Skip to main content
NCBI home page
3 Biotech logoLink to 3 Biotech
. 2025 Jul 12;15(8):253. doi: 10.1007/s13205-025-04371-w

Anti-bacterial activity of silibinin-functionalized silica-coated Fe3O4 magnetic nanocomposites on Pseudomonas aeruginosa

Nazanin Pasandideh Kordmahaleh 1, Mirsasan Mipour 1,✉, Najmeh Ranji 2, Mahdi Shahriarinour 2, Mohammad Nikpassand 2
PMCID: PMC12255613  PMID: 40661143

Abstract

The nanocomposites were synthesized and characterized using Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA), transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), and X-ray diffraction (XRD). The physicochemical analyses confirmed the structural integrity, uniform particle size (34–58 nm), thermal stability (> 600 °C), and magnetic properties of the nanocomposites. The nanocomposites exhibited strong synergistic effects with ciprofloxacin, reducing the minimum inhibitory concentration (MIC) in combination by 4- to 16-fold compared to ciprofloxacin alone. Furthermore, the nanocomposites in combination with ciprofloxacin significantly inhibited biofilm formation in clinical isolates. The gene expression analysis revealed downregulation of key efflux pump genes (mexX, mexY, and oprM), resulting to increased intracellular accumulation of ciprofloxacin. This mechanism potentiated the bactericidal effects of ciprofloxacin, even against resistant strains. These findings highlight the potential of Fe3O4@SPN@Silibinin as a biodegradable, stable, and water-soluble nanocarrier for silibinin delivery, offering a novel therapeutic strategy for combat multidrug-resistant bacterial infections. This study presents a promising approach to addressing the growing threat of antibiotic resistance through targeted drug delivery and genetic modulation.

Keywords: Biofilm, Silica-coated magnetic nanoparticles, Pseudomonas aeruginosa, Quantitative real time-PCR, Silibinin

Introduction

Silibinin (C25H22O10) is a polyphenolic flavonoid compound isolated from the seeds of milk thistle (Silybum marianum), a plant (Hossainzadeh et al. 2019; Khakinezhad Tehrani et al. 2020) belonging to the Asteraceae family and native to Southern Europe, Southern Russia, Asia Minor, and North Africa (Reed et al. 2020). Also known as silybin, silymarin I, flavobin, and silliver, silibinin has a molecular weight of 482.441 and comprises two structural domains: a flavanonol group and a phenylpropanoid group. Silibinin possesses antibacterial, anti-inflammatory, anti-tumoral, and hepatoprotective properties (Fallah et al. 2021). However, its hydrophobic and non-ionizable nature results in poor water solubility (≤ 50 μg/mL), which significantly limits its bioavailability and therapeutic effectiveness following oral administration (Silybin and a Major 1942). The antibacterial, anti-inflammatory, anti-tumoral, and hepatoprotective effects of silibinin have been reported (Lee et al. 2012). Despite its promising therapeutic potential, silibinin suffers from low aqueous solubility and poor cellular uptake (Hossainzadeh et al. 2019; Tuon et al. 2022).

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, poses a major challenge in healthcare due to its multidrug resistance (MDR). It is commonly associated with severe infections, particularly in patients intensive care unit, at surgical sites, and in burn wounds (Tuon et al. 2022). The intrinsic resistance of P. aeruginosa stems from multiple factors, including (Murray et al. 2015), low outer membrane permeability (Chevalier et al. 2017a) and robust biofilms formation (Tuon et al. 2022), and the overexpression of efflux pump systems, most notably MexAB-OprM and MexXY-OprM which actively expel antibiotics, contribute to resistance (Neidig et al. 2023; Parham et al. 2020).

Given the escalating threat of antibiotic resistance, there is an urgent need to develop alternative therapeutic strategies. Recent advances in nanotechnology have shown that nanomaterials can enhance the delivery and efficacy of bioactive compounds. The magnetic nanoparticles, in particular, offer several advantageous: high chemical and colloidal stability, small particle size, rapid and efficient separation, cost-effective synthesis, responsiveness to magnetic fields, and excellent biocompatibility (Chevalier et al. 2017b). Functionalized magnetic nanoparticles further improve therapeutic potential by enabling targeted drug delivery. Among these, magnetic iron oxide nanoparticles (Fe3O4) are especially notable for their ease of synthesis, magnetic responsiveness, and surface functionalization capabilities. These properties allow them to be directed to specific sites using external magnetic fields, enabling precise drug delivery and efficient recovery or separation of therapeutic agents (Mouraki et al. 2018; Aguilar et al. 2007; Lee et al. 2014). Silibinin-functionalized Fe3O4 nanoparticles represent a novel strategy for overcoming the solubility and bioavailability limitations of silibinin while simultaneously harnessing its antibacterial properties. The silica coating on Fe3O4 nanoparticles improves their stability (Madhavi et al. 2145) and provides a functionalization platform for silibinin, facilitating its efficient delivery to bacterial cells. Previous studies have shown that nanocomposites can potentiate antibiotic activity by inhibiting efflux pumps (Dey et al. 2022) and reducing biofilm formation (Ferreres et al. 2023), thereby targeting two major resistance mechanisms in Pseudomonas aeruginosa (Fernández-Billón et al. 2023). This synergistic interaction suggests that Fe3O4@SPN@Silibinin nanocomposites may serve as a promising tool against multidrug-resistant pathogens.

The objective of this study was to design and synthesize silibinin-functionalized silica-coated Fe3O4 (Fe3O4@SPN@Silibinin) nanocomposites and evaluate their antibacterial properties. Specifically, the aimed were to: (1) characterize the physicochemical properties of the synthesized nanocomposites using advanced techniques such as FTIR, TEM, and XRD; (2) assess their antibacterial efficacy against clinical isolates of P. aeruginosa, with a focus on biofilm inhibition; (3) evaluate their ability to enhance the antibacterial effect of ciprofloxacin through synergistic interactions; and (4) investigate the downregulation of key efflux pump genes (mexX, mexY, and oprM) as a potential mechanism of action. These objectives support the broader goal of developing innovative therapeutic strategies to combat antibiotic resistance in high-priority bacterial pathogens.

Materials and methods

Materials

Silibinin (C25H22O10, 3,5,7-trihydroxy-2-[3-(4-hydroxy-3-methoxyphenyl)−2(hydroxymethyl)−1,4-benzodioxan-6-yl]−4-chromanone) was purchased from Sigma-Aldrich Co. (Germany) with a purity of ≥ 94% (molecular weight: 482.4 g/mol). Reagents used for nanocomposite preparation, including 3-(chloropropyl)-trimethoxysilane and xylene, were obtained from Merck (Germany, Hohenbrunn). All solvents used in this study were of HPLC grade (Merck) and were used without further purification, dilution or modification.

