Milk Thistle (Silybum marianum L.) Nanoparticles Exhibited Protective Effects on Cisplatin-Induced Cardiotoxicity in Experimental Rats

1. INTRODUCTION

Cisplatin (cis-diamminedichloroplatinum II) is a cornerstone chemotherapeutic agent with strong antitumour activity. Clinically, it is prescribed for a wide range of malignancies, including ovarian, lung, head and neck, testicular, and bladder cancers.^[1] However, its clinical utility is restricted by several dose-limiting adverse effects, most notably nephrotoxicity, hepatotoxicity, and cardiotoxicity. Cardiovascular diseases (CVDs), which are a spectrum of disorders involving the heart and vasculature, remain the foremost cause of illness and death globally.^[2] The cardiotoxic effects of cisplatin (CD) are thought to arise mainly from excessive production of reactive oxygen species (ROS), depletion of the antioxidant glutathione (GSH), and the resulting oxidative stress. These events are accompanied by increased serum levels of cardiac injury biomarkers such as creatine kinase (CK) and troponin I.^[3]

Botanical extracts are rich reservoirs of bioactive molecules, especially antioxidant constituents such as carotenoids, tocopherols, anthocyanins, and diverse phenolic compounds.^[4] Roughly one-fifth of all known plant species have been incorporated into traditional and contemporary medical practices over centuries, and their therapeutic value is largely linked to secondary metabolites, including alkaloids and phytoestrogens, which exhibit well-documented anticancer, anti-inflammatory, antiviral, and antimicrobial properties.^[5]

Milk thistle (Silybum marianum L.), also referred to as St. Mary’s thistle or Scotch thistle, is a widely distributed medicinal plant found across the Mediterranean basin, East Asia, Europe, Australia, and the Americas. It has long been employed as a traditional herbal remedy, particularly in the management of liver disorders.^[6,7] Extracts of milk thistle are the main natural source of silymarin, a flavonolignan complex composed predominantly of silybin A, silybin B, isosilybin A, isosilybin B, and silychristin A. Among these, silybin is considered the principal and most pharmacologically active constituent; it acts as a strong antioxidant capable of efficiently scavenging free radicals and ROS, thereby boosting cellular antioxidant defences and attenuating the harmful consequences of free radical–mediated reactions. More recently, silymarin has been shown to exert hypocholesterolemic, cardioprotective, antidiabetic, hypolipidemic, anti-inflammatory, neuroprotective, and nephroprotective effects. In addition, it demonstrates a favourable safety profile in experimental animals, and human studies have not reported serious adverse events; nevertheless, its broader use in oncology is hampered by its hydrophobic character and poor bioavailability.^[8]

Despite encouraging in vitro findings, many phytomedicines and plant extracts do not achieve meaningful in vivo efficacy because of limitations such as low water solubility, insufficient lipid solubility, and variable particle size, all of which contribute to poor absorption and reduced bioavailability. Nanotechnology provides a promising approach to address these issues by minimising particle size and transforming bioactive agents into nanostructured forms, thereby enhancing their solubility, bioavailability, and uptake.^[9,10] Accordingly, plant-derived compounds can be incorporated into a variety of nano delivery platforms, including liposomes, nanospheres, and nano capsules.^[11,12]

In recent years, a wide array of nanoparticulate formulations has been introduced; nonetheless, only a limited number of investigations have specifically addressed the development of Silybum marianum (silymarin; Sily) nanoparticles designed to improve cellular uptake and cardioprotective efficacy. In this context, the present study prepared silymarin nanoparticles (Sily-NPs) employing Tween 60 as a stabilising agent to establish an efficient drug-delivery system and evaluated their cardioprotective potential against cisplatin-induced cardiac injury.

