Comparative Anti-inflammatory and Oxidative Effects of Milk Thistle and Royal Jelly Against Hepatotoxicity of Imipramine in Rats

Comparative Anti-inflammatory and Oxidative Effects of Milk Thistle and Royal Jelly Against Hepatotoxicity of Imipramine in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Anti-inflammatory and Oxidative Effects of Milk Thistle and Royal Jelly Against Hepatotoxicity of Imipramine in Rats Daniah Ahmed Altuma, Manal Abdul Khaliq Ibrahim, Manal N Alhayder This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7442584/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Imipramine is an antidepressant drug that with repeated administration can cause hepatic toxicity, which results in hepatic injury; hence the need for liver protection from toxicity by the use of hepatoprotactants, like milk thistle and royal jelly. Objective This study aims to comparatively evaluate the antioxidant and anti-inflammatory effects of milk thistle and royal jelly, both as monotherapies and in combination, in a rat model of imipramine-induced hepatotoxicity. Materials and Methods Thirty adult female Albino Wistar rats were divided into six groups (n = 6).Group A: Negative control (1ml D.W.); Group B imipramine (10 mg/kg/day) ;Group C milk thistle (140 mg/kg/day ) and royal jelly (150 mg/kg/day); Group D milk thistle (140 mg/kg/day ) and imipramine (10 mg/kg); Group E royal jelly (150 mg/kg/day) and imipramine (10 mg/kg); Group F milk thistle (140 mg/kg/day ), royal jelly (150 mg/kg/day) and imipramine (10 mg/kg). All treatments were taken orally. At 4 weeks of treatment, rats will be sacrificed and anesthetized. Serum for the assessment of Tumor Necrosis Factor Alpha (TNF-α), Interleukin-10 (IL-10), Glutathione (GSH), and Malondialdehyde (MDA) will be collected. All the parameters were determined by ELISA. Results Imipramine group exhibited hepatotoxicity as reflected by the elevation of MDA and TNF alpha and reduction of GSH compared to the control group. In contrast, the change in IL-10 was insignificant. Royal jelly and milk thistle had a remarkable antioxidant and anti-inflammatory effect by way of reduction of MDA and TNF alpha and elevation of GSH when compared to imipramine. Conclusion Both milk thistle and royal jelly have an anti-inflammatory and antioxidant action against imipramine hepatotoxicity. Milk Thistle Imipramine Hepatotoxicity Hepatoprotective Royal jelly Comparative Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Imipramine is a tricyclic antidepressant, a dibenzoazepine derivative, FDA approved imipramine as an antidepressant drug in the 1950s [ 1 ]. Imipramine is a second-line drug primarily used for severe depression with atypical features [ 2 ]. In addition to its antidepressant effect, imipramine has proven to be a good off-label medication for the management of nocturnal enuresis in children over six years of age [ 3 ]. Imipramine has also been prescribed by doctors as an off-label medication for the management of chronic neuropathic pain [ 4 ]. As for other tricyclic drugs, toxicity with imipramine is associated with serious side effects like critical hypotension, cardiac dysrhythmia, convulsions, respiratory depression, hepatotoxicity, and coma [ 5 ]. Imipramine causes hepatotoxicity primarily through oxidative stress and inflammatory processes, the therapy with this drug significantly enhances pro-inflammatory cytokines such as TNF-α, which is held accountable for inducing liver injury. Hepatotoxicity is also linked with loss of antioxidant defense strategies, e.g., decreased reduced glutathione (GSH), these changes reflect oxidative damage to the liver and redox homeostasis imbalance. [ 6 ] Silybum marianum L. (Milk thistle), a plant of the Carduus marianum group, is an earliest medicine plant has been used for times to treatment diseases for example gallbladder and liver disorders, protecting the liver from mushroom toxins, insect bites, snake bites, and alcoholism [ 7 ]. This is found in Kashmir, Mexico, Canada, and the United States, it has big leaves and purple-red thorny flowers, also the drug parts of the plant are seeds or fruits [ 8 ]. Milk thistle also has antifibrotic, anti-inflammatory, and immunomodulatory effects in addition to its antioxidant effect by increasing the glutathione content by