Efficient synthesis of aqueous soluble Silymarin nanocrystals from Silybum marianum and assessment of their antibacterial and cytotoxicity insights

Silybum marianum, generally named Milk Thistle, is a botanical species belonging to the genus Leucanthemum¹. Silybum marianum, as an herb, facilitates the process of detoxification². Silymarin represents an extract derived from the seeds of Milk Thistle, which are known to contain a suite of biologically active flavonolignan compounds. The primary isomeric forms of flavonolignan in silymarin include silybin, isosilybin, silydianin, and silychristin, with silybin being the most pharmacologically potent. Because of its structure, silybin is a tiny, highly functionalized molecule resistant to reduction. Silybin comprises five hydroxyl functional groups, which are categorized based on their chemical characteristics into phenolic, secondary, and primary alcoholic groups³. The lipid composition of Silymarin is characterized by critical phospholipids that exhibit a substantial concentration of unsaturated fatty acids, including oleic, linoleic, and palmitic acids⁴. The lipid component additionally encompasses α-tocopherol, which modulates signal transduction pathways, gene expression, and cellular functionality to mitigate the likelihood of oncogenesis⁵. Silymarin possesses considerable pharmaceutical attributes encompassing antioxidant, anti-inflammatory, anti-fibrotic, and immune system-modulating activities⁶. However, because of its significant phase II metabolism, limited permeability across intestinal epithelial cells, low water solubility, and quick excretion in bile and urine, it has poor oral bioavailability^7,8. Therefore, it becomes necessary to introduce a robust formulation to overcome these challenges.

In the past, different nanotechnology-based approaches have been employed to enhance the bioavailability and solubility of different phytochemicals, and also offer robust formulation against environmental, chemical, and physical degradation^9,10. When compared to traditional biomaterials, nanostructured biomaterials offer several benefits, including their eco-friendly and cost-effective properties, enhanced bioavailability, better cellular interactions, and tailored functionalities¹¹. They are distinguished by their nanoscale architecture and size^12,13,14. Nanocrystals possess a robust, typically crystalline drug core within the nanometer dimension spectrum, accompanied by a stabilizing outer layer. NCs are frequently synthesized in aqueous solutions or non-aqueous solvent systems that are stabilized through the use of surfactants, polymers, or a combination of both. NCs improve the solubility and bioavailability of poorly soluble drugs and have several other benefits, including sustained release action, dose reduction, and tissue targeting¹⁵. Thus, the formation of silymarin NCs could enhance silymarin’s solubility and boost its herbal properties.

Improper antibiotic consumption has resulted in the development of multidrug-resistant (MDR) bacteria. Bacterial resistance, antibiotic complications, and the lack of new antibiotic agents necessitate achieving a new, efficient, alternative, and safe antibacterial drug¹⁶. Many studies have been done to investigate silymarin’s antibacterial effects. El-Sapagh et al. conducted a comprehensive evaluation of the efficacy of various extracts derived from S.marianum seeds in combating multidrug-resistant (MDR) bacteria associated with wound infections. Their findings indicated that the ethanol extract of S. marianum seeds exhibited a significant bacteriostatic performance against both gram-positive and gram-negative bacterial strains¹⁶. According to de Oliviera et al. assessment of silymarin and silybin’s antibacterial activity, silibinin exhibited notable activity against E. coli, with an MIC of 64 µg/mL (minimum inhibitory concentration). These substances show promising performance for working in concert with antibiotics¹⁷. As a result, silymarin NCs may be a natural alternative to conventional antibiotic medications, enhancing the antibacterial properties of silymarin.

Furthermore, silybin has demonstrated significant antitumor properties, as evidenced by not only the reduction of tumor cell adhesion, migration, intracellular glutathione (GSH) concentrations, and total antioxidant capacity (T-AOC) but also by the enhancement of the apoptotic index, caspase that silymarin has a strong anticancer impact while also protecting normal cells¹⁸. So far, no reported research has been conducted to prepare aqueous soluble silymarin nanocrystals using solvent evaporation and evaluate their antibacterial and cytotoxicity effects. Therefore, this study aims to prepare silymarin NCs and characterize them through several evaluations, including XRD, FESEM/TEM, EDX, FT-IR, UV-Vis, and lastly assess their antibacterial performance and cytotoxicity effects on the MDA-MB-231 cell line.

