction between the HBD and HBA while developing a eutectic mixture. Fig. 1 shows the FTIR-ATR spectra of pure ChCl, TEG, 1,4-But, and their subsequent developed DESs. A comparative analysis of the functional groups of the individual components and the final eutectic solvents was conducted. The absorbance peaks in FTIR spectra corresponded to various functional groups such as C—N stretching at 952 cm⁻¹, stretching bands of C—O and C—C between 1000 and 1300 cm⁻¹, C—H bending at 1478 cm⁻¹, N—H bending at 1638 cm⁻¹, C O stretching of carboxylic acid groups at 1720 cm⁻¹, C—H stretching between 3026 and 2900 cm⁻¹ and O—H stretching in the range of 3650–3100 cm⁻¹ which are seen in Fig. 1 (Lanjekar & Rathod, 2021). In the pure ChCl spectrum, important bands were observed as vibrational bands around 3200 cm⁻¹ and 1200–880 cm⁻¹ assigned to hydroxyl or amino groups, where amino indicates C—N symmetric and N—H stretching, while vibrational bands at 2990–2850 cm⁻¹ and 1485–1420 cm⁻¹ corresponded to alkyl groups, and bands around at 1485 cm⁻¹ (Alfaleh & Sindi, 2024). The pure choline chloride spectrum depicts several functional groups, but few of them co-exist after the DESs formation. Figs. 1A & 1B show FTIR spectra of TEG and 1,4-But-based DESs, and their individual components, respectively. In the FTIR spectrum of pure 1,4-But and TEG DESs (Fig. 1A & 1B), the presence of an O—H group is shown by vibrational bands at 3291 cm⁻¹ and 3380 cm⁻¹, whereas C—H stretching is shown by the peaks between 2868 and 2935 cm⁻¹, respectively. Furthermore, the O—H bending is shown in the region of 1400–1437 cm⁻¹ for both types of DESs, but the C—O stretching of the primary alcohol is seen at 1056 cm⁻¹ and 1063 cm⁻¹ for 1,4-But and TEG DESs, respectively. FTIR spectra of formulated DESs (TEG and 1,4-But) consist of some dominating functional groups derived from their parent components (Fig. 1A &1B). A peak representing O—H stretching band has observed a band shift to a higher wavenumber 3338 cm⁻¹ and 3314 cm⁻¹ with a broad hump in the formulated DESs, TEG, and 1,4 But respectively compare to their parent components. These changes can be attributed to strong H-bonds forming between the chloride anion and hydroxyl groups when the eutectic solvent is being formed. Similarly, the C—H stretching band in formulated DESs (TEG and 1,4-But) around 2946–2973 cm⁻¹ is inherited from pure TEG and 1,4 But respectively, whereas the N—H bending band at 1652–1648 cm⁻¹ in TEG and 1,4-But DESs originates from pure ChCl, respectively. Since the dominant peaks of both parent components show up with minor variations, indicating an interaction between them, the FTIR data confirms the formulation of TEG and 1,4-But DESs (Fig. 1A and B). The ChCl spectra are in accordance with published research (Alfaleh & Sindi, 2024).

Fig. 1.

FTIR spectra of pure components and their subsequent DESs (A) ChCl, TEG and ChCl: TEG (B) ChCl, 1, 4- Butanediole and ChCl:1,4-But.

