Preparation of silymarin-loaded zein polysaccharide core–shell nanostructures and evaluation of their biological potentials

2.3.1 Analysis by gas chromatography–mass spectrometry (GC–MS)

For active constituent’s detection, 1 µl of extracted oil was injected into the Thermo Scientific GC Focus Series DSQ model within the GC–MS instrument (Shimadzu-Lab) and undergone analysis utilizing a column with dimensions of 0.1 mm diameter, 0.5 μm film thickness, and 50 m length, using helium as the carrier gas flowing at a rate of 1 ml·min⁻¹. The column temperature ranged from 80°C to 310°C at a rate of 10°C·min⁻¹, facilitating the separation of silymarin constituents based on their volatility and interaction with the stationary phase, composed of a polar-embedded material polyethylene glycol (PEG).

2.3.2 Fourier transform infrared (FT-IR) analysis

FT-IR analysis was performed via IRTracer-100, Shimadzu, Japan, instrument to assess functional groups present in extracted oils at variable IR spectra, i.e., 400–45,000 cm⁻¹. The analysis was performed utilizing a specific attenuated total reflectance (ATR) acquisition mode due to its suitability for analyzing oils directly without extensive sample preparation.

2.3.3 Extraction of silymarin

For silymarin extraction from both pink and white flowering S. marianum seeds, 70 g of dried powder of S. marianum seeds were dissolved in 120 ml of 70% ethanol in a blue-capped bottle. The solution was kept at room temperature for 48 h and then filtered into a flask using filter paper. Moreover, 10% (w/v) of NaCl solution was prepared and added to the ethanolic extract drop wise with continuous stirring till precipitation of silymarin occurred. Following that, the resulting precipitate was washed with distilled water to remove residual NaCl and impurities. The precipitates were collected by passing through filter paper, dried, and stored in a suitable container at 25°C for further use.

2.4.1 Preparation of pectin/sodium alginate solution

Aqueous solutions (0.2% w/v) of pectin alginate were prepared by mixing pectin and sodium alginate in different ratios, i.e., (100–0), (70–30), (50–50), (30–70), and (0–100). For proper dissolution, the solutions were stirred at 700 rpm using a magnetic stirrer for 30 min at 70°C. The solutions were then cooled in tap water and stirred for another hour. Non-soluble particles were removed from the solutions through filtration and then pH was adjusted at 4.0 with 1 mol·l⁻¹ of HCL.

2.4.2 Preparation of silymarin-loaded zein-pectin/alginate core–shell NPs

Silymarin-loaded NPs were prepared following the literature method, specifically pH-induced antisolvent precipitation, with slight modification [29]. Initially, a silymarin–zein stock solution was prepared. About 20.0 ml of this stock solution was mixed with 80.0 ml of acidified water (pH 4.0) under constant stirring at 900 rpm for 3 min in the dark. This step aimed to disperse the silymarin–zein solution uniformly in the aqueous medium. The resultant dispersion solution underwent ethanol evaporation using a rotary evaporator (Model RE-2000A, Yarong Bio-instrument, Shanghai, China). This process removed ethanol from the solution, leaving behind a mixture of silymarin and zein in the aqueous phase. Following ethanol evaporation, the remaining solution was adjusted to a final volume of 100 ml by adding more acidified water (pH 4.0) with constant stirring at 900 rpm for 3 min in the dark. This step ensured the desired concentration and pH for subsequent reactions. The synthesis of the core–shell NPs involved combining portions of this solution with pectin–alginate solutions to form the desired silymarin-loaded zein–polysaccharide core–shell NPs.

2.5.1 Characterization by transmission electron microscope (TEM) and scanning electron microscope (SEM)

The size and morphology of SZPCS-NPs were analyzed via TEM (FEI Techni, G2, 300 kV) and SEM (Hitachi-s 3400 N, Japan). A thin film of test solution was prepared on the carbon-coated copper grid, which was then vaporized using a mercuric vapor lamp for 5 min. Finally, a 2-D micrograph of the test NPs manifesting their size was observed through TEM. Furthermore, a sample of SZPCS-NPs was placed on a copper-coated grid and allowed to dry. After drying, the SEM was operated at a high voltage of 100 kV. SEM images of prepared SZPCS-NPs, which were prepared in different concentrations of pectin and sodium alginate solution, were depicted.

2.5.2 Characterization by X-ray diffractometer (XRD)

The nature of SZPCS-NPs was analyzed through an XRD (Jeol JDX-3532, Japan), which contains an Ni filter and CuKα as a source of monochromatic radiation at 1.5418 A° wavelength. The machine was operated at 40 kV voltage and 30 mA current. For scanning, 2θ/θ range was selected, and for precise determination, the scanning speed of 10 min⁻¹ was used.

2.5.3 Characterization by FT-IR

For the identification of functional groups, the SZPCS-NPs were subjected to infrared spectroscopic analysis. Infrared spectrometer Prestige FT-IR spectrophotometer (IRTracer-100, Shimadzu, Japan) was used for this purpose. The wavelength used for analysis was set in the 4,000–600 cm⁻¹ range.

2.5.4 Particle size and charge measurements

The zeta potential of the particle size distribution of the colloidal dispersions was measured by dynamic light scattering using a commercial instrument (Malvern Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, UK). The Z-average diameter and polydispersity index (PDI) were calculated from the light scattering measurements. Each sample was analyzed in duplicate. The electrical properties (ζ-potential) of the particles in the colloidal dispersions were characterized using a micro-electrophoresis device (Malvern Zetasizer Nano-ZS, Malvern Instruments, Worcestershire, UK). Colloidal dispersions were diluted with 5 mM phosphate-citric acid buffers at the same pH as the samples prior to analysis to avoid multiple scattering effects. Each sample was analyzed in duplicate.

