Combined anti-inflammatory effects of piperine and curcumin in mitigating hepatic toxicity induced by malathion in rats

Authors

Abstract

AimMalathion (Mal) is one of the most used organophosphorus pesticides that are used for many reasons, such as agriculture and industry. Human exposure to malathion may occur through various means, such as eating food that has been treated with it. Malathion not only increases oxidative stress but also decreases the antioxidant capacity. That is why the current study was designed to emphasize the function of piperine and curcumin alone or combined as antioxidants and anti-inflammatory natural products in mitigating or eliminating the biochemical and histopathological problems resulting from the intoxication of the rats with malathion.
MethodsForty-five Sprague Dawley rats were divided into five groups: control, malathion, malathion + piperine, malathion + curcumin, and malathion + piperine + curcumin groups.
ResultsPiperine and curcumin significantly improved the serum activities of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) enzymes after malathion exposure and significantly restored the oxidant/antioxidant balance. Piperine and curcumin exerted their protective effects and restored the histological architecture of the liver.
ConclusionThe results of the current study showed the anti-inflammatory and synergistic preventive effects of piperine and curcumin against hepatic toxicity induced by malathion.

Keywords

Introduction

Extensive use of organophosphorus pesticides in agriculture and industry can cause significant health and environmental problems. Malathion is one of the most commonly used compounds.¹ Humans are exposed through contaminated food and water, and due to its lipophilic nature and rapid absorption, malathion distributes widely in tissues.² Its toxicity primarily affects the liver via oxidative stress mediated by reactive oxygen species (ROS), leading to tissue damage.³ Medicinal plants have been used for years as a source of food, spices, and, in traditional medicine, as a remedy for numerous diseases. Curcumin is a hydrophobic polyphenol derived from the rhizome (turmeric) of the herb Curcuma longa. It is a spice found in carry powder. It has been used traditionally for many diseases due to its wide spectrum of pharmacological activities. Curcumin has potent antioxidant, anti-inflammatory, anti-diabetic, antiviral, anti-infectious, antimutagenic, and anticarcinogenic properties.⁴
In recent years, functional compounds from food sources have attracted much attention owing to their disease prevention and health-promoting benefits.⁵Piper nigrum is one of the most widely used spices all over the world. It has a distinct, sharp flavor attributed to the presence of the phytochemical piperine. Piper longum has been used in Asia as a natural treatment for poor peripheral blood circulation. Piperine is also a promising natural source with potential for clinical use. An extract of the active phenolic component, piperine, is well known to provide beneficial physiological effects.⁶ It stimulates the digestive enzymes of the pancreas, protects against oxidative damage, lowers lipid peroxidation, is anti-inflammatory, and enhances the bioavailability of a number of therapeutic drugs.⁷ Therefore, the main goal of this work was to highlight the importance of piperine and curcumin as antioxidants and anti-inflammatories in improving biochemical and histopathological complications following exposure of rats to malathion.

