The effects of crocin nanoparticle on trace elements levels in paraquat-induced oxidative stress in rat

Abstract

Paraquat, a non-spesific herbicide, induces toxicity primarily through oxidative stress, with no specific antidote available. Evidence suggests paraquat disrupts ion homeostasis, affecting essential trace elements involved in antioxidant defense. Crocin, a saffron-derived antioxidant, may counteract these effects, though its instability limits therapeutic use. Niosomes, as nanocarriers, enhance crocin delivery and efficacy. This study investigated the effects of crocin and crocin-loaded niosomes on serum levels of calcium, iron, copper, zinc, magnesium, total antioxidant capacity (TAC), total thiol groups (TTG) and Lipid peroxidation (LPO) in paraquat-induction oxidative stress in male rats. Thirty male Wistar rats were allocated to six groups (n = 5): normal saline, paraquat (5 mg/kg orally), crocin (200 mg/kg IP), crocin-niosomes (200 mg/kg IP), paraquat + crocin, or paraquat + crocin-niosomes for one week. Serum levels of calcium, iron, copper, zinc, and magnesium were measured via inductively coupled plasma optical emission spectrometry) ICP-OES( after acid digestion. also, we evaluated serum TAC, TTG and LPO levels. Paraquat increased serum LPO, TAC, and TTG levels. Both crocin and niosomal crocin reduced LPO elevation, with niosomes showing superior efficacy in normalizing TAC and TTG. Notably, crocin alone occasionally increased oxidative parameters. Paraquat increased serum iron and calcium while decreasing copper and zinc (p < 0.05). Crocin treatment attenuated the paraquat-induced rise in calcium, whereas crocin-niosomes counteracted the paraquat-induced alterations in calcium, iron, copper, and zinc, bringing their serum levels closer to those of the control group. Neither paraquat nor niosomes altered magnesium levels. Niosomal delivery enhances crocin’s efficacy in mitigating paraquat-induced oxidative stress and trace element dysregulation in serum. These results underscore niosomal crocin’s as a promising nano-antioxidant therapy, warranting further investigation into its organ-protective effects and mechanistic pathways.

Introduction

Paraquat (1,1′-dimethyl-4,4′-bipyridinium chloride) is a potent herbicide extensively used in farming for its quick action and affordability^1,2. Despite its agricultural benefits, paraquat poses significant risks to human health. This is particularly evident in cases of intentional ingestion for suicidal purposes, which leads to acute poisoning. Such poisoning results in multi-organ failure and has a fatality rate exceeding 50% within 24 to 72 h. The absence of an effective antidote underscores the urgent need for therapeutic interventions to mitigate its toxic effects^3–5.

Paraquat exerts its toxic effects primarily through oxidative stress mechanisms, undergoing cyclic reduction-oxidation reactions that generate highly reactive superoxide radicals. This process begins when cellular reductants like NADPH donate electrons to paraquat, which then transfers these electrons to molecular oxygen, creating a destructive cycle of free radical production⁶. The resulting oxidative damage represents the hallmark of paraquat toxicity.

Trace elements, despite their minute concentrations, are indispensable for physiological balance, serving as enzymatic cofactors and stabilizers for protein structures. Their deficiency can lead to significant metabolic disturbances and disease^7–9.

Metals like iron, copper, and zinc are pivotal in oxidation-reduction reactions. Iron and copper can exacerbate oxidative stress via Fenton-type reactions, and copper is particularly vital for mitochondrial function as a component of cytochrome c oxidase^10,11. Paradoxically, copper can also activate apoptotic pathways, while iron maintains a dual role as both an essential nutrient and a potential pro-oxidant^12,13.

Magnesium deficiency weakens cellular defenses by impairing mitochondrial integrity¹⁴. Zinc regulates over 100 metalloenzymes for antioxidant defense and immune function, but the body lacks specialized storage systems for it. During inflammation, zinc is redistributed from the plasma to the liver and spleen, and its renal excretion is increased^15–19. In the lungs, zinc deficiency can disrupt lipid metabolism and contribute to edema²⁰.

