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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Fabrication, characterization and biological evaluation of silymarinnanoparticles against carbon tetrachloride-induced oxidative stress andgenotoxicity in rats

Mohamed H.A. Aboshanaba, Mohamed A. El-Nabarawia, Mahmoud H. Teaimaa,Aziza A. El-Nekeetyb, Sekena H. Abdel-Aziemc, Nabila S. Hassand, Mosaad A. Abdel-Wahhabb,⁎

a Pharmaceutics & Industrial Pharmacy Dept., Faculty of Pharmacy, Cairo University, Cairo, Egyptb Food Toxicology & Contaminants Dept., National Research Center, Dokki, Cairo, Egyptc Cell Biology Dept., National Research Center, Dokki, Cairo, Egyptd Pathology Dept., National Research Center, Dokki, Cairo, Egypt

A R T I C L E I N F O

Keywords:SilymarinNanoparticlesHepatoprotectiveIonotropic gelation, oral bioavailabilityPolymeric nanoparticlesDrug deliverySilymarin nanoparticles

A B S T R A C T

This study aimed to synthesize silymarin nanoparticles (SILNPs) using chitosan nanoparticles as a deliverysystem and to evaluate their protective effects against CCl4 in rats. Eight groups of male Sprague-Dawley ratswere treated for three weeks included the control group, CCl4-treated group (100 mg/kg b.w twice a week); SIL-treated group (50 mg/lg b.w); the groups treated daily with low dose (LD) or high dose (HD) of SILNPs (25,50 mg/kg b.w) and the groups treated with CCl4 plus SIL, SILNPs (LD) or SILNPs (HD). Blood and tissue sampleswere collected for different assays. The synthesized SILNPs showed a smooth rounded shape with averageparticle size of 100 ± 2.8 nm. SILNPs contain the same compounds found in raw SIL and the in vitro release ofSILNPs continues till 24 h. The in vivo study revealed that SIL and SILNPs at the low or high dose induced asignificant improvement in the hematological parameters, liver and kidney function, lipid profile, serum cyto-kines, gene expression DNA fragmentation and histology of liver and kidney tissue resulted from CCl4. It could beconcluded that SILNPs can be applied in oral delivery formulations with a potential application value for liverdisease therapy.

1. Introduction

Approximately two million people die annually worldwide due toliver diseases. Liver cirrhosis rank the 11th prevalent cause of mortalitywith more than 1.16 million deaths (Asrani et al., 2019). In in-dustrialized Western countries, Alcohol or NAFLD (non-alcoholic fattyliver disease) is the main cause of cirrhosis; however, in several Asianand African countries, hepatitis B and C are the major causes (Asraniet al., 2019). The therapeutic role of the natural components has got aconsiderable interest in the last 2 decades. Silybum marianum (Milkthistle) is reported as a safe, natural and traditional herbal medicine fortreatment of several liver disorders. Previous reports revealed that thebioactive compounds in silymarin (SIL) showed potential antioxidantand hepatoprotective properties (Kwon et al., 2013). Several formula-tions of SIL are currently available in the markets for oral administra-tion and their pharmacodynamic and pharmacokinetic properties arewell characterized (Siegel and Stebbing. 2013). Crude SIL is lipophilic,

poorly soluble in the water and only 20–50% of SIL extract is absorbedin the intestine after administration (Abenavoli et al., 2011; Zhu et al.,2013). Consequently, the improvement of the solubility and the oralbioavailability of SIL is of great demand. Several methods have beenattempted to improve the bioavailability of silybin or SIL and providethem a robust strength against physical, chemical and environmentaldegradation such as using a buccal liposomal delivery system (El-Samaligy et al., 2006; Ding et al., 2018, Van Tran et al., 2019), formingpolyhydroxy phenyl chromanone salts, soluble derivatives and com-plexes with phospholipids (Giacomelli et al., 2002), incorporation intosolid /semisolid dispersions with a hydrophilic polymer such as PEG6000 (Li et al., 2003). Moreover, Elmowafy et al. (2013) encapsulatedSIL into liposomes. Additionally, other methods were reported such assolubilization in a self-micro-emulsifying drug delivery system(SMEDDS), nanoparticles, nano-capsulation by using polymers, andalbumin (Esfanjani and Jafari, 2016; Fan et al., 2018; Guo et al., 2018;Parveen et al., 2011; Xu et al., 2018). The application of nano-

https://doi.org/10.1016/j.ijpharm.2020.119639Received 17 June 2020; Received in revised form 7 July 2020; Accepted 8 July 2020

⁎ Corresponding author.E-mail address: mosaad_abdelwahhab@yahoo.com (M.A. Abdel-Wahhab).

International Journal of Pharmaceutics 587 (2020) 119639

Available online 14 July 20200378-5173/ © 2020 Elsevier B.V. All rights reserved.

T

technological strategies seems to be a promising method to enhance thetherapeutic role and to promote the continuous release of the bioactivecomponents of herbal extract. Nanostructured biomaterials, exhibit ananoscale size and morphology, featuring a vast range of characteristiccompared to the traditional biomaterials, like improved cellular inter-action, high bioavailability, improve the therapeutic value of applieddrugs by addition of retention time, and pass the biological barriers(Javed et al., 2011). Polymeric nanoparticles are widely used as de-livery systems because they able to overcome the physiological barriersand induce protection and targeting the loaded compounds to specificcells in the organ (Singh et al., 2009). Chitosan (CS) is a natural cationicamino polysaccharide and an ideal biological material due to its safety,biodegradability, and biocompatibility (Abdel-Wahhab et al., 2015,2016; Prabaharan and Mano, 2005; Duceppe and Tabrizian, 2010). CShas attracted a lot of attention as a biomedical material because it hasseveral biological activities, including antitumor activities, immunestimulating effects, antiallergic effects, anticoagulant effects; wound-healing effects; antimicrobial effects; antiviral activity; anti-in-flammatory activities and radical scavenging properties (Abdel-Gawadet al., 2016; Abdel-Wahhab et al., 2017; Ziwei et al., 2011; Thanh-Sanget al., 2012; Ukun et al., 2012; Ai et al., 2012; Chung et al., 2012;Anraku et al., 2008). Therefore, this study was conducted to formulateand optimize a stable formulation of SIL by incorporating in chitosannanoparticles (SILNPS) for passive targeted delivery, therebyprolonging its retention time and evaluate its therapeutic potentialprotective effects of silymarin nanoparticle (SILNPs) against carbontetrachloride-induced hepatonephrotoxicity, oxidative stress and gen-otoxicity in the rats.