2.6.1 2,2-Diphenyl-1-picrylhydrazyl (DPPH) scavenging activity

The antioxidant activity of SZPCS-NPs was evaluated using literature with slight modifications [30]. For this purpose, differently concentrated ethanolic solutions of the encapsulated silymarin NPs as well as of pure silymarin were prepared. Then, 100 μM DPPH solutions in ethanol were prepared. From the DPPH–ethanol solution, 2 ml were mixed with equivalent volumes of the differently concentrated ethanolic solutions of encapsulated silymarin and pure silymarin. The resulting solutions were subsequently incubated for 30 min in the dark at room temperature. After 30 min, the solutions were filtered and subjected to spectrophotometric absorbance at 517 nm. The free radical scavenging activity from the sample was calculated using Eq. 1:

where A _s and A _c are the absorbance of sample and control (ethanol) solutions, respectively. The scavenging activity (SC50) was used to compare the levels of free radical scavenging activity of different samples, which was taken to be the concentration of sample required to reduce the scavenging by 50%. The experiment was performed in triplicate.

2.6.2 ABTS^·+ scavenging capacity

The scavenging activity towards the ABTS˙⁺ radical was measured by modifying a previously used methodology. First, the ABTS˙⁺ radical was produced by the reaction of ABTS stock solution (7 mM) with 2.45 mM of potassium persulfate in darkness at room temperature for 16 h before use. Subsequently, radical ABTS˙⁺ solution was diluted with buffer (5 mM, pH 7.4) to adjust the absorbance to 0.700 ± 0.002 AU measured at 734 nm wavelength and 30°C. The mixtures in the 96-well microplate contained the following reagents: 250 µl of ABTS˙⁺ radical solution and 100 µl of S. marianum extract serially diluted from 0.014 to 0.0014 µg·ml⁻¹ in 50% aqueous EtOH was incubated at room temperature in the absence of the light for 45 min. The absorbance readings were measured at 734 nm and 30°C. Trolox was used as the reference standard. ABTS˙⁺ inhibition percentage from S. marianum extracts was calculated according to the formula:

where ABTS control is the absorbance of ABTS˙⁺ radical in buffer at t = 0 min; A sample is the absorbance of an ABTS˙⁺ solution mixed with extracts, and A sample blank is the absorbance of samples without ABTS˙⁺. Comparisons between extracts were done by IC₅₀ value representing the concentration.

2.7.1 Design of study

Adult rats of both sexes weighing 200–220 g were indiscriminately allocated into 5 equal groups (A–E), having 15 rats in each class. Each group was allocated to separate cages and labeled accordingly. Seven days of acclimatization were allowed. The therapeutic groups, denoted as Groups C, D, and E, received a dose of 200 mg·kg⁻¹ body weight of silymarin, a dosage selected based on a comprehensive review of literature and established practices in hepatoprotection research [31,32]. The rats were euthanized using the physical method of cervical dislocation. No anesthetic agents were employed in the euthanasia procedure [33].

Group A. Normal untreated control

Group B. Carbon tetrachloride (CCl₄)-treated control

Group C. Commercial silymarin

Group D. Crude silymarin

Group E. SZPCS-NPs

2.7.2 Biochemical assay of blood

To compare the hepatoprotective effects of crude silymarin, commercially available and SZPCS-NPs, blood biochemical tests were carried out every 2, 4, and 6 weeks. Rats were anesthetized, and 2 ml of blood was drawn by cardiac puncture from every experimental rat and transferred into labelled tubes. Samples were made to spin to separate as much serum as possible. The collected serum was kept at 4°C until it was time to use it for the standard test procedures for measuring the parameters of the liver function tests (LFTs).

2.7.3 Biochemistry of blood

Serum biochemical parameters, such as amino alanine transferase (ALT), serum alkaline phosphatase (ALP), serum albumin, total proteins, serum globulin, urea, and creatinine, were determined through commercially accessible kits of RANDOX.

1. Ethical approval: The research related to animals’ use has been complied with all the relevant national regulations and institutional policies for the care and use of animals.

3.7.1 TEM analysis

In this study, different ratios of alginate and pectin were used to form the shell around zein NPs to get uniform size and polydispersity. Figure 4a–e shows TEM images of SZPCS-NPs produced in various concentrations of pectin and sodium alginate solution. The dark areas in the photos represent the size of the NPs. According to the TEM study in Figure 4a, the size of silymarin NPs produced in a polysaccharide solution with a pectin ratio of 100% is uneven and not uniform in size ranges of 20–80 nm. Figure 4b demonstrates the best results, the shape of the created NPs was spherical and uniform in size, around 80 nm in diameter (average), and they were made in a 70% pectin and 30% alginate (P70–A30) solution. Furthermore, in Figure 4c, SZPCS-NPs were prepared in a ratio of 50% pectin and 50% sodium alginate solution, possessed an average diameter of 50 nm; in Figure 4d, SZPCS-NPs were prepared in P70–A30 solution possessed an average diameter of 70 nm; and in Figure 4e, the NPs had an irregular shape with an average diameter of 80 nm. Our results are similar to previous studies. Muzamil et al. determined the morphology for both NP1 and NP3. In the case of both NP1 and NP3, small rounded or spherical-shaped morphology with regular shapes was noticed. The particles were highly monodispersed [46]. Huang et al. also used a combination of alginate and pectin to form the shell around zein NPs. They concluded that the replacement of 30% of pectin with alginate greatly improved aggregation stability at pH 5–7. It was observed that the shape of the prepared NPs was spherical and uniform in size, about 80 nm in diameter [20].