Materials and Methods

Chemicals and MaterialsMalathion (purity; 99.9%) is available in a white liquid formula, obtained from Al Nasr Company (Cairo, Egypt) for chemical industries in, Egypt. Piperine (extracted from black pepper fruit) was purchased as a dietary supplement from Natures Plus, NY, USA. Curcumin (Extracted from turmeric root) was purchased as a dietary supplement from Now Foods, IL., USA. All other reagents were commercial products of standard chemical grade.
Experimental AnimalForty-five adult male Sprague-Dawley albino rats weighing 182 and 197 g were used in this investigation. Following a week of acclimation, the animals were placed in groups (9 rats/group) and kept in climate-controlled settings (22 ± 2 C, 45% humidity).
Design for Experiments After the acclimatization period, rats were divided randomly into 5 groups (nine rats/group). They were allowed free access to food (standard pellet diet) and tap water ad libitum for 42 days (6 weeks).
Group 1 (Control): Healthy, untreated, and received distilled water.
Group 2 (malathion group): Rats received malathion by oral gavage at a dose of 100 mg/kg/day.
Group 3 (piperine + malathion group): received piperine solution by oral gavage at a dose of 30 mg/kg/day and, three hours later, received malathion (100 mg/kg/day).
Group 4 (curcumin + malathion group): received curcumin solution by oral gavage at a dose of 250 mg/kg/day and, three hours later, received malathion (100 mg/kg/day).
Group 5 (piperine + curcumin + malathion group): received piperine solution at a dose of 30 mg/kg/day plus curcumin solution by oral gavage at a dose of 250 mg/kg/day and, three hours later, received malathion (100 mg/kg/day).
Sample Collection and Biochemical AssessmentOn the last day of the experimental period, rats were fasted overnight, then anesthetized with ether, blood samples were drawn from the hepatic portal vein, and then transported into centrifuge tubes. Tubes were centrifuged at 5000 x g for 15 minutes at 24 °C to collect serum for the biochemical examination. Serum samples were stored at -20°C until used for various biochemical analyses. To determine hematological parameters (WBC count, platelet count, hemoglobin concentration, and prothrombin time), additional blood samples (approximately 2 ml) were collected into test tubes containing ethylenediaminetetraacetic acid (EDTA). The liver samples were separated, rinsed, and washed with cold saline solution, then blotted on filter paper. The liver samples were rapidly freeze-clamped and stored at -20 ºC for histopathological examination.
Serum AnalysisSerum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), direct bilirubin, total cholesterol (TC), total proteins, albumin (A) were evaluated spectrophotometrically using Biodiagnostic kits, Cairo, Egypt. Globulin (G) was calculated theoretically according to the following formula: globulin = total proteins- albumin.
Oxidative Stress BiomarkersSuperoxide dismutase (SOD), malondialdehyde (MDA), reduced glutathione (GSH), nitric oxide (NO), and catalase (CAT) were evaluated in serum spectrophotometrically using the Biovision kit, CA. USA.
Inflammatory CytokinesTumor necrosis factor alpha (TNF-α), interleukin-10 (IL-10), nuclear factor kappa-B (NF-κB), and interleukin-1β (IL-1β) were determined in serum by enzyme-linked immunosorbent assay following the instructions of the manufacturer using ELISA kits (Kamiya Biomedical Co., CA, USA).
Ethical ApprovalThe study was approved by the Ethics Committee of Ain Shams University (Date: 23.12.2024, Decision No: sci1432503001).
Statistical AnalysisAll data were expressed as mean value ± standard deviation (SD), SPSS program version 21 (SPSS Inc., Chicago, Illinois, USA). Comparison between groups was carried out using one-way ANOVA; the method used to assign significance letters (a, b, c…) is least significance difference (LSD). between mean values. P-value p ≤ 0.01 was considered significant.
Reporting GuidelinesThis study is reported in accordance with the ARRIVE guidelines for animal research.

Results

As shown in Supplementary Figure 1 (A, B, C, and D) , the findings of the current research revealed that serum concentrations of ALT, AST, ALP, and direct bilirubin respectively were significantly increased in malathion rats (G2) in comparison with the control group There was a statistically significant improvement (reduction) in rats treated with the piperine (G3) or curcumin (G4). This effect was reversed in G5, which received curcumin combined with piperine, as compared to rats exposed to malathion alone.
Data from Table 1 clearly revealed that treatment with piperine and curcumin improves and helps to nearly normal the levels of serum TNF-α, NF-kB, IL-1β, and IL-10 when compared with untreated groups subjected to malathion. Rats treated with both piperine and curcumin (G5) showed the greatest improvement in inflammatory biomarker levels when compared to other treatment groups.

The pretreatment effect of curcumin, piperine, and curcumin plus piperine on Mal-induced hepatic toxicity is given in Supplementary Figure 2 (A, B, C, D, and E). Malathion treatment is associated with hepatotoxicity in male rats, as revealed by a decrease in serum levels of total proteins, albumin, globulin, and A/G ratio. This effect was reversed in G3, G4, and G5, which received piperine and curcumin. The result also showed a significant increase in the total cholesterol values in rats exposed to malathion when compared with the controls. Piperine and curcumin, administered alone, had similar effects on serum total cholesterol. However, treatment with both curcumin plus piperine resulted in a significant synergistic reduction in total cholesterol levels.
A considerable increase (p ≤ 0.01) in NO and MDA values and a substantial drop in serum SOD, CAT, and GST enzyme activities as compared to the healthy group are indicative of malathion-induced oxidative stress. All treated groups' levels of oxidative stress indicators were significantly improved by piperine and curcumin treatment. The remarked effects compared well with healthy control rats, and rats treated with a mixture of piperine and curcumin produced a substantial improvement in these parameters (Table 2).