3.1.2. Extraction efficiency and Total phenolic content (TPC)

The PBCs extraction efficiency of the different DESs is influenced by parameters such as viscosity, surface tension, diffusion rate, the composition of raw materials, and time of extraction (Rathnasamy et al., 2019). Additionally, extraction temperature plays an important role in reducing the viscosity of DESs, thereby lowering surface tension and increasing molecular diffusion, which enhances the extraction efficiency of bioactive compounds (Rodríguez-Martínez et al., 2022). Earlier, advanced techniques using different types of DESs were implemented and evaluated for their extraction efficiency in extracting PBCs (Airouyuwa et al., 2023; Osamede Airouyuwa et al., 2022). Therefore, before testing antibacterial action, we were keen to explore the extraction efficiency of the DESs that were used in this study. The extraction efficiency of PBCs using DESs was assessed by measuring the total phenolic content (TPC). The results related to the extraction of PBCs indicated that ChCl: fructose, ChCl: lactic acid and ChCl: urea showed a TPC of 44.59, 43.64, and 41.74 ± 1.71 mg GA/g, respectively. While ChCl: TEG and ChCl:1,4-But showed 27.28 and 30.05 mg GA/g of total phenolic content (Table 1), respectively. In previous work on microwave-assisted extraction of bioactive compounds from date seeds, the maximum TPC was recorded as 147.92 mg GAE/g for choline chloride-lactic acid (ChCl-LA) at a temperature of 80 °C, irradiation time of 5 min, and DES contents of 63.0 %. The TPC for choline chloride-xylose (ChCl-Xylo) was 234.65 mg GAE/g at a temperature of 80 °C, irradiation time of 20 min, and DES contents of 50.0 % (Airouyuwa et al., 2023). In another study, carboxylic acid-based DES (ChCl-lactic acid and ChCl-malic acid) showed the highest recovery of total polyphenol compounds, followed by sugar-based DESs (ChCl-xylose, ChCl-sucrose, ChCl-glucose) and polyols-based DES (ChCl-1,4-Butanediol) with yield varying from 125.26 ± 3.31 to 76.57 ± 2.96 mg GAE/g powder (Osamede Airouyuwa et al., 2022). This study supports the use of carboxylic acid-based DES for achieving a high recovery rate of TPC and maximum extraction yields, due to their higher polarity and lower pH as compared to sugar- and polyol-based DES when using ultrasound-assisted techniques (Bi et al., 2013; Osamede Airouyuwa et al., 2022). It should be noted that the differences in the TPC values observed between the present study and those reported in the literature are likely due to variations in factors such as the types of DESs, viscosity, solid-to-solvent ratio, molar ratio of DESs, extraction parameters, and techniques employed (Bakirtzi et al., 2016).

3.1.3. Antioxidant activity

Further, results related to antioxidant capacity of various DESs based extract indicated higher DPPH radical scavenging among ChCl: TEG (33.46 mmol TE/g), ChCl:1,4-But (35.02 mmol TE/g), and ChCl: fructose (31.24 mmol TE/g). ChCl: TEG based DES extract also showed highest scavenging of ABTS radicals as indicated by its TE values of 206.51 mmol TE/g, followed by ChCl: lactic acid (202.36 mmol TE/g), and ChCl: fructose (200.95 mmol TE/g) based extracts. Maximum FRAP activity was observed in ChCl: fructose, ChCl: lactic acid followed by ChCl: TEG (Table 1). The antioxidant activity of bioactive compounds from natural biowaste is influenced by the availability of various phenolic acids and flavonoids and their ability to neutralize free radicals (Barakat et al., 2020). Therefore, we explored the antioxidant activity of TEG and 1,4-But-based DES extracts of DSP (Table S1). TEG-based DES extract recorded more ABTS and FRAP activity, whereas 1,4 But-based DES extract showed higher DPPH activity 35.02 ± 0.34 mmol TE/g powder). Previously, water content (%) in DESs was noted to influence the antioxidant activity of five different types of DES-based extracts from date seeds (Airouyuwa et al., 2023). DESs with 70 % water content exhibited higher radical scavenging activity (DPPH), and ferric-reducing activity (FRAP) than DESs with 50 % water content. For DPPH activity, carboxylic acid-based DESs extract recorded the highest activity in the sequence: ChCl-lactic acid> ChCl-citric acid > ChCl-malic acid (Osamede Airouyuwa et al., 2022). Regarding the FRAP assay, the highest activity FRAP was observed in the arrangement of ChCl-lactic acid> ChCl-xylos > ChCl-1,4-butanediol. The variations in the extraction efficiencies of TPC and their antioxidant activity are expected because these parameters mainly depend on the composition of DESs and the extraction method. Another study investigated the effect of four different types of DESs (ChCl: lactic acid, ChCl: citric acid, ChCl: glycerol, and ChCl: fructose) and conventional solvents (water, 70 % methanol, and 70 % ethanol) on the extraction of polyphenols from date seed. The DESs, composed of ChCl: lactic acid and ChCl: citric acid, exhibited the highest antioxidant activity (DPPH) with RSA% values of 93.26 ± 0.13 and 93.25 ± 0.13, respectively (Alfaleh & Sindi, 2024).