2. Material and methods

2.1. Chemicals and kits

Silymarin (purity > 98%) was purchased from Arab Company forPharmaceuticals and Medicinal plant (MEPACO- MED), Cairo, Egypt;Chitosan (75–85% deacetylation, 20–300 cP, mol. wt. 50–190 kDa) waspurchased from Sigma Aldrich (St. Louis, MO, USA). Kits for aspartateaminotransferase (AST) and alanine aminotransferase (ALT) were pur-chased from spectrum-diagnostics, Cairo, Egypt. Kits for alkalinephosphatase (ALP), gamma- glutamyl transferase (GGT), creatinine,urea, uric acid, cholesterol, triglycerides (TG), high-density lipoproteincholesterol (HDL), alpha-fetoprotein (AFP), malondialdehyde (MDA),nitric oxide (NO), total antioxidant capacity (TAC) and glutathioneperoxidase (GPX) were purchased from Spectrum Diagnostics Co.(Cairo, Egypt). Kits for RNase-free DNase and preMix cDNA were pur-chased from iNtRON Biotechnology, Korea. Trizol solvent, DNase kitwas purchased from Promega Corporation (WI, USA) and DNA ladderwas purchased from Invitrogen (Massachusetts, USA). Carbon tetra-chloride (CCl4) was purchased from Morgan, Cairo, Egypt.

2.2. Formulation of silymarin nanoparticles (SILNPs)

SILNPs were formulated by the ionotropic gelation method de-scribed by Chuah et al. (2013) and Nagpal et al. (2013). In brief, aconcentration of CS (2%) was dissolved in acetic acid solution (2%) andthe pH was adjusted to 5 using 1 M sodium hydroxide. The SIL solutionwas prepared by dissolving 500 mg in 50 ml ethanol and the solutionwas added dropwise into the chitosan solution with continuous stirringon a magnetic stirrer for 2 h. Then, sodium tripolyphosphate (TPP)solution, (pH 5) was added dropwise to the silymarin-chitosan mixturewith stirring at 1000 rpm for 2 h. The formulated nanoparticles (NPs)were collected by the centrifugation for 45 min at 7000 rpm and 10 °Cusing cooling centrifuge. The obtained NPs were resuspended in a vo-lume of 50 ml distilled water and then sonicated for 45 min using anultrasonic bath. The prepared SILNPs were freeze dried and kept in asealed vial.

2.3. HPLC analysis of silymarin components

Crude SIL was analyzed using high resolution preparative HPLCDionex Summit systems (Sunnyvale, CA, USA) equipped with P680HPLC pump, solvent delivery module, autosampler, automatic samplerinjector, (117-well capacity), controller module, column oven, photo-diode array detector (PDA), with data collected and analyzed using StarChromeleon chromatography managing system software (version7.2.0.3765). Phenomenex (Luna C18 AXIAP 5 µm) column of 250 mmin length and 21.2 mm in diameter was used for the separation.Fractionation was carried out with an isocratic mobile phase of me-thanol: 0.1% formic acid in water (60:40, V/V) at a flow rate of 0.6 ml/min. The purity of the individual components was evaluated usingmass, ultraviolet spectral data, retention times, and co-chromatographywith Sigma standard chemicals (of silychristin, silydianin, silybin A,silybin B, isosilybin A and isosilybin B).

2.4. In vitro release of SIL and SILNPs

The in vitro drug release study of raw SIL and SILNPs was performedby paddle method as described by Lu et al. (2007) with slight mod-ification. SIL (500 mg) and SILNPS (containing 500 mg SIL) were im-mersed in 900 ml of phosphate buffer solution (pH 7.5, including 0.5%SDS) and the mixture was stirring at 100 rpm at 37.0 ± 0.5 °C. Then,5 ml of the mixture was taken after 5 min, 15 min, 30 min, 45 min, 1 h,2 h, 4 h, 8 h, 12 h and 24 h, centrifuged at 8000 rpm for 5 min andfiltrated through a membrane filter (0.45 μm, nylon syringe filter) forHPLC analysis at different time points. Meanwhile, the same volume offresh release medium was supplemented to keep constant volume. Thefiltrate samples were directly injected into the HPLC system, and thenthe SIL concentration was assayed.

2.5. Characterization of SILNPs

The particle size and polydispersity index (PDI) of drug loaded NPswas analyzed by dynamic light scattering technique (DLS) with aZetasizer™ 3000E (Malvern Instruments Worcestershire, UK). Eachsample was diluted using double distilled water until the appropriateconcentration of the particles then it was kept constant to avoid multiscattering events. The particle size and size distribution of equivalenthydrodynamic spheres were calculated using the Malvern associatedsoftware by the exponential sampling method. FTIR spectra of chitosan,SIL and its nanoparticles were determined using an infrared spectro-meter (FTIR) (Thermo Fisher Scientific Inc., Nico-let iS10, USA).

2.6. Experimental animals

Three months old male Sprague-Dawely rats (100–120 g) were ob-tained from Animal House Colony, National Research Center, Giza,Egypt. Animals were maintained on standard lab diet in artificially il-luminated and temperature-controlled room free from any chemicalcontamination at the Animal House Lab, NRC, Dokki, Cairo, Egypt. Allanimals were received humane care in compliance with the guidelinesof the Animal Care and Use Committee of the National Research Center,Dokki, Cairo, Egypt and the National Institutes of Health (NIH pub-lication 86–23 revised 1985).

2.7. Experimental design

After 1 week of acclimatization, the animals were divided into eightgroups (10 rats/group) and treated orally for 3 weeks as shown in(Table 1). At the end of the treatment period (i.e. day 21), all animalswere fasted for 12 h, then blood samples were collected from the retro-orbital venous plexus under diethyl ether anesthesia. The sera wereseparated by cooling centrifugation and stored at 20 °C until analysis.The sera were used for the determination of AST, ALT, ALP, GGT, Cho,

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TG, LDL, HDL, urea, creatinine, uric acid and AFP according to the kitsinstructions using a spectrophotometer. All animals were sacrificedafter the collection of blood samples by cervical dislocation and samplesof the liver and kidney were collected. Samples of the liver and kidneywere collected from each animal within different treatment groups. TAsample of each of the liver and kidney for each rat was dissectedweighed and homogenized in phosphate buffer (pH 7.4) as described byLin et al. (1998) and the homogenate was used for MDA and NO (20%)then it was dilated for the determination of TAC level and GPX activity.Another sample of the liver from each animal was used for the de-termination of DNA fragmentation and cytogenetic studies. Othersamples from each organ were fixed in 10% neutral formalin and par-affin embedded. Sections (5 mm thickness) were stained with hema-toxylin and eosin (H & E) for the histological examination (Drury andWallington, 1980).