Results presented in Table 3 revealed a correlation between malathion-induced spleen toxicity and the increased white blood cell count and time of prothrombin and reduced platelet count. Administration with piperine and curcumin (G3- G5) caused an increase in platelet count and significantly improved leukocyte count, hemoglobin (Hb) concentration, and prothrombin time when compared to malathion-intoxicated rats (G2).


In control rats (Supplementary Figure 3A), the histopathological examination of the liver showed no pathological alterations. Liver tissues of rats showing normal structure, normal hepatocytes with normal nuclei, and normal blood sinusoids appeared between the liver cords. However, liver tissues of animals treated with malathion (Supplementary Figure 3B) showed vacuolization of hepatocytes and congested blood sinusoids in between the liver cords with intense mononuclear inflammatory cellular infiltration. Liver cells appeared swollen, and areas of hemorrhage were noticed between the cells. The number of rats displaying histopathological changes in the liver decreased (Supplementary Figure 3C and 3D), which had received a piperine or curcumin only, and the rats in this group revealed only mild necrotic changes and moderate degenerative changes in the hepatocytes. The liver tissues of rats treated with piperine plus curcumin (Supplementary Figure 3E) showed normal structure, normal hepatocytes with normal nuclei, and normal blood sinusoids, as in the control.

Discussion

Data from the current study revealed that rats, after malathion intoxication, showed a significant increase in serum levels of both NO and MDA and a significant decrease in CAT, GSH, and SOD. The generation of ROS is typically regulated under normal physiological conditions. Cellular oxidative stress happens when there is a disruption to the balance between the production of ROS and the antioxidant capacity of the cells. The oxidative stress is created by malathion biotransformation into malaoxon and via malathion detoxification through conjugation with glutathione, which leads to the generation of ROS and depletion of antioxidant markers, as evidenced by the formation of MDA, and depletion of GSH.^8,9
Liver damage after malathion treatment was generally considered to result from two main effects: firstly, a decrease in some hepatic antioxidative enzymatic activity, followed by an increase in some free radicals, such as NO and lipid peroxidation. Secondly, hepatocyte apoptosis occurs because free radicals interact with physiological signaling mechanisms and initiate apoptosis, which activates proapoptotic regulators such as cytochrome c and caspases.¹⁰
In the present study, the antioxidant and anti-inflammatory effects of curcumin alone and with adjunct piperine have been investigated against malathion-mediated toxicity. There was a reduction in elevated NO and MDA in piperine and curcumin-treated groups as compared to the malathion-treated group, with a subsequent increase in the antioxidant parameters (CAT, GSH, and SOD). The hepatoprotective effect of curcumin is related to its potent antioxidant activity and ROS-scavenging properties. Curcumin could exert antioxidative effects either directly as a chemical antioxidant due to its ability to scavenge reactive oxygen and nitrogen free radicals or by modulating cellular defenses, which themselves exert antioxidant effects.¹¹
The results of the present study investigated the protective effect of piperine on antioxidant enzyme activity. Rats treated with piperine exhibited a statistically significant increase in antioxidant enzymatic activity compared to those exposed to malathion. The administration of piperine restored antioxidant enzyme activity to baseline levels. SOD activity is a reliable biomarker for hepatic damage, as it neutralizes superoxide anions and produces hydrogen peroxide, which helps alleviate harmful effects. Hepatic cells rely on catalase actions to eliminate peroxides during detoxification processes.¹²The results showed a significant decrease in SOD and catalase levels in rats treated with malathion. However, the administration of piperine led to a notable recovery of these enzymes. This suggests that piperine plays a critical role in promoting the efficient removal of reactive oxygen species.¹³
Furthermore, piperine has been shown to enhance the bioavailability of both synthetic drugs and bioactive phytochemicals. For instance, piperine is used to improve the absorption of silybin, a flavonoid known for its liver-protective properties, in a carbon tetrachloride-induced liver damage model. Additionally, the bioavailability of ginsenoside, a compound known to modulate the immune system, was significantly enhanced when co-administered with piperine.¹⁴ Moreover, coadministration of piperine with curcumin showed promising effects against benzo[a]pyrene-mediated toxicity in male albino mice. Treatment resulted in lowering lipid peroxidation and protein carbonyl content along with an increase in endogenous antioxidants like SOD, catalase, glutathione peroxidase, and reductase in the liver.¹⁵
Malathion induces hepatotoxicity by promoting the release of liver enzymes into the bloodstream. AST and ALT have been released into the blood after the cellular destruction, which is proportional to the intensity of cellular aggression. The recorded increase in AST and ALT levels may be due to increased lipid peroxidation.¹⁶ The decrease in AST and ALP activities supports the hepatoprotective effects of curcumin, consistent with the findings that curcumin modulated the increased activity of marker enzymes and plasma lipid levels in nicotine-treated rats.¹⁷ In our study, cholesterol levels increased after the administration of malathion. An increase in serum cholesterol as a result of pesticide exposure may indicate loss of membrane integrity. The curcumin treatment caused a significant reduction in cholesterol levels. A possible explanation for this observed effect may be due to the prooxidant effects of curcumin.¹⁸
The administration of piperine effectively reduced the elevated liver enzyme levels. This suggests that piperine may protect hepatocytes from damage induced by malathion. The protective effect could involve maintaining the structural integrity of hepatocyte membranes and preventing the release of enzymes. Our findings are consistent with several studies that report the hepatoprotective effects of piperine.¹⁹ The findings revealed the effectiveness of piperine for preventing liver damage, offering protection against necrosis and oxidative stress. The positive effects of piperine on liver health have been reported in various studies based on hepatic health biomarkers. Several researchers indicated that piperine lowers ALT, AST, and ALP levels in sera of cholesterol-fed albino mice.²⁰
It was reported that the reduction in plasma protein, particularly albumin, in animals treated with pesticides could be attributed to changes in protein and free amino acid metabolism and their synthesis in the liver. The decrease in serum protein also may be due to loss of protein either by reduced protein synthesis, increased proteolytic activity, or degradation.²¹ The treatment of malathion-exposed rat with piperine or curcumin caused a significant increase in their activity of them compared to malathion-treated rats. This suggests that piperine protects the liver by preventing cell death caused by malathion.²²
Hematological and biochemical blood profiles provide important insight into the internal environment of the organism. Malathion treatment increased total leukocyte count compared to controls, likely reflecting immune activation through stimulated lymphopoiesis or disruption of nonspecific immunity. Hepatocyte regeneration is essential for liver cell renewal following injury.²³Tumor necrosis factor-alpha (TNF-α) is a key cytokine involved in liver cell death signaling. In the malathion-only group, TNF-α levels were significantly increased, indicating liver toxicity and hepatocyte necrosis. In contrast, piperine-pretreated groups showed lower TNF-α levels, suggesting an anti-inflammatory effect as part of its protective mechanism.²⁴ These findings indicate that piperine may attenuate TNF-mediated inflammation during liver injury and exert a hepatoprotective effect against pesticide-induced damage.²⁵

