Antioxidant Mediated Hepato-Renal Protection by Punica granatum Peel Extract in Fipronil-treated Rats

Introduction

Pesticide (especially insecticides) use has gradually increased over the past few decades in both agricultural and non-agricultural areas, in the form of sprays, liquids, and powders, to get rid of rats, ticks, cockroaches, and other dangerous bugs (WHO. 1990) [67] . India is the leading producer of pesticides in Asia and ranks 12 th on a global scale (Indira et al., 2007) [34] . Around 90,000 MT of pesticides are used annually in India as an effective pest control management. But indiscriminate and inappropriate use of these insecticides leads to excess leaching and surface runoff into aquatic ecosystems, which will hamper the non-target species and eventually disturb their food chain (David et al., 2015) [21] . Over the past forty years, pesticides have found widespread use in horticulture and veterinary activities around the world. (De et al., 2014) [22] . Through an ongoing cycle of evaporation and deposition, persistent pesticides that are applied in one part of the planet might spread through the air to other parts. (Koirala et al., 2007) [40]. Fipronil is one among the group of "new generation" insecticides which differs from other groups of insecticides like pyrethroids (sodium channel blockers), organophosphates and carbamates (cholinesterase inhibitors) in their mode of action (Aajoud et al., 2007) [1] . Being a broad-spectrum insecticide, fipronil is used in veterinary (Medleau et al., 2002) [45] households, topical pet care products, and agricultural practices to control ants, wasps, flies, cockroaches, termites, thrips, rootworms, weevils, fleas, ticks, and beetles (Badgujar et al., 2015) [15] . Fipronil causes hyperexcitation and mortality in insects by noncompetitively blocking glutamate-activated chloride channels and g-aminobutyric acid A receptors in the central nervous system.

According to reports, the parent molecule is less harmful to fish, birds, mammals, and insects than its sulfone metabolites and fipronil desulfinyl, a byproduct of photodegradation. (Hainzl et al., 1998) [29] . The kidney and liver are the most vulnerable organs to pesticide toxicity and injury, and they are also key players in the biotransformation of pesticides. These tissues' vulnerability to pesticide stress results from a disruption in the equilibrium between the level of oxidative stress and antioxidant capacity. Most pesticides cause toxicity that results in cell and DNA damage from free radicals, and oxidative stress is a major contributing factor in this process. (John et al., 2001) [35] . In addition to managing oxidantantioxidant balance, oxidative processes play a crucial role in insecticide-induced tissue damage by preventing neutrophil infiltration and controlling inflammatory mediators. (Muniz et al., 2008) [49] . Thus, measuring antioxidant indicators such as catalases, superoxide dismutase, glutathione peroxidase, and lipid peroxidase can assist in determining the extent of oxidative damage to the liver and kidney (Li et al., 2003) [41]. Estimation of biochemical parameters like liver enzymes (AST, ALT, Total Protein, Albumin, globulin levels) and renal enzymes (BUN, Creatinine) was also an effective indicator for determining the extent of toxic effects caused by pesticides such as fipronil in respective organs (Pimpao et al., 2007) [52] . Herbal medicines are now widely used as complementary and alternative medicine in many developing countries (Anquez-Traxler, 2011) [13]. Because of their low side effects and compatibility with physiological flora, herbal medications are used by almost 80% of the world's population (Pandy et al., 2011) [51]. Pomegranate (Punica granatum) is one among them, and it has been widely used in traditional medicine for many years (Ajai Kumar et al., 2005) [8] , in which the peel extract of pomegranate has been reported to have higher antioxidant potential compared to other parts (Wang et al., 2011 ) [66] . PPE enriched with polyphenolic compounds like (gallagic acid), flavonoids (catechins and quercetin), and anthocyanidin which are responsible for the natural antioxidant potential of pomegranate (Gil et al., 2000) [28] . Protective effects of pomegranate peel on brain injury have been widely studied by (Belal et al., 2020) [16] . The hepatoprotective and nephroprotective (Emam et al., 2020) [25] actions of PPE have also been reported. Hence, the current study aimed to investigate the toxic effects of fipronil on the liver and kidney of Wistar rats and to check the hepato-renal protective role of Pomegranate peel extract (PPE) in it.