Synthesis of Fe3O4@SPN@Silibinin nanocomposites

Fe3O4@SPN magnetic nanoparticles (MNPs) were synthesized via a co-precipitation method based on previously established protocols with slight modifications (Nikpassand et al. 2022, 2015, 2017). Initially, Fe3O4 nanoparticles were synthesized by mixing FeCl3·6H2O and FeSO4·4H2O in a 2:1 molar ratio in deionized water under a nitrogen atmosphere. The pH was adjusted to ~ 10 using ammonia solution, and the mixture was stirred at 80 °C under light exposure for 1 h using a Heidolph Hei-Tec stirrer to form Fe3O4 nanoparticles. The resulting nanoparticles were magnetically separated, washed with deionized water, and dried at 50 °C. To coat the Fe3O4 nanoparticles with silica, a sol–gel method was employed. Fe3O4 nanoparticles (0.2 g) were dispersed in a solution of ethanol and deionized water containing ammonium hydroxide. Tetraethyl orthosilicate (TEOS) was added dropwise under continuous stirring, and the reaction was allowed to proceed for 6 h at room temperature. The resulting silica-coated Fe3O4 nanoparticles (Fe3O4@SPN) were then washed with ethanol and dried.

For silibinin, functionalization, Fe3O4@SPN nanoparticles (0.2 g) were suspended in a solution containing silibinin (0.2 g), NaOH (10% w/v, 5 mL), and deionized water (20 mL). The mixture was stirred for 24 h, with triethylamine (5 mL) added after 5 h to enhance the reaction. Following magnetic separation, the functionalized nanoparticles were washed with ethanol and deionized water to remove unreacted silibinin and then dried at 50 °C for 24 h. The successful synthesis and functionalization of the Fe3O4@SPN@Silibinin nanocomposites were confirmed using various characterization techniques, including FTIR, TEM, FE-SEM, XRD, TGA, and VSM. These analyses were conducted at BimGostar Taban Co. (Tehran, Iran), to evaluate the structural, thermal, and magnetic properties of the nanocomposites.

Physicochemical characterization

The physicochemical properties of the Fe3O4@SPN@Silibinin nanocomposites were thoroughly investigated using multiple characterization techniques. Fourier transform infrared (FTIR) spectroscopy was used to identify the functional groups present in the synthesized nanocomposites (Amr et al. 2023; Farouk et al. 2023; Nikpassand et al. 2021). The spectra were recorded in the range of 400 to 4000 cm−1 to confirm the characteristic peaks associated with silibinin and silica-coated Fe3O4 core.

Transmission electron microscopy (TEM) analysis was performed using a Zeiss-EM10C-100 kV instrument (Germany). The sample was prepared by diluting the nanocomposites in distilled water (1 mg/mL), then placing a 20 μL drop on a charcoal-coated copper grid for 2 min. Excess liquid was removed using filter paper, and the sample was stained with 2% uranyl acetate. The grid was air-dried at room temperature before microscopic imaging.

Surface morphology and crystal structure were examined using a MIRA3 TESCAN field emission scanning electron microscope (FE-SEM, Czech Republic) equipped with energy-dispersive X-ray (EDX) analysis. FE-SEM was also employed to determine particle size distribution (Nikpassand et al. 2024). X-ray diffraction (XRD) analysis confirmed the crystalline structure of the nanoparticles (Pourasgar et al. 2024). The diffraction patterns showed distinct peaks corresponding to Fe2O4 with a silica coating, consistent with a face-centered cubic structure. XRD measurements were carried out using a Philips Xpert X-ray powder diffractometer (CuKα, radiation, λ = 0.154056) scanned at 2°/min over a 2θ range of 10°–80°.

Thermal stability was evaluated via thermogravimetric analysis (TGA) using a TA Q600 instrument (USA) (Pourasgar et al. 2024). The TGA profile revealed decomposition of the organic components, while thermal stability was maintained up to 600 °C. Magnetic properties were measured using vibrating sample magnetometry (VSM;Lakeshore 7403, USA) (Nikpassand et al. 2024). The nanocomposites exhibited a saturation magnetization of approximately 30 emu/g, indicating that the magnetic properties were retained despite silica and silibinin surface modifications. Overall, these analyses confirmed the successful synthesis and functionalization of Fe3O4@SPN@Silibinin nanocomposites, validating their structural integrity, stability, and suitability for subsequent antibacterial evaluations.

Antibacterial analysis

Antibacterial agents and susceptibility testing

The clinical samples were obtained from sputum, urine, burns wounds, general wounds and blood, collected from various hospitals and laboratories in Tehran Province. Pseudomonas aeruginosa ATCC 9027 was used as the reference strain. The antibiotic susceptibility patterns of P. aeruginosa clinical isolates were determined using the Kirby–Bauer disk diffusion method (Abdelhai et al. 2024), following the Clinical and Laboratory Standards Institute (CLSI) 2023 guideline. The isolates were tested against nine antibiotics: piperacillin (100 µg), imipenem (10 µg), meropenem (10 µg), gentamicin (10 µg), ceftazidime (30 µg), amikacin (30 µg), levofloxacin (5 µg), colistin (10 µg) and ciprofloxacin (5 µg). Additionally, the minimum inhibitory concentrations (MICs) (Mohamed et al. 2024) of ciprofloxacin and Fe3O4@SPN@Silibinin nanocomposites were determined using the broth dilution method, also according to the CLSI2023 guideline.

Phenotypic and genotypic characterization of P. aeruginosa isolates

Clinical isolates were cultured on McConkey and Cetrimide agar media. Phenotypic identification was performed using standard biochemical assays, including catalase, oxidase, urease, SIM, TSI, and growth at 42 °C, as previously described (Mohamed et al. 2024). For genotypic confirmation, genomic DNA was extracted from all 22 clinical isolates using the SinaPure DNA extraction kit (SinaClon, Iran).

The gyrA gene was amplified by polymerase chain reaction (PCR) to confirm the identity of P. aeruginosa. The PCR reaction mixture included genomic DNA, gene-specific primers, and a commercial master mix. Amplification was carried out using a BioRad thermocycler (USA) under the following conditions: initial denaturation at 92 °C for 3 min, 30 cycles of denaturation at 92 °C for 30 s, annealing at 52 °C for 30 s, and extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. The primers sequences targeting the gyrA gene were: Forward 5'-GTGTGCTTTATGCCATGAG-3'and Reverse 5'-GGTTTCCTTTTCCAGGTC-3'.

PCR products were visualized using agarose gel electrophoresis.

Checkerboard dilution method

The checkerboard dilution method was employed to evaluate the synergistic effects of Fe3O4@SPN@Silibinin MNCs in combination with ciprofloxacin against Pseudomonas aeruginosa isolates. The final inoculum concentration was adjusted to 5–7.5 × 105 CFU/mL in 96-well microtiter plates, with a total volume of 200 µl per well. Both Fe3O4@SPN@Silibinin MNCs and ciprofloxacin were serially diluted in Mueller–Hinton Broth (MHB) to yield eight concentrations levels ranging from 4 to 1024 µg/mL.