Limitations

The most significant limitation of our study lies in its design. The relatively short follow-up period limits our ability to assess long-term durability and potential late complications associated with malathion toxicity. Future prospective studies with extended follow-up durations will be essential to validate these preliminary findings and determine the long-term performance of piperine and curcumin treatment.

Conclusion

In summary, data obtained from this study showed that piperine and curcumin possess a combined remarkable broad spectrum of protective activities in addition to pharmacological potentials to treat numerous diseases and ailments. Furthermore, this natural compound can provide a safer alternative to pharmaceuticals to target hepatic and splenic diseases.

Declarations

Abbreviations

ALP: alkaline phosphatase
ALT: alanine transaminase
ANOVA: analysis of variance
ARRIVE: animal research: reporting of in vivo experiments
AST: aspartate transaminase
CAT: catalase
EDTA: ethylenediaminetetraacetic acid
ELISA: enzyme-linked immunosorbent assay
GSH: reduced glutathione
Hb: hemoglobin
IL-10: interleukin-10
IL-1β: interleukin-1 beta
LSD: least significant difference
Mal: malathion
MDA: malondialdehyde
NF-κB: nuclear factor kappa b
NO: nitric oxide
ROS: reactive oxygen species
SD: standard deviation
SOD: superoxide dismutase
SPSS: statistical package for the social sciences
TC: total cholesterol
TNF-α: tumor necrosis factor alpha

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About This Article

Received:

March 15, 2026

Accepted:

April 24, 2026

Published Online:

May 13, 2026