Although, fructose, lactic acid and urea based DESs were found to be more efficient than TEG and 1,4 But. However, we selected two DESs (TEG and 1,4 But) and their extracts for further screening despite their lower TPC content, considering their pH levels (pH of around 4.5), instead of using DESs with low pH (0.44 for ChCl: lactic acid) and high pH (9.3 for ChCl, urea), as bacteria are sensitive to pH and possibly interfere with the assay and this hypothesis of ours is also evident from Table 1, where ChCl: lactic acid based DES itself was highly antimicrobial against Salmonella Typhimurium DT104. Therefore, this DES was not considered for further studies. We also considered viscosity in selection of DES for further study, as extracts made with ChCl: fructose was very viscous and hard to handle. While adding more water could reduce viscosity, it might also affect our experimental design and potentially disrupt DES's stability and decrease its extraction efficiency. Therefore, we selected DESs that would not complicate our analysis and thus were investigated further and presented here in this study.

3.2. Phenolic compounds and antimicrobial acitivity

3.2.1. Identification and quantification of phenolic compounds

The identification and quantification of the major phenolic compounds present in the TEG and 1,4-But-based DESs crude extracts of date seeds are depicted in Table S1. The phenolic compounds listed in Table S1 are divided into three main categories including hydroxybenzoic (3,4-dihydroxy benzoic acid, benzoic acid, 4 hydroxybenzoic acid, syringic acid, vanillic acid, and gallic acid), hydroxycinnamic (chlorogenic acid, cinnamic acid, ferulic acid, caffeic, and p-coumaric) and flavanol (catechin, rutin, and quercetin). In the TEG-based extract, 6 distinct phenolic compounds were identified and quantified, while 11 compounds were recorded in samples extracted with 1,4-But DES. Similar types of phenolic compounds have been reported earlier in date seeds by HPLC detection techniques (Airouyuwa et al., 2023; Airouyuwa et al., 2022). TEG DES-based extract showed chlorogenic acid and rutin hydrate as primary phenolic compounds. However, 4-hydroxybenzoic acid and rutin hydrate were found as major phenolic compounds in the 1,4-But DES-based extract. Our findings align with some of the reported trends. For instance, a study by (Barakat et al., 2020), reported that rutin and syringic acid are the major components, while gallic and catechins are found in small quantities. In contrast, another study investigated the extraction of polyphenols from date seeds using various DESs (ChCl: Lactic acid, ChCl: Citric acid, ChCl: Glycerol, and ChCl: Fructose), in addition to conventional solvents like water and ethanol. This study found that the water-based extract had the maximum level of gallic acid (860.99 mg/100 g), then 70 % ethanol (349.56 ± 15.79 mg/100 g) and ChCl: Lactic acid (46.48 ± 0.68 mg/100 g) extracts. On the other hand, catechin was found to be highest in ChCl: citric acid and ChCl: glycerol-based extracts. Additionally, the ethanolic extract contained the highest quantities of syringic acid and p-coumaric acid, at 128.38 mg/100 g and 128.18 mg/100 g, respectively (Alfaleh & Sindi, 2024). The differences between our findings and those in other studies could be due to some factors such as plant variety, environmental conditions, collection site, extraction solvents, and methods. The results presented in this study are based on the preliminary study which needs further in-depth analysis to confirm the results regarding the different type and content of phenolics present in the DES-based extracts obtained from date seeds.

3.2.2. Antimicrobial activity

Here, our primary aim was to evaluate the antimicrobial potential of two polyol-based DESs date seed extracts against multidrug-resistant Salmonella Typhimurium DT104. Salmonella Typhimurium DT104 was selected due to its penta-resistance to conventional antibiotics (ACSSuT), making it a suitable model for testing novel antimicrobial interventions (Phasaludeen et al., 2025). The antibacterial activity of TEG and 1,4-But-based DESs and their DES-based extracts was assessed using agar well diffusion, broth microdilution, and dot plating assays. Additionally, to assess the antimicrobial effect of individual DES components (ChCl, TEG, and 1,4-But), each was separately dissolved in distilled water at the same concentration and tested using the same methods.