2.8. Molecular genetics analyses

2.8.1. Gene-expression analysisThe frozen tissues of liver were thawed, homogenized in Trizol re-

agent, and RNA was extracted according to the manufacturer’s in-structions. The yield and quality of RNA were analyzed usingNanoDrop™ 1000 spectrophotometer (Thermo Fisher Scientific, USA)and RNA integrity and level of degradation were assessed by agarosegel electrophoresis. A 2 μg of RNA was treated with RNase-free DNasekit to remove any genomic DNA contamination and cDNA was syn-thesized using PreMix cDNA kit according to the manufacturer’s in-structions. The program of PCR cycles was operated as follows: initiallydenaturing for 20 s at 95 °C, then 40 cycles (3 s at 95 °C, 30 s at 60 °C).The mRNA expressions of Caspase-3, p53, Bcl-2 and Bax were de-termined by quantitative reverse transcription-PCR and normalizedagainst GAPDH mRNA levels. The primer sequences used in this studyand cycling conditions are summarized in the supplementary material

file (Table 1S). The PCR product was run on a 2% agarose gel in Tris-borate–EDTA buffer and visualized on a UV Trans-illuminator. Theethidium bromide-stained gel bands were scanned and the signal in-tensities were quantified by use of the computerized Gel-Pro (version3.1 for Windows 3). The ratio between the levels of the target gene-amplification product and GAPDH (internal control) was calculated tonormalize for initial variation in sample concentration as a control forreaction efficiency (Raben et al., 1996).

2.8.2. DNA fragmentation assays for apoptosisApoptotic changes in the liver were evaluated colorimetrically by

DNA fragmentation and by agarose gel electrophoresis according to theprocedure of Lu et al. (2002).

2.8.3. DNA gel electrophoresis laddering assayExtracted DNA was electrophoresed on 1% agarose gels containing

0.71 g/ml ethidium bromide. At the end of the runs, gels were ex-amined using UV transillumination. The diphenyl amine (DPA) assayreaction suggested by Gibb et al. (1997) and modified by Abdel-Wahhab et al. (2017).

2.9. Statistical analysis

All data were statistically analyzed by analysis of variance (ANOVA)using the general linear model procedure of the statistical analysissystem (SAS Institute Inc., 1982). Duncan’s multiple range tests (Wallerand Duncan, 1969) was used to clarify the significance between theindividual groups at p ≤ 0.05. The values in this study were expressedas mean ± standard.

3. Results

3.1. Characterization of SILNPs

Scanning electron microscope (SEM) images revealed that both SILand SILNPS synthesized by ionotropic gelation method using e (Fig. 1).The size distribution of SILNPS by DLS measurements showed that theaverage chitosan as a polymer which was cross-linked with sodium TPPshowed a smooth rounded shapparticles sizes were 100 ± 2.8 nm asshown in the Supplementary material file (Fig. 1S). The FTIR analysis ofchitosan (COS) showed peak at 1038.45 cm−1 which indicated thepresence of saccharide structure. The peaks of stretching vibrations ofOH, amide I, and amide III appeared at 3455.35 cm−1, 1585.15 cm−1

and 1380.16 cm−1, respectively. However, SIL showed several peaks at3341.52 cm−1, 1666.15 cm−1 and 1439.27 cm−1 represented thephenolic (OH) vibrations, mixed (C = O) amide and (C = C) vibrations,and the symmetric aromatic ring stretching vibration (C = C ring),respectively. On the other hand, SILNPs showed peaks at 1550.89 cm−1

and 1485.36 cm−1 indicating C = C aromatic, N–H/C–H stretching,and CH2 wagging coupled with OH groups of COS. The absence ofcharacteristic peak of SIL indicates the successful entrapment of drug

Table 1Experimental group and their respective treatments.

Groups Treatment

Group (1) Control groupGroup (2) CCl4Group (3) SIL aloneGroup (4) SILNPs (LD)Group (5) SILNPs (HD)Group (6) CCl4 + SILGroup (7) CCl4 + SILNPs (LD)Group (8) CCl4 + SILNPs (HD)

CCl4: Carbon tetra chloride (100 mg/kg b.w.) in corn oil.SIL: silymarin alone (50 mg/kg b.w.) in distilled water.SILNPs (LD): low dose of silymarin nanoparticles (25 mg/kg b.w.)SILNPs (HD): high dose of silymarin nanoparticles(50 mg/kg b.w.)

Fig. 1. Scanning electron microscope (SEM) images of SIL (A), SILNPS (B) and (C) TEM of SILNPs.

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within the polymeric matrix as shown in the supplementary materialfile (Fig. 3S).

3.2. Drug release and the cumulative percent of SIL and SILNPs

The composition of main SIL components (silybin A and B, sily-dianin, isosilybin and silychristin) was analyzed using HPLC. The re-sults revealed that the retention times (Rt) of silychristin, silydianin,silybin A, silybin B, isosilybin A and isosilybin B were 5.6, 8.1, 8.7,13.03, 14.03, 17.5 min, respectively (Table 3). The in vitro release ofSILNPs and SIL as determined by HPLC was gradual overtime after5 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 8 h, 12 h and 24 h. Theresults showed a burst release for SIL and SILNPs from 5 min to 1 h, andafter 2 h, the release of SIL becomes slowly and completely stoppedthereafter; however, the release of SILNPs continued to release at 4, 8,12 and 24 h as shown in the Supplementary material file (Fig. 2S-A).The cumulative percent of the drug release (Fig. 3B) showed that therelease percentage of SIL was increased gradually to reach the peak at45 min then decreased and completely stopped after 2 h. However, thepercent of SILNPs release was almost stable from the zero time till 24 has shown in the Supplementary material file (Fig. 2S-B).