Each column (vertical series) of wells was filled with sub-MIC concentrations of Fe3O4@SPN@Silibinin (Drug A), while each row (horizontal series) contained sub-MIC concentrations of cicrofloxacin (Drug B). The synergistic effects were assessed by calculating the fractional inhibitory concentration index (FICI) using the following Eq. (Pourasgar et al. 2024):

FICI=MICdrug A in combination/MICdrug A alone+MICdrug B in combinatione/MICdrug B alone 1

Interactive of FICI values was as follows:

FICI ≤ 0.5 Synergistic effect, 0.5 < FICI ≤ 0.75 Partial synergy, 0.76 < FICI ≤ 1 Additive effect, 1 < FICI ≤ 4 No significant interaction, FICI > 4 Antagonistic effects (Pourasgar et al. 2024).

Biofilm formation assay

Biofilm formation in P. aeruginosa isolates was evaluated using crystal violet staining, with slight modifications to a previously described protocol (Gomaa et al. 2024). Twenty clinical isolates of P. aeruginosa and the reference strain ATCC 9027 were adjusted to a 0.5 McFarland standard (5–7.5 × 105 CFU/mL) and inoculated into sterile 96-wells polystyrene microtiter plates. The plates were incubated at 37 °C for 24 h to allow biofilm formation. Following incubation, non-adherent cells were removed by washing the wells with phosphate-buffer saline (PBS, pH = 7). The wells were then fixed with 96% methanol for 5 min and stained with 1% (w/v) crystal violet for 10 min. Excess dye was removed and the bound dye was solubilized using 33% glacial acetic acid. Absorbance was measured at 570 nm, with a reference wavelength of 630 nm, using an ELISA plate reader (Hiperion, Germany). To assess the effect of Fe3O4@SPN@Silibinin MNCs and ciprofloxacin on biofilm formation, a subset of clinical isolates (isolates 1, 8, 10 and 14, selected at random) and the ATCC 9027 strain were treated under two conditions:

1) Ciprofloxacin (sub-MIC) in combination with Fe3O4@SPN@Silibinin (sub-MIC).

2) Ciprofloxacin (sub-MIC) alone.

Treated cultures were incubated at 37 °C for 24 h, after which biofilm formation was analyzed.

Time-kill kinetics

Four selected clinical isolates of P. aeruginosa (isolates NO. 1, 8, 10 and 14), along with the ATCC 9027 reference strain, were adjusted to a concentration of 5–7 × 105 CFU/mL in Mueller–Hinton Broth (MHB). These were incubated in 96-well microtiter plates with sub-MIC concentrations of ciprofloxacin (CIP), both in the presence and absence of Fe3O4@SPN@Silibinin for 0–24 h at 37 °C (Zainin et al. 2013). At designated time intervals, 10 μL from each treatment group was spotted onto nutrient agar plates and incubated for 24 h. Following incubation, colony counts were performed to assess bacterial viability.

Anti-pseudomonal treatment with Fe3O4@SPN@Silibinin MNCs and ciprofloxacin

A standardized suspension of P. aeruginosa ATCC 9027 and the four selected clinical isolates (NO. 1, 8, 10 and 14) was prepared at a 0.5 McFarland (approximately 0.5 × 108 CFU/mL). These suspensions were diluted to 5–7.5×105 CFU/mL and exposed to the following treatment conditions before incubation at 37 °C for 24 h:

  1. 1/2 MIC(Fe3O4@SPN@Silibinin)+1/2 MIC(CIP)

  2. 1/4 MIC(Fe3O4@SPN@Silibinin)+/2 MIC (CIP)

  3. 1/2 MIC(CIP) alone

  4. Untreated control

Quantitative analysis of mexX, mexY, rpsL and oprM genes expression

Quantitative real-time PCR (Q-RT-PCR) was conducted to evaluate the relative expression levels of mexX, mexY, and oprM in P. aeruginosa isolates under the following treatment conditions:

  1. 1/2 MIC ((Fe3O4@SPN@Silibinin) + 1/2 MIC(CIP)

  2. 1/4 MIC (Fe3O4SPN@Silibinin)+ 1/2 MIC(CIP)

  3. 1/2 MIC (CIP) alone

Total RNA was extracted using the Total RNA Extraction Mini-Kit (Yekta Tajhiz Azma, Iran). cDNA synthesis was carried out with a commercial cDNA synthesis kit (Yekta Tajhiz Azma, Iran). q-RT-PCR was performed using YTA SYBR Green qPCR MasterMix 2X (Yekta Tajhiz Azma, Iran) on a Rotor-Gene® Q instrument (QIAGEN). Cycling conditions were as follows: 95 °C for 3 min, followed by 40 cycles at 95 °C for 5 s, 60 °C for 5 s, and 75 °C for 5 s (for 100–150 bp amplicons) or 10 s (for 150–300 bp amplicons). The rpsL gene served as the internal reference gene. Primer sequences are listed in Table 1. All reactions were run in triplicate, and data were analyzed using the ΔΔCT method. Fold-change in gene expression was calculated using the 2−ΔΔCT formula (Rao et al. 2013):

ΔCT=CTtarget gene(A)-CTreference gene(B)
ΔΔCTtest=ΔCTtest-ΔCTcontrol=CTA.test-CTB.test-CTA.control-CTB.control 2
Table 1.

Sequence of primers used for quantitative RT-PCR in P. aeruginosa

name sequence Length of production
mexX-F 5'- GTCAAGGTGGTCAACCCAAA- 3' 123 bp
mexX-R 5'-GTTCTCGACGATCACCCACT—3'
mexY-F 5'-TCTCGGTGTTGATCGTGTTC—3' 156 bp
mexY-R 5'-GGCCGACCTTGAAGTAGATG −3'
oprM-F 5'- TTCGGGTTCCTGGTTGTTCC- 3' 236 bp
oprM-R 5'-GCTGGTAGTACTCGTCGCTG—3'
rpsL-F 5'- CTCGGCACTGCGTAAGGTAT- 3' 216 bp
rpsL-R 5'-CCGTACTTCGAACGACCCTG—3'
Open in a new tab

Statistical analysis

Statistical significance between groups was determined using student's t-test, as well as one-way and two-way ANOVA followed by Tukey's post-hoc test. A p value ≤ 0.05 was considered statistically significance. All statistical analyses were performed using Prism®9.4.1 Software (GraphPad Software Inc., La Jolla, California). All experiments were independently conducted in triplicate.