Material

The bacterial strains used in this research comprise Escherichia coli, classified as gram-negative, and Staphylococcus aureus, which is categorized as gram-positive, were prepared by the infectious diseases department of the hospital, whereas the MDA-MB-231 cell line was acquired from the Cell Bank located in Tehran, Iran. Silybum marianum powder was obtained from the local market.

Characterization

The crystal nature of the silymarin NCs was determined through XRD patterns (D8 ADVANCE, Germany). Also, the morphology and size of silymarin NCs were assessed through FESEM (HITACHI, Japan) and TEM (LEO 910 Model, ZEISS, Germany) images. The purity of silymarin NCs was evaluated via EDX/Mapping (JCM electron microscope operating at 10 kV). Moreover, FT-IR analysis (Shimadzu) was utilized to identify the functional groups existing in the silymarin NCs. The optical properties of silymarin NCs were evaluated using UV-Vis analysis (CE 9500, CECIL, England).

Preparation of silymarin nanocrystals

To prepare silymarin nanocrystals, first, 0.5 g of Silybum marianum (fruits) powder was dissolved in 10 mL of acetone. Then the resulting solution was stirred for 20 min at room temperature until the powder was completely dissolved in acetone. In continuation, 25 mL of hexane was added dropwise to the above solution, and the resulting mixture was stirred for 30 min at 25 °C. The resulting solution became almost two-phase (light brown phase at the bottom and colorless phase at the top). In the next step, the solvent was removed with a rotary evaporator device¹⁹, and brown aqueous soluble silymarin NCs were obtained. The synthesis schematic of silymarin NCs is shown in Fig. 1.

Synthesis of silymarin nanocrystals.

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Assessment of antibacterial activity

The antibacterial properties of silymarin NCs were assessed using the diffusion agar methodology against Escherichia coli, classified as gram-negative bacteria, and Staphylococcus aureus, which is categorized as gram-positive^20,21. Initially, 1 mg of silymarin NCs was agitated in 1 mL of sterile distilled water to establish a homogenous solution.

Furthermore, to assess the antibacterial attributes of silymarin NCs against Escherichia coli and Staphylococcus aureus strains, an appropriate amount of bacteria was cultivated for 24 h in a broth medium, and the resultant suspension was calibrated to a 0.5 McFarland standard²². A sterilized swab was then placed into the bacterial suspension and applied to the surface of the agar plate²³. Following this, 100 µL aliquots of silymarin NCs were administered into the wells formed within the agar plates. Ultimately, the dimension of the inhibition zone was quantified after a 24-hour incubation period. The antibiotics kanamycin served as positive controls, while sterile distilled water acted as the negative control. In conclusion, the antibacterial efficacy of silymarin NCs was interpreted.

Cytotoxicity

Cell culture

In vitro, the cytotoxicity of silymarin NCs was assessed by employing the MTT test in conjunction with the MDA-MB-231 cell line. Dulbecco’s Modified Eagle Medium (DMEM), which contained 10% fetal bovine serum (FBS) and 1% streptomycin and penicillin, was employed as the culture medium for the MDA-MB-231 line²⁴.

MTT test

The cellular density was established at 5000 cells for the cancer MDA-MB-231 line within each well of 96-well plates, which underwent incubation for 24 h under conditions of 5% CO₂, and 95% humidity at 37 °C. Cell suspensions were subsequently amalgamated with varying concentrations of silymarin NCs (0–1000 µg/mL). Following this, 100 µL of the resulting sample mixture was carefully dispensed into each well of the 96-well plate, which was maintained in a 95% humidity, 5% CO₂ environment and incubated at 37 °C for a total duration of 24 h.