The toxicant paraquat disrupts ion homeostasis by altering neuronal calcium and zinc flux, modifying ion channels, and impairing immune cell proliferation through zinc-dependent mechanisms^21–23. The body’s antioxidant defenses, including enzymes like superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), plus non-enzymatic molecules, are fundamentally reliant on trace elements as cofactors. Zinc activates key enzymes, copper is integral to SOD, and iron is a component of CAT and cytochromes^24–28. Consequently, the activity of these antioxidant enzymes directly reflects the body’s trace element status, making micronutrient profiling a valuable indicator of oxidative stress²⁹. However, a significant research gap exists in developing interventions that effectively modulate these trace element pathways to mitigate paraquat-induced toxicity.

Crocin, a water-soluble carotenoid derived from saffron (Crocus sativus L.), has demonstrated potent antioxidant and anti-inflammatory properties³⁰. It scavenges free radicals, upregulates endogenous antioxidants (e.g., SOD, glutathione peroxidase), and modulates signaling pathways such as NF-κB^31–33. However, its clinical application is limited by poor stability and bioavailability. To address this, nanoformulations like niosomes, non-ionic surfactant-based vesicles, have emerged as promising carriers. Niosomes enhance drug delivery by improving cellular uptake, protecting encapsulated compounds from degradation, and enabling targeted release, thereby amplifying therapeutic efficacy^34,35.

Despite crocin’s established antioxidant activity, its therapeutic potential is frequently constrained by low bioavailability and chemical instability. Furthermore, despite the recognized role of oxidative stress in paraquat toxicity, comprehensive studies investigating the simultaneous impact on a broad profile of essential trace elements (calcium, iron, copper, zinc, magnesium) are scarce. Based on our comprehensive review of the existing literature, this is the first investigation to systematically evaluate the protective effects of both free crocin and a novel crocin-loaded niosome formulation against paraquat-induced dysregulation of these specific serum trace elements and oxidative stress biomarkers. We hypothesized that the niosomal delivery system would not only enhance crocin’s antioxidant capacity but also provide superior efficacy in restoring trace element homeostasis compared to the free compound.

Materials and methods

Ethical approval and animal care

This study received approval from Hamadan University of Medical Sciences (IR.UMSHA.REC.1399.793 and IR.UMSHA.REC.1400.272).

A total of thirty adult male Wistar rats, weighing between 180 and 250 g, were sourced from the animal lab of Hamadan University of Medical Sciences. The animals were kept in a controlled environment maintained at 22 ± 1 °C with a relative humidity of 60 ± 5% and a standardized 12-hour light/dark cycle. They were provided with standard rodent feed and water ad libitum. A one-week acclimatization period was observed for all animals prior to the commencement of the experimental procedures.

For all surgical procedures, including blood collection, rats were deeply anesthetized with an intraperitoneal injection of ketamine/xylazine (50 mg/kg/5 mg/kg) mixture. At the end of the experimental period, euthanasia was performed humanely by exsanguination from the inferior vena cava while the animals were under deep anesthesia^36,37.

Experimental design

Rats were randomly assigned to six groups (n = 5 each). The doses of paraquat (agroxir, Iran) (5 mg/kg, orally) and crocin (Sigma-Aldrich, USA) (200 mg/kg, intraperitoneally) were selected based on a preliminary pilot study conducted by our research group. This pilot investigation established that the chosen paraquat dose effectively induces significant oxidative stress and trace element dysregulation over a 7-day period while maintaining animal viability. Similarly, the crocin dose demonstrated significant antioxidant efficacy without adverse effects in this model. The specific groups were as follows: (1) N, control (normal saline (saha, Iran)), (2) PQ (5 mg/kg, oral), (3) C (200 mg/kg, i.p.), (4) NC (200 mg/kg, i.p.), (5) PQ + C, (6) PQ + NC. All treatments were administered daily for 7 days^36,37.

Sample collection and processing

Blood was collected via the inferior vena cava into clot activator tubes. After a 30-minute incubation period at room temperature, the tubes were centrifuged at 3000 rpm for 15 min. The separated serum was then carefully aliquoted and stored at − 80 °C until all analyses could be performed.