In the agar well diffusion assay, both DES-based extracts exhibited inhibition zones of approximately 3–4 mm against Salmonella Typhimurium DT104 (Table 2, Fig. 2). However, the observed inhibition zones were relatively small, indicating poor diffusion and limited antimicrobial activity. To confirm that the observed activity was attributable to the extracted PBCs and not the DES solvents, control 70 % DES formulations (ChCl-TEG and ChCl-1,4-But) and individual components of DESs (ChCl, TEG, and 1,4-But) were also tested. No inhibition zones were observed for the control DES formulations and individual components of DESs, confirming that the antimicrobial activity originated from the PBCs within the date seed extracts rather than the DESs themselves or a synergistic effect between the two. However, the individual components of DESs (ChCl, TEG, and 1,4-But) did not show any inhibition against Salmonella Typhimurium DT104.

Table 2.

The result of the well diffusion test, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) of the individual components of selected DES, DESs, and DESd based extract against Salmonella Typhimurium DT104.

Codes	Antimicrobial Activity
	Diameter of inhibition zone (mm)	MIC (v/v)	MBC (v/v)
ChCl	NI	NE	NE
TEG	NI	50 %	NE
1,4-But	NI	50 %	NE
ChCl: TEG	NI	35 %	35 %
ChCl:1,4-But	NI	35 %	35 %
TEG Based extract	∼3 mm	17.5 %	35 %
1,4-But based extract	∼ 4 mm	17.5 %	35 %

NI: No Inhibition, NE: No effect at maximum tested concentration of material, MIC and MBC are expressed as a percent of DESs providing the growth inhibition of bacteria.

Fig. 2.

Zone of inhibition against Salmonella Typhimurium DT104 (A) 1,4 But based DES (B) 1,4 But DES based Extract (C) TEG based DES (D) TEG DES based extract.

The minimum inhibitory concentration (MIC) of the TEG and 1,4-But-based DES extracts against Salmonella Typhimurium DT104 was determined using the broth microdilution method. Both extracts exhibited antimicrobial activity at a dilution of 17.5 % (Table 2). This level of activity was relatively low compared to other natural antimicrobial agents. Furthermore, the control DES formulations alone did not exhibit significant antimicrobial effects at concentrations below 35 %, further supporting the role of PBCs in the DES-based extracts or the possible synergistic effect of both.

To determine the minimum bactericidal concentration (MBC), a dot plate screening assay was conducted. The individual components of DESs (ChCl, TEG, and 1,4-But) did not exhibit any bactericidal effect at this concentration. The results indicated that both TEG and 1,4-But-based extracts exerted bactericidal effects at 35 % dilution (Table 2). However, similar antimicrobial activity was observed at 35 % for the control DES formulations, suggesting that at higher concentrations, the solvents themselves might contribute to some antibacterial effects. This corroborates the MIC results and highlights the need for further optimization to enhance the antimicrobial efficacy of these extracts.

Although the observed antimicrobial activity of both DES-based extracts was minimal, these findings confirm that the PBCs from the date seeds contribute to bacterial inhibition. The antibacterial effects of date seed extract could be attributed to their polyphenol content, which has been well-documented in the literature for its antimicrobial properties (Osaili et al., 2024). Polyphenols are known to disrupt bacterial cell membranes, interfere with enzyme activity, and induce oxidative stress, ultimately leading to bacterial growth inhibition and cell death (Hussain et al., 2019).

3.3. Investigation of DESs and DES-based extract as antibiotic adjuvants

Despite their limited direct antibacterial activity, polyol-based DES extracts (polyphenol-rich) of date seeds could still play a significant role as antibiotic adjuvants. Polyphenols, as plant-derived secondary metabolites, have been extensively studied for their antimicrobial activity against various pathogens (Bae et al., 2022). These compounds, although exhibiting modest antimicrobial effects on their own, may enhance the effectiveness of conventional antibiotics against multidrug-resistant pathogens (Cho et al., 2017). Similarly, a subcategory of Deep Eutectic Solvents (DESs) known as Therapeutic DESs (THEDESs) has been explored to enhance the physicochemical properties of drugs, including improved dissolution, permeability, and bioavailability (Abdelquader et al., 2023). Numerous investigations have demonstrated the potent antibacterial properties of DESs, particularly those derived from organic acids. However, their toxicity varies depending on the bacterial strain, most likely as a result of interactions with bacterial membranes (Bedair et al., 2024).

Therefore, our next focus was to evaluate their (DESs and DES-based extracts) synergistic interactions with antibiotics to assess their potential in combating antimicrobial resistance, contributing to the development of green solvent-based polyphenol-rich bioactive compounds as potential interventions against drug-resistant bacteria.