Materials and Methods

Purchase of lab animals, fipronil and pomegranate peel extract A total of 24 male Wistar albino rats used for this study were purchased from Sri Venkateshara Enterprises, Bangalore. The procured rats were randomly grouped and housed in standard polypropylene cages after two weeks of acclimatization. They were maintained at 25

Study design

The study was designed such that a total of 24 male Wistar albino rats were randomly assigned into four groups with six animals in each group as shown below. a) Group 1 (Negative control) -six rats fed with ad libitum feed and distilled water. b) Group 2 (Fipronil control) -six animals treated with fipronil @ 10 mg/kg b. wt. orally in distilled water for a period of 45 days. c) Group 3 (Punica granatum control) -six animals treated with Punica granatum orally @ 200 mg/kg b. wt. for a period of 45 days. d) Group 4 (Fipronil + Punica granatum) -six animals treated with both fipronil @ 10 mg/kg b. wt. and Punica granatum @ 200 mg/kg b. wt. orally in distilled water for a period of 45 days.

Animal parameters studies

Oxidative stress Tissue pieces of liver and kidney were minced and homogenized in 0.05 M ice-cold phosphate buffer (pH 7.4) by using a Virtis homogenizer to make a 10% homogenate. 0.2 ml of the homogenate was used for the lipid peroxidation assay (Yagi, 1976) [68]. The remaining part of the homogenate was mixed with 10% trichloroacetic acid in the ratio of 1:1, centrifuged at 5000 rpm for 10 min at 4 °C and the supernatant was used for the estimation of Reduced glutathione (Moron et al., 1979) [47]. The remaining part of the homogenate was further centrifuged at 15000 rpm for 60 min at 4 °C and the supernatant obtained was used for estimation of Superoxide dismutase (Marklund and Marklund, 1974) [44], Catalase (Caliborne, 1985) [17] and Glutathione peroxidase (Rotruck et al., 1973) [56] in liver and kidney of all rats in all groups.

Gross and histopathology

A detailed post-mortem examination was conducted on all the sacrificed rats in all the experimental groups. The gross lesions were recorded and representative tissue pieces from liver and kidney were collected and preserved in 10% neutral buffered formalin for histopathological studies. Fixed tissues were processed by the routine paraffin embedding technique. Sections of 5-6 (µ) thickness were cut and stained with the routine Hematoxylin and Eosin method (H&E) (Culling, 1974) [18].

Statistical analysis

By performing one-way ANOVA, the results were statistically analysed (Snedecor and Cochran, 1994) [61].

Results and Discussion

Oxidative stress Lipid Peroxidation (LPO)

The mean LPO values in the liver of Group I to IV rats were 171. [60], (Albasher et al., 2020) [9] and (Elshony et al., 2021) [24] . The increased LPO value might be due to alterations of lipid bilayer configuration in the cell membrane by reactive oxygen species (ROS) generated by fipronil, which in turn produces an excess of lipid peroxides (malondialdehyde) from the damaged membrane (Liu et al., 1996) [43], (Scandalios, 2005) [57] and (Kanbur et al., 2008) [36] . Hence, malondialdehyde is a secondary product of lipid peroxidation, and is used as a marker of cell membrane damage (Esterbauer et al., 1991) [26] and Pizzimenti et al., 2013) [53] .

Superoxide Dismutase (SOD)

The mean liver SOD values in the Group I to IV rats were 13.8, 7.7, 12.9, and 12.2 (U/ mg of protein) respectively, and are given in Table 1. In kidney, SOD values were 14.2, 6.4, 13.8 and 13.4 respectively, which are given in Table 2. A significant decrease (p < 0.05) in the liver and kidney SOD values was observed in fipronil-treated rats compared to the control group. Ameliorated (Group Ⅳ) rats showed a significant increase (p < 0.05) in the SOD values when compared to fipronil-treated rats.

