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

: Silymarin-loaded zein polysaccharide core – shell nanoparticles (SZPCS-NPs) were synthesized where sodium alginate and pectin o ﬀ er stability and controlled release qualities to zein, a maize protein, having excellent biocompatibility. The present study is an attempt to develop zein – sily-marin polysaccharide core – shell nanostructures to enhance water solubility, thereby improving bioavailability and producing enhanced biological responses in living systems. SZPCS-NPs were prepared using pH-induced antisolvent precipitation method. Five di ﬀ erent types of SZPCS-NPs were synthesized using di ﬀ erent combinations of sodium alginate and pectin, namely P100 – A00 (non-uniform size ranging from 20 to 100 nm), P70 – A30 (spherical and uniform size measuring approximately 80 nm in diameter), P50 – A50, P30 – A70, and P00 – A100 exhibited irregular shapes with the presence of some triangular and oval structures and non-uniform size ranging from 20 to 100 nm. The SZPCS-NPs P70 – A30 possessed the best results in terms of shape, size, and other characterization studies. Furthermore, the SZPCS-NPs possessed a percent drug loading of 72.5% and entrapment e ﬃ ciency of 51.7%, respectively. The resulting SZPCS-NPs exhibited an excellent relative bioavailability percentage of 97.4% in comparison to commercial silymarin, having 58.1%, and crude silymarin, having 46.97% bioavailability percentage, correspondingly. In addition, SZPCS-NPs possessed an almost two folds ’ increase in antioxidant activity in comparison to crude and commercially available silymarin. Similarly, SZPCS-NPs also showed better stabilization in hepatic bio-marker enzymes and possessed better hepatoprotective activity for a period of 6 weeks, in contrast to commercial and crude silymarin formulations.