Figure 4

TEM images of ZSPCS-NPs made with different ratios of pectin and alginate. (a) 50% pectin and 50% alginate solution, (b) 70% pectin and 30% alginate solution, (c) 30% pectin and 70% alginate solution, (d) 100% alginate solution, and (e) 100% pectin solution.

3.7.2 SEM analysis

Figure 5a–e shows the SEM images of prepared SZPCS-NPs, which were prepared in different concentrations of pectin and sodium alginate solution. The white spots indicate the size of NPs in the images. According to SEM analysis in Figure 5a, the size of SZPCS-NPs prepared in a polysaccharide solution with a pectin ratio of 100% is irregular and not uniform in size, ranging from 20 to 100 nm. While Figure 5b shows the best results, it was observed that the shape of the prepared SZPCS-NPs was spherical and uniform in size, about 80 nm in diameter, and that they were prepared in P70–A30 solution. Moreover, in Figure 5c, in which the SZPCS-NPs were prepared in a ratio of 50% pectin and 50% sodium alginate solution, in Figure 5d, where the SZPCS-NPs were prepared in P70–A30 solution, and in Figure 5e, where the SZPCS-NPs were synthesized in 100% sodium alginate solution, they also showed an irregular shape; some triangle and oval shapes were also observed; and in the above-mentioned figures, the NPs were not all of the same size, ranging from 20 to 100 nm. Paul and Yadav prepared silver NPs having 50 nm size. On SEM analysis, it was observed that the generated silver NPs were spherical in shape with a size ranging from 3 to 36 nm [49]. Silybum et al. synthesized the silymarin-loaded TiO₂ NPs having an average size of 20 nm. The size of our prepared SZPCS-NPs was a little larger than silymarin-loaded TiO₂ NPs [50]. While silibinin-loaded nanoparticles (SILNPs) prepared by Guhagarkar et al. were ranging from 60 to 70 nm [51], Sajadi et al. synthesized silymarin-loaded Cu/Fe₃O₄ NPs having spherical shape and size ranging from 8.5 to 60 nm [52]. Li et al. demonstrated that plain zein colloidal NPs showed a spherical shape and smooth surface with diameters ranging from 80 to 100 nm [49]. Abbasi et al. determined the size and surface morphology of the prepared ZnO-NPs, P-ZNPs, C-ZNPs, and S-ZNPs of S. marianum extract through SEM analysis. The average size of ZnO NPs evaluated from SEM analysis was 61.3, 51.7, 64, and 61.9 nm synthesized for W-ZNPs, P-ZNPs, C-ZNPs, and S-ZNPs, respectively [53]. The average size of silymarin-zein NPs (SMN-Zein) evaluated from SEM analysis was around 100 nm in diameter, having spherical and uniform size [54].

Figure 5

SEM images of ZSPCS-NPs made with different ratios of pectin and alginate. (a) 50% pectin and 50% alginate solution, (b) 70% pectin and 30% alginate solution, (c) 30% pectin and 70% alginate solution, (d) 100% alginate solution, and (e) 100% pectin solution.

3.7.3 XRD analysis

Powder XRD was used to verify the crystallinity and phase distribution of the synthesized SZPCS-NPs. Figure 6a–e represents the photograph of the XRD pattern. Sharp peaks for SZPCS-NPs (P100–A00) were recorded at 20.0, 25.0, and 40.0 (Figure 6a). For these NPs, the presence of distinct peaks at 20.0, 25.0, and 40.0 implies a particular crystalline structure. The peaks for SZPCS-NPs (P70–A30) were recorded at 30.0, 39.0, and 45.0, showing sharper peaks, although some additional peaks were also noted at 20.5, 21.0, and 39.0. Increased peak sharpness at 30.0, 39.0, and 45.0 indicates a distinct crystalline structure. Extra peaks at 20.5, 21.0, and 39.0 indicate possible differences in phase distribution as well as composition (Figure 6b). The peaks for SZPCS-NPs (P50–A50) were recorded at 25.0, 28.0, and 31.5 (Figure 6c). The peaks located at 25.0, 28.0, and 31.5 indicate a characteristic crystalline pattern; however, it may not be as prominent as in other compositions as mentioned above. The peaks for SZPCS-NPs (P30–A70) were recorded at 13.5, 20.0, 30.0, and 45.0. In contrast to other compositions, peaks at 13.5, 20.0, 30.0, and 45.0 imply a distinct crystalline structure, perhaps signifying changes in the composition or crystalline phases present (Figure 6d). Major peaks for SZPCS-NPs (P00–A100) were recorded at 29.5, 31.5, and 45.0. Prominent peaks at 29.5, 31.5, and 45.0 have a particular crystalline structure, which may suggest an alternate phase distribution in this structure (Figure 6e).

Figure 6

XRD spectra of ZSPCS-NPs made with different ratios of pectin and alginate. (a) 50% pectin and 50% alginate solution, (b) 70% pectin and 30% alginate solution, (c) 30% pectin and 70% alginate solution, (d) 100% alginate solution, and (e) 100% pectin solution.