We investigated whether our DESs and DES-based date seed extracts could function as antibiotic adjuvants to enhance the susceptibility of Salmonella Typhimurium DT104 to ACSSuT antibiotics. Since Salmonella Typhimurium DT104 exhibits resistance to five antibiotics (ACSSuT phenotype), we confirmed its resistance profile by determining its MIC values using the broth microdilution method. The MICs for ampicillin (>512 μg/mL), chloramphenicol (128 μg/mL), sulfamethoxazole (>512 μg/mL), streptomycin (128 μg/mL), and tetracycline (128 μg/mL) were consistent with previously reported studies (De Jong et al., 2009; Wang et al., 2019).

TEG-based DES extract caused a significant reduction in the MICs of chloramphenicol, ampicillin, tetracycline, and streptomycin. The MIC of chloramphenicol decreased 32-fold (from 128 to 4 μg/mL), shifting Salmonella Typhimurium DT104 from resistant to sensitive according to CLSI standards. Similarly, ampicillin MIC decreased by 256-fold (>512 to 2 μg/mL), restoring ampicillin sensitivity in Salmonella Typhimurium DT104. TEG-based extract also induced a > 128-fold reduction in tetracycline MIC (from 128 to 1 μg/mL), rendering Salmonella Typhimurium DT104 susceptible to the antibiotic. Furthermore, TEG-based DES extracts caused a > 256-fold decrease in streptomycin MIC (from 128 to <0.5 μg/mL), converting DT104 to a streptomycin-sensitive phenotype, whereas no adjuvant effect was observed with sulfamethoxazole (Table 3).

Table 3.

The synergies result of DESs and DES-based extract with the tested antibiotics.

Samples	Sample's MIC (%)	MIC drop of Antibiotics with DESs and DES based extracts
		Chloramphenicol				Ampicillin				Tetracycline
		MIC (μg/mL)	MIC Drop	Fold Change	FICI	MIC (μg/mL)	MIC Drop	Fold Change	FICI	MIC (μg/mL)	MIC Drop	Fold Change	FICI
TEG DES	4.35	128	2	64	0.13	>512	2	256	0.12	128	>0.5	>256	0.12
TEG DES-Based Extract	8.7	128	4	32	0.52	>512	2	256	0.49	128	1	128	0.49
1,4- But DES	4.35	128	4	32	0.15	>512	>0.5	>1024	0.12	128	>0.5	>256	0.12
1,4- But DES Based Extract	4.35	128	2	64	0.25	>512	>0.5	>1024	0.24	128	>0.5	>256	0.24
Samples	Sample's MIC (%)	Sulfamethoxazole				Streptomycin
		MIC (μg/mL)	MIC Drop	Fold Change	FICI	MIC (μg/mL)	MIC Drop	Fold Change	FICI
TEG DES	4.35	>512	512	1	1.12	128	4	32	0.15
TEG DES-Based Extract	8.7	>512	128	4	0.74	128	>0.5	>256	0.49
1,4- But DES	4.35	>512	512	1	1.12	128	1	1	0.13
1,4- But DES Based Extract	4.35	>512	128	4	0.49	128	2	64	0.25

Since the antibacterial effects of our tested DES-based extracts were not significantly different from their respective DES formulations, we further explored whether DES alone could exhibit synergy with conventional antibiotics in inhibiting Salmonella Typhimurium DT104. Notably, compared to the TEG-based extract, TEG-DES itself contributed to a substantial reduction in MIC values. Specifically, TEG-DES induced a 64-fold decrease in the MIC of chloramphenicol (from 128 to 2 μg/mL) and a > 256-fold reduction in the MIC of tetracycline (from 128 to <0.5 μg/mL), confirming its synergistic potential as a polyol-based DES. For ampicillin, TEG-DES exhibited similar MIC reductions as the TEG-based extract, reinforcing its role in enhancing antibiotic efficacy. However, for streptomycin, TEG-DES demonstrated a comparatively lower MIC reduction, yielding a 32-fold decrease (from 128 to 4 μg/mL). As observed previously, no interaction was detected between TEG-DES and sulfamethoxazole, indicating that its synergistic effects were antibiotic-specific. (Table 3).