Introduction
Plant-based natural products have been widely used as treatments against numerous diseases since ancient times [1].Around 40% of the marketed medicines have hydrosolubility issues, making it difficult to achieve enough drug absorption from the gastrointestinal tract (GIT) [1,2].Drugs having low hydro-solubility and cytopermeability have lower absorption and bioavailability, which has an impact on drug safety and efficacy.To produce a therapeutic product with better pharmacokinetic features from these active pharmaceutical ingredients, innovative formulation efforts are required [3].The poor water solubility of the majority of therapeutic medicines might decrease their dissolution rate and oral bioavailability, posing a problem for pharmaceutical development [4].In order to get the appropriate drug concentration in the systemic circulation, solubility is an important attribute [5].Over 90% of the drugs that have been manufactured possess poor oral bioavailability due to limited solubility in water and/or a slow disintegration rate [6].Using poorly soluble drugs has several limitations, including the need to increase the dose and administration frequency of the drug product as well as the likelihood of side effects.The solubility of poorly soluble drugs can be improved using various techniques [7].
One method is the application of nanotechnology, which reduces the size of the drug particle to the nanoscale.This increases the surface area and enhances the solubility and dissolution rate, leading to an increase in bioavailability [8].Pharmaceutical nanoparticles (NPs) are solid except spongosomes, submicron-sized (<100 nm in diameter), and submicron-sized biodegradable drug carriers [9,10].The latest nanotechnology technique plays a significant role in improving the water dispersibility, bioavailability, and therapeutic properties of compounds, particularly plant compounds [11,12].The protection of drugs or biological compounds by incorporating them in the form of nanostructures to enhance the solubility and dissolution rate, leading to an increase in bioavailability, has been extensively investigated [13].Furthermore, few colloidal carrier solutions have been reported to understand the problem of poor hydro-solubility and low bioavailability of active pharmaceutical ingredients.However, these colloidal carrier systems possess numerous drawbacks, such as inadequate physical stability, employment of residual organic solvents, drug expulsion during storage, low drug loading, and polymer cytotoxicity [14].According to previous studies, the benefits of encapsulation of the drug compounds into zein polysaccharide core-shell nanostructures are specifically shown to possess excellent loading capacity and encapsulation efficiency, good dispersion in water, better stability in hard conditions, control release and improved bioavailability, and maintenance or improvement of biological activities of the encapsulated drug or biological compound [15][16][17].According to Shuai et al., under in vitro GIT conditions, zein polysaccharide NPs significantly delayed the release and the photoand thermal-degradation of the encapsulated curcumin and piperine [18].Quercitin-loaded zein polysaccharide NPs demonstrate good physicochemical stability and biological activity.The resulting NPs showed good stability and slow release throughout simulated GIT digestion in vitro, and it was found that they have increased antioxidant and antibacterial activity [19].Huang et al. incorporated curcumin into core-shell zein polysaccharide NPs by pH-induced antisolvent precipitation to improve the water dispersibility and antioxidant activity of curcumin [20].
Zein is a prominent biological carrier that has effective self-assembly and monodispersive characteristics when it comes to encapsulating bioactive components.The Food and Drug Administration (FDA), USA, has recognized zein as a safe human food ingredient that has the capacity to act as an edible carrier, making it a drug delivery system [21].Many functional and nutritional ingredients have been successfully encapsulated in zein, and zein NPs have been synthesized to increase their physicochemical properties [22].By embedding active compounds in the zein carrier, their microstructure, chemical stability, and bioavailability are considerably increased.All these efficacies make it a preferable option for developing NP delivery systems that encapsulate lipophilic medicinal compounds [21,22].However, zein as a solitary material is often unsuitable for technological applications due to its weak water solubility and inability to effectively regulate the release of encapsulated substances.Widespread usage of polysaccharides as natural materials in the food business, which are very stable, biocompatible, and degradable.Polysaccharides may contribute to constructing nanocarriers with proteins through the following mechanisms: chemical cross-linking, physical cross-linking, poly-ion complex formation, and self-assembly [23].Combining zein with polysaccharides to generate nanodelivery systems offers greater opportunities for the use of hydrophobic bioactivities than polymers alone [24].Various polysaccharides, including chitosan, alginate, and pectin, have been combined with zein to develop nanodelivery systems.Silybum marianum is a plant that belongs to the family "Asteraceae" and is trivially known as milk thistle.It is native to Europe, Asia, and Northern Africa.Parts of plants can be consumed as raw vegetables and cultivated as ornamental plants.It is thought to be an ancient medicinal weed possessing ethno-pharmacological significance for the treatment of various liver ailments [25].Many therapeutic compounds such as silymarin and their functional analogues like silybins, isosilybins, silychristin, and silydianin are documented to possess effective hepatoprotective properties by neutralizing the effects of harmful free radicals [26].Along with this, silymarin has been reported to have excellent anti-arthritic, anti-cancer, anti-diabetic, anti-viral, antimicrobial, and immunemodulatory properties [27].It has been reported that about 23% of the total silymarin is accessible to be absorbed from GIT due to very low aqueous solubility, hence resulting in very little bioavailability.Tea preparations of silymarin are less preferred due to their reduced hydro-solubility; therefore, oral encapsulated forms are readily administered by herbal practitioners to treat the ailment [28].
Keeping in view the importance of zein and silymarin, the present study aimed to develop zein-silymarin polysaccharide core-shell nanostructures to increase its water solubility that will enhance the bioavailability and biological responses in the living system.

Plants collection
The S. marianum plants of white flowering and pink flowering were collected from two different regions of Peshawar (Hayatabad and University town Peshawar) and dried in shade separately.The plants and seeds were properly authenticated by Prof. Dr. Farhat Ali Khan and Prof. Dr. Muhammad Zahoor.The seeds of white and pink flowering plants were collected and ground into pulverized form (pulverization) separately.The ground samples were kept in a cool, dry place for the extraction procedure.

Extraction of oil
In blue-capped bottles, 250 g of powdered seeds of white and pink flowering S. marianum were soaked separately in 150 ml of n-hexane and kept at room temperature for 48 h.Each extract was then filtered by using filter paper and transferred into a rotary evaporator for oil extraction.Both the oils were then shifted into separate amber glass containers and stored at 25°C for further analysis.This oil extraction step is intended to obtain a comprehensive understanding of the overall composition of the seed extract, including lipids and all other non-polar components, and the percent yield by white and pink flowering S. marianum varieties.