Catalase

The mean values of catalase in liver of Group I to IV rats were 0.21, 0.08, 0.18, and 0.15 (nM of H2O2 decomposed/min/ mg of protein) respectively, and given in Table . 1. In kidney, CAT values were 0.18, 0.09, 0.16 and 0.15 respectively, which are given in Table 2. There was a statistically significant (p < 0.05) decrease in the mean liver and kidney catalase value of fipronil-treated (Group II) rats when compared to control rats. Meanwhile, the mean catalase values of control and Punica granatum-treated (Group Ⅲ) rats were not statistically significant. A significant (p < 0.05) increase was observed in Punica granatum ameliorated rats (Group IV) when compared to fipronil-treated rats.

Reduced glutathione

The 2. There was a significant (p < 0.05) decrease in GPx values of fiproniltreated rats (Group II) when compared to the control group (Group I). No significant difference was noticed between Punica granatum-treated (Group Ⅲ) and control rats. A significant improvement was recorded in mean liver GPx values in the ameliorated rats (Group IV) compared to the fipronil-treated rats (Group II). In the current research, a significant (p < 0.05) decrease in SOD, CAT, GPx, GST, and GSH was observed in the liver and kidney of fipronil treated animals when compared to corresponding controls. These findings were in agreement with (Mossa et al., 2015) [

, (Badgujar et al., 2016) [14] , (Kartheek and David, 2016) [37] , (Singh et al., 2019) [60] , (Albasher et al., 2020) [9] and (Elshony et al., 2021) [24] . The decreased antioxidant enzymes activities in different organs in the current study might be due to fipronil-induced excessive generation of reactive oxygen species (ROS) and reduced cell defence mechanism. Significant reduction in LPO values along with increased levels of SOD, CAT, GPx, GST, and GSH was observed in the Punica granatum ameliorated group (Group IV). The restoration of these values near to normal range in Punica granatum ameliorated rats might be due to anti-lipid peroxidation property of Punica granatum (Dkhil et al., 2013) [23] and (AI-Gubory et al., 2015) [10] and its antioxidant activity (Li et al., 2006) [42] , (Shabtay et al., 2008) [58] , (Moneim et al., 2012) [46] , (Hasan et al., 2016) [30] and (Farideh et al., 2017) [27] .