Results

Physicochemical characterization of Fe3O4@SPN@ Silibinin nanoparticles

Fe3O4@SPN@Silibinin magnetic nanocomposites (MNCs) were synthesized using readily available and cost-effective materials (Fig. 1). The FT-IR spectra of Silibinin and Fe3O4@SP@Silibinin (Fig. 2) were analyzed to identify functional groups present in the synthesized nanoparticles. A broad absorption band in the 3300–3550 cm−1 range corresponds to the O–H stretching vibrations of phenolic or alcoholic groups from both Silibinin and the Fe3O4@SPN@Silibinin nanocomposite. A peak at 1702 cm−1 is attributed to the C=O stretching vibration of Silibinin ketone group. Additionally, characteristic peaks at 1445 cm−1 and 1510 cm−1 represent C=C stretching vibrations.

Fig. 1.

Fig. 1

Open in a new tab

Structure of Fe3O4@SPN@Silibinin MNCs

Fig. 2.

Fig. 2

Open in a new tab

The FT-IR spectra of Silibinin and Fe3O4@SPN@Silibinin MNCs to identify the functional groups of the synthesized nanoparticles

The bands centered at 1150 cm−1 and 1220 cm−1 are associated with Si–O–Si stretching vibrations from the SiO2 shell. A peak near 605 cm−1 corresponds to Fe–O bending vibrations, confirming the presence of Fe3O4 in the Fe3O4@SPN@Silibinin nanocomposite (Fig. 2). Transmission electron microscopy (TEM) and field emission scanning electron microscopy (FE-SEM) were employed to assess the morphology and size of the nanoparticles. TEM images (Fig. 3A) reveal a degree of aggregation, suggesting chemical bonding between Silibinin and the Fe3O4@SPN macromolecule on the magnetic nanoparticles. The FE-SEM image (Fig. 3B) indicates that the nanoparticles have a size range of approximately 34–58 nm, with a homogenous structure showing uniform, symmetrical, and regular distribution (Fig. 3B).

Fig. 3.

Fig. 3

Open in a new tab

A TEM image of Fe3O4@SP@Silibinin MNCs and B FE-SEM image of Fe3O4@SP@Silibinin MNCs to determine the morphology and size of the nanoparticles. The particles size was estimated by FE-SEM ranging from 34–58 nm with a highly homogenous structure exhibiting a uniform, symmetrical, and regular distribution

The X-ray powder diffraction (XRD) patterns of Fe3O4@SPN@Silibinin (Fig. 4) further confirm the successful synthesis of Fe3O4 magnetic nanoparticles. Distinct diffraction peaks appear at 2θ values of 31.1° (311), 35.7° (220), 43.5° (400), 54.2° (422), 57.7° (511), and 63.4° (440), which correspond to the face-centered cubic structure of (Fe3O4 and match standard reference data. A broad peak in the 10–20° range is attributed to amorphous silica (SiO2), verifying the incorporation of a silica layer in the nanocomposite (Fig. 4). The magnetic properties of the synthesized MNCs were analyzed using vibrating sample magnetometry (VSM) at room temperature (Fig. 5A). The VSM profile shows a rapid increase in magnetization with increasing external magnetic field strength, reaching saturation at approximately 5000 Oe.

Fig. 4.

Fig. 4

Open in a new tab

The X-ray powder diffraction (XRD) patterns. The magnetic properties of uncoated magnetic iron oxide (Fe3O4), Fe3O4@SPN, and Fe3O4@SPN@Silibinin. XRD analysis confirmed formation of Fe3O4 MNPs

Fig. 5.

Fig. 5

Open in a new tab

A The evaluation of magnetic properties of nano-Fe3O4 (Black), Fe3O4@SPN (Red) and Fe3O4@SPN@Silibinin MNCs (Blue) with Vibrating Sample Magnetometry (VSM) analysis. The VSM spectrum revealed that the magnetic response of the material increases with the strength of the external magnetic field, reaching saturation at approximately 5000 Oe. B Energy-dispersive X-ray spectroscopy (EDX) analysis was determined the components of Fe3O4@SPN@Silibinin MNCs including Fe (30.55 w/w %), O (26.63 w/w %), N (4.05 w/w %), Si (12.31 w/w %), and C (26.46 w/w %).

The saturation magnetization was approximately 30 emu/g, indicating strong magnetic behavior.

The symmetrical drop in magnetization upon reversal of the magnetic field indicates a reversible magnetic response. Notably, the presence of organic layers around the (Fe3O4e core caused only a slight reduction in magnetic performance, maintaining the material's classification as a ferromagnetic nanocomposite. Thus, the synthesized Fe3O4@SPN@Silibinin nanocomposite is classified as a ferromagnetic material (Fig. 5A).

Energy-dispersive X-ray spectroscopy (EDX) analysis confirmed the elemental composition ofFe3O4@SPN@Silibinin MNCs (Fig. 5B). The EDX spectrum revealed the presence of Fe (30.55 w/w %), O (26.63 w/w %), N (4.05 w/w %), Si (12.31 w/w %), and C (26.46 w/w %), validating the successful incorporation of Fe3O4 as the core component of the nanocomposite.

Antimicrobial analysis

Antibiotic susceptibility profiles

A total of 20 clinical P. aeruginosa isolates were analyzed (Fig. 6A). The minimum inhibitory concentration (MIC) values of ciprofloxacin revealed that all clinical isolates were resistant, with MIC ranging from 64 µg/mL to 256 µg/mL (Fig. 6B). Based on the disk diffusion method, 80% of the strains were identified as multidrug-resistant (MDR) (Fig. 6C).

Fig. 6.

Fig. 6

Open in a new tab

Antibiotic susceptibility profile. A Distribution of P. aeruginosa clinical samples which isolated from various sources including urine and burns. B MIC of ciprofloxacin in 20 resistant isolates of P. aeruginosa using the broth microdilution method. C Antibiotics susceptibility profiling by the disc diffusion method. D Percentage of biofilm formation in 20 clinical isolates of P. aeruginosa. E The presence of gyrA gene in the isolates to identify P. aeruginosa samples. Lanes M and Ns contain 1 kb DNA ladder marker and PCR products, respectively

Synergistic effects of Fe3O4@SPN@Silibinin and ciprofloxacin in P. aeruginosa

The synergistic interactions between Fe3O4@SPN@Silibinin and ciprofloxacin were evaluated using the fractional inhibitory concentration (FIC) index. The results of the checkerboard assay, summarized in Table 2, indicate that the combination treatment produced a synergistic effect in all tested P. aeruginosa isolates.

Table 2.

Interactive effects of Fe3O4@SP@Silibinin with ciprofloxacin on isolates of P. aeruginosa

Strains
No		 MIC alone (µg/ml) MIC combined (µg/ml) FIC(µg/ml)
CIP Fe3O4@SP@SIL CIP Fe3O4@SP@SIL
ATCC 9027 512 64 64 4 0.1875
No.1 1024 64 256 4 0.3125
No.8 512 512 64 64 0.25
No.10 1024 64 64 8 0.1875
No.14 512 64 64 4 0.1875
Open in a new tab

FICI ≤ 0.5 means synergy

0.5 < FICI ≤ 0.75 means partial synergy

0.76 < FICI ≤ 1 means additive

Biofilm formation assay

Biofilm formation by the 20 clinical isolates of P. aeruginosa was quantified (Fig. 6D). Treatment with 1/2 MIC(Fe3O4@SPN@Silibinin) + 1/2 MIC(CIP) and 1/4 MIC(Fe3O4@SPN@Silibinin) + 1/2 MIC(CIP) significantly reduced biofilm formation compared to treatment with ciprofloxacin alone (½MIC) in both the ATCC 9027 reference strain and four drug-resistant clinical isolates. Furthermore, untreated control samples exhibited significantly higher levels of biofilm formation compared to all treated groups (Fig. 7).