Similarly, exposure to the 1,4-ButDES-based extract resulted in a 64-fold reduction in the MIC of chloramphenicol (from 128 to 2 μg/mL), which was comparable to the effect observed with TEG-based extracts. However, in the case of ampicillin, 1,4-But DES-based extract demonstrated a significantly greater effect, reducing the MIC by 1024-fold (>512 to <0.5 μg/mL), surpassing the activity of TEG-based extracts. For tetracycline, the MIC reduction was similar to that of TEG-based extracts, with a > 256-fold decrease (from 128 to 0.5 μg/mL). Compared to TEG-based extracts, 1,4-But DES extracts exhibited slightly lower activity against streptomycin, with a 64-fold reduction in MIC (from 128 to 2 μg/mL). Notably, unlike TEG-based extracts, 1,4-But DES extracts demonstrated adjuvant activity with sulfamethoxazole, leading to a fourfold MIC reduction (from >512 to 128 μg/mL) (Table 3).

When testing 1,4-But DES alone, chloramphenicol exhibited slightly lower activity compared to its corresponding extract. Ampicillin and tetracycline showed similar MIC reductions to their respective extracts, while no synergy was detected with sulfamethoxazole or streptomycin (Table 3).

Overall, the tested DES-based extracts and their respective DES formulations effectively lowered the MICs of antibiotics, with reductions ranging from 32- to >1024-fold and 64- to >1024-fold, respectively, highlighting their potential as powerful antibiotic adjuvants. Synergy and antagonism of antibiotic adjuvants are quantitatively assessed using the traditional fractional inhibitory concentration index (FICI). A FICI ≤0.5 indicates synergy, a FICI between >0.5 and 4 represents indifference or no interaction, and a FICI >4 signifies antagonism (Phasaludeen et al., 2025).

Next, we conducted a checkerboard assay to identify the minimum concentration at which the compounds showed a significant adjuvant effect with the antibiotics and to assess the synergistic potential of the DES-based extracts and their corresponding DES formulations against the antibiotics to which Salmonella Typhimurium DT104 was resistant. Overall, both the DES-based extracts and DESs demonstrated a synergistic adjuvant effect at 0.5× MIC. However, when concentrations were reduced to 0.25× MIC, the extracts and DESs were too diluted to exhibit any significant adjuvant activity.

Although both extracts caused a reduction in MICs, our results revealed that among the tested DES-based extracts, only the 0.5× MIC 1,4-But DES extract exhibited a significant synergistic effect with chloramphenicol, with a FICI of 0.26. Similarly, both 0.5× MIC 1,4-But DES and TEG-based DES demonstrated synergy with chloramphenicol, with FICI values of 0.13 and 0.15, respectively. However, for the TEG-based DES extract, no interaction was observed, as indicated by a FICI >0.5 (Table 3).

In the case of ampicillin, the FICI index was ≤0.5 for the (0.5× MIC) 1,4-But DES extract, 1,4-But DES, and TEG DES, suggesting synergy. For TEG-based DES extract, however, the FICI was >0.5, indicating no interaction (Table 3).

Regarding tetracycline and streptomycin, similar patterns were observed. 0.5× MIC TEG-based DES extract showed a FICI >0.5, indicating no interaction, while 1,4-But DES extract, 1,4-But DES, and TEG DES exhibited FICI values ranging from 0.12 to 0.26, indicating synergy with these antibiotics. In contrast, for sulfamethoxazole, only the 1,4-But DES extract showed a FICI value of 0.4 (≤ 0.5), indicating synergy. For all other combinations, including TEG DES and TEG-based DES extracts, the FICI was >0.5, showing no significant interaction (Table 3). These findings suggest that 1,4-But DES extract and its corresponding DES exert a stronger adjuvant effect with several antibiotics, including chloramphenicol, ampicillin, tetracycline, and streptomycin, compared to TEG-based DES extracts. However, for sulfamethoxazole, no significant synergy was observed except with the 1,4-But DES extract.