Gross and histopathology

At the end of 6th week of experimental period, liver of fipronil treated rats (group II) showed paleness (Fig: 1). Whereas some of the Punica granatum ameliorated (group IV) rats showed slight paleness of the liver and most of the ameliorated rats regained almost normal appearance compared to fipronil treated rats. Histopathology of the liver of fipronil treated rats revealed congested and dilated blood vessels, sinusoidal congestion (Fig: 2), moderate to severe degenerative changes in hepatocytes with areas of loss of architecture, disruption of hepatic cords, dilated sinusoids, and atrophied hepatocytes with infiltration of MNC's (Fig: 3). Mild to moderate fibroblasts proliferation (Fig: 4), bile duct epithelial hyperplasia and proliferation of bile ducts (Fig: 5) along with mononuclear cell infiltration were observed in portal and periportal areas. In multi-focal areas, dilatation of sinusoids with haemorrhages, focal loss of hepatocytes with infiltration of MNCs, and mild to moderate fatty degenerative changes (Fig: 6) were conspicuous in the majority of rats whereas some rats showed the microvesicular type of fatty change. Perivascular infiltration of mononuclear cells, centrilobular necrotic changes (Fig: 7) and micronodule like lesions with fibroblasts and MNC's infiltrations were more evident by the end of 6th week of the experiment. These findings are in line with (De Oliveira et al., 2012) [50] , (Badgujar et al. 2016) [14] , (da Cunha et al., 2017) [19] , (Abdel-Daim and Abdeen 2018) [4] , (Kartheek and David 2018) [38] and (Anber et al., 2021) [12] . The liver alterations in the present study might be due to free radicals produced by fipronil and its primary metabolite (fipronil sulfone) in the liver impairing mitochondrial respiratory function, membrane potential, and the electron transport chain complex (Das et al., 2006 ) [20] leading to greater leakage of mitochondrial Ca 2+ into the cytoplasm and stimulates proteases, phospholipases, and endonucleases, which disrupt cytoskeleton-plasma membrane connections and destabilize the liver's lipid bilayer structure. Ultimately, the histologic architecture of the liver becomes altered (Trump and Berzesky, 1992) [63]. Histopathological findings of liver in group IV rats were similar to that of group II with reduced intensity like mild sinusoidal congestion, mild infiltration of MNC's in portal and perivascular areas (Fig: 8) and mild degenerative changes in hepatocytes with regained architecture of hepatic cords near to normal appearance by the end of the 6 th week of experiment was observed. Similar lesions with reduced intensity were observed in Punica granatum ameliorated rats (group IV) and this might be due to the hepatoprotective action of pomegranate peel extract (Ahmad et al., 2016) [7] , (Al-Shaaibi et al., 2016) [11] , (Ibrahim et al., 2016 ) [33] and (Ramzy et al., 2019) [54] . Punica granatum has oxygen free radical scavenging activity and antioxidant property, which helps to scavenge the excess free radicals generated due to fipronil toxicity in the liver (Moneim et al., 2012 ) [46] , (Shiban et al., 2012) [59] , Dkhil et al., 2013) [23] and (Hasan et al., 2016) [30] . Histopathology of kidney of the fipronil rats revealed severely degenerated and desquamated tubular epithelium with the presence of cellular debris in the renal tubules (Fig: 9), mild to moderate infiltration of fibroblasts in the interstitial spaces (Fig: 10), intertubular hemorrhages, congested and thickened blood vessels, perivascular edema and pockets of hemorrhages in the cortex and medullary region. Congested, degenerated, atrophied, and cystic glomeruli (Fig: 11) were more evident in all treated rats. The majority of the tubules lost their architectural details with complete distortion and loss of basement membrane (Fig: 34) accompanied by the presence of hyaline cast in some tubules. Moderate to severe infiltration of mononuclear cells in between the tubules and periglomerular spaces (Fig: 12) was more conspicuous in the majority of treated rats. Similar findings were observed by (Uzunhisarcikli et al., 2003) [64] , (Mossa et al., 2015) [48] , (Abdel Daim and Abdeen, 2018) [4] , (Abdel Daim et al., 2019) [5] , (Khalaf et al., 2020) [39] , (Abou zeid et al., 2021) [6] , (Hossam El din et al., 2022) [32] and (Soliman et al., 2023 ) [62] . These kidney lesions might be due to the generation of oxygen free radicals by fipronil and its metabolite. Fipronil primarily modifies NADPH oxidase activity in the inner mitochondrial membrane, which results in alterations in the electron transport chain and lipid peroxidation in cell membrane. Fipronil also damages the cellular proteins, lipids, and DNA structure and causes complete histologic alterations of renal tissue (Walmsley and White, 1994) [65] and Abdel-Daim and Abdeen, 2018) [3] . Histopathological findings of kidney in group IV rats were similar to that of group II rats with reduced intensity like focal areas of mild renal tubular degenerative changes, mild haemorrhages (Fig: 13) and mild infiltration of MNC's in periglomerular spaces. The kidney regained its normal architecture near to the normal with slight variation in the size of the glomerulus by the end of experimental period. These reduced kidney alterations might be due to high polyphenolic compounds, particularly ellagitannins present in Punica granatum, which can easily pass through the mitochondrial membrane and decrease the production of excess free radicals that damage the renal tissue (Gil et al., 2000) [28], (Rosenblat et al., 2015) [55].

Conclusion

The present investigation clearly demonstrates that subchronic exposure to fipronil induces marked hepato-renal toxicity in male Wistar albino rats, as evidenced by enhanced oxidative stress, and pronounced gross and histopathological lesions in the liver and kidney. Oral administration of Punica granatum peel extract (PPE) concomitantly with fipronil effectively mitigated these toxic effects by restoring hepatic and renal function markers, normalizing antioxidant defence systems, and markedly reducing lipid peroxidation. The substantial improvement in gross morphology and near-complete restoration of normal histoarchitecture of the liver and kidney further confirm the cytoprotective potential of PPE. Overall, the findings suggest that Punica granatum peel extract exerts significant hepato-renal cytoprotective and antioxidant effects against fipronil-induced toxicity, highlighting its potential as a natural therapeutic agent for managing pesticide-associated oxidative organ damage.