Fig. 7.

Fig. 7

Open in a new tab

The evaluation of Biofilm formation in P. aeruginosa isolates. Biofilm formation was decreased in the ATCC 9027 strain and four clinical isolates in four groups treated with 1/2 or 1/4 MIC(Fe3O4@SPN@Silibinin) + 1/2 or 1/4 MIC(CIP), compared to 1/2 or 1/4 MIC(CIP) and untreated cells (control). The results were represented as mean±SD. Significant differences between the test and control were calculated based on P<0.0001

Time-kill assay

To further confirm the synergistic antibacterial effects of Fe3O4@SPN@Silibinin and ciprofloxacin, time-kill kinetics were evaluated. The combination therapy produced a time-dependent reduction in viable bacterial counts. Among the clinical isolates, the most pronounced bacterial killing was observed at 2 h post-treatment (Fig. 8A–D). In the ATCC9027 strain, the most significant reduction in colony counts occurred at 4 h following treatment with either 1/2 MIC (Fe3O4@SPN@Silibinin) + 1/2 MIC(CIP) or 1/4 MIC(Fe3O4@SPN@Silibinin) (Fig. 8E).

Fig. 8.

Fig. 8

Open in a new tab

Time-kill assays were conducted on clinical and standard isolates treated with 1/2 MIC(Fe3O4@SPN@Silibinin) + 1/2 MIC(CIP), 1/4 MIC(Fe3O4@SPN@Silibinin) + 1/2 MIC(CIP), and 1/2 MIC(CIP) compared to untreated cells to determine the antibacterial activity of these agents over time

MexXY-oprM expression pattern

Our results demonstrated that the expression levels of oprM (Fig. 9A), mexX (Fig. 9B), and mexY (Fig. 9C) were significantly downregulated in both the ATCC9027 and the four clinical isolates treated with either 1/2 MICFe3O4@SPN@Silibinin MNCs + 1/4 MICCIP and 1/2 MICFe3O4@SPN@Silibinin MNCs + 1/2 MICCIP, in comparison to isolates treated with 1/2 MICCIP alone.

Fig. 9.

Fig. 9

Open in a new tab

Downregulation of efflux pump related genes in the presence of Fe3O4@SPN@Silibinin. Downregulation of A oprM, B mexX, and C mexY, in bacterial cells treated with 1) 1/2 or 1/4 MIC(Fe3O4@SPN@Silibinin) + 1/2 MIC(CIP)(test groups) compared to treated cells with 1/2 MIC(CIP, control group).The results are represented as mean ± SD. Significant differences between the test and control were calculated based on *P<0.05, **P<0.01, and ***P<0.001

Discussion

This study successfully demonstrated the synthesis, characterization, and antibacterial evaluation of Fe3O4@SPN@Silibinin nanocomposites. The following discussion elaborates on the key findings, aligning them with the study objectives and initial hypotheses. The bioactive components present in medicinal plants, such as flavonoids, alkaloids, terpenoids, carotenoids, polyphenols, saponins, tannins, volatile oils, minerals, and vitamins, exhibit a wide range of therapeutic benefits. The medicinal plants offer a promising alternative to synthetic antibacterial agents due to their safety profile, cost-effectiveness, and potential to address antibiotic resistance (Shakeri et al. 2012). Silybum marianum (L.), commonly known as milk thistle, is a wild, annual herb (Lahlah et al. 2012) with well-documented medicinal applications. The major active constituent of milk thistle, silibinin, has been shown to possess detoxifying, anticancer and antibacterial properties (Wagoner et al. 2010; Polyak et al. 2013; Safarpoor et al. 2018; Ali et al. 2020). Despite these pharmacological benefits, the clinical application of silibinin is limited by its poor solubility in biological systems, which often necessitates prolonged oral administration to achieve therapeutic effects (Christodoulou et al. 2015).

To overcome this challenge, we functionalized silibinin onto a Fe3O4 nanocomposite coated with silica, then investigated its physicochemical and antibacterial properties. This aim was to preserve silibinin’s chemical integrity while enhancing its bioavailability and therapeutic efficacy as the active molecule in the nanocomposite. Numerous studies have demonstrated the utility of nanoparticles to enhancing drug delivery, improving stability, and increasing bioavailability. In our study, Fe3O4@SPN nanoparticles were employed as a nanocarrier system to optimize silibinin delivery and boost its antibacterial activity against bacterial pathogens. Pourasgar et al. synthetized curcumin-functionalized silica-coated Fe3O4 nanoparticles with particle sizes ranging from 40 to 80 nm, exhibiting high thermal stability (>600 °C) and paramagnetic properties (Rao et al. 2013). Sugumaran et al. demonstrated the anticancer potential of curcumin-conjugated dextran-coated Fe3O4 nanoparticles, which had particle size range from 147 to 216 nm (Sugumaran et al. 2022). Salimi et al. reported that curcumin-loaded Fe3O4/GO and Fe3O4/ZnO/GO nanocomposites exhibited anticancer activity against U87 glioblastoma cells, with particle sizes of 42.36 nm and 53.21 nm, respectively (Salimi et al. 2024). Pourgholi et al. showed that silibinin loaded polymeric nanoparticles demonstrated anticancer activity against breast cancer cells, with particle size between approximately 150 and 220 nm (Pourgholi et al. 2021). In the presence study, the successful functionalization of silibinin onto silica-coated Fe3O4 nanoparticles was confirmed by the identification of characteristic functional groups. The synthesized nanoparticles exhibited a uniform morphology, with an average size range of 34–58 nm and high thermal stability (>600 °C), making them well-suited for drug delivery applications as ferromagnetic carriers.