2. MATERIALS AND METHODS

3. RESULTS

3.1. Physicochemical characterisation of Silybum marianum nanoparticles

3.1.1. Solubility and UV-visible spectroscopy

Several approaches have been explored to prepare Silybum marianum nanoparticles (Sily-NPs). In this study, Sily-NPs were successfully produced using an ultrasound-assisted technique with Tween 60 as the stabilising agent, without adding any extra carrier materials [Figure 1]. This method generated physically stable nanoparticles through a simple three-step procedure: sonication, evaporation, and freeze-drying of the aqueous nano-extract solution. A clear difference in water solubility was noted between the freeze-dried S. marianum extract (Sily) and the newly prepared Sily-NPs. The conventional extract showed very poor solubility in water and remained largely undissolved when dispersed in an aqueous medium. In contrast, the Sily-NPs were fully soluble in water, forming a transparent and stable dispersion. This marked improvement in solubility is mainly due to two factors: i) the greatly increased surface area-to-volume ratio resulting from the nanoscale particle size, and ii) the presence of Tween 60 at the particle surface, which promotes interactions with water. These results align with earlier reports indicating that native silymarin is highly hydrophobic and non-ionisable, and therefore exhibits poor water solubility.^[13,17] Such poor aqueous solubility typically leads to low bioavailability, since dissolution becomes the rate-limiting step for absorption in the gastrointestinal tract and subsequent biological activity.^[18] UV-visible spectroscopic analysis [Figure 1] provided further evidence of successful nanoparticle formation. The Sily-NPs showed a distinct absorption band at around 270 nm, which is characteristic of silymarin in a nano-dispersed state. By comparison, the conventional Sily powder suspended in water displayed very low absorbance across the UV-visible spectrum and lacked a defined peak at 270 nm. This difference in spectral profile confirms that silymarin was effectively incorporated into a nanoscale, water-dispersible system. The appearance of the 270 nm absorption peak in the Sily-NPs suggests that the active components are molecularly dispersed and able to interact with light, whereas in the conventional extract, hydrophobic aggregates hinder proper dispersion and limit chromophore exposure. These observations are consistent with previously reported spectroscopic analyses of silymarin nano-formulations.^[19,20] The water-soluble Sily-NPs were then tested in vivo by oral administration to albino rats [Figure 1, Steps 3-4]. Blood samples were subsequently collected, and histological examinations were performed to evaluate bioavailability and assess the resulting biological effects.

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Figure 1:

Preparation workflow and characterisation of Silybum marianum nanoparticles (Sily-NPs).

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3.1.2. Particle size distribution, zeta potential, and morphological characterisation

Figure 2 compares the crude Silybum marianum extract with its nano-formulated form (Sily-NPs) in terms of particle size distribution, surface charge, morphology, and elemental composition to verify successful nanoscale formulation. Dynamic light scattering (DLS) showed that Sily-NPs have an average hydrodynamic diameter of about 55 nm with a relatively narrow, monomodal size distribution, whereas the conventional extract consists of much larger, polydisperse particles in the submicron to micron range. The close match between the DLS size and that observed by TEM confirms the formation of uniformly nanosized silymarin particles rather than heterogeneous aggregates. Zeta potential measurements further highlighted these differences. The normal extract exhibited a low surface charge, indicating poor colloidal stability and a higher tendency to form aggregates in aqueous media. In contrast, Sily-NPs showed a zeta potential of approximately −31.2 mV, representing a sufficiently high negative surface charge to promote electrostatic repulsion between particles and thus reduce aggregation. This relatively high negative value is consistent with effective steric and electrostatic stabilisation provided by Tween 60 adsorbed on the nanoparticle surface, indicating that the nanosuspension is physically stable and suitable for biological and in vivo applications. Morphological examination by SEM and TEM supported the observed size and structural differences between the two formulations. SEM images of the crude extract revealed large, irregular agglomerates with rough surfaces, whereas SEM micrographs of Sily-NPs showed discrete nanostructures with smoother, more regular outlines. TEM images of the normal extract displayed dense, plate-like structures, while TEM micrographs of Sily-NPs at different magnifications (b1, b2) demonstrated predominantly spherical, regular, and monodispersed particles with sizes in the tens-of-nanometres range, consistent with the DLS data. Collectively, these observations confirm that the applied organic solvent dispersion followed by sonication and high-pressure homogenisation effectively converted the crude silymarin-rich extract into stable, uniformly distributed nanoparticles. Elemental energy-dispersive X-ray spectroscopy (EDX) analysis of both the conventional and nano-formulated samples showed that carbon and oxygen remained the main elements, confirming that the organic, phytochemical nature of the S. marianum extract was preserved after nano-processing. The absence of unexpected elements in the nano-formulation suggests that the preparation method did not introduce inorganic contaminants and that Tween 60 functions primarily as a surface stabiliser rather than altering the core composition. Overall, the combination of small particle size (∼55 nm), uniform spherical morphology, and sufficiently negative zeta potential (∼−31.2 mV) indicates that the prepared Sily-NPs are physically stable, well dispersed, and suitable as a nano-delivery system to enhance the cardioprotective effect of silymarin against cisplatin-induced cardiotoxicity in experimental rats.