Biochemical analyses

TAC

The measurement of TAC was performed using the manual FRAP (Ferric Reducing Antioxidant Power) method. In this assay, antioxidant compounds reduce ferric ions, generating a colored complex quantified spectrophotometrically at 593 nm. In this study The spectrophotometer (Bel UV/Vis, Italy) was zeroed at 593 nm using the FRAP reagent, and sample absorbance was measured after adding 50 µL sample to 1.5 mL FRAP reagent. The TAC for each sample was measured in duplicate, quantified against a standard curve, and reported in units of nmol/mL serum³⁸.

TTG

DTNB (5,5′-dithiobis-(2-nitrobenzoic acid))-based colorimetric assay was utilized for TTG measurement. In this reaction, DTNB interacts with thiol groups to generate a yellow complex at 412 nm. All measurements were performed in duplicate. A standard curve was used for quantification, and results were expressed as nmol/mL serum ³⁹.

LPO

LPO levels were measured using the fluorometric thiobarbituric acid (TBA) method according to Yagi’s protocol⁴⁰. The fluorescence intensity of samples were measured at 532 nm. Samples were analyzed in duplicate, and LPO concentration was determined using a standard curve and expressed as nmol/mL serum.

Trace element analysis

Serum samples were subjected to acid digestion using a buffer containing nitric acid (Merck, Germany), butanol (Merck, Germany), and Triton X-100 (Sigma-Aldrich, USA) at boiling temperature for 1 h, followed by 1:15 dilution with deionized water. Concentrations of calcium, iron, copper, zinc, and magnesium were quantified through ICP-OES, with the analyses conducted by Zar Azma lab in Tehran, Iran⁴¹.

Data analysis method

The sample size was determined a priori using a power analysis based on CAT activity data from a previous study with a comparable design Ranjbar et al.⁴². Input parameters for the power analysis included an effect size of 2 IU/L, a standard deviation of 0.9, an alpha (α) level set at 0.05, and a target statistical power (1-β) of 90%. The analysis estimated a minimum group size of 3.6 animals. To ensure robust detection of significant differences across all evaluated parameters, a final sample size of five animals per group (n = 5) was implementedAll experimental data are expressed as the Mean ± SE and were statistically processed using GraphPad Prism software, version 6.The normality of data distribution was confirmed using the Kolmogorov-Smirnov test. For multiple group comparisons, a one-way analysis of variance (ANOVA) was applied, followed by Duncan’s post hoc test to identify specific differences between groups. A p-value below 0.05 was established as the threshold for determining statistical significance.

Results

TAC

Paraquat significantly decreased serum TAC compared to the control group (p < 0.5). Co-treatment with crocin-loaded niosomes (Group PNC) resulted in a significant improvement in TAC compared to the paraquat + crocin group (Group PC, p < 0.05), with levels in the PNC group not differing significantly from the control (Fig. 1).

Fig. 1.

Serum TAC levels following treatment with crocin and crocin-loaded niosomes in rats exposed to paraquat. Experimental groups: N (normal saline control), P (paraquat), C (crocin), NC (crocin niosomes), PC (paraquat + crocin), PNC (paraquat + crocin niosomes). “S” indicate statistically significant differences between groups (p < 0.5).

TTG

A significant decrease in TTG was observed in the paraquat group (p < 0.05 vs. control). Niosomal crocin (Group PNC) partially counteracted this reduction, showing a significant improvement over the paraquat group (p < 0.05), whereas free crocin (Group PC) had no restorative effect (Fig. 2).

Fig. 2.

Serum TTG concentrations in response to crocin and crocin-loaded niosome administration in paraquat-intoxicated rats. Group designations: N (normal saline control), P (paraquat), C (crocin), NC (crocin niosomes), PC (paraquat + crocin), PNC (paraquat + crocin niosomes). “S” denote significant intergroup differences (p < 0.5).

LPO

Paraquat administration significantly increased LPO level (p < 0.05 vs. control). Both free crocin and niosomal crocin effectively attenuated this increase, normalizing LPO to levels indistinguishable from the control, with a more pronounced reduction in the niosomal crocin group (Fig. 3).