Silibinin, a compound with notable pharmacological properties, high bioavailability, and low toxicity, exhibits a range of antibacterial effects (El-Sapagh et al. 2023).It has demonstrated synergistic activity when combined with antibiotics such as oxacillin and ampicillin, suggesting its potential role in modulating resistance and controlling MRSA infections (Kang et al. 2011). Lee et al. reported that silibinin exhibited synergistic effects with ampicillin and gentamicin against various oral bacteria including S. sanguinis, S. anginosus, S. sobrinus, S. mutans, S. criceti, and S. ratti (Lee et al. 2012). Furthermore, Holasová et al. demonstrated that silybin, a major component of silibinin, acts as a non-toxic modulator of multidrug resistance and can reduce bacterial pathogenicity (Holasová et al. 2022). In clinical isolates of Acinetobacter baumannii, curcumin-functionalized magnetic nanoparticles shown to decrease the MIC of ciprofloxacin by 4- to 32-fold when used in combination therapy (Pourasgar et al. 2024). In this present study, the checkerboard dilution method revealed a significant synergistic interaction between Fe3O4@SPN@Silibinin and ciprofloxacin, reducing the MIC of ciprofloxacin to 4 to16-fold and the MIC of Fe3O4@SPN@Silibinin by 8 to16-fold. These findings suggest that silibinin, when combined with antibiotics, can enhance antibacterial efficacy and reduce the required antibiotic dosage in resistant isolates. Therefore, we propose that silibinin may serve as a promising complementary agent in the treatment of drug-resistant bacterial infections.

Evren et al. demonstrated the antibacterial and anti-adhesion/anti-biofilm activity of silymarin against standard bacterial strains, suggesting its potential use as a food supplement or medicine agent (Evren and Yurtcu 2015). Holasová et al. further confirmed that silybins exert a dose-dependent anti-adhesion effect (Holasová et al. 2022). Vimberg et al. reported that flavonolignans derived from Silybum marianum (milk thistle) effectively inhibited Staphylococcus aureus biofilm formation (Vimberg et al. 2015). Additionally, Hurtová et al. found that silybin A and silybin B inhibit biofilm formation in Pseudomonas aeruginosa (Hurtová et al. 2022), while Makled et al. demonstrated that silver (Ag) nanoparticles possess both antibacterial and anti-biofilm activities against P. aeruginosa (Makled et al. 2023). Fekri Kohan et al. also showed that silibinin exhibits antibiofilm activity against uropathogenic E. coli isolates (Fekri Kohan et al. 2024).Pourasgar et al. revealed that curcumin-functionalized silica-coated Fe3O4 magnetic nanoparticles reduced biofilm formation by downregulating efflux pomp genes and the bap gene in Acinetobacter baumannii isolates (Rao et al. 2013). In the present study, our findings indicate that silibinin-functionalized silica-coated Fe3O4 nanocomposites (Fe3O4@SP@Silibinin) significantly reduced biofilm formation in P. aeruginosa isolates when used in combination with ciprofloxacin compared to ciprofloxacin alone. This enhanced anti-biofilm activity may be attributed, at least in part, to the downregulation of efflux pump genes and increased intracellular accumulation of ciprofloxacin, thereby improving its efficacy against drug-resistant isolates.

Rahbar Takrami et al. reported that Curcumin, a flavonoid derived from the rhizome of Curcuma longa, downregulated the expression of efflux pump genes mexX and oprM in clinical isolates of Pseudomonas aeruginosa (Rahbar Takrami et al. 2019). Fekri Kohan et al. demonstrated that silybin (silibinin), when loaded into polymersome nanoparticles, suppressed the expression of d AcrAB-TolC efflux pump genes and upregulated ompC and ompF porin genes at the transcriptional level in combination with ciprofloxacin uropathogenic Escherichia coli (Fekri Kohan et al. 2024). Wang et al. showed that inhibition of efflux pump genes expression by silybin restored antibiotic sensitivity in Staphylococcus. aureus (Wang et al. 2018). Similarly, Holasová et al. found that 2,3-dehydrosilybin B reduced the antibiotic-induced overexpression of the norA and mepA efflux pumps to basal levels and functioned as a non-competitive inhibitor of both pumps (Holasová et al. 2022). In our study, quantitative analysis indicated that Fe3O4@SP@Silibinin enhanced ciprofloxacin susceptibility in P. aeruginosa isolates by downregulation the efflux pump genes mexX, mexY, and oprM. This downregulation likely led to increased intracellular accumulation of ciprofloxacin, thereby enhancing its antibacterial efficacy. In drug-resistant isolates, reduced expression of efflux pump genes resulted in the intracellular trapping of higher amounts of the antibiotic, ultimately leading to bacterial cell death due to the elevated antibiotic concentration.

The results of this study highlight the potential of silibinin-functionalized nanoparticles as a novel strategy for combatting antibiotic resistance. The combination of magnetic targeting, improved solubility, and modulation of bacterial gene expression offers a multifaceted approach to addressing multidrug-resistant (MDR) pathogens. Future research should focus on in vivo studies to evaluate the therapeutic efficacy and safety of these nanocomposites in clinical settings.

Conclusions

Our physicochemical analyses confirmed the successful functionalization of the nanocomposites, which exhibited uniform particle size, high thermal stability, and notable magnetic properties. The Fe3O4@SPN@Silibinin nanocomposites demonstrated enhanced antibacterial activity when combined with ciprofloxacin, as evidenced by biofilm inhibition assays and checkerboard dilution methods. This combination showed significant synergistic effects, leading to reduced biofilm formation and increased bacterial susceptibility to the antibiotic. Time-kill kinetics studies revealed a marked reduction in bacterial cell count within the first 2–4 h following combination treatment in both clinical isolates and the ATCC 9027 strain. Additionally, the observed downregulation of the mexX, mexY, and oprM efflux pump genes suggests that Fe3O4@SPN@Silibinin facilitates increased intracellular accumulation of ciprofloxacin, thereby enhancing its antibacterial effects. These findings underscore the potential of Fe3O4@SPN@Silibinin nanocomposites as a potent adjuvant therapy for managing MDR bacterial infections. Their ability to boost antibiotic efficacy and target resistant strains positions this nanoplatform as a promising strategy for future clinical applications.

Declarations

Conflict of interest

On behalf of all authors, the corresponding author declares that there is no conflict of interest.