P70–A30 exhibits sharper and extra peaks among the five different types of SZPCS-NPs, which may indicate a more complicated structure and composition. It might serve effectively in situations when a combination of multiple phases or enhanced properties is required. Given that, the goal of the present study was to improve water solubility and dispersibility of silymarin, the XRD pattern clearly indicated that P70–A30 provided the most favorable outcomes. Abbasi et al. studied the XRD pattern of S. marianum extract-loaded NPs, namely W-ZNPs, S-ZNPs, C-ZNPs, and P-ZNPs, peaks for W-ZNPs were recorded at 31.6, 34.2, 36.2, 47.3, 56.4, 62.7, 67.8, and 68.9, while peaks for S-ZNPs were recorded at 31.7, 34.3, 36.1, 47.4, 56.5, 62.8, 66.2, 67.9, 69.1, and 76.9. C-ZNPs showed peaks at 31.5, 34.2, 36.0, 47.3, 56.3, 62.5, 67.8, and 68.9. P-ZNPs showed peaks at 31.6, 34.3, 36.1, 47.3, 56.4, 62.7, 66.2, 67.8, 69.0, 72.4, and 76.8 [53]. The XRD pattern of Fe₃O₄ S. marianum NPs showed characteristic peaks at 2θ = 30.5, 35.8, 43.2, 53.7, 57.2, and 62.9 [55]. Similarly, the XRD pattern of silymarin-loaded Ag-NPs distinguished diffraction peaks at 2θ of 38.5° (111), 44.1° (200), 64.5° (220), and 77.3° (311) that strongly indicate the face-centered cubic crystalline structure of Ag-NPs [56].

3.7.4 FT-IR analysis

Figure 7a shows that the FT-IR spectrum of SZPCS-NPs displays strong absorption bands at 3,264.3, 2,923.0, 1,729.3, 1,610.4, 1,526.7, 1,195.1, 1,010.1, and 861.5 cm⁻¹. Figure 7b demonstrates that the FT-IR spectrum of SZPCS-NPs displays strong absorption bands at 2,997.6, 1,603.4, 1,492.2, 1,217.7, 1,017.1, 853.6, and 601.8 cm⁻¹. Figure 7c reveals that the FT-IR spectrum of SZPCS-NPs displays strong absorption bands at 2,923.0, 2,849.1, 1,595.6, 1,538.5, 1,195.7, 1,017.1, and 846.8 cm⁻¹. Figure 7d shows that the FT-IR spectrum of SZPCS-NPs displays strong absorption bands at 2,916.01, 2,856.9, 1,610.4, 1,484.44, 1,017.1, and 853.61 cm⁻¹. Figure 7e shows the FT-IR spectrum of SZPCS-NPs displays strong absorption bands at 3,220.0, 2,916.0, 2,856.9, 1,751.1, 1,603.4, 1,543.5, 1,239, 846.81, and 609.6 cm⁻¹.

Figure 7

Overlaped images of FTIR spectra of ZSPCS-NPs made with different ratios of pectin and alginate. (A) 50% pectin and 50% alginate solution, (B) 70% pectin and 30% alginate solution, (C) 30% pectin and 70% alginate solution, (D) 100% alginate solution, and (E) 100% pectin solution.

The FT-IR spectrum obtained for silymarin Zein (SMN-Zein) NPs showed characteristic peaks at 1,693 and 1,702 cm⁻¹ [57]. Our results were pretty much closure with that of Gopalakrishnan and Raghu, and the FT-IR spectrum of gold NPs of S. marianum extract prepared during their study displayed strong absorption bands at 3,416, 2,924, 1,630, 1,383, and 1,031 cm⁻¹ and the spectrum of silver NPs of the S. marianum extract displays strong bands at 3,421, 2,925, 1,654, 1,399, 1,102, and 706 cm⁻¹ [58]. Iqbal et al. determined that the FT-IR spectra of Cu NPs of S. marianum produced during polymerization exhibit peaks at 2,163 and 1,990 cm⁻¹. The peak at 1,004 cm⁻¹ may be associated with ether, pyranose ring, and glycosidic linkage, as demonstrated by the C–O(C–O–C) stretch. Cu–O stretching is observed by the peaks at the fingerprint areas at 518, 523, 528, and 542 cm⁻¹ [59]. In addition, Gopalakrishnan et al. determined the FT-IR analysis of S. marianum seed extract, which shows prominent peaks located at 3,415, 2,925, 1,654, 1,383, and 1,115 cm⁻¹. While S. marianum-based Au–Ag bimetallic nanocomposites exhibit robust absorbance bands at 3,423, 2,918, 1,603, 1,383, 1,105, and 824 cm⁻¹. Strong and wide bands at 3,415 and 3,423 cm⁻¹ are the stretching vibrations of the O–H group in alcohols and phenols that are intermolecular hydrogen bound. They can also be attributed to the stretching of the hydrogen-bonded N–H group of proteins [55]. For chemically synthesized S. marianum-based ZnO-NPs, sharp absorption peaks were seen at 3,220.63, 1,551.90, 1,415.86, 1,088.28, 1,033.43, 848.48, 776.23, 716.20, 620.72, and 610.06 cm⁻¹. For WPE-mediated ZnO-NPs, intense absorption peaks were seen at 3,244.37, 2,930.31, 1,416.63, 1,088.89, 1,036.02, 854.96, 778.42, 715.84, and 611.70 cm⁻¹ [60].

3.7.5 Analysis by thermal gravimetric analysis (TGA)

The thermal stability and mass loss of SZPCS-NPs were monitored using thermogravimetry differential thermal analysis in the temperature range of 0–800°C (Figure 8). The prepared NPs from selected plants were thermally stable ≤200°C; the loss in particle aptitude and mass was witnessed by a gradual increase in temperature (200–800°C), thus affirming it as a thermally sensitive compound.