Acknowledgement

AcknowledgementsThe authors are thankful to College of Veterinary Science, Tirupati, Andhra Pradesh for providing necessary facilities for research work.

References

1. Aajoud A, Ravanel P, Tissut M. Fipronil metabolism and dissipation in a simplified aquatic ecosystem. J Agric Food Chem. 2003;51:1347-1352.
2. Abdel-Daim MM, Abdeen A. Antioxidant and antiapoptotic effects against fipronil. Food Chem Toxicol. 2018;114:69-77.
3. Abdel-Daim MM, Abdeen A. Rosuvastatin and vitamin E protect against fipronil toxicity. Food Chem Toxicol. 2018;114:69-77.
4. Abdel-Daim MM, Abdeen A. Vitamin E protection against fipronil toxicity. Food Chem Toxicol. 2018;114:69-77.
5. Abdel-Daim MM, Dessouki AA, Abdel-Rahman HG, et al. Taurine and NAC protect against fipronil injury. Sci Total Environ. 2019;650:2063-2073.
6. Abou-Zeid SM, Tahoun EA, AbuBakr HO. Jojoba oil against fipronil-induced toxicity. Environ Sci Pollut Res. 2021;28:25959-25971.
7. Ahmad N, Tahir M, Lone K. Hepatoprotective effect of pomegranate peel extract. J Pak Med Assoc. 2016;66:859-863.
8. Ajaikumar KB, Asheef M, Babu BH, Padikkala J. Inhibition of gastric mucosal injury by Punica granatum methanolic extract. J Ethnopharmacol. 2005;96:171-176.
9. AlBasher G, Abdel-Daim MM, Almeer R, et al. Synergistic antioxidant effects of resveratrol and curcumin against fipronil toxicity. Environ Sci Pollut Res. 2020;27:6505-6514.
10. Al-Gubory K, Blachier F, Faure P, Garrel C. Pomegranate peel extract reduces intestinal lipid peroxidation. J Sci Food Agric. 2016;96:2727-2733.
11. Al-Shaaibi SN, Waly MI, Al-Subhi L, et al. Pomegranate peel extract in NAFLD. Prev Nutr Food Sci. 2016;21(1):14-23.
12. Anber HA, Abdo W, Abd El-Raof TK, et al. Hepatorenal toxicity of fipronil in mice. Egypt J Plant Prot Res. 2021;9(1):39-53.
13. Anquez-Traxler C. The legal and regulatory framework of herbal medicinal products in the European Union. Drug Inf J. 2011;45:15-23.
14. Badgujar PC, Chandratre G, Pawar NN, et al. Fipronil alters SOD1 and catalase gene expression. Environ Toxicol. 2016;31(9):1147-1158.
15. Badgujar PC, Pawar NN, Chandratre GA, Telang AG, Sharma AK. Fipronil-induced oxidative stress in kidney and brain of mice: protective effect of vitamin E and vitamin C. Pestic Biochem Physiol. 2015;118:10-18.
16. Belal SKM, Afifi OK, Afeefy AA. Protective role of pomegranate peel extract against mobile phone radiation-induced brain changes in rats. Int J Radiat Res. 2020;18(4):753-764.
17. Claiborne AL. Assay of catalase. In: Greenwald RA, editor. Handbook of Oxygen Radical Research. Boca Raton: CRC Press; 1985.
18. Culling CFA. Handbook of histopathological and histochemical techniques. 3rd ed. London: Butterworths; 1974. p. 361.
19. Da Cunha ELR, da Silva Matos R, Pereira NRC, et al. Histopathological effects of fipronil in mice. J Histol Histopathol. 2017; 4:9.
20. Das PC, Cao Y, Cherrington N, Hodgson E, Rose RL. Fipronil induces CYP isoforms in human hepatocytes. Chem Biol Interact. 2006;164:200-214.
21. David M, Kartheek RM. Malathion acute toxicity in tadpoles of Duttaphrynus melanostictus: morphological and behavioural study. J Basic Appl Zool. 2015;72:1-7. International Journal of Advanced Biochemistry Research https://www.biochemjournal.com