In the subsequent step, 40 µL of MTT (aq.) was supplemented to each well, and the mixture was incubated for a further 4 h. Ultimately, 80 µL of DMSO was introduced into each well, and the absorbance was measured using a plate reader at a wavelength of 570 nm²⁵. The viability (%) of the silymarin NCs was calculated utilizing Eq. (1). The MTT test was deployed to evaluate cell viability in each well after intervals of 24 h. Each assay was conducted in triplicate.

Statistical section

Statistical investigation of this study was completed by the application of IBMSPSS^® and Prism^® software of variance (ANOVA), while the p-value < 0.0001 was determined significant.

UV–Vis/bandgap

The optical identification of produced silymarin NCs was performed utilizing UV–Vis spectrophotometry in the range of 200–800 nm. UV–Vis spectra of silymarin NCs demonstrated absorbance bands at two wavelengths of 230 nm and 285 nm (Fig. 2a), which are ascribed to its flavonoid components²⁶. The linear extrapolation technique was utilized to ascertain the bandgap of silymarin NCs. This methodology entails the analysis of the correlation between (αhν)² and hν, as a derivative from Eq. (2) (Tauc equation)²⁷, where α denotes the absorption coefficient, hν, and Eg signifies the photon and band gap energies, respectively. The band gap energy for silymarin NCs was determined to be 3.7 eV (Fig. 2b).

UV–Vis a chart and bandgap energy, b of silymarin NCs.

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XRD

The structure and nature of the silymarin NCs were analyzed using the X-ray diffractometry technique. The XRD pattern underwent assessment at a 2theta range extending from 10 to 80°¹⁰. As shown in Fig. 3, the XRD pattern of the prepared silymarin NCs exhibited a singular broad peak at 2θ = 21.32°, indicating that the silymarin nanocrystals possess an amorphous phase²⁸.

XRD pattern of silymarin NCs.

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FT-IR

FT-IR analysis determined the structural attributes and functional groups of silymarin and silymarin NCs. FTIR spectra were acquired in the range of 4000–400 cm⁻¹, and the resulting graphical representations are displayed in Fig. 4. For bioactive materials authentication, the initial step involved the analysis of the silymarin spectrum. This spectrum revealed extensive bands at 3452 cm⁻¹ (indicative of O–H stretching), 2925 cm⁻¹ (CH group), 1639 cm⁻¹ (representing –C=O stretching associated with reactive flavonolignan), 1511 cm⁻¹ (reflecting the stretching vibration of the aromatic C=C ring), 1365 cm⁻¹ (corresponding to –C–C stretching), 1271 cm⁻¹ (of polyols C–O stretching), 1083 cm⁻¹ (demonstrating the stretching of the benzopyran ring), 1032 cm⁻¹ (related to the stretching of the C–O group), and 824 cm⁻¹ (indicative of out-of-plane –C–H bending of the alkene). The silymarin spectrum was congruent with existing literature^29,30. The FTIR spectra of silymarin NCs and silymarin powder resemble each other, suggesting that silymarin was successfully incorporated into the formulation.

FT-IR spectra of Silymarin and Silymarin NCs.

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Mapping/EDX/FESEM

As illustrated in Fig. 5a–c, the silymarin NCs exhibited a spherical morphology. At the same time, the elemental constituents, including carbon (C), oxygen (O), and nitrogen (N), were approved via EDX analysis (Fig. 5g). Furthermore, the use of Mapping images substantiated the elements’ uniform and homogeneous distribution in synthesized silymarin NCs (Fig. 5d–f).

FESEM (a–c), mapping (d–f), and EDX (g) of silymarin NCs.

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TEM

The morphological characteristics and dimensions of silymarin NCs were assessed utilizing TEM and PSA analyses. The TEM imagery (Fig. 6a) of silymarin NCs revealed a predominantly spherical morphology³¹. The PSA histogram (Fig. 6b) of silymarin NCs indicated a mean size of approximately 23.14 nm.