Figure 8

Overlapped image of TG/DTA of ZSPCS-NPs made with different ratios of pectin and alginate. (A) 50% pectin and 50% alginate solution, (B) 70% pectin and 30% alginate solution, (C) 30% pectin and 70% alginate solution, (D) 100% alginate solution, and (E) 100% pectin solution.

The thermal stability of SZPCS-NPs (P100–A00) sample weight 6.5 mg was evaluated via TGA by subjecting the NPs to a temperature range of 0–800°C. Major weight loss was observed at 280–380°C, and loss of weight in this event was ∼75%.

In Figure 8, A–D represents the thermal stability of SZPCS-NPs. (A) represents the thermal stability of SZPCS-NPs (P70–A30) sample weight 6.5 mg was evaluated via TGA by subjecting the NPs to a temperature range 0–800°C. Major weight loss was observed at 220–320°C; the loss of weight in this event was ∼75%. (B) The thermal stability of SZPCS-NPs (P50–A50) sample weight 6.5 mg was evaluated via TGA by subjecting the NPs to a temperature range of 0–800°C. Major weight loss was observed at 200–310°C, which is attributed to the decomposition of the compound, while the loss of weight in this event was ∼70–78%. The minor decomposition occurred between 500°C and 700°C. (C) The thermal stability of SZPCS-NPs (P30–A70) sample weight 6.5 mg was evaluated via TGA by subjecting the NPs to a temperature range of 0–800°C. Initial weight loss was observed at 200–310°C, which is attributed to the loss of moisture, while the loss of weight in this event was ∼50%. The minor decomposition occurred between 500°C and 700°C. (D) The thermal stability of SZPCS-NPs (P0–A100) sample weight 6.5 mg was evaluated via TGA by subjecting the NPs to a temperature range of 0–800°C. Initial weight loss was observed at 200–400°C, which is attributed to the loss of moisture, while the loss of weight in this event was ∼70%. The second minor decomposition occurred between 500°C and 700°C. (E) A minor weight loss was observed for all SZPCS-NPs below 100°C, which is attributed to the loss of moisture.

According to these results, SZPCS-NPs maintained their thermal stability up to 160°C. Nonetheless, the loss of particle mass and aptitude increased gradually over 200°C. This means that the compound loses bulk and structural integrity when the temperature rises over 200–800°C since it becomes more sensitive to heat. P70–A30 maintains an appropriate balance between stability and decomposition temperatures. Many applications might benefit from this balance, particularly those that need stability in temperatures that are moderately high. According to the results, P70–A30 appears to be a promising option that maintains a balance between thermal stability and the beginning of decomposition. As such, it is a candidate that is worth researching further for certain applications where this balance is important. In the meanwhile, P30–A70 exhibits little degradation at higher temperatures and moderate stability with initial moisture loss (200–310°C). Even though P100–A00 loses weight at temperatures between 280°C and 380°C, it may still be taken into consideration if greater thermal stability is a top concern. P50–A50, on the contrary, decomposes at lower temperatures (200–310°C) and loses weight at a faster rate (70–78%), making it less suitable for applications that demand strong stability. Abbasi et al. also studied the thermal stability of the green synthesized silymarin NPs via TGA by subjecting the NPs to a temperature of 0–1,100°C. Weight loss was observed at 130–140°C [61].

3.7.6 Analysis of zeta potential

Figure 9a shows zeta potential of SZPCS-NPs (P100–A0). The zeta potential analysis of SZPCS-NPs (P100–A0) revealed a % intensity of 15> and a particle size ranging below 100 nm. Figure 9b shows zeta potential of SZPCS-NPs (P70–A30). The zeta potential analysis of SZPCS-NPs (P70–A30) revealed a % intensity of 15> and a particle size ranging from 90 to 100 nm. The observed zeta potential suggests a suitable surface charge for stability. Figure 9c shows the zeta potential of SZPCS-NPs (P50–A50). The zeta potential analysis of SZPCS-NPs (P50–A50) revealed an intensity of 16% and a particle size ranged from 100 nm. Figure 9d shows the zeta potential of SZPCS-NPs (P70–A30). The zeta potential analysis of SZPCS-NPs (P70–A30) revealed an intensity of the NPs of 17%, indicating the relative strength of their scattering signals. Furthermore, the size distribution of the NPs ranged from 100 nm. Figure 9e shows the zeta potential of SZPCS-NPs (P0–A100). The zeta potential analysis of SZPCS-NPs (P0–A100) revealed an intensity of the NPs of 17%, indicating the relative strength of their scattering signals. Furthermore, the size distribution of the NPs ranged from 100 nm. According to Mohammadi et al., the particle size of silymarin-loaded silver NPs was 1–25 nm, having a zeta potential of −24.2 mV [62]. According to Ma et al., the zeta potential analysis of silymarin-loaded self-assembled NPs of Bletilla striata polysaccharide revealed a % intensity of 12 > and a particle size ranging from 50 to 200 nm [63]. The average particle size of gold and silver NPs was found to be 120 and 64 nm, having a zeta potential distribution of −15.4 and −15.8, respectively [55].

Figure 9

Zeta potential of SZPCS-NPs made with different ratios of pectin and alginate. (a) 50% pectin and 50% alginate solution, (b) 70% pectin and 30% alginate solution, (c) 30% pectin and 70% alginate solution, (d) 100% alginate solution, and (e) 100% pectin solution.