Synergistic compounds exhibit diverse mechanisms of action that can enhance antimicrobial effectiveness, which can broadly be classified into three categories: congruous synergy, syncretic synergy, and coalism. Congruous synergy occurs when both compounds independently possess antimicrobial activity, while syncretic synergy involves one compound enhancing the effectiveness of another without exhibiting antimicrobial properties itself. Coalism refers to a combination of two compounds, both lacking individual antimicrobial activity, but together demonstrating effectiveness (Tyers & Wright, 2019). A study recorded that curcumin (50 mg/L) significantly reduced antibiotic MICs (carbenicillin > ceftazidime > meropenem) in multidrug-resistant strains (P. aeruginosa) while lacking antibacterial activity on its own, comparable to the standard inhibitor PAβN (phe-arg-beta-naphthylamide) (Negi et al., 2014). Similarly, phytochemicals including naringenin, 5-methoxypsoralen, and licarin A were found to be AcrAB-TolC efflux pump inhibitors in in silico experiments on Salmonella enterica, with licarin A exhibiting the highest affinity (Oyedara et al., 2023).

Since our tested polyol-based DESs extract and DESs did not show significant inherent antimicrobial activity, we classify the observed antibiotic synergy as syncretic synergy. Notably, the 1,4-But DES extract exhibited syncretic synergy with all five tested antibiotics, suggesting its potential as a broad-spectrum antibiotic adjuvant. A similar example from the literature is the polyphenol-rich complex derived from green propolis, Tabebuia avellanedae bark, and Olea europaea leaf extracts. These extracts were synergistic with azithromycin, clarithromycin, and amoxiclav. Synergistic activity between the extracts and antibiotics was noted against Staphylococcus aureus, Haemophilus influenzae, and Klebsiella pneumoniae (Ramata-Stunda et al., 2022).

3.4. Growth kinetics studies

Here, we conducted a time-kill assay to examine the synergistic effects of different DESs and DES-based extracts in combination with chloramphenicol on the growth inhibition of Salmonella Typhimurium DT104. Initially, Salmonella Typhimurium DT104 was inoculated in MHB supplemented with a sub-inhibitory concentration (0.25× MIC) of chloramphenicol (32 μg/mL), and bacterial growth kinetics were monitored over 24 h. Due to the presence of chloramphenicol, the bacteria exhibited a prolonged lag phase of approximately 4 h, followed by an exponential growth phase from 10 to 20 h, and then a stationary phase. To assess the impact of DES-based extracts (TEG and 1,4-But) and their respective DES on bacterial growth kinetics, these compounds were introduced at the mid-exponential phase (MEP), the most actively growing phase of the strain, which was reached 12 h post-inoculation. At this point, DESs and DES-based extracts (TEG and 1,4-But) were added at three concentrations -1×, 0.5×, and 0.25× MIC, and bacterial counts were measured using standard dilution and plating methods.

Overall, a 2–3-fold reduction in viable bacterial cell counts was observed when both 1,4-But DES extract (0.25× MIC) and chloramphenicol (0.25× MIC) were present in the medium. Similarly, the corresponding 1,4-But DES (0.25× MIC) with chloramphenicol (0.25× MIC) resulted in a 2-fold decrease in bacterial counts. A more pronounced reduction was observed with the combination of 0.5× MIC 1,4-But DES extract or DES with 0.25× MIC chloramphenicol, leading to a significant (P < 0.05) (Fig. S1C) 4–5-fold and 3–4-fold drop in viable counts, respectively (Fig. 3). The highest reduction in viable counts was recorded with 1× MIC 1,4-But DES extract or DES combined with 0.25× MIC chloramphenicol, where no viable cells were detected.

Fig. 3.

Growth kinetics showing DT104 inhibition in the presence of the (A) 0.25× MIC of 1,4 But DES alone with Salmonella Typhimurium DT104, 0.25× MIC of 1,4 But DES, and 0.25× MIC of 1,4 But DES-based extract with 0.25× MIC of chloramphenicol (B) 0.25× MIC of TEG DES alone with bacteria, 0.25× MIC of TEG DES, and 0.25× MIC of TEG DES-based extract with 0.25× MIC of chloramphenicol (C) 0.5× MIC of 1,4-But DES alone with bacteria, 0.5× MIC of 1,4-But DES, and 0.5× MIC of 1,4-But DES-based extract with 0.25× MIC of chloramphenicol (D) 0.5× MIC of TEG DES alone with bacteria, 0.5× MIC of TEG DES, and 0.5× MIC of TEG DES-based extract with 0.25× MIC of chloramphenicol. The standard deviation is shown by the error bars, and all counts are averaged from three separate replicates. According to the color key provided in the figure, the asterisk and plus signify statistical significance based on based on two-way ANOVA followed by Tukey's post hoc test P > 0.05 (∗), P > 0.01–0.02 (∗∗),P > 0.02–0.05 (∗∗∗), P > 0.05–0.0009(∗∗∗∗) and P < 0.0001 (∗∗∗∗∗).