22. De A, Bose R, Kumar A, Mozumdar S. Targeted delivery using biodegradable polymeric nanoparticles. SpringerBriefs Mol Sci. 2014.
23. Dkhil MA. Antioxidant and antiparasitic activity of pomegranate peel extract. Parasitol Res. 2013;112:2639-2646.
24. Elshony N, Nassar AM, El-Sayed YS, et al. Cerium oxide nanoparticles against fipronil-induced brain toxicity. Front Neurosci. 2021;15:651471.
25. Emam NM, Anjum S, Okail HA, Ibrahim MAR, Ahmad T. Pomegranate peel extract protects against CCl₄-induced nephrotoxicity in mice. Biomed Rep. 2020;13(3):1-11.
26. Esterbauer H, Schaur RJ, Zollner H. Chemistry of lipid peroxidation aldehydes. Free Radic Biol Med. 1991;11:81-128.
27. Farideh D, Roxana V, Parvin Z, et al. Effects of pomegranate peel on methotrexate-induced oxidative stress. Adv Pharm Bull. 2017;7(2):269-274.
28. Gil MI, Tomás-Barberán FA, Hess-Pierce B, Holcroft DM, Kader AA. Antioxidant activity of pomegranate juice and its relationship with phenolic composition. J Agric Food Chem. 2000;48:4581-4589.
29. Hainzl D, Cole LM, Casida JE. Mechanisms for selective toxicity of fipronil insecticide and its sulfone metabolite and desulfinyl photoproduct. Chem Res Toxicol. 1998;11:1529-1535.
30. Hasan S, Elrahman AA, Abou-Rawash A, et al. Protective effect of pomegranate peel against lead toxicity. J Toxicol Sci. 2016;41:207-215.
31. Hasan S, Elrahman AA, Abou-Rawash A, et al. Protective role of Punica granatum peel on leadinduced anemia. J Biol Sci. 2016;16:102-110.
32. Hossam El-Din HA, Abdallah AA, El-Dahshan AA, et al. Dates phytochemicals mitigate fipronil toxicity. Toxicology. 2022;480:153313.
33. Ibrahim MAR, Okail HAM, Emam NMM. Ameliorative effects of pomegranate peel on CCl₄ hepatotoxicity. Int J Res Stud Biosci. 2016;4:23-33.
34. Indira DP, Bellamy R, Shyam Sunder P. Facing hazards at work-agricultural workers and pesticide exposure in Kuttanad, Kerala. South Asian Network for Development and Environmental Economics. 2007;1907:1-4.
35. John S, Kale M, Rathore N, Bhatnagar D. Protective effect of vitamin E in dimethoate-and malathioninduced oxidative stress in rat erythrocytes. J Nutr Biochem. 2001;12:500-504.
36. Kanbur M, Atalay O, Ica A, Eraslan G, Cam Y. Curative efficiency of doramectin in rabbits. Res Vet Sci. 2008;85:291-293.
37. Kartheek R, David M. Subchronic fipronil toxicity in male Wistar rats. World J Pharm Sci. 2016;5(2):26-32.
38. Kartheek RM, David M. Hepatotoxic effects of fipronil in Wistar rats. Toxicol Rep. 2018;5:448-456.
39. Khalaf AA, Ibrahim MA, Galal MK, et al. Terminalia laxiflora protects against fipronil toxicity. Environ Sci Pollut Res. 2020;27:39507-39515.
40. Koirala P, Khadka DB, Mishra A. Pesticide residues as environmental contaminants in foods in Nepal. J Agric Environ. 2007;8:96-100.
41. Li W, Yin D, Zhou Y, Hu S, Wang L. 3,4Dichloroaniline-induced oxidative stress in liver of crucian carp (Carassius auratus). Ecotoxicol Environ Saf. 2003;56:251-255.
42. Li Y, Guo C, Yang J, et al. Antioxidant properties of pomegranate peel extract. Food Chem. 2006;96:254260.
43. Liu J, Wang X, Shigenaga M, et al. Oxidative damage and antioxidants. FASEB J. 1996;10:1532-1538.
44. Marklund SL, Marklund G. Involvement of superoxide anion radical in pyrogallol auto-oxidation. Eur J Biochem. 1974;47:469-474.