References

  1. Abdelhai MF, Shabaan RH, Kamal NM, Elemary EA, Abd-Elhalim BT, Hassan EA (2024) Copper nanoparticles biosynthesis by Stevia rebaudiana extract: biocompatibility and antimicrobial application. AMB Express 14(1):59 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aguilar MR, Elvira C, Gallardo A, Vazquez B, Román J (2007) Smart polymers and their applications as biomaterials. Top Tissue Eng 3(6):1–27 [Google Scholar]
  3. Ali BS, ElSayed AI, Doheem MA, Eita AMA, Omar AA. Effect of Milk thistle (Silybum marianum (L.) Gaertn) seed extract on bacterial activities and growth of human liver cancer cells. Journal of Biotechnology Research. 2020;6(5):27–33.
  4. Amr M, Abu-Hussien SH, Ismail R, Aboubakr A, Wael R, Yasser M et al (2023) Utilization of biosynthesized silver nanoparticles from Agaricus bisporus extract for food safety application: synthesis, characterization, antimicrobial efficacy, and toxicological assessment. Sci Rep 13(1):15048 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chevalier S, Bouffartigues E, Bodilis J, Maillot O, Lesouhaitier O, Feuilloley MG et al (2017a) Structure, function and regulation of Pseudomonas aeruginosa porins. FEMS Microbiol Rev 41(5):698–722 [DOI] [PubMed] [Google Scholar]
  6. Chevalier S, Bouffartigues E, Bodilis J, Maillot O, Lesouhaitier O, Feuilloley MGJ et al (2017b) Structure, function and regulation of Pseudomonas aeruginosa porins. FEMS Microbiol Rev 41(5):698–722 [DOI] [PubMed] [Google Scholar]
  7. Christodoulou E, Kechagia I-A, Tzimas S, Balafas E, Kostomitsopoulos N, Archontaki H et al (2015) Serum and tissue pharmacokinetics of silibinin after per os and iv administration to mice as a HP-β-CD lyophilized product. Int J Pharm 493(1–2):366–373 [DOI] [PubMed] [Google Scholar]
  8. Dey N, Kamatchi C, Vickram AS, Anbarasu K, Thanigaivel S, Palanivelu J et al (2022) Role of nanomaterials in deactivating multiple drug resistance efflux pumps—a review. Environ Res 204:111968 [DOI] [PubMed] [Google Scholar]
  9. El-Sapagh S, Allam NG, El-Sayed MNE-D, El-Hefnawy AA, Korbecka-Glinka G, Shala AY. Effects of Silybum marianum L. Seed Extracts on Multi Drug Resistant (MDR) Bacteria. Molecules. 2023;29(1):64. [DOI] [PMC free article] [PubMed]
  10. Evren E, Yurtcu E (2015) In vitro effects on biofilm viability and antibacterial and antiadherent activities of silymarin. Folia Microbiol 60:351–356 [DOI] [PubMed] [Google Scholar]
  11. Fallah M, Davoodvandi A, Nikmanzar S, Aghili S, Mirazimi SMA, Aschner M et al (2021) Silymarin (milk thistle extract) as a therapeutic agent in gastrointestinal cancer. Biomed Pharmacother 142:112024 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Farouk SM, Abu-Hussien SH, Abd-Elhalim BT, Mohamed RM, Arabe NM, Hussain AAT et al (2023) Biosynthesis and characterization of silver nanoparticles from Punica granatum (pomegranate) peel waste and its application to inhibit foodborne pathogens. Sci Rep 13(1):19469 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fekri Kohan S, Nouhi Kararoudi A, Bazgosha M, Adelifar S, Hafezolghorani Esfahani A, Ghaderi Barmi F, et al. Determining the potential targets of silybin by molecular docking and its antibacterial functions on efflux pumps and porins in uropathogenic E. coli. International Microbiology. 2024. [DOI] [PubMed]
  14. Fernández-Billón M, Llambías-Cabot AE, Jordana-Lluch E, Oliver A, Macià MD (2023) Mechanisms of antibiotic resistance in Pseudomonas aeruginosa biofilms. Biofilm 5:100129 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ferreres G, Ivanova K, Ivanov I, Tzanov T. Nanomaterials and Coatings for Managing Antibiotic-Resistant Biofilms. Antibiotics (Basel). 2023;12(2). [DOI] [PMC free article] [PubMed]
  16. Gomaa HH, Amin DY, Ahmed AR, Ismail NA, El Dougdoug KA, Abd-Elhalim BT. Antimicrobial, antibiofilm, and antiviral investigations using egyptian phoenix dactylifera L. pits extract. AMB Express. 2024;14(1):44. [DOI] [PMC free article] [PubMed]
  17. Holasová K, Křížkovská B, Hoang L, Dobiasova S, Lipov J, Macek T et al (2022) Flavonolignans from silymarin modulate antibiotic resistance and virulence in Staphylococcus aureus. Biomed Pharmacother 149:112806 [DOI] [PubMed] [Google Scholar]
  18. Hossainzadeh S, Ranji N, Naderi Sohi A, Najafi F (2019) Silibinin encapsulation in polymersome: a promising anticancer nanoparticle for inducing apoptosis and decreasing the expression level of miR-125b/miR-182 in human breast cancer cells. J Cell Physiol 234(12):22285–22298 [DOI] [PubMed] [Google Scholar]
  19. Hurtová M, Káňová K, Dobiasová S, Holasová K, Čáková D, Hoang L et al (2022) Selectively halogenated flavonolignans—preparation and antibacterial activity. Int J Mol Sci 23(23):15121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kang HK, Kim HY, Cha JD (2011) Synergistic effects between silibinin and antibiotics on methicillin-resistant Staphylococcus aureus isolated from clinical specimens. Biotechnol J 6(11):1397–1408 [DOI] [PubMed] [Google Scholar]
  21. Khakinezhad Tehrani F, Ranji N, Kouhkan F, Hosseinzadeh S (2020) Apoptosis induction and proliferation inhibition by silibinin encapsulated in nanoparticles in MIA PaCa-2 cancer cells and deregulation of some miRNAs. Iran J Basic Med Sci 23(4):469–482 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lahlah ZF, Meziani M, Maza A (2012) Silymarin natural antimicrobial agent extracted from Silybum marianum. J Acad 2:164–169 [Google Scholar]
  23. Lee YS, Jang KA, Cha JD (2012) Synergistic antibacterial effect between silibinin and antibiotics in oral bacteria. J Biomed Biotechnol 2012:618081 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lee J-J, Jeong KJ, Hashimoto M, Kwon AH, Rwei A, Shankarappa SA et al (2014) Synthetic ligand-coated magnetic nanoparticles for microfluidic bacterial separation from blood. Nano Lett 14(1):1–5 [DOI] [PubMed] [Google Scholar]
  25. Lee Y-S, Jang K, Cha J-D. Synergistic antibacterial effect between silibinin and antibiotics in oral bacteria. BioMed Research International. 2012;2012.
  26. Madhavi, Kumar M, Ansari JR, Kumar V, Nagar S, Sharma A. Fe3O4 Coated SiO2 Magnetic Nanoparticles for Enhanced Antibacterial Activity and Electrochemical Sensing. Metals. 2022;12(12):2145.
  27. Makled AF, Mohamed SA, Teima AAZ, Mohammed Nabil Shaban D, Dawoud AM. Effect of silver nanoparticles on biofilm forming Pseudomonas aeruginosa Isolates from Menoufia University Hospitals. Microbes and Infectious Diseases. 2023;4(4):1355–68.