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Figure 2:

Physicochemical characterisation of conventional and nano-formulated Silybum marianum extract (Sily NPs) prepared by organic solvent dispersion-sonication-homogenisation technique. SEM: Scanning electron microscopy, TEM: Transmission electron microscopy. EDX: Energy dispersive X-ray spectroscopy

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3.2. In vivo evaluation of cardioprotective effects

3.2.1. Serum cardiac enzyme activities

Cisplatin administration produced clear evidence of cardiotoxicity, as shown by marked increases in all measured serum cardiac biomarkers compared with the normal control group [Figure 3 and Table 1]. In the cisplatin-only rats, CK-total activity rose to around 960 U/L, CK-MB and LDH activities were likewise elevated, and troponin I converted from negative to positive, collectively reflecting substantial myocardial cell injury. The table and corresponding bar charts indicate that treatment with the different silymarin formulations significantly blunted these cisplatin-induced rises, although the extent of protection varied according to both the preparation and the specific biomarker. For CK-total, every silymarin-treated group showed lower enzyme activity than the cisplatin group, indicating reduced leakage of this cytosolic marker from damaged cardiomyocytes. The crude S. marianum extract (100 mg/kg) decreased CK-total to roughly 783 U/L, while Sily-NPs at 100 mg/kg and 50 mg/kg produced values of about 837 U/L and 932 U/L, respectively. Of note, the silymarin tablet formulation reduced CK-total to near-normal levels (approximately 430 U/L), suggesting a particularly strong protective influence on this relatively nonspecific marker of muscle injury. These tendencies are clearly illustrated in the CK-total bar graph, where all treated groups appear below the cisplatin bars but above the normal control, with distinct lettering denoting statistically significant differences. For CK-MB, a more cardiac-specific isoenzyme, the pattern of protection shifted somewhat. The Sily-NPs 100 mg/kg group provided the most pronounced effect, lowering CK-MB activity to about 941.6 U/L, clearly below the cisplatin-only value. The silymarin tablet and crude extract groups showed slightly higher CK-MB levels (around 980 U/L and 1072.2 U/L, respectively), while the Sily-NPs 50 mg/kg group achieved the smallest reduction (approximately 1125.6 U/L), remaining closer to the cisplatin group. This ranking is evident in the CK-MB bar chart, where the Sily-NPs 100 mg/kg bars fall below those of the other treated groups, underscoring the superior capacity of the higher-dose nano-formulation to preserve cardiomyocyte membrane integrity and limit CK-MB release.

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Figure 3:

Serum creatine-kinase (CK)-total, CK-MB, and lactate dehydrogenase (LDH) activities in cisplatin-induced cardiotoxic rats treated with Silybum marianum tablets, conventional extract, and nanoparticles.

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Table 1: Effects of Silybum marianum conventional extract and nanoparticles on serum cardiac biomarkers in cisplatin-induced cardiotoxicity in rats.

Groups	Ck total (U/L)	Ck-Mb (U/L)	LDH (U/L)	Tn I
Normal (+ ve control)	576±8.25	304±7.26	298±2.45	Negative
Cispt (5mg/kg/bw), (-ve control)	960±9.75 a	1260±9.25 a	1100±4.37 a	Positive
Cispt+ Tablets (100 mg)	430±10. 13 b	980±12.30 b	720±9.13 b	Positive
Cispt +Nano Ext (100mg)	873±5.20 c	941.6±8.23 c	674±5.14 c	Positive
Cispt+Nano (50 mg)	932±11.02 d	1125.6±10.35 d	791±6.38 d	Positive
Cispt+Normal Ext (100 mg)	783±7.42 d	1072.2±5.22 d	1031±10.2 d	Positive

CK: Creatine kinase, LDH: Lactate dehydrogenase, Data are mean ± SD of four animals for each group¸ a. Significantly different from the control group, b. Significantly different from the cisplatin group, c. Significantly different from tablets group, d Significantly different from normal group.

A comparable pattern was seen for LDH, another marker of cellular necrosis. Cisplatin greatly elevated LDH activity, whereas all silymarin formulations reduced it to varying extents. Among the nano-formulations, Sily-NPs 100 mg/kg again produced the largest decrease, followed by Sily-NPs 50 mg/kg, with the tablet and crude extract groups showing intermediate values. The LDH bar graph reflects these relationships, demonstrating that although all treatments lowered LDH relative to cisplatin alone, the high-dose nano-formulation consistently delivered the most robust protection. Troponin I remained positive in all cisplatin-exposed groups; however, its elevation (as inferred from the overall biomarker profile) was attenuated in the silymarin-treated animals, indicating partial preservation of myocardial integrity. Overall, the combined data from the table and figure show that all Silybum marianum preparations—tablet, crude extract, and nanoparticles—alleviated cisplatin-induced cardiotoxicity by reducing serum CK-total, CK-MB, LDH, and troponin I compared with the cisplatin-only group. The magnitude of this protection was both biomarker-specific and formulation-dependent: the tablet formulation was particularly effective in normalising CK-total, whereas Sily-NPs at 100 mg/kg generally provided the strongest cardioprotective effect on the more cardiac-specific markers CK-MB and LDH. These results support the concept that converting the silymarin-rich extract into a nano-sized form—via organic solvent dispersion followed by sonication and high-pressure homogenisation—enhances its cardioprotective efficacy against cisplatin-induced myocardial damage, likely by improving its bioavailability and interaction with cardiac tissue.