Fig. 3.

Serum LPO concentrations following intervention with crocin and crocin-loaded niosomes in paraquat-challenged rats. Treatment groups: N (normal saline control), P (paraquat only), C (crocin only), NC (crocin niosomes only), PC (paraquat + crocin), PNC (paraquat + crocin niosomes). denote significant intergroup differences (p < 0.5).

Effects of crocin and crocin-loaded niosomes on serum iron and calcium levels

Iron & Calcium: Paraquat significantly elevated serum iron and calcium levels (p < 0.05). While crocin partially reduced calcium, only crocin-loaded niosomes significantly reduced both iron and calcium, restoring them to baseline levels (Fig. 4A and B).

Fig. 4.

Serum levels of trace elements in paraquat-intoxicated rats treated with crocin or crocin-loaded niosomes. (A) Serum iron, (B) calcium, (C) copper, (D) zinc, and (E) magnesium levels. Values are presented as mean ± SEM (n = 5 per group). Group abbreviations: N: control (normal saline); P: paraquat; C: crocin; PC: paraquat + crocin; PNC: paraquat + crocin-loaded niosomes. “S” denote significant intergroup differences (p < 0.5).

Effect of crocin and crocin-loaded niosomes on serum copper and zinc levels

Exposure to paraquat significantly decreased copper and zinc levels (p < 0.05). Crocin-loaded niosomes achieved a partial recovery of copper and restored zinc to control levels, showing superior efficacy compared to free crocin (Fig. 4C and D).

Effect of crocin and crocin-loaded niosomes on serum magnesium levels

No significant changes in serum magnesium levels were detected across all experimental groups. Neither crocin nor crocin-loaded niosomes exhibited a significant impact on magnesium concentration (Fig. 4E).

Discussion

A central finding of this research was the significantly greater effectiveness of crocin when delivered via niosomal nanoparticles compared to its free form in countering paraquat poisoning in rats. Compared to free crocin, the nano-encapsulated form showed enhanced efficacy in rebalancing disturbed trace element levels (lowering iron and calcium, elevating copper and zinc) and producing greater improvements in oxidative stress markers.While the antioxidant nature of crocin is known, this research specifically highlights a novel benefit of its nano-encapsulated version. correcting the dysregulation of multiple metals, an under-explored aspect of paraquat’s toxic effects. This combined action on both oxidative damage and elemental imbalance points to a broad therapeutic potential for the niosomal delivery strategy.

The experimental results validate the fundamental mechanism of paraquat toxicity, which is initiated by the generation of reactive oxygen species that cause oxidative damage to cellular components^6,43. Biochemical analysis revealed this damage through a significant increase in serum LPO, confirming lipid peroxidation, and a concurrent decrease in TAC and TTG, indicative of a systemic loss of antioxidant protection and protein oxidation^44–46.

Crocin, a bioactive compound derived from saffron, exhibited notable antioxidant effects in this study³¹. Treatment with crocin alone significantly reduced LPO levels, suggesting its ability to scavenge ROS and mitigate lipid peroxidation. However, crocin alone did not fully restore TAC and TTG levels to baseline, indicating partial efficacy in enhancing systemic antioxidant capacity. This observation corroborates existing literature indicating that crocin enhances the activity of key endogenous antioxidant enzymes, including SOD, CAT, and GPx, consequently alleviating oxidative stress-induced cellular injury. (68). The partial restoration of TTG levels by crocin may reflect its limited bioavailability or rapid metabolism in vivo, which could hinder its therapeutic potential when administered in free form³⁵.

The therapeutic profile of crocin was substantially enhanced through its encapsulation into niosomes. These nanoparticle carriers improve drug stability, bioavailability, and delivery^47,48. Consequently, the niosomal formulation of crocin outperformed the free compound, effectively normalizing LPO levels and producing a significantly greater restoration of both TAC and TTG^34,47. This marked improvement is attributed to the niosomes’ role in facilitating sustained release and increased cellular uptake of the active compound. The finding that crocin alone did not alter oxidative markers in healthy subjects confirms its safety at the tested dose, while the success of the niosomal system highlights the promise of nanotechnology in advancing natural antioxidant therapies.