Overall, based on the previous characterization results, SZPCS-NPs that had a 70% pectin/30% alginate (P70–A30) were selected for further assessments, including antioxidant activity and hepatoprotective activity. The SZPCS-NPs (P70–A30) demonstrated the best results; the shape of the created NPs was spherical and uniform in size, around 80 nm in diameter. The XRD analysis suggests that the P70–A30 formulation may have the best combination of crystallinity and phase distribution among the tested formulations, as it exhibited sharper peaks compared to P100–A00 and P50–A50. In addition, the FT-IR analysis demonstrated a prominent absorption band at 2,997.6 cm⁻¹, indicating C–H stretching vibrations. The band at 1,603.4 cm⁻¹ suggests the presence of C═C stretching vibrations. Peaks at 1,492.2, 1,217.7, 1,017.1, 853.6, and 601.8 cm⁻¹ may correspond to other functional groups.

3.7.7 % Drug loading and % entrapment efficiency

The data provided represent the percent drug loading and percent entrapment efficiency of silymarin into SZPCS-NPs at different time points, as derived from the percent absorbance measurements (Table 1).

After 10 min, the percent drug loading was found to be 54.67%, indicating the amount of silymarin loaded into SZPCS-NPs. Simultaneously, the percent entrapment efficiency was calculated to be 38.54%, representing the efficiency of trapping or encapsulating silymarin within the NPs at this time point. At 20 min, there was an increase in both percent drug loading and percent entrapment efficiency. The percent drug loading reached 65.43%, indicating a higher amount of silymarin incorporated into SZPCS-NPs. The percent entrapment efficiency also increased to 44.67%, suggesting an improved efficiency in trapping silymarin within the NPs.

The trend of increasing percent drug loading and percent entrapment efficiency continued at subsequent time points. After 30 min, the percent drug loading was 72.43%, while the percent entrapment efficiency was 51.78%. After 40 min, the values were 72.42% and 51.54% for percent drug loading and percent entrapment efficiency, respectively. At 50 min, the percent drug loading and percent entrapment efficiency were measured to be 72.55% and 51.46%, respectively. Similarly, at 60 min, the values were 72.46% for percent drug loading and 51.76% for percent entrapment efficiency. These findings indicate that the zein silymarin NPs effectively loaded silymarin, with the percent drug loading consistently increasing over time. The percent entrapment efficiency remained relatively stable throughout the studied time points, suggesting consistent trapping of silymarin within the NPs.

Overall, the results highlight the capability of SZPCS-NPs to efficiently load and entrap silymarin, offering a promising approach for the delivery and controlled release of silymarin in various applications. Our observations regarding the entrapment efficiency align with previous studies in the field. A study conducted by Gupta et al. investigated the loading and encapsulation efficiency of silymarin chitosan NPs. Entrapment efficiency of silymarin chitosan NPs was observed in the range of 52.68–85.28% [64]. In addition, solid lipid NPs of silymarin possessed % drug loading of 79.5–92.4% and entrapment efficiency of 83.9–92.3%, respectively [65].

Eqs. 3 and 4 were used to determine drug loading and encapsulation, efficiency, respectively.

4.1.1 DPPH scavenging capacity

The percent absorbance of commercially available silymarin, crude silymarin, and SZPCS-NPs at a sample concentration of 10 µl·ml⁻¹ was recorded at 39.2%, 37.8%, and 81.8%, respectively (Figure 10). Similarly, at sample concentration 20 µl·ml⁻¹, the % absorbance (517 nm) of commercially available silymarin, crude silymarin, and SZPCS-NPs were 43.4%, 41.7%, and 91.3%, correspondingly. In addition, at sample concentrations 30 and 60 µl·ml⁻¹, the % absorbance (517 nm) of commercially available silymarin, crude silymarin, and SZPCS-NPs were 42.2%, 39.9%, 92.1%, and 62.8%, 58.2%, and 96.3%, individually. Finally, at sample concentrations 70 and 80 µL·ml⁻¹, the % absorbance (517 nm) of commercially available silymarin, crude silymarin, and SZPCS-NPs were 66.3%, 63.4%, and 97.8% and 67.0%, 65.8%, and 98.8%, respectively. An aliquot of the samples was mixed with DPPH solution (5 ml, 23.6 μg·ml⁻¹ in ethanol), followed by incubation of 30 min. It can be concluded from the results that zein silymarin NPs consistently exhibit the highest percent absorbance at all sample concentrations. This indicates that the SZPCS-NPs possess higher solubility or better optical properties at the tested wavelength compared to the other forms of silymarin. In addition, SZPCS-NPs showed better antioxidant activity compared to both commercially available silymarin and crude silymarin. The absorbance of each sample was read at 517 nm [66]. Seeds of S. marianum with purple flower showed 62.67%, 74.02%, and 78.34% activity after 10, 20, and 30 min, respectively, while white flowering seeds exhibited 37.79%, 42.97%, and 49.18% absorbance activity, respectively [34]. DPPH free radical scavenging activity of methanol extracts of S. marianum at sample concentrations 0.25, 0.05, 0.1, 0.25, and 0.5 µg·ml⁻¹ was 25%, 30%, 55%, 75%, and 85%, respectively [66]. Seeds of S. marianum with purple flower showed 62.67%, 74.02%, and 78.34% activity after 10, 20, and 30 min, respectively, while white flowering seeds exhibited 37.79%, 42.97% and 49.18% activity, respectively [67].