3.2.2. Histopathological evaluation of cardiac tissue

Histopathological examination of the heart strongly supports the biochemical biomarker findings, together confirming that cisplatin causes cardiotoxicity and that this damage is alleviated by Silybum marianum treatments [Figure 4]. Heart sections from cisplatin-treated rats show disrupted cardiac architecture, cytoplasmic degeneration, and inflammatory cell infiltration, in clear contrast to the normal control hearts, which display well-aligned muscle fibres and preserved cellular morphology. Giving silymarin, whether as a tablet, crude extract, or nano-formulation, after cisplatin injection markedly improves the overall histological picture, with less myocyte degeneration, fewer inflammatory cells, and better preservation of myocardial structure. Among all treated groups, Sily-NPs at 100 mg/kg show the most pronounced improvement, with myocardial architecture nearly resembling that of the control group and only minimal remaining lesions, indicating that the nanoparticle formulation provides superior structural protection to cardiac tissue against cisplatin-induced injury. These microscopic findings are consistent with the significant decreases in serum CK-total, CK-MB, LDH, and troponin I seen in Sily-NP-treated rats, further supporting the cardioprotective action of silymarin and emphasising the enhanced effectiveness of its nano-encapsulated form in reducing cisplatin-associated myocardial damage.

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Figure 4:

Histopathological changes in cardiac tissue of cisplatin-induced cardiotoxic rats treated with different Silybum marianum formulations (Hematoxylin and Eosin, x400). Red and black (arrows) indicate the changes occurred.

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4. DISCUSSION

Cisplatin is a very effective chemotherapeutic drug, but its clinical use is restricted by several serious side effects, including nephrotoxicity, ototoxicity, neurotoxicity, gastrointestinal disturbances, bone marrow suppression, and various hypersensitivity reactions.^[21,22] Although kidney damage is generally regarded as the main dose-limiting toxicity.^[22] Several studies have also reported that cisplatin treatment can be accompanied by cardiotoxic complications.^[23] These cardiac events may present as electrocardiographic abnormalities, arrhythmias, myocarditis, cardiomyopathy, or even congestive heart failure, particularly when cisplatin is combined with other cardiotoxic drugs such as methotrexate, 5-fluorouracil, bleomycin, or doxorubicin.^[23] The cardiotoxicity induced by cisplatin is believed to be driven, at least in part, by excessive production of reactive oxygen species (ROS), weakening of antioxidant defences (e.g., depletion of glutathione), and increased lipid peroxidation in cardiac tissue. Together, these changes compromise membrane integrity, impair mitochondrial function, and ultimately cause leakage of cardiac enzymes such as CK, CK-MB, LDH, and troponin I into the bloodstream. The pronounced elevations in these biomarkers seen in the cisplatin-only group in our study are in line with this proposed mechanism and agree with previous experimental observations.^[21,24]

Silymarin is a flavonolignan mixture with well-established antioxidant and cytoprotective properties.^[8] Its main component, silybin, functions as a strong free radical scavenger and metal chelator and has been shown to inhibit lipid peroxidation, preserve glutathione stores, and boost the activity of endogenous antioxidant enzymes such as superoxide dismutase (SOD). Through these mechanisms, silymarin stabilises cellular membranes and limits the permeability changes associated with oxidative injury. Earlier studies have demonstrated that oral silymarin (e.g., 100 mg/kg for 10 days) can protect the heart from cisplatin-induced damage by lowering serum LDH, CK-MB, and cardiac troponin I levels, largely through its anti-lipid-peroxidation effects.^[21,23] In the current study, silymarin—whether given as a crude extract, tablet formulation, or nanoparticle preparation—reduced cisplatin-induced increases in CK-total, CK-MB, LDH, and troponin I and improved the histological appearance of the myocardium. These results support the idea that silymarin helps preserve the structural and functional integrity of cardiac myocytes, thereby preventing enzyme leakage and limiting myocardial necrosis.