Characterization of oil 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 polarembedded material polyethylene glycol (PEG).

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.

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.

Preparation of silymarin-zein stock solution
The stock solution of the silymarin-zein complex was prepared by dissolving 2.0 g zein in 100 ml of ethanol (85% v/v) using a magnetic stirrer at 500 rpm (IKA R05, USA) for 1 h.Then, dried powdered silymarin (0.24 g) was added to the solution and stirred for 30 min in the dark.The insoluble impurities were removed from the solution through centrifugation at 1,006 × g for 15 min.
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 silymarinzein 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 Characterization of silymarin-loaded zein polysaccharide core-shell nanoparticles (SZPCS-NPs) The prepared SZPCS-NPs were then passed through different sophisticated instruments for characterization.

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.

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°w avelength.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.

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.

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.

Antioxidant potential of SZPCS-NPs
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.

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 IC 50 value representing the concentration.

Hepatoprotective activity
Silymarin and SZPCS-NPs were formulated in carboxymethyl cellulose (0.5%) to be administered through the oral route (oral gavages).

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].

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).

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.

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.

Determination of oil content from the seed (capitulum) of S. marianum
From 1 kg of pink flower plant seed, it was observed that 24.3 ml of oil was extracted, and from 1 kg of white flower plant seed, 22.7 ml of seed was extracted.It is evident that the difference is 1.6 ml, so there is no significant difference between these two different flowering seeds of S. marianum.Lahlah et al. studied the estimation of active principle pharmacological entities of white and blue capitulum and the whole plant of S. marianum.They also found no significant difference and concluded that S. marianum (blue and white flowering varieties) showed nearly the same values of phytochemical constituents and possessed similar antimicrobial activities [34].Shaker et al. also concluded that there was less phenotypic difference in purple and white varieties of S. marianum [35].

GC-MS analysis of oil
The composition analysis of oil extracted from S. marianum seedsboth white and pink flowering varietieswas conducted using GC-MS.The GC-MS spectra for both extracts are illustrated in Figure 1a (white flowering seeds) and Figure 1b (pink flowering seeds).In Figure 1a, the GC-MS analysis revealed prominent peaks at specific retention times (RTs) of 18.85, 20.28, 21.97, and 25.96.These peaks corresponded to significant constituents present in the S. marianum oil extracted from white flowering seeds.The respective peak areas associated with these RTs were 56,066,569.30,120,574,395.29,56,546.66,and 8,915,044.66,indicating the relative abundance of these compounds within the oil extract.In contrast, Figure 1b  Additionally, the exact composition of the oil obtained from S. marianum has previously been reported in the literature.In the fatty acid fraction, for example, linoleic (50.5%) and oleic (30.2%) acids were found to be predominant.In contrast, essential oil contains 14 volatile components, with the two main ones being γ-cadinene (49.8%) and α-pinene (24.5%).The main ingredients in the seed oil extracts were linoleic acid (43.5-64.4%),oleic acid (20.8-29.8%),palmitic acid (7.2-9.7%), and stearic acid (2-6.6%).A dwarf S. marianum genotype from India was used to produce around 20% of the yellow oil, with the seeds having 9.7% palmitic, 4.7% stearic, 30.9% oleic, 45.8% linoleic, and 5.2% linolenic acids [36].
The comparison of the major peaks observed through GC-MS analysis between the white and pink flowering seed extracts provides insights into the differences in chemical composition and relative abundance of key compounds present in the two varieties of S. marianum seeds.Our GC-MS results closely correspond with the results of the GC-MS analysis of Iraqi S. marianum flowers, leaves, and seed extracts by Eldalawy and Al-ani [37].3.4 Quantification of silymarin content in the seeds of S. marianum

FT-IR analysis of oil
From 1 kg of pink flower seed, it was observed that 38.6 g of silymarin was extracted, and from 1 kg of white flower seed, 35.7 g of silymarin was extracted.It can be observed that the difference is only 2.9 g, so there is no significant difference between these two different flowers of S. marianum.No significant difference was found among S. marianum blue and white varieties and they possess almost similar amounts of phytochemical constituents.It has been reported that the contents of silymarin in blue and white capitulum seeds of S. marianum show a very negligible variation, and the difference was noted as insignificant [40].
Studies on blue and white flower varieties of S. marianum with special reference to the silymarin content were carried out by Maryam et al., which confirmed nearly the same values of silymarin content in the seeds of both S. marianum varieties [41].