Figure 10

DPPH scavenging capacity of commercially available silymarin, crude silymarin, and SZPCS-NPs.

4.1.2 ABTS radical scavenging assay

The ABTS radical cation decolorization test was employed to assess the free radical scavenging activity of commercial, crude, and SZPCS-NPs (Figure 11). The ABTS cation radical was developed by combining 10 mg of ABTS and 2 mg of potassium persulfate in water. The solution was stored in the dark at room temperature for 12–16 h before use. After that, the ABTS solution (1 ml) was diluted with 60 ml of methanol. The measurements were then gradually performed in triplicate. The percent inhibition was determined using the equation as follow;

Figure 11

DPPH scavenging capacity of commercially available silymarin, crude silymarin, and SZPCS-NPs.

Sample SM (S. marianum extracts; commercial, crude, and zein silymarin NPs)

% Absorbance (734 nm) of commercially available silymarin, crude silymarin, and SZPCS-NPs at sample concentration 10 µl·ml⁻¹ was recorded at 31.45%, 30.67%, and 78.98%, respectively (Figure 11). Similarly, at sample concentration 20 µl·ml⁻¹, the % absorbance (734 nm) of commercially available silymarin, crude silymarin, and SZPCS-NPs were recorded as 38.17%, 33.76%, and 91.3%, respectively. In addition, at sample concentrations 30 and 60 µl·ml⁻¹, the % absorbance (734 nm) of commercially available silymarin, crude silymarin, and SZPCS-NPs increased to 42.27%, 39.98%, 92.12% and 62.89, 58.23, and 96.34, respectively. Finally, at sample concentrations 70 and 80 µl·ml⁻¹, the % absorbance (734 nm) of commercially available silymarin, crude silymarin, and SZPCS-NPs further increased to 64.78%, 63.47%, 96.98% and 68.56%, 66.87%, and 98.67%, respectively. The maximum values for total antioxidants in terms of ascorbic acid equivalents per mg were found to be 67.6 ± 1.44 and 72.6 ± 1.32 for the tested concentration of 1,000 μg·ml⁻¹ for ZnO and Ag–ZnO, respectively [60].

4.4.1 Week 1 study

Light microscopic studies of the liver tissues, stained with hematoxylin–eosin (H&E) in each group. After 1 week, Group A untreated control group shows normal lobular architecture and cell structure with no evidence of portal inflammation and hepatocellular necrosis or liver tissue damage. Group B CCl₄-treated control group shows extensive hepatocellular damage with the presence of portal inflammation, hepatocellular necrosis, and liver tissue damage. Group C treated with commercial silymarin, and Group D treated with crude silymarin show less hepatocellular damage in comparison to Group B. Group E, treated with SZPCS-NPs, possesses much lesser hepatocellular damage in comparison to Groups B, C, and D. All the hepatocytes are visible within the microscopic pictures shown above, closely resemble Group A subjects showing normal lobular architecture and cell structure with no evidence of portal inflammation and hepatocellular necrosis. SZPCS-NPs hold a better hepatoprotective effect than commercial and crude silymarin (Figure 12).

Figure 12

Histopathological assessment of hepatotoxicity after 1 week, through light microscopic studies of the liver tissues, stained with H&E in each group. Specifically, (a) represents (H&E) stained hepatocytes of normal untreated control, (b) CCl₄ treated control, (c) commercial silymarin, (d) crude silymarin, and (e) SZPCS-NPs.

4.4.2 Week 2 study

Light microscopic studies of the liver tissues, stained with H&E in each group. After 2 weeks, Group A untreated control group shows normal hepatocellular physiology with no evidence of necrosis or liver tissue damage. Group B CCl₄-treated control group shows much more hepatocellular damage (red encircled) in comparison to week 1, with the presence of portal inflammation, cellular necrosis, and liver tissue damage. Group C treated with commercial silymarin and Group D treated with crude silymarin show a little bit of hepatocellular damage in comparison to Group B. Group E treated with SZPCS-NPs possesses much lesser hepatocellular damage in comparison to Groups B, C, and D. All the hepatocytes are visible within the microscopic pictures shown above, closely resemble Group A subjects showing normal lobular architecture and cell structure with no evidence of portal inflammation and hepatocellular necrosis. SZPCS-NPs holds a better hepatoprotective effect than commercial and crude silymarin (Figure 13).

Figure 13

Histopathological assessment of hepatotoxicity after 2 weeks, through light microscopic studies of the liver tissues, stained with H&E in each group. Specifically, (a) represents (H&E) stained hepatocytes of normal untreated control, (b) CCl₄-treated control, (c) commercial silymarin, (d) crude silymarin, and (e) SZPCS-NPs.

4.4.3 Week 3 study

Light microscopic studies of the liver tissues, stained with H&E in each group. After 3 weeks, Group A untreated control group shows normal hepatocellular physiology with no evidence of necrosis or liver tissue damage (TN; tissue necrosis). Group B CCl₄-treated control group shows much more hepatocellular damage (red encircled) in comparison to weeks 1 and 2, with the presence of portal inflammation, cellular necrosis, and liver tissue damage. Group C treated with commercial silymarin and Group D treated with crude silymarin show a little bit hepatocellular damage (red encircled) in comparison to Group B. Group E treated with SZPCS-NPs possesses much lesser hepatocellular damage in comparison to Groups B, C, and D. All the hepatocytes are visible within the microscopic pictures shown above, closely resemble Group A subjects showing normal lobular architecture and cell structure with no evidence of portal inflammation and hepatocellular necrosis. SZPCS-NPs hold a better hepatoprotective effect than commercial and crude silymarin (Figure 14).