At the molecular level, the free hydroxyl groups on the silymarin molecule (especially at positions C5 and C7) are thought to react with peroxide radicals, thereby interrupting lipid peroxidation chain reactions. Silymarin has also been reported to lessen oxidative damage to mitochondrial DNA and to enhance the overall antioxidant capacity of cells, further contributing to cardio protection. Despite these favourable biological activities, conventional silymarin is hampered by poor water solubility and low oral bioavailability due to its hydrophobic, non-ionisable nature.^[17] These physicochemical drawbacks limit its therapeutic efficacy, particularly when high local or systemic concentrations are needed. Nanotechnology-based delivery platforms offer a logical strategy to overcome these limitations by shrinking particle size, increasing surface area, improving solubility, and enhancing absorption.

In this study, silymarin was successfully formulated as Tween 60–stabilised nanoparticles with an average diameter of about 55 nm, complete dispersibility in water, and a zeta potential of −31.2 mV. Such physicochemical features are expected to promote better dissolution in the gastrointestinal tract, improved mucosal penetration, and potentially more favourable tissue distribution. Our in vivo results are consistent with this expectation. All silymarin preparations reduced cisplatin-induced cardiotoxicity, but the degree of protection varied between groups. For CK-total, the tablet formulation produced the largest shift toward normal levels, while the crude extract and Sily-NPs (100 and 50 mg/kg) also lowered enzyme activity compared with cisplatin alone. For CK-MB and LDH, however, Sily-NPs at 100 mg/kg consistently showed the greatest improvement, followed by the tablet and crude extract, with the 50 mg/kg nanoparticle dose exerting a moderate effect. Troponin I followed a similar pattern. Altogether, these findings suggest that nano-encapsulation enhances the cardioprotective potential of silymarin, most likely by improving its bioavailability and facilitating more efficient delivery to cardiac tissue. The observation that the higher nanoparticle dose (100 mg/kg) generally outperformed the lower dose further supports a dose-dependent protective effect. Overall, the data indicate that Sily-NPs represent a promising approach to augment the therapeutic benefits of silymarin while overcoming its intrinsic solubility and absorption constraints.

The present results underscore the potential of silymarin nanoparticles as a cardioprotective adjuvant during cisplatin chemotherapy. Nonetheless, several issues require further study. First, although serum enzyme profiles and histopathology strongly indicate reduced myocardial injury, additional mechanistic markers, such as detailed oxidative stress indices, mitochondrial function assays, and analyses of apoptotic signalling pathways, would provide deeper insight into how Sily-NPs exert their protective effects. Second, pharmacokinetic comparisons between free silymarin and Sily-NPs are needed to quantitatively confirm improvements in bioavailability and tissue distribution. Finally, long-term safety, optimal dosing, and potential interactions with standard chemotherapeutic regimens should be assessed in future preclinical and clinical investigations. Cisplatin-induced cardiotoxicity causes extensive biochemical and structural changes in the heart, including myocardial necrosis, inflammation, and impaired cardiac function, as reflected by elevated serum CK-total, CK-MB, LDH, and troponin I levels and by histopathological damage. In this study, silymarin administered as crude extract, tablet, or Tween 60–stabilised nanoparticles attenuated these cisplatin-induced alterations, confirming its role as a natural antioxidant and cardioprotective agent.^[8,21,24]

5. CONCLUSION

The nano-formulation developed in this study successfully transformed poorly water-soluble silymarin into a stable, completely water-dispersible system with a nanoscale particle size (∼55 nm) and a zeta potential of −31.2 mV. These characteristics indicate improved solubility and colloidal stability, which are expected to enhance its bioavailability. Among the various treatment protocols evaluated, Sily-NPs at 100 mg/kg generally produced the most marked protective effect on cardiac biomarkers, particularly CK-MB and LDH, while histopathological examination of myocardial tissue showed better preservation of cardiac architecture in Sily-NP–treated rats compared with those receiving cisplatin alone. Overall, the current findings demonstrate that silymarin nanoparticles stabilised with Tween 60 constitute a promising cardioprotective formulation capable of attenuating cisplatin-induced cardiotoxicity by lowering serum markers of cardiac injury and improving myocardial histology. By overcoming the constraints imposed by the hydrophobic nature and poor bioavailability of conventional silymarin, this nano-delivery system may serve as a valuable adjunct to cisplatin-based chemotherapy. Future work should address detailed mechanistic pathways, pharmacokinetic profiling, and long-term safety assessment, as well as the potential translation of this strategy into clinical practice.