3.3. In vivo studies of SIL and SILNPS

3.3.1. Biochemical resultsTreatment with CCl4 resulted in a significant increase in serum AST,

ALT, ALP and GGT; however, the animals received SIL alone were si-milar to the control group in these tested parameters. Treatment withSILNPs at the low or high dose induced a significant decrease in ASTlevel in a dose dependent manner compared to the control group(Table 2). Treatment with CCl4 plus SIL or SILNPs at the two testeddoses produced a significant improvement in the activity of ALT andAST. It is worthy to report that co-treatment with CCl4 plus SIL alonewas more effective in the improvement of ALP followed by the groupsreceived CCl4 plus the high dose of SILNPs then those received CCl4 plus

the low dose of SILNPs (Table 2). Treatment with CCl4 alone induced asignificant increase in the kidney function indices. The level of creati-nine and uric acid in the group received SIL was similar to the controlgroup. Meanwhile, SILNPs (LD) or SILNPs (HD) alone did not induceany significant effect on the level of urea, creatinine and uric acid(Table 2). Additionally, the data presented revealed that CCl4 plus thehigh dose of SILNPs was the most effective against CCl4-induced ele-vation in the kidney function. The effect of different treatment on lipidprofile (Table 3) revealed that animals treated with CCl4 alone showeda significant increase in cholesterol, triglycerides and LDL and a sig-nificant decrease in HDL compared to the control group. A significantincrease in triglycerides level and LDL was observed in the groupstreated with SIL, SILNPs (LD) or SILNPs (HD) compared to the controlgroup. Co-treatment with CCl4 and SIL, SILNPs (LD) or SILNPs (HD)resulted in a significant reduction in on serum lipid profile parameterscompared to the CCl4-treated group although none of these treatmentscould normalize the level of triglycerides and LDL. It is of interest tomention that SILNPs (HD) gave the best result compared to the othergroups regarding TG (Table 3).

The data presented in Fig. 2 revealed that CCl4 administration sig-nificantly increased the level of AFP. Treatment with SIL, SILNPs (LD)of SILNPs (HD) did not induce any significant changes than the control.Moreover, SILNPs at the low or the high dose induced more improve-ment and the group received the high dose was nearly normal. Theeffect of different treatments on the oxidative stress parameters(Table 4) revealed that treatment with CCl4 induced a significant in-crease in MDA and NO accompanied with a significant decrease in TACand GPx in the liver and kidney tissues. The hepatic NO, MDA and GPXin the rats received SIL or SILNPs (LD) were not significantly differentfrom the control group; however, the high dose of SILNPs induced asignificant decrease in NO level in the hepatic and renal tissues. On thecontrary, renal GPx was reduced significantly in the groups treated withSIL, SILNPs at the two tested doses. It is of interest to mention thatSILNPs (LD) was more effective than SILNPs (HD) in the reduction ofNO level in the renal tissue (Table 4) and could normalize the hepatic

0.001.002.003.004.005.006.007.008.009.00

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Fig. 2. Effect of SIL, SILNPs (LD) and SILNPs (HD) on Alpha fetoprotein level (AFP) in rats treated with CCl4 Within each column, means superscript with differentletters are significantly different (P ≤ 0.05).

Table 2Effect of SIL, SILNPs (LD) and SILNPs (HD) on liver and kidney function parameters in rats treated with CCl4.

Parameter Groups ALT (IU/L) AST (IU/L) ALP (IU/L) GGT (mg/dl) Urea (mg/dl) creatinine (mg/dl) Uric acid (mg/dl)

Control 54.33 ± 1.80a 87.00 ± 2.31a 144.50 ± 5.22a 9.001 ± 0.37a 29.33 ± 0.80a 1.32 ± 0.07a 1.40 ± 0.05a

CCl4 160.17 ± 4.00b 215.67 ± 0.67b 259.83 ± 5.03b 42.50 ± 0.99b 48.67 ± 1.02b 5.50 ± 0.29b 5.97 ± 0.21b

SIL 56.67 ± 1.12a 85.17 ± 1.22a 164.00 ± 7.14c 9.67 ± 0.42a 34.00 ± 0.86c 1.33 ± 0.03a 1.30 ± 0.07a

SILNPs (LD) 58.33 ± 2.81c 83.50 ± 0.43ac 153.83 ± 2.40d 10.00 ± 0.58a 27.33 ± 1.28a 1.23 ± 0.05a 1.22 ± 0.06a

SILNPs (HD) 59.67 ± 1.02c 78.83 ± 1.11d 144.17 ± 3.77a 9.67 ± 0.33a 26.67 ± 1.28a 1.27 ± 0.04a 1.23 ± 0.06a

CCl4 + SIL 83.50 ± 1.43d 162.00 ± 1.78e 139.67 ± 3.42e 26.33 ± 0.76c 38.83 ± 1.70d 3.37 ± 0.15c 3.35 ± 0.08c

CCl4 + SILNPs (LD) 56.67 ± 2.04a 137.83 ± 1.56f 207.50 ± 17.04f 21.67 ± 0.92d 30.33 ± 0.92a 2.30 ± 0.03d 2.38 ± 0.08d

CCl4 + SILNPs (HD) 52.17 ± 0.79a 80.50 ± 4.88cg 148.33 ± 2.76 g 16.33 ± 0.61e 24.67 ± 1.17e 1.28 ± 0.02a 1.27 ± 0.06a

Within each column, means superscript with different letters are significantly different (P ≤ 0.05).

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NO level.

3.3.2. The cytogenetic resultsThe gene expression analysis (Fig. 3) showed that CCl4 induced

severe genotoxicity since it increased the expression of the proapoptoticBcl-2 (Fig. 3A), decreased mRNA expression of the antiapoptotic Bax(Fig. 3B), mRNA expression of p53 (Fig. 3C) and caspase-3 (Fig. 3D).SIL alone did not induce any significant change in mRNA expression ofBcl-2; however, SILNPs (LD) or (HD) increased the expression of Bcl-2mRNA and SILNPs (LD) were effective than SILNPs (HD). Co-treatmentwith CCl4 plus SIL or SILNPs at both doses improved significantlymRNA expression of Bcl-2 and SILNPs (LD) showed the best resultcompared to SIL or SILNPs (HD). Moreover, SLI or SILNPs (LD) alonedid not show any significant effect on mRNA expression of Bax; how-ever, SILNPs (HD) decreased Bax mRNA expression. Treatment withCCl4 plus SIL or SILNPs improved significantly mRNA expression of Baxand SILNPs (LD) also was more effective than the other treatments.Additionally, SIL and SILNPs alone induced a significant decrease inmRNA of p53 and caspase-3. Furthermore, both SIL and the low andhigh dose of SILNPs induced a significant decrease in the mRNA ex-pression of p53 and caspase-3 in rats treated with CCl4 and the groupreceived SILNPs (LD) plus CCl4 showed the best result compared tothose received SIL or SILNPs (HD). In addition, SILNPs (LD) showed asignificant decrease in DNA fragmentation compared to the control

group. Co-treatment with SIL, SILNPs at the low or high dose plus CCl4induced a significant improvement in DNA fragmentation percent to-wards the control value (8.6 ± 0.72, 7.5 ± 0.65 and 10 ± 0.98)although this percentage was still above the control level (Table 5).Moreover, the fragmentation of DNA in different treatment groups wasdetected by the gel electrophoresis as a DNA ladder representing aseries of fragments (Fig. 4).