FT-IR analysis of silymarin
FT-IR analysis of silymarin extracted from seeds of S. marianum of two different flowerings, i.e., pink and white flowers, is shown in Figure 3a and b, respectively.FT-IR of silymarin extracted from S. marianum (pink flower capitulum seeds) displays strong absorption bands at 3,449.72,

Preparation of SZPCS-NPs
SZPCS-NPs were prepared by using different ratios of pectin and alginate solutions and then characterized by TEM, XRD, and FT-IR to select the best ratio.

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   Preparation of silymarin-loaded zein polysaccharide core-shell nanostructures  9 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].

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].[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].

XRD analysis
Powder XRD was used to verify the crystallinity and phase distribution of the synthesized SZPCS-NPs.Figure 6a-e    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

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.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 Preparation of silymarin-loaded zein polysaccharide core-shell nanostructures  13 temperature of 0-1,100°C.Weight loss was observed at 130-140°C [61].

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].
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.

% 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.
Amount of drug in NPs mg Amount of NPs mg 100 %Entrapment efficiency Amount of drug in NPs mg Initial amount of drug mg 100 4 Potential activities of SZPCS-NPs (P70-A30)

Antioxidant activity
Finally, the impact of the encapsulation of silymarin within SZPCS-NPs on its antioxidant activity was determined.This kind of analysis is important because any strategy used to improve the water dispersibility and physical stability of silymarin should not adversely affect its functional attributes.In these experiments, SZPCS-NPs that had a P70-A30 coating were prepared since this system gave the best physical stability and were compared with the antioxidant activity of commercially available silymarin and crude silymarin.Preparation of silymarin-loaded zein polysaccharide core-shell nanostructures  15

Hepatoprotective activity
Up to date, many studies have revealed that S. marianum extracts possess potential hepatoprotective activity [67][68][69][70][71][72].However, we were interested in checking the relative hepatoprotective effect of commercial, crude, and SZPCS-NPs.For induction of hepatotoxicity and liver damage, CCl 4 was used to induce liver fibrosis in the experimental animals (rats).In order to evaluate the liver cell damage, the level of the hepatocytolytic marker enzymes, such as ALT, AST, ALP, and TP levels of bilirubin and albumin, which leak into the blood stream from the liver tissue, was assessed.In addition, the effects of commercial, crude, and SZPCS-NPs were assessed for 6 weeks from hepatocytolytic marker enzymes and concentration of bilirubin and albumin proteins.Hermenean et al. [73] and Kim et al. [74] injected the experimental mice with CCl 4 to induce hepatotoxicity, while Mandegary et al. used hydrogen sulfide (H 2 S) [68].Clichici et al. also used the same method (ALT and AST levels) for evaluating the hepatoprotective effect of silymarin-loaded gold NPs [75].Kalantari et al. used AST, ALT, and ALP levels for the assessment of the hepatoprotective action of S. marianum [76].In addition, some studies also focused on evaluating the hepatoprotective effects of S. marianum from levels of gamma-glutamyl transferase (GGT), ALT, AST [73,77], lactate dehydrogenase and creatine kinase [78], malondialdehyde (MDA), and CYP450 levels [79].On the contrary, Abuzaiton investigated the hepatoprotective activity from serum glutamate pyruvate transaminase and serum glutamic-oxaloacetic transaminase levels of experimental animals [80].
Group A. Normal untreated control Group B. CCl 4 -treated control Group C. Commercial silymarin Group D. Crude silymarin Group E. SZPCS-NPs Tables 2-7 show a 6-week detailed analysis of the levels of hepatocytolytic marker enzymes such as ALT, AST, ALP, and TP, and levels of bilirubin and albumin, which leak into the blood stream from the liver tissue.Group A animals correspond to normal untreated control and physically healthy rats, with no administration of CCl 4 or either silymarin (crude, commercial, or SZPCS-NPs).In addition, Group B animals relate to CCl 4 -treated control rats, with no administration of silymarin (crude, commercial, or SZPCS-NPs were done for this group).Similarly, Group C animals relate to CCl 4 -treated rats along with coadministration of commercial silymarin.Group D animals relate to CCl 4 -treated rats along with co-administration of crude silymarin.Finally, Group E animals relate to CCl 4treated rats along with co-administration of SZPCS-NPs.
It is evident from the results (Tables 2-7) that SZPCS-NPs showed a maximal hepatoprotective effect in comparison to crude silymarin and commercial silymarin.Clichici et al. also concluded that the effect of silymarin gold NPs was significantly better than silymarin alone in terms of its hepatoprotective effects [75].The concentrations of the liver enzymes such as AST and ALT were significantly reduced by the S. marianum extract [80].Pre-treatment with a self-micro-emulsifying drug delivery system containing S. marianum native seed oil (SMEDDS-SM) decreases the CCl 4induced elevation both in biochemical and in morphological parameters associated with liver damage or toxicity [81].Guhagarkar et al. also concluded that polyethylene sebacatesilymarin nanoparticles (PES-SIM NPs) possess better hepatoprotection in comparison to crude silymarin [51].Gupta et al. also concluded that Silymarin chitosan NPs successfully enhance its hepatoprotective effect by passive targeting and sustained release [66].Yang et al. determined that silymarin-loaded NPs significantly reduced carbon tetrachloride-induced hepatotoxicity, indicating improved bioactivity compared with silymarin powder and the commercial product [82].