An intriguing observation was made in the healthy control groups, where administration of crocin-loaded niosomes (Group NC), but not free crocin, led to a significant reduction in serum TTG. This suggests that the nano-formulation may induce a mild pro-oxidant shift in the redox balance under normal physiological conditions. This phenomenon finds context in the broader ‘antioxidant paradox’ observed in nutritional and therapeutic studies, where high-dose or highly bioavailable antioxidant supplements have sometimes failed to confer benefits or have even shown paradoxical adverse effects, contrary to the protective effects of antioxidant-rich diets^49,50. We hypothesize that the enhanced bioavailability and cellular uptake afforded by the niosomal system lead to a localized concentration of crocin that may transiently overwhelm the basal redox equilibrium in healthy cells. While this effect was observed only in a specific biomarker and not in a pathological context, it underscores the importance of dosage optimization and careful toxicological profiling for nano-antioxidant therapies.

The study documented a specific pattern of serum trace element dysregulation following paraquat exposure, characterized by elevated iron and calcium and depleted copper and zinc. These alterations are interpreted as a complex consequence of oxidative damage and the body’s compensatory response. The elevated iron may stem from both hemolysis and oxidative release from storage proteins, potentially exacerbating further oxidative injury via the Fenton reaction⁵¹. Increased serum calcium suggests disrupted cellular calcium homeostasis linked to apoptotic pathways^52–54, while the reduction in copper and zinc likely reflects their increased utilization as cofactors for antioxidant enzymes and possible stress-induced redistribution^21,24.

The superior ability of niosomal crocin to normalize these elemental imbalances points to a mechanism beyond simple antioxidant activity. We propose that by more effectively quenching the initial oxidative storm, niosomal crocin interrupts the vicious cycle of metal-ion-mediated radical generation (e.g., by preventing iron liberation) and mitigates the cellular damage that leads to calcium dysregulation and trace element depletion^53,55. This prevents the excessive demand for copper and zinc, thereby stabilizing their serum levels. This hypothesis, however, remains to be directly tested. Future investigations measuring the activity of metal-dependent enzymes like SOD, examining the expression of metal transporters (e.g., ZIP/ZnT families for zinc), and assessing tissue metal levels are crucial to establish a direct mechanistic link between crocin’s antioxidant action and the normalization of trace element homeostasis.

The superior efficacy of niosomal crocin in normalizing serum levels of the affected trace elements compared to free crocin highlights a potential advantage of the nanoparticle delivery system. It is important to note, however, that these serum measurements are an indicator, not a direct confirmation, of restored cellular or systemic homeostasis. Notably, niosomal crocin completely normalized iron levels, which is particularly significant given iron’s central role in paraquat-induced oxidative damage through hydroxyl radical generation.The niosomal formulation’s enhanced performance highlights nanotechnology’s potential in toxicological interventions where rapid, targeted action is crucial^35,47. Interestingly, magnesium homeostasis remained stable despite paraquat exposure, possibly due to its involvement in less immediately vulnerable metabolic pathways⁵⁶, though chronic exposure might yield different results .

From a clinical perspective, these findings suggest trace element monitoring could serve as both diagnostic markers and therapeutic targets in paraquat poisoning. The ability of niosomal crocin to favorably modulate serum levels of multiple trace elements supports the rationale for further investigating its potential as an adjunct therapy. Future studies are essential to determine if these changes in serum biomarkers translate to improved organ function and survival.