A similar pattern to that observed with 1,4-But DES extract and DES was noted for TEG DES extract and TEG DES in combination with 0.25× MIC chloramphenicol. At 0.5× MIC of TEG DES extract and TEG DES with 0.25× MIC chloramphenicol, an initial steady decline of up to 3-fold was observed, followed by a gradual significant (P < 0.05) (Fig. S1D) drop reaching 4–5-fold. Similar to 1,4-But DES extract and DES, no viable bacterial counts were detected when treated with 1× MIC of both TEG DES extract and TEG DES, indicating complete bactericidal activity.

Our time-kill assay using a combination of polyol-based DES extracts and DESs with chloramphenicol on continuous Salmonella Typhimurium DT104 cultures indicates that both the extracts and DESs sensitized DT104 to low subinhibitory concentrations (0.25×) of the antibiotic. Upon repeating the experiment with progressively lower concentrations of these compounds, we observed that the PBCs in the DES-DSP extract, together with DES, maintained their adjuvant effect by synergistically sensitizing DT104 to 0.25× chloramphenicol, even at concentrations as low as 0.5× MIC.

This indicates that the combination of TEG-based (extracts and DES) and 1,4-But-based (extract and DES) at 0.5× MIC with chloramphenicol at 32 μg/mL (0.25× MIC) effectively inhibited the growth of Salmonella Typhimurium DT104. These findings align with our MIC results, which showed bacterial inhibition in the 0.5× DES-based extract and 0.25× antibiotic combination. Growth kinetics assays are essential to confirm and expand upon the MIC-based results and provide further insights into the dynamics of antibiotic efficacy in the presence of these polyphenol-rich bioactive compounds.

4. Conclusions

Overall, our study highlights the significant potential of polyol-based DESs formulations and DES-based extracts derived from date seeds as effective antibiotic adjuvants in combating multidrug-resistant Salmonella Typhimurium DT104. This approach, utilizing microwave-assisted extraction techniques, represents a sustainable and efficient method for extracting polyphenol-rich bioactive compounds with potent antioxidant properties from date seed powder. The 1,4-But DES-based extract demonstrated the highest total phenolic content (TPC) and antioxidant activity (DPPH). These DES-based extracts and their corresponding DES formulations exhibited strong synergistic effects when combined with conventional antibiotics, significantly enhancing their antimicrobial efficacy. Notably, 1,4-But-based extract showed broad-spectrum synergy with all tested antibiotics against Salmonella Typhimurium DT104, highlighting their potential to target various antibiotic classes. Based on various antimicrobial assays, we conclude that 1,4-But DES-based date seed extracts exhibited superior antimicrobial activity compared to DES alone. This enhanced activity is likely attributed to the synergistic interaction between DES and the polyphenol-rich bioactive compounds present in the date seed crude extract. While TEG-based DES extract also reduced the MICs of antibiotics, they did not meet the synergy criteria based on the FICI index. These findings underscore the importance of further research into DESs and DES-based extracts, which, even without direct antimicrobial properties, can serve as valuable adjuvants in antibiotic therapy. This approach offers a promising solution to the global challenge of antibiotic resistance, opening new avenues for improving the effectiveness of existing antibiotics and combating foodborne pathogens. Additionally, this adjuvant technique has potential for use in clinical care as a supplement to current antimicrobials and in food systems (as part of a multi-hurdle preservation approach), provided that food-grade formulation, processing/storage stability, safety, and regulatory compliance are validated.

CRediT authorship contribution statement

Hina Khan: Writing – original draft, Methodology, Investigation, Formal analysis. Priti Mudgil: Writing – review & editing, Supervision, Methodology, Investigation. Bismi Phasaludeen: Writing – review & editing, Formal analysis. Shabarinath Srikumar: Writing – review & editing, Investigation, Funding acquisition, Conceptualization. Sajid Maqsood: Writing – review & editing, Investigation, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material

Data availability

Data will be made available on request.