Bioavailability
The results obtained from the percent absorbance measurements at different contact times provide valuable information about the rate and extent of absorption of commercial silymarin, crude silymarin, and SZPCS-NPs (Table 8).
After 10 min of contact time, the percent absorbance values were 22.34%, 15.87%, and 45.85% for commercial silymarin, crude silymarin, and SZPCS-NPs, respectively.These values indicate that SZPCS-NPs exhibited the highest absorbance, suggesting a more efficient and rapid absorption compared to the other two formulations.At 20 min, the percent absorbance values increased to 34.87%, 25.76%, and 66.64% for commercial silymarin, crude silymarin, and SZPCS-NPs, respectively.Again, SZPCS-NPs showed the highest absorbance, indicating a continued increase in their absorption rate.After 30 min, the percent absorbance values further increased to 45.32%, 38.67%, and 82.14% for commercial silymarin, crude silymarin, and zein silymarin NPs, respectively.SZPCS-NPs continued to exhibit the highest absorbance, demonstrating a greater extent of absorption compared to the other formulations.The absorbance values at 50 min were 54.43%, 46.44%, and 96.55% for commercial silymarin, crude silymarin, and zein silymarin NPs, respectively.Once again, SZPCS-NPs displayed the highest absorbance, indicating a significant increase in their absorption compared to the other two formulations.After 60 min of contact time, the recorded percent absorbance values were 58.15%, 46.97%, and 97.48% for commercial silymarin, crude silymarin, and SZPCS-NPs, respectively.SZPCS-NPs maintained their superior absorbance, demonstrating the highest rate and extent of absorption among the three substances.
Based on the percent absorbance results at different contact times, it can be inferred that zein silymarin NPs exhibit a faster and more extensive absorption compared to commercial silymarin and crude silymarin.These findings suggest that SZPCS-NPs have the potential for enhanced bioavailability and improved therapeutic efficacy.These findings are consistent with other studies that have investigated the absorption of silymarin NPs.A study by Zhao et al. reported similar results, showing that silymarin-loaded NPs had higher absorption rates compared to raw formulation [83].Additionally, a study by Onodera et al. demonstrated that silymarin NPs exhibited improved bioavailability in in vitro release studies [84].