While the current study is focused on serum biomarkers to provide a systemic overview of paraquat intoxication and the therapeutic effects of crocin formulations, it is part of a broader research endeavor by our group to comprehensively understand this pathology. It is important to note that the organ-level protective effects of crocin-loaded niosomes have already been substantiated in independent studies. Specifically, our parallel research demonstrated that nanocrocin significantly attenuates paraquat-induced lung injury by reducing oxidative stress markers LPO, enhancing antioxidant capacity TAC in lung tissue homogenates, diminishing hydroxyproline content (a marker of fibrosis), and improving histopathological alterations³⁷. Furthermore, the efficacy of this nano-formulation is not limited to the lung. Naderi et al. independently demonstrated that crocin-loaded niosomes effectively protect against ischemia-reperfusion injuries in rat kidney tissue, reducing histological damage and oxidative stress⁵⁷.most critically for neurotoxicity, it protects brain tissue by preventing neuronal loss in the hippocampal CA1 region and normalizing cerebral oxidative stress markers⁵⁸. The concordance observed, wherein niosomal crocin effectively normalized serum oxidative stress markers and trace element levels, aligns with its documented tissue-level efficacy in the lung, brain, and kidney. This provides compelling evidence that the systemic improvements detected in blood are indicative of authentic, multi-organ protective actions. Therefore, the serum data presented here provide a validated, non-invasive correlate of the therapeutic response, reinforcing the potential of niosomal crocin as a systemic and organ-protective agent.

This study acknowledges limitations that warrant consideration. The absence of an empty niosome control group precludes definitive attribution of all observed effects solely to crocin. Furthermore, while alterations in serum biomarkers provide valuable systemic insights, they do not establish causal mechanisms or confirm true restoration of tissue homeostasis. The current findings are constrained by the absence of data on specific molecular mechanisms, such as Nrf2 activation, NF-κB signaling, and apoptotic regulation. Incorporating these analyses in future studies is essential to fully elucidate niosomal crocin’s therapeutic potential.

Collectively, these findings showed that niosomal encapsulation can enhances crocin’s ability to mitigate paraquat-induced systemic oxidative stress and trace element dysregulation in serum, positioning it as a promising candidate for antidote development.

Conclusion

This study showes that crocin-loaded niosomes effectively mitigate paraquat-induced elevations in LPO and modulate serum TAC. Furthermore, our findings indicate that niosomal delivery can improves crocin’s ability to normalize paraquat-induced alterations in serum trace element levels. The favorable modulation of serum iron, calcium, and copper by niosomal crocin, compared to free crocin, suggests a promising approach worthy of further investigation. However, establishing true therapeutic efficacy and the restoration of systemic homeostasis requires future studies incorporating histological and functional endpoints. These results provide new insights into the metal-dependent mechanisms underlying paraquat toxicity and establish a conceptual framework for designing targeted therapeutic interventions that can modulate antioxidant pathways.The efficacy of the niosomal formulation highlights the potential of nanoparticle delivery systems in treating acute poisonings. these results augment the expanding repository of scientific data validating the medicinal potential of crocin, while simultaneously highlighting the transformative impact of sophisticated delivery technologies in enhancing its pharmacological profile.Future research should focus on optimizing dosing protocols, evaluating organ-specific protection, and exploring clinical translatability of this approach. The promising results with niosomal crocin warrant further investigation as a potential treatment strategy for paraquat poisoning, particularly in combination with existing therapeutic modalities.

Author contributions

Sepideh Abaszadeh: Data curation; Formal analysis; Validation; Visualization.Mehdi Beyrami & Ashkan Karbasi: Data curation; Formal analysis; Validation; Visualization.Akram Ranjbar: Conceptualization; Data curation; Formal analysis; Project administration; Software; Supervision; Validation; review & editing.Farzin Firouzian: Conceptualization; Data curation; Formal analysis; Project administration; Software; Supervision; Validation; review & editing.Fatemeh bahramibanan: Data curation; Formal analysis; Validation; Visualization; review & editing.Anahita Abbasifard: Data curation; Formal analysis; Validation; Visualization.Sepideh abbaszadeh: Writing - original draft; and Writing - review & editing.

Funding

The research received financial support from the Vice-chancellor for Research and Technology at Hamadan University of Medical Sciences, Hamadan, Iran (Grant No:140008046399 and 140004153310).

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

This study is a research project at Hamadan University of Medical Sciences. All ethical concerns, including issues related to plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, and redundancy, were fully adhered to by the authors. The study’s ethical procedures were sanctioned by the Ethics Committee of Hamadan University of Medical Sciences (Ethical code: IR.UMSHA.REC.1399.793 and IR.UMSHA.REC.1400.272). All procedures were conducted in compliance with ethical guidelines and standard protocols.