3.3.3. The histological resultsThe microscopic examination of the liver and kidney sections con-

firmed the biochemical results of the current study. The liver section ofthe control group showed normal hepatic architecture (Fig. 5A). Theliver sections of rats treated with CCl4 (Fig. 5B) showed disorganizedhepatic architecture with dense fibrosis septa, mononuclear cellularinfiltration around the blood vessels, proliferation, bile ducts destruc-tion, fatty degeneration, focal areas necrosis with apoptotic cell,apoptotic body distinguished by dense eosinophilic cytoplasm and py-knotic nucleus as well as nucleus with irregular contour and densechromatin structure. The examination of liver sections of SIL alone-administrated animals showed nearly normal hepatocytes with minimalfibrous tissues around blood vessels (Fig. 5C). The group treated withSILNPs (LD) or (HD) showed that the hepatocytes were nearly normalwith polyhedral shape, central rounded vesicular nuclei and an acid-ophilic granular cytoplasm (Fig. 5D, E). The liver of animals treated

Table 3Effect of SIL, SILNPs (LD) and SILNPs (HD) on serum lipid profile in rats treated with CCl4.

Groups cholesterol (mg/dl) Triglycerides (mg/dl) HDL-CHL (mg/dl) LDL (mg/dl)

Control 179.04 ± 1.75a 161.04 ± 10.44a 41.78 ± 0.85a 133.93 ± 4.55a

CCl4 277.46 ± 3.17b 227.31 ± 2.90b 29.47 ± 1.44b 247.98 ± 3.56b

SIL 182.83 ± 2.18a 196.64 ± 2.96c 41.31 ± 1.89a 162.86 ± 8.03c

SILNPs (LD) 160.26 ± 1.13c 190.96 ± 4.52d 35.06 ± 0.40c 174.86 ± 5.40d

SILNPs (HD) 171.62 ± 4.14d 176.73 ± 6.16e 33.80 ± 0.57d 170.90 ± 2.73d

CCl4 + SIL 217.86 ± 2.89e 212.50 ± 7.05f 37.80 ± 0.64c 180.05 ± 3.18e

CCl4 + SILNPs (LD) 130.35 ± 4.52f 201.40 ± 6.50cg 36.14 ± 1.34c 176.25 ± 2.26d

CCl4 + SILNPs (HD) 126.65 ± 1.23 g 193.98 ± 10.50dh 35.08 ± 1.03c 164.69 ± 1.47c

Within each column, means superscript with different letters are significantly different (P ≤ 0.05).

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Fig. 3. Effect of SIL, SILNPs (LD) and SILNPs (HD) on (A) Bax, (B) Bcl-2, (C) p53 and (D) Caspase-3 mRNA expression in the liver of rats treated with CCL4.

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with CCl4 plus SIL showed disorganized hepatocytes and pyknotic nu-clei few periportal fibrosis (Fig. 5F). On the other hand, the liver sec-tions in the group treated with SILNPs at low or high dose showednearly normal hepatocytes and marked decrease in fibrous tissues(Fig. 5G, H)

The examination of the kidney sections of the control rats showednormal renal distal and proximal convoluted tubules and intact glo-meruli with Bowman capsule (Fig. 6A). However, the sections of CCl4-treated rats showed tubular epithelial cells degeneration in the form ofvacuolation, fatty degeneration and pyknotic nuclei (Fig. 6B). Thekidney of SIL-treated rats showed swollen renal tubules with pyknoticnuclei and glomerular mesangial proliferation (Fig. 6C). However, thekidney of SILNPs (LD)-treated rats showed nearly normal tubules, glo-merular mesangial proliferation, some tubules have hyaline casts andinterstitial hemorrhage (Fig. 6D). The animals treated with SILNPs (HD)showed glomerular mesangial proliferation, tubular dilation and widelumen (Fig. 6E). The examination of kidney sections of rats treated withCCl4 plus SIL showed nearly normal glomerular mesangial and Bowmancapsule and normal renal tubules (Fig. 6F). However, the kidney of ratstreated with CCl4 plus SILNPs (LD) showed the renal tubules withnormal nuclei, some tubules lumen are narrow and diffuse mildlyswollen tubules (Fig. 6G). The kidney of rats treated with SILNPs (HD)plus CCl4 showed glomerular mesangial proliferation and Bowmancapsule obliteration and normal renal tubules (Fig. 6H).

4. Discussion

SIL suffers several limiting factors, including its low solubility inwater, low bioavailability and poor absorption in the GIT, so the pre-sent investigation is an effort for improving its characteristics andprolonged its release to promote the therapeutic effects in terms ofhepatoprotective activity. Several attempts were made to solubilize SILin order to overcome the biopharmaceutic limitations; however, noneof these attempts have shown any pharmacological successes (Wooet al., 2007). In the current study, we synthesized and characterizedSILNPs by incorporating it in chitosan nanoparticles (CNPs) and eval-uate its efficiency against CCl4-induced toxicity, genotoxicity, oxidativestress and histological changes in the liver and kidney.

The synthesized SILNPs showed a smooth rounded shape with anaverage particle size of 100 nm. The particles size of the synthesizedSILNPs were similar to those reported by Zhao et al. (2016) althoughthe shape of the particles was different from that reported by theseauthors since they found nearly spherical shape particles. The FTIRanalysis revealed the successful entrapment of drug within the poly-meric matrix. The HPLC analysis of raw SIL revealed the occurrence ofthe main six compounds included silychristin, silydianin, silybin A, si-lybin B, isosilybin A and isosilybin B similar to those reported in theliterature (Mudge et al., 2015). The current results also showed that therelease of SILNPs was sustained and continue till 24 h; whereas, therelease of SIL was stopped completely after 4 h (Das et al., 2011). Aprevious study indicated that SILNPS exhibited a sustained releaseTa

ble4

Effectof

SIL,

SILN

Ps(LD)an

dSILN

Ps(H

D)on

antiox

idan

ten

zymeactivity

andox

idativestress

marke

rsin

kidn

eyof

rats

treatedwithCCl 4.