TEM (a) and PSA of silymarin NCs (b).

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Antibacterial evaluation of silymarin nanocrystals

The agar diffusion approach was utilized to assess the antibacterial potency of silymarin NCs. The study focused on Escherichia coli as gram-negative bacteria and Staphylococcus aureus as gram-positive bacteria. The formation of inhibition zones demonstrated the antibacterial efficacy of silymarin NCs in comparison with the kanamycin antibiotic. The diameters of the inhibition zones are exhibited in Table 1, while the result of the antibacterial efficacy of silymarin NCs is illustrated in Fig. 7. While there hasn’t been a definitive mechanism provided for the antibacterial properties of silymarin NCs, one of the methods employed by nanoparticles to eliminate pathogens is the generation of ROS and free radicals³².

Antibacterial efficacy of silymarin NCs.

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Cytotoxicity evaluation of silymarin nanocrystals

The cytotoxicity of silymarin NCs (0–1000 µg/mL) was assessed on a cancerous MDA-MB-231 cell line for 24 h using an MTT assay (Fig. 8). The findings indicated that silymarin NCs exhibited minimal toxicity toward the MDA-MB-231 cells. Furthermore, the IC₅₀ value of silymarin NCs was determined to be 420.3 µg/mL. The findings demonstrated a concentration-dependent inhibition on the viability of MDA-MB-231 cells. This implies that silymarin NCs possess a relative safety profile for potential biological applications.

Cytotoxicity efficacy of silymarin NCs.

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In the present investigation, our foremost aim was to realize the eco-friendly synthesis of aqueous soluble silymarin NCs. The characterization of the silymarin NCs was performed through a comprehensive suite of analyses, including XRD, FESEM/TEM, EDX, FT-IR, and UV–Vis. The XRD analysis of NCs indicated the presence of amorphous structures. The spherical shape of the silymarin NCs was substantiated by the TEM and FESEM images, while the TEM analysis of silymarin NCs exhibited a homogeneous distribution via a size of about 23.14 nm.

FTIR spectra confirmed the occurrence of distinct functional groups in both silymarin and its nanocrystals. Additionally, UV–Vis spectrophotometry of silymarin NCs revealed significant absorption bands within the wavelength range of 230 to 285 nm. Silymarin NCs exhibited significant cytotoxicity effects on the cancerous MDA-MB-231 cell line (IC₅₀ = 420.3 µg/mL). Furthermore, it became apparent that silymarin NCs possessed noteworthy antibacterial activity. They revealed superior efficacy against harmful strains such as Staphylococcus aureus (ATCC) and Escherichia coli (ATCC) compared to clinical strains. Collectively, these findings emphasize the potential of silymarin NCs and elucidate the promising applications of these nanomaterials within biomedical sciences. In conclusion, future research could examine the potential modifications to the nanocrystal formulation that could enhance its antibacterial qualities and lessen its cytotoxicity. To completely comprehend the therapeutic potential and safety profile of these NCs in clinical applications, in vivo research will also be essential.

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Authors and Affiliations

1. Department of Medical Biotechnology and Nanotechnology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

   Iman Sirvani, Zahra Sabouri & Asma Mostafapour

2. Student Research Committee, Mashhad University of Medical Sciences, Mashhad, Iran

   Zahra Sabouri

3. Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran

   Majid Darroudi

Contributions

All authors have contributed to the manuscript and have agreed on the final versionIman Sirvani: Data acquisition, Analysis, Methodology, Investigation, Software, and writing an original draft. Zahra Sabouri: Data acquisition, Analysis, Methodology, Investigation, Software, and Writing – Review & Editing. Asma Mostafapour: Data acquisition, Analysis, and Writing – Review & Editing. Majid Darroudi: Supervision, Project administration, Funding, Methodology, Resources, and Review & Editing.

Corresponding author

Correspondence to Majid Darroudi.

Competing interests

The authors declare no competing interests.

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