Histopathological evaluation
All animals from Group A showed normal liver histology.The animals assigned in the other three experimental groups such as Groups B, C, D, and E presented marked morphological changes, characteristic of a liver injury associated with the administration of CCl 4 .

Week 1 study
Light microscopic studies of the liver tissues, stained with hematoxylin-eosin (H&E) in each group.After 1 week, 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).

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 CCl 4 -treated control group shows     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).Preparation of silymarin-loaded zein polysaccharide core-shell nanostructures  23 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).

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 CCl 4 -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).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 CCl 4induced 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 CCl 4 -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 CCL 4 -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].

Conclusion
In conclusion, there were no significant differences noted in the contents of oil and silymarin in the pink and white flowering S. marianum seeds can be used in any variety for oil and silymarin extraction.SZPCS-NPs were found best in pectin/sodium alginate solution with a ratio of 30% pectin and 70% alginate, whereas the antioxidant activity and hepatoprotective activity were enhanced with many folds.
These findings suggest that SZPCS-NPs hold promise as a potential therapeutic approach for liver diseases and as a drug delivery system for silymarin.Further research and development in this area are warranted to explore their full potential in clinical applications.
depicts the GC-MS analysis of S. marianum oil obtained from pink flowering seeds, exhibiting major peaks at RTs of 18.89, 20.27, 20.32, 21.99, and 25.98.These RTs signify the presence of significant chemical components within the pink flowering seed extract.The corresponding peak areas for these RTs were measured at 13,987,946.94, 176,668,263.01,63,484.01,952,602.82,and 2,663,794.40,indicating the relative quantities of these compounds in the oil extract from the pink flowering seeds.

Figure 1 :
Figure 1: GC-MS of oil extracted from (a) white and (b) pink flowering seeds of S. marianum.

Figure
Figure 2a and b represents the FT-IR analysis of oil derived from S. marianum of two distinct flowering seeds, namely pink and white flowers.FT-IR of oil extracted from S. marianum (pink flower capitulum seeds) displays strong absorption bands at 3,009.1, 2,923.4,2,886.46,2,853.8,1,737.9,1,710.7,1,557.6,1,463.6,1,243.8,1,182.7,and 466.0 cm −1 .FT-IR of oil extracted from S. marianum (white flower capitulum seeds) displays strong absorption bands at 3,009.0, 2,923.4,2,853.8,1,737.9,1,710.0,1,556.8,1,463.5, 842.7, and 466.4 cm −1 .The existence of saturated hydrocarbon chains is indicated by strong absorption bands at around 3,009.1, 2,923.4,2,886.46,and 2,853.8cm −1 .These bands most likely correspond to the C-H stretching vibrations of aliphatic hydrocarbons.The stretching vibration of carbonyl (C]O) groups, perhaps originating from esters or other compounds containing carbonyl, might be represented by the absorption band at 1,737.9 cm −1 .The absorption bands at 1,557.6 cm −1 , 1,463.6, 1,243.8,1,182.7,and 466.0 cm −1 may be indicative of aromatic chemicals or unsaturated hydrocarbons that have C]C stretching.The bands located at 1,243.8 and 1,182.7 cm −1 may be linked to C-O stretching, which might suggest the existence of alcohols or esters.The band at 710.7 cm −1 may be associated with aromatic compounds or substituent outof-plane bending vibrations.Both the FT-IR spectra of white and pink flowering S. marianum seeds oil show approximately the same values as already reported by Fadhil et al.[38].Our results are in close accordance with the study conducted by Radusheva

Figure 2 :
Figure 2: FT-IR spectra of oil extracted from seeds of (a) white and (b) pink flowering S. marianum.

Figure 3 :
Figure 3: FT-IR spectra of silymarin extracted from (a) white and (b) pink flowering S. marianum seeds.

4. 4 . 4
Week 4 studyLight 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 CCl 4 -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 (Figure15).

4. 4 . 5
Week 5 studyLight 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 CCl 4treated 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

Table 8 :
Percent absorbance measurements at different contact times about the rate and extent of absorption of commercial silymarin, crude silymarin, and zein silymarin NPs 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 CCl 4 -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 (Figure12).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 CCl 4treated 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 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