Parameter

Group

Live

rKidne

y

MDA

(nmol/g

)NO

(µM/m

g)TA

C(µM/g

)GPX

(U/m

g)MDA

(nmol/g

)NO

(µM/m

g)TA

C(µM/g

)GPX

(U/m

g)

Con

trol

55.64

±2.57

a45

5.33

±10

.105

a20

.47

±0.82

a28

8.59

±1.07

a66

.75

±1.53

a39

9.33

±32

.71a

20.00

±1.00

a52

9.90

±24

.88a

CCl 4

149.53

±1.56

b52

9.33

±5.17

b46

.23

±0.79

b41

.28

±2.30

b13

6.54

±6.4b

554.67

±51

.87b

15.00

±0.00

b17

7.46

±8.54

b

SIL

55.53

±2.51

a45

0.00

±8.89

a20

.37

±0.91

a29

0.87

±0.92

a72

.20

±2.74

c38

5.67

±6.39

a18

.00

±1.00

c50

3.06

±12

.49c

SILN

Ps(LD)

46.20

±1.97

c45

5.67

±3.71

a17

.71

±0.81

c29

3.57

±2.95

a65

.40

±1.83

a46

3.67

±8.67

c19

.00

±1.00

ac51

8.76

±23

.1d

SILN

Ps(H

D)

41.92

±1.09

d43

1.00

±8.33

c19

.54

±1.94

a29

0.50

±3.75

a61

.97

±1.38

d50

1.33

±15

.50d

23.00

±0.00

d48

9.02

±9.56

e

CCl 4

+SIL

134.48

±0.86

e44

2.00

±2.65

a17

.70

±1.18

c16

3.57

±3.57

d11

1.76

±0.41

e49

2.00

±31

.05e

16.00

±0.00

be

383.60

±6.31

f

CCl 4

+SILN

Ps(LD)

127.37

±1.24

f44

0.33

±11

.57a

14.70

±0.12

d18

9.57

±2.57

e85

.81

±1.80

f43

1.67

±7.86

f17

.00

±0.00

ce39

0.09

±22

.31f

CCl 4

+SILN

Ps(H

D)

75.83

±2.72

g39

6.00

±15

.51d

16.30

±0.35

cd28

4.50

±3.95

f76

.72

±0.57

cg49

7.33

±33

.58e

23.00

±1.00

d37

3.94

±36

.2fg

Withineach

column,

means

supe

rscriptwithdifferen

tlettersaresign

ificantly

differen

t(P

≤0.05

).

Table 5Effect of either SIL or SILNPs on the percentage of DNA fragmentation in liver ofrats treated with CCl4.

Groups DNA fragmentation %(M ± SE)

% of Change compared tocontrol

Control 9.8 ± 0.86 –CCl4 23.5 ± 1.5 +13.7SIL 10.3 ± 0.11 +0.5SILNPs (LD) 8.9 ± 0.97 −0.9SILNPs (HD) 11.3 ± 0.68 +1.5CCl4 + SIL 8.6 ± 0.72 −1.2CCl4 + SILNPs (LD) 7.5 ± 0.65 −2.3CCl4 + SILNPs (HD) 10 ± 0.98 +0.2

M.H.A. Aboshanab, et al. International Journal of Pharmaceutics 587 (2020) 119639

6

profile during 8 h of the in vitro study period. The difference betweenthe current results and those reported earlier may be due to the polymermatrix used in the preparation since this release is affected by nano-particles surface-adsorbed molecules and the insoluble nature of thepolymer (Liang et al., 2018). Moreover, the slow release of the drugmay result from electrostatic interaction between the drug and self-assembled micelles (Wang et al., 2011). Several factors may be involvedin the controlled release of the drug-loaded nanoparticles, including thesize of the nanoparticles, the percentage of drug-loaded particles, pH,temperature and the concentration of dissolution medium (Heikkilaet al., 2007; Ukmar et al., 2011). These results suggested that in-corporation of SIL into compounds with greater bioavailability enhanceits absorption and control the release (Javed et al., 2011). The dis-solution results indicated that the drug release from the formulation

follows diffusion and the erosion of polymer. When the formulation isin contact with the dissolution media, the media penetrates the latticenetwork of the polymer leading to polymer chain disentanglementcausing polymer erosion and subsequent release of SIL. During thisstudy, the burst release of raw SIL over a period from 5 min to 1 h wasobserved but was not available for prolonged release which mainly dueto the heterogeneous of SILNPs distribution (Igartua et al., 1997; Rafatiet al., 1997). On the other hand, the release of SILNPs from the for-mulation was sustained and continue till 24 h that can be attributed tothe high polymer concentration that delays SILNPs diffusion withinpolymer droplets (Bodmeier and McGinity, 1988) beside to chitosanproduced a partially hydrophobic matrix, which has less affinity forwater resulting in retardation of drug release form the formulation

In the in vivo study, we investigated the protective role of SILNPs in

M Control CCl4 SIL SILNPs (LD)

SILNPs (HD)

SIL + CCl4

SILNPs (LD) + CCl4

SILNPs (HD) + CCl4

Fig. 4. Effects of SIL or SILNPs at the low or high dose on DNA fragmentation in hepatic tissue of rats treated with CCl4. Agarose gel electrophoresis pattern of DNAisolated from liver tissue of different groups. Lane M: 100-bp ladder marker, Lane 1: Control, Lane 2: CCl4 Lane 3: SIL; Lane 4 SILNPs (LD); Lane 5: SILNPs (HD); Lane6: SIL plus CCl4; lane 7: SILNPs (LD) plus CCl4 and Lane 8: SILNPs (HD) plus CCl4.

Fig. 5. Photomicrograph of a liver section of (A) control rat showing normal hepatocytes structure; (B) rats treated with CCl4 showing Fatty degeneration, focal areasnecrosis with apoptotic cell and apoptotic body distinguished by dense eosinophilic cytoplasm and pyknotic nucleus, the nucleus showed irregular contour and densechromatin structure. Mononuclear cellular infiltration and fibrosis around portal tracts; (C) Animals treated with SIL showing nearly normal hepatocytes withminimal fibrous tissues around blood vessels; (D) Animals treated with SILNPs at low dose showing the hepatocytes are polyhedral in shape with central roundedvesicular nuclei and an acidophilic granular cytoplasm; (E) animals treated with SILNPs at high dose showing the hepatocytes are polyhedral in shape with centralrounded vesicular nuclei and an acidophilic granular cytoplasm; (F) animals treated with CCl4 plus SIL showing disorganized hepatocytes and pyknotic nuclei fewperiportal fibrosis; (G) animals treated with CCl4 plus SILNPs at the low dose showing nearly normal hepatocytes and marked decrease in fibrous tissues and (H)animals treated with CCl4 plus SILNPs at the high dose showing nearly normal hepatocytes and marked decrease in fibrous tissues.