  28. Mohamed SH, Othman BA, Abd-Elhalim BT, Seada MNA (2024) Copper nanoparticles biosynthesis by Priestia megaterium and its application as antibacterial and antitumor agents. Sci Rep 14(1):23615 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mouraki A, Sanjabi S, Mahdavian AR (2018) Magnetic polymeric composites and gels containing magnetite nanoparticles. Basparesh 8(3):3–13 [Google Scholar]
  30. Murray JL, Kwon T, Marcotte EM, Whiteley M. Intrinsic antimicrobial resistance determinants in the superbug Pseudomonas aeruginosa. MBio. 2015;6(6):10.1128/mbio. 01603–15. [DOI] [PMC free article] [PubMed]
  31. Neidig A, Strempel N, Waeber NB, Nizer WSdC, Overhage J. Knock‐out of multidrug efflux pump MexXY‐OprM results in increased susceptibility to antimicrobial peptides in Pseudomonas aeruginosa. Microbiology and Immunology. 2023;67(9):422–7. [DOI] [PubMed]
  32. Nikpassand M, Fekri ZL, Karimian L, Rassa M (2015) Synthesis of biscoumarin derivatives using nanoparticle Fe3O4 as an efficient reusable heterogeneous catalyst in aqueous media and their antimicrobial activity. Curr Org Synth 12(3):358–362 [Google Scholar]
  33. Nikpassand M, Fekri LZ, Nabatzadeh M (2017) Fe3O4@SiO2@KIT-6 as an efficient and reusable catalyst for the synthesis of novel derivatives of 3,3’-((Aryl-1-phenyl-1H-pyrazol-4- yl)methylene)bis (1H-indole). Comb Chem High Throughput Screen 20(6):533–538 [DOI] [PubMed] [Google Scholar]
  34. Nikpassand M, Fekri LZ, Varma RS, Hassanzadi L, Pashaki FS (2021) Green synthesis of novel 5-amino-bispyrazole-4-carbonitriles using a recyclable Fe(3)O(4)@SiO(2)@vanillin@thioglycolic acid nano-catalyst. RSC Adv 12(2):834–844 [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nikpassand M, Keyhani A, Fekri LZ, Varma RS (2022) Mechanochemical synthesis of azo-linked 2-amino-4H-chromene derivatives using Fe3O4@SiO2@KIT-6-NH2@Schiff-base complex nanoparticles. J Mol Struct 1251:132065 [Google Scholar]
  36. Nikpassand M, Rafat F, Zare FL (2024) Greener Synthesis of Novel Azo-linked 1,2,4-Triazolidine-3-ones (thiones) using recyclable Fe3O4@SP@vanillin@thioglycolic acid magnetic nanocomposite. Org Prep Proced Int 56(3):234–242 [Google Scholar]
  37. Parham S, Kharazi AZ, Bakhsheshi-Rad HR, Nur H, Ismail AF, Sharif S et al (2020) Antioxidant, antimicrobial and antiviral properties of herbal materials. Antioxidants 9(12):1309 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Polyak SJ, Ferenci P, Pawlotsky JM (2013) Hepatoprotective and antiviral functions of silymarin components in hepatitis C virus infection. Hepatology 57(3):1262–1271 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Pourasgar S, Ranji N, Asadpour L, Shahriarinour M, Nikpassand M. Antibacterial and Anti-cancer Properties of Curcumin-Functionalized Silica-Coated Fe3O4 Magnetic Nanoparticles. Arabian Journal for Science and Engineering. 2024.
  40. Pourgholi A, Dadashpour M, Mousapour A, Firouzi Amandi A, Zarghami N (2021) Anticancer potential of silibinin loaded polymeric nanoparticles against breast cancer cells: insight into the apoptotic genes targets. Asian Pac J Cancer Prev 22(8):2587–2596 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rahbar Takrami S, Ranji N, Sadeghizadeh M (2019) Antibacterial effects of curcumin encapsulated in nanoparticles on clinical isolates of Pseudomonas aeruginosa through downregulation of efflux pumps. Mol Biol Rep 46(2):2395–2404 [DOI] [PubMed] [Google Scholar]
  42. Rao X, Huang X, Zhou Z, Lin X (2013) An improvement of the 2ˆ(-delta delta CT) method for quantitative real-time polymerase chain reaction data analysis. Biostat Bioinforma Biomath 3(3):71–85 [PMC free article] [PubMed] [Google Scholar]
  43. Reed D, Raina K, Agarwal R. Anti-cancer Effects of silibinin: the current status in cancer chemoprevention. natural products for cancer chemoprevention: Single Compounds and Combinations. 2020:161–208.
  44. Safarpoor M, Ghaedi M, Asfaram A, Yousefi-Nejad M, Javadian H, Khafri HZ et al (2018) Ultrasound-assisted extraction of antimicrobial compounds from Thymus daenensis and Silybum marianum: antimicrobial activity with and without the presence of natural silver nanoparticles. Ultrason Sonochem 42:76–83 [DOI] [PubMed] [Google Scholar]
  45. Salimi N, Mohammadi-Manesh E, Ahmadvand N, Danafar H, Ghiasvand S (2024) Curcumin-loaded by Fe3O4/GO and Fe3O4/ZnO/GO nanocomposites for drug delivery applications: synthesis, characterization and anticancer assessment. J Inorg Organomet Polym Mater 34(3):1256–1271 [Google Scholar]
  46. Shakeri A, Hazeri N, Vlizadeh J, Ghasemi A, Tavallaei FZ. Phytochemical screening, antimicrobial and antioxidant activities of Anabasis aphylla L. extracts. Kragujevac Journal of Science. 2012;34(34):71–8.
  47. Bijak M. Silybin, a Major Bioactive Component of Milk Thistle (Silybum marianum L. Gaernt.)—Chemistry, Bioavailability, and Metabolism. Molecules. 2017;22(11):1942. [DOI] [PMC free article] [PubMed]
  48. Sugumaran A, Sadhasivam J, Gawas P, Nutalapati V, Pandian R, Kumar PS (2022) Curcumin conjugated dextran coated Fe3O4 nanoparticles: cytotoxic effect on lung cancer cell line A549. Mater Sci Eng, B 286:116047 [Google Scholar]
  49. Tuon FF, Dantas LR, Suss PH, Tasca Ribeiro VS (2022) Pathogenesis of the Pseudomonas aeruginosa biofilm: a review. Pathogens 11(3):300 [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Vimberg V, Kuzma M, Stodulkova E, Novak P, Bednarova L, Sulc M et al (2015) Hydnocarpin-type Flavonolignans: semisynthesis and inhibitory effects on Staphylococcus aureus biofilm formation. J Nat Prod 78(8):2095–2103 [DOI] [PubMed] [Google Scholar]
  51. Wagoner J, Negash A, Kane OJ, Martinez LE, Nahmias Y, Bourne N et al (2010) Multiple effects of silymarin on the hepatitis C virus lifecycle. Hepatology 51(6):1912–1921 [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wang D, Xie K, Zou D, Meng M, Xie M (2018) Inhibitory effects of silybin on the efflux pump of methicillin-resistant Staphylococcus aureus. Mol Med Rep 18(1):827–833 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zainin N, Lau K, Zakaria M, Son R, Razis AA, Rukayadi Y. Antibacterial activity of Boesenbergia rotunda (L.) Mansf. A. extract against Escherichia coli. International Food Research Journal. 2013;20(6):3319.

Articles from 3 Biotech are provided here courtesy of Springer

RESOURCES