Figure 14

Histopathological assessment of hepatotoxicity after 3 weeks, through light microscopic studies of the liver tissues, stained with H&E in each group. Specifically, (a) represents H&E stained hepatocytes of normal untreated control, (b) CCl₄-treated control, (c) commercial silymarin, (d) crude silymarin, and (e) SZPCS-NPs.

4.4.4 Week 4 study

Light microscopic studies of the liver tissues, stained with H&E in each group. After 4 weeks, Group A untreated control group shows normal hepatocellular physiology with no evidence of necrosis or liver tissue damage (TN; tissue necrosis). Group B CCl₄-treated control group shows much more hepatocellular damage in comparison to weeks 1 and 2, with the presence of portal inflammation, cellular necrosis, and liver tissue damage (red encircled). Group C treated with commercial silymarin and Group D treated with crude silymarin show a little bit hepatocellular damage in comparison to Group B. Group E treated with SZPCS-NPs possesses much lesser hepatocellular damage in comparison to Groups B, C, and D. All the hepatocytes are visible within the microscopic pictures shown above, closely resemble Group A subjects showing normal lobular architecture and cell structure with no evidence of portal inflammation and hepatocellular necrosis. SZPCS-NPs hold better hepatoprotective effects than commercial and crude silymarin (Figure 15).

Figure 15

Histopathological assessment of hepatotoxicity after 4 weeks, through light microscopic studies of the liver tissues, stained with H&E in each group. Specifically, (a) represents (H&E) stained hepatocytes of normal untreated control, (b) CCl₄-treated control, (c) commercial silymarin, (d) crude silymarin, and (e) SZPCS-NPs.

4.4.5 Week 5 study

Light microscopic studies of the liver tissues, stained with H&E in each group. After 5 weeks, Group A untreated control group shows normal lobular architecture and cell structure with no evidence of portal inflammation and hepatocellular necrosis or liver tissue damage. Group B CCl₄-treated control group, showing extensive hepatocellular damage (encircled red) with the presence of portal inflammation, hepatocellular necrosis, and liver tissue damage. Group C treated with commercial silymarin and Group D treated with crude silymarin showed less hepatocellular damage in comparison to Group B. While Group E treated with SZPCS-NPs possesses much lesser hepatocellular damage in comparison to Groups B, C, and D. All the hepatocytes are visible within the microscopic pictures. SZPCS-NPs hold better hepatoprotective effects than commercial and crude silymarin (Figure 16).

Figure 16

Histopathological assessment of hepatotoxicity after 5 weeks, through light microscopic studies of the liver tissues, stained with H&E in each group. Specifically, (a) represents (H&E) stained hepatocytes of normal untreated control, (b) CCl₄-treated control, (c) commercial silymarin, (d) crude silymarin, and (e) SZPCS-NPs.

4.4.6 Week 6 study

Light microscopic studies of the liver tissues, stained with H&E in each group. After 6 weeks, Group A untreated control group shows normal lobular architecture and cell structure with no evidence of portal inflammation and hepatocellular necrosis or liver tissue damage. Group B CCl₄-treated control group shows extensive hepatocellular damage with the presence of portal inflammation, hepatocellular necrosis, and liver tissue damage. The damaged cells are not properly stained with H&E stained (red encircled). Group C treated with commercial silymarin and Group D treated with crude silymarin show less hepatocellular damage in comparison to Group B. Group E treated with SZPCS-NPs possesses much lesser hepatocellular damage in comparison to Groups B, C, and D. All the hepatocytes are visible within the microscopic pictures. SZPCS-NPs hold better hepatoprotective effects than commercial and crude silymarin (Figure 17).

Figure 17

Histopathological assessment of hepatotoxicity after 6 weeks, through light microscopic studies of the liver tissues, stained with H&E in each group. Specifically, (a) represents (H&E) stained hepatocytes of normal untreated control, (b) CCl₄-treated control, (c) commercial silymarin, (d) crude silymarin, and (e) SZPCS-NPs.

Overall, these findings suggest that SZPCS-NPs hold promise as a potential therapeutic approach for liver diseases and as a drug delivery system for silymarin. It is evident from the results that SZPCS-NPs demonstrated a maximal hepatoprotective effect in comparison to crude silymarin and commercial silymarin.

Clichici et al. also concluded that the effect of silymarin gold NPs possessed significantly better hepatoprotective effects in contrast to crude silymarin [75]. Yang et al. determined that silymarin-loaded NPs significantly reduced CCl₄-induced hepatotoxicity, indicating improved bioactivity compared with silymarin crude powder and the commercial silymarin product [82]. In addition, pre-treatment with a self-micro-emulsifying drug delivery system containing SMEDDS-SM decreases the CCl₄-induced elevation both in biochemical and in morphological parameters associated with liver damage or toxicity [81]. In addition, Guhagarkar et al. also concluded that PES-SIM NPs possess better hepato-protection in comparison to crude silymarin [51]. Gupta et al. also concluded that silymarin chitosan NPs successfully enhance its hepatoprotective effect [66]. Shriram et al. revealed that the nano-delivery system of silymarin offers enhanced hepatoprotective activity [85]. Yang et al. found that silymarin-loaded NPs dramatically reduced CCL₄-induced hepatotoxicity in contrast to commercial and crude silymarin formulations [82]. Nasr et al. also concluded that silymarin-loaded mesoporous silica NPs possess better hepatoprotective activity in comparison to crude silymarin [86].