M.H.A. Aboshanab, et al. International Journal of Pharmaceutics 587 (2020) 119639

7

against the toxicity, genotoxicity, oxidative stress and histologicalchanges in the liver and kidney in rats treated with CCl4. The selecteddose of SIL and SILNPs was based on the work of El-Denshary et al.(2012); however, the selected dose of CCl4 was based on the work ofSingab et al. (2005). CCl4 is well known as a model of hepatotoxicityexperiments (Cemek et al., 2010) and for the evaluation of the hepa-toprotective role of several herbs and drugs (Ahsan et al., 2009). Theformation of its reactive trichloromethyl radicals (%CCl3) by Cyt P450 isthe pivotal role of the mode of action of CCl4-induced its toxicity (Ipand Ko, 1996). CCl3 is rapidly transformed into CCl3O2% (tri-chloromethyl peroxyl radical) in the presence of O2. This CCl3O2%

covalently binds with the cellular lipids and proteins and initiates theperoxidation of lipids in the cell membrane resulting in the release ofthe enzymes ending by the cellular necrosis and apoptosis (Manibusanet al., 2007). Hence, the release of cytosolic enzymes is used to estimatethe hepatotoxicity of CCl4 (Opoku et al., 2007). The current resultsrevealed that CCl4 induced a significant increase in ALT, AST and ALPindicating the severe hepatotoxicity of CCl4. The elevation of theseserum enzymes indicated the disturbance in hepatocytes membraneleading to enzyme leakage into the circulation (Kim et al, 2012, Martinset al., 2012, Abdel-Wahhab et al., 2015, Salim et al., 2018, Xiao et al.,2015). These results were in accord with numerous studies indicatinghepatotoxicity of CCl4 in rats (Hassan et al., 2019, Oke et al., 2019). Thecurrent results also showed that CCl4 increased triglycerides, choles-terol and LDL and decreased HDL. The elevation of cholesterol levelindicated the increase of the fatty acids esterification process whichdecreases the cellular lipid excretion and inhibits b-oxidation of fattyacids (Fernandez and West 2005). Additionally, CCl4 stimulates theacetate transfer into hepatocytes, enhances the cholesterol, triglycer-ides and fatty acids synthesis from acetate (Weber et al. 2003). Ad-ditionally, CCl4 was also reported to inhibit apo-lipoprotein synthesisconsequently, reduces the lipoprotein synthesis (Kamalakkannan et al.,2005).

Administration of CCl4 increased AFP level, which is consideredspecific biomarkers for liver cancer (Sarhan et al., 2012). The elevationof AFP reported herein indicated that CCl4 is a potent hepatocarci-nogen, enhance reactive oxygen species (ROS) formation and causesoxidative DNA damage, which may play a role in its carcinogenicity(Yang et al., 2015; Abdel-Aziz et al., 2005; Abdel-Wahhab et al., 2007;El-Denshary et al., 2012). In addition, MDA and NO level in the liverand kidney tissues elevated significantly upon the exposure to CCl4, the

production of MDA induces the damage of mitochondria, suppresses themitochondrial electron transport chain and promote the production offurther ROS (Abdel-Wahhab et al., 2017). Moreover, the generation ofROS activates the stellate cells in these organs, resulting in the de-position of extracellular matrix which contribute to the development offibrosis or cirrhosis (Abdel-Wahhab et al., 2017; Baiocchini et al., 2016;Iacob et al., 2020). The decrease in TAC and GPx in hepatic and renaltissue in CCl4-exposed rats indicated the stressful and oxidative stress inthese organs. Furthermore, these disturbances in the enzymatic anti-oxidant systems were correlated positively with the impairment of liveror kidney injury (Hassan and Yousef, 2010, Abdel-Wahhab et al., 2016)resulted from the increased production of ROS. These events as well asthe machinery of protein synthesis, lead to the damage of macro-molecules inside the mitochondria, especially protein, DNA and lipids;consequently, the mitochondrial dysfunction, energy depletion and thedeath of cells (Casini et al., 1997; Hassan et al., 2019).

The current study also indicated that exposure to CCl4 resulted insignificant disturbances in the gene expression in the hepatic tissue byincreasing the hepatic mRNA expression of Bax, p53 and caspase-3 anddown-regulated the expression of Bcl2 mRNA. ROS disturbs the mi-tochondrial membrane potential (MMP) due to the physiological re-sponse to apoptosis (Ramachandran et al., 2000) and Bax promotes theformation of mitochondrial apoptosis-induced channel due to the re-lease of mitochondrial cytochrome c into the cytoplasm which activatescaspase-9 then Caspase-3 and thus promotes the apoptosis process(Dejean et al. 2006; Antonsson, 2004). Additionally, the up-regulationof p53 mRNA expression in addition to the Bax mRNA expression, as anapoptosis promoting gene, maybe opposed the effects of p53 on apop-tosis as suggested by Singh et al. (2019). Hence, the expression of p53mRNA in CCl4-treated rats did not suppress cell apoptosis but it en-hanced the proliferation and the malignant conversion of the cells(Kruiswijk et al., 2015).

The histopathological findings in the liver and kidney are correlatedwith the biochemical and cytogenetical estimations of studied experi-mental groups. The liver sections of rats treated with CCl4 showed severdisorganized hepatic architecture with dense fibrosis septa and mono-nuclear cellular infiltration around the blood vessels and proliferation.These histological changes were similar to those reported earlier (Songet al., 2007; El-Denshary et al., 2012). The kidney sections of CCl4-treated rats showed tubular epithelial cells degeneration in the form ofvacuolation, fatty degeneration and pykotic nuclei. Similar pathological

Fig. 6. Photomicrograph of a liver section of kidney section of (a) control rats showing normal renal distal and proximal convoluted tubules and intact glomeruli withBowman capsule; (b) animals treated with CCl4 showing tubular epithelial cells degeneration in the form of vacuolation, fatty degeneration and pyknotic nuclei; (c)animals treated with SIL showing swollen renal tubules with pyknotic nuclei and glomerular mesangial proliferation; (d) animals treated with SILNPs at the low doseshowing nearly normal tubules, glomerular mesangial proliferation, some tubules have hyaline casts and interstitial hemorrhage; (e) animals treated with SILNPs atthe high dose showing glomerular mesangial proliferation, tubular dilation and wide lumen; (f) animals treated with CCl4 plus SIL showing nearly normal glomerularmesangial and Bowman capsule and normal renal tubules and (h) animals treated with CCl4 plus SIL showing nearly normal glomerular mesangial and Bowmancapsule and normal renal tubules.