45. Medleau L, Hnilica KA, Lower K, Alva R, Clekis T, Case J, et al. Effect of topical application of fipronil in cats with flea allergic dermatitis. J Am Vet Med Assoc. 2002;221:254-257.
46. Moneim AEA. Pomegranate peel and aluminuminduced brain injury. Biol Trace Elem Res. 2012;150:328-336.
47. Moron MS, Depierre JW, Mannervik B. Levels of glutathione and related enzymes in rat lung and liver. Biochim Biophys Acta. 1979;582(1):67-78.
48. Mossa ATH, Swelam ES, Mohafrash SM. Fipronilinduced oxidative stress in rats. Toxicol Rep. 2015;2:775-784.
49. Muniz JF, McCauley L, Scherer J, et al. Biomarkers of oxidative stress and DNA damage in agricultural workers: a pilot study. Toxicol Appl Pharmacol. 2008;227:97-107.
50. Oliveira PR, Bechara GH, Morales MAM, Mathias MIC. Action of fipronil on tick reproduction. Food Chem Toxicol. 2009;47:1255-1264.
51. Pandey M, Debnath M, Gupta S, Chikara SK. Phytomedicine: an ancient approach turning into future potential source of therapeutics. J Pharmacogn Phytother. 2011;3:27-37.
52. Pimpão CT, Zampronio AR, de Assis HC. Effects of deltamethrin on hematological parameters and enzymatic activity in Ancistrus multispinis. Pestic Biochem Physiol. 2007;88:122-127.
53. Pizzimenti S, Ciamporcero E, Daga M, et al. Interaction of lipid peroxidation aldehydes with proteins. Front Physiol. 2013;4:242.
54. Ramzy M. Role of pomegranate peel in hyperglycemia and hypercholesterolemia. J Med Sci Res. 2019;2(3):185-190.
55. Rosenblat M, Volkova N, Borochov-Neori H, et al. Anti-atherogenic properties of date and pomegranate polyphenols. Food Funct. 2015;6:1496-1509.
56. Rotruck JD, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hekstra WG. Selenium as a component of glutathione peroxidase. Science. 1973;179:588-590.
57. Scandalios JG. Oxidative stress and antioxidant gene defenses. Braz J Med Biol Res. 2005;38:995-1014.
58. Shabtay A, Eitam H, Tadmor Y, et al. Nutritive and antioxidant potential of pomegranate by-products. J Agric Food Chem. 2008;56:10063-10070.
59. Shiban M, Al-Otaibi M, Al-Zoreky N. Antioxidant activity of pomegranate peels. Food Nutr Sci. 2012;3:991-996.
60. Singh D, Anand S, Swarnkar R, Choudhary A. Antioxidant potential of Pithecellobium dulce fruit against fipronil toxicity. J Pharmacogn Phytochem. 2019;8(4):1362-1367.
61. Snedecor WG, Cochran GW. Statistical methods. 6th ed. New Delhi: Oxford & IBH; 1967. p. 258-268.
62. Soliman N, El-Beltagy MA, Abdelrazek HMAA, Gouda SG. Reno-protective effects of Uncaria tomentosa. Egypt J Histol. 2023;46(1):460-477.
63. Trump BF, Berezesky IK. Role of cytosolic calcium in cell injury. Curr Opin Cell Biol. 1992;4:227-232.
64. Uzunhisarcikli M, Apaydin FG, Bas H, Kalender Y. Quercetin and curcumin against fipronil nephrotoxicity. Toxicol Res. 2023;12:1-12.
65. Walmsley RN, White GH. A guide to diagnostic clinical chemistry. 3rd ed. London: Blackwell Scientific; 1994.
66. Wang Z, Pan Z, Ma H, Atungulu GG. Extract of phenolics from pomegranate peels. Open Food Sci J. 2011;5:1-7.
67. World Health Organization. Public health impact of pesticides used in agriculture. Geneva: WHO; 1990.
68. Yagi K. Simple fluorimetric assay for lipid peroxides in blood plasma. Biochem Med. 1976;15:212-216.