M.H.A. Aboshanab, et al. International Journal of Pharmaceutics 587 (2020) 119639

8

changes were reported in the renal sections of rats treated with CCl4 for7 weeks (Dogukan et al., 2003). It was suggested that histopathologicalchange in the kidney may be linked with the absorption power of thetubules and nephrons functional overloading with subsequent kidneydysfunction (Adewole et al., 2007).

Co-administration of SIL or SILNPs showed a protective role of theseagents against CCl4. Animal treated with SIL or SILNPs alone did notshow any significant changes in all the biochemical parameters testedor the histological picture of the liver and kidney. Moreover, both SILand SILNPs exhibited a protective role against CCl4 regarding the bio-chemical, the genetic and the oxidative stress markers as well as thehistology of the liver and kidney although SILNPs was more effectivethan the raw SIL. As mentioned earlier, the efficacy of SIL as a ther-apeutic agent was hindered by its limited bioavailability due to its pooraqueous solubility, low intestinal permeability, extensive metabolismand rapid excretion (Javed et al., 2011). Although orally administeredSIL can be absorbed rapidly with a tmax of about 2–4 h and a t1/2 of 6 h,only 20–50% of it can get absorbed during the process which laterundergoes enterohepatic circulation (Wu et al., 2009). Besides, about83% of total SIL absorbed can get metabolized to glucuronide andsulfate conjugates in 1–3 h and eliminated from the body throughbiliary route (Wen et al., 2008). SILNPs showed a higher protectiveeffect against CCl4 intoxication compared to raw SIL. Previous reportsindicated that SIL posse antioxidant and free radical scavenging ac-tivity. In this concern, the proton-radical scavenging action has beenknown as a vital mechanism of antioxidation. Zhao et al. (2016) re-ported that SILNPs was more effective than raw SIL in the scavenging ofDPPH radicals in vitro. These authors found that the IC50 value ofSILNPs on DPPH radical scavenging assay was 0.052 mg/ml comparedwith 0.097 mg/ml of raw SIL. The same authors concluded that to reacha similar extent of the DPPH-scavenging effect, the concentration re-quired for raw SIL should be higher than that required for SILNPs.Additionally, SILNPs can exert the antioxidant property efficiently be-cause of their higher dissolution rate and solubility than the raw SIL.SILNPs demonstrated a proton donating ability and can serve as freeradical inhibitors or scavengers, thereby potentially acting as primaryantioxidants. Furthermore, SILNPs showed a significant reduction ofthe Fe3+/ferricyanide complex to its ferrous form (Chung et al., 2005).Besides, SILNPs showed a higher antioxidant activity than raw SILwhen incubated in vitro with plasma and the oral bioavailability of si-lybin in SILNPs was significantly higher than raw SIL in vivo since a veryhigh concentration of silybin was found in the liver with a long reten-tion time in different organs (Zhao et al., 2016). Therefore, SILNPs mayoffer a great potential value to become a new oral SIL formulation. Onthe other hand, SILNPs was found to induce rapid regeneration of thehepatic GSH levels along with down regulation of serum enzymeparameters, and marked increase in survival even when administeredafter acetaminophen (APAP)-induced hepatic damage suggesting thatthe formulation of nanoparticles improved solubilization of SIL (Daset al., 2011).

In the current study, chitosan nanoparticles were used in the fab-rication of SILNPs. Hence, we proposed additional mechanism for theenhancement of the protective role of SILNPs. The potential therapeuticeffects of chitosan against hepatic fibrosis (HF) and other medical ap-plications such as the antitumor, antiulcer, immunostimulatory andantibacterial properties have been reported (Liu et al., 2017; Abdel-Gawad et al., 2016; Abdel-Wahhab et al., 2016; Abdel-Aliem et al.,2019). Chitosan is known for its anti-inflammatory effects, which maybe protecting against CCl4-induced infiltration of inflammatory cells inthe liver. Wang et al. (2018) reported that chitosan significantly im-proved hepatic fibrosis through the decreasing of serum AST, ALT, andALP and decreasing the expression levels of COl3. Positive charge aminogroups in chitosan can adhere to cell membranes, which are negativelycharged and evoke some biological change in cell membrane and pre-vent a leakage of intracellular enzymes into the blood (Hasegawa et al.,2001). Furthermore, the antioxidant property of chitosan nanoparticles

was reported in a series of reports (Abdel-Aziem et al., 2011; El-Denshary et al., 2015; Abdel-Gawad et al., 2016; Abdel-Wahhab et al.,2015, Abdel-Wahhab et al., 2016, Abdel-Wahhab et al., 2017). It wasreported that chitosan nanoparticles themselves may be advantageousin the alleviation of NAFLD (Si et al., 2017; Subhapradha et al., 2017)through promotion of expression of proteins that have roles in the lipidmetabolism in the liver (Liu et al., 2011) and activation of the anti-oxidant enzymes like glutathione peroxidase (Santhosh et al., 2007;Liang et al., 2018). Taken together, the results from the biochemical,cytogenetic and histopathological analysis revealed that the developedSILNPs showed greater protective effects in ameliorating CCl4 toxicityin rats and in turn, could have increased the drug bioavailability andthen its therapeutic effects.

5. Conclusion

The current study succeeded to develop the silymarin nanoparticles(SILNPs) through the incorporation of raw silymarin (SIL) with chitosannanoparticles. The developed SILNPs showed a smooth rounded shapewith an average particle size of 100 ± 2.8 nm. The in vitro release ofraw SIL was slowly and completely stopped after 4 h; however, therelease of SILNPs was continuing to release up to 24 h. The HPLCanalysis showed that SILNPs have the same compounds in raw SIL.SILNPs showed greater protective effects in ameliorating CCl4 toxicityin rats and in turn could have increased the drug bioavailability andthen its therapeutic effects. CCl4 induced disturbances in the bio-chemical and cytogenetical parameters as well as histological changesin the liver and kidney typical to those reported in the literature. SILNPsshowed greater protective effects than raw SIL due to the higher dis-solution rate and solubility. Additionally, the antioxidant effect ofchitosan nanoparticles used in the fabrication of SILNPs may induce asynergistic effect and enhanced the protective role of SILNPs. Takentogether, the results from the biochemical and histopathological ana-lysis provide evidence that the developed SILNPs possess the potentialfor future clinical application as demonstrated by its improved watersolubility and antioxidant efficiency.

Declaration of Competing Interest

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.

Acknowledgments

This work was supported by the Faculty of Pharmacy, CairoUniversity and The National Research Centre, Dokki, Cairo, Egyptproject # 12050305

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijpharm.2020.119639.

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