neutralizing free radicals, so it can be used in the management of hepatic cirrhosis and hepatitis, as well as mushroom poisoning [ 9 , 10 ]. Royal jelly (RJ) a whitish natural fluid secreted by bees (by hypopharyngeal glands of bees) has been shown to reduce nephrotoxicity induced by most drugs, such as cisplatin [ 11 ], RJ comprises fatty acids (such as 10-HDA), minerals, proteins (such as royalactin), free amino acids, monosaccharides, lipids, and trace vitamins. The major activities of RJ are credited to its anti-inflammatory activity, strong antioxidants, and free radical scavenging activity [ 12 ]. So far, there is no study that has assessed the hepatoprotective action of milk thistle and royal jelly on imipramine -induced toxicity. Additionally, long-term use of imipramine in depression has been linked to liver damage. As such, this study sought to demonstrate the comparative protective action of milk thistle and royal jelly against imipramine -induced hepatotoxicity, through an assessment of inflammatory cytokines and oxidative parameters in female albino Wistar rats. Methods and Materials Imipramine tablets (Accord, UK), milk thistle capsules (Mason Natural, USA), royal jelly capsules (Basic Nutrition, UK). distilled water and normal saline 0.9% (Pioneer, Iraq). diethyl ether (Thomas Baker, India), and formaldehyde solution (SDFC Limited, India) Animals: Thirty healthy albinos female Wistar rats were used in the current study, that were within the age of 10-12 weeks and weighed between 180-250 grams. They were shipped from Iraq, Babylon, AL Raqeem office, and housed in polypropylene cages with sawdust at Pharmacy College, University of Basrah animal home. In order to acclimatize them to the environment, they kept the room temperature at 21±4°C and light/dark photoperiods (12L-12D) for two weeks. This possibly avoided making them unnecessarily stressed out, commercial pellet diet and water were offered to trial rats. Experimental Method: Group A: (Negative Control) The control rats were given distilled water (1 ml). Group B: (Positive control) animals were treated with imipramine (10 mg/kg/day) [13]. Group C: Milk thistle was given 140 mg/kg daily [14]. Then, rats were treated by royal jelly (150 mg/kg/d) [15]. Group D: Administration of milk thistle (140 mg/kg/day) to the rats as a pretreatment, imipramine (10 mg/kg/day) was then given to the rats. Group E: Pretreatment was given in the form of royal jelly (150 mg/kg/day), and then imipramine (10 mg/kg/day). Group F: Milk thistle was administered to the rats, followed by royal jelly, and then imipramine All of the milk thistle, royal jelly, and imipramine were dissolved in distilled water before administration. Within treated groups, the dosing interval was 60 min. Rats were fasted for 24 h after the final dose(28days). Rats were killed with diethyl ether anesthesia, dissected, and blood drawn. Blood was taken from rats by cardiac puncture, put in tubes free of anticoagulant, and allowed to stand at area temperature for 30 min to permit clot retraction [16]. Serum was got from the blood samples via centrifugation for 15 min (Genex, FL, USA). Subsequently centrifugation, the serum was collected intended for biochemical study [17]. Rat-specific ELISA kits (Reed Biotech Ltd, China) were employed for the determination of serum MDA levels that have sensitivity of 9.15 ng per mL, an average of 2000ng/mL and a detection range of (31.25-2000ng/mL).The GSH content was measured using the (Reed Biotech Ltd, China) kit through a detection limit of 0.48 ng/ml, a standard of 100 ng/ml, and a detection variety of (1.56–100 ng/ml).TNF alpha was measured using (Reed Biotech Ltd, China) with a detection limit of 0.15 pg./ml, a standard of 50 pg./ml, and a detection range of 0.78 - 50 pg/ml. IL-10 was measured by using the (Reed Biotech Ltd, China), with a sensitivity of 1.70 Pg/ml, a standard of 500 (Pg/ml) and an assay range of 7.81- 500pg/ml. All assays followed the sandwich ELISA principle and optical density at 450 nm (OD450). [18-21] Statistical analysis: An average value ± SE of study data is provided. SPSS Statistics package was used in the statistical analysis of this study. In estimation of differences among test groups, a one-way ANOVA comparison was used [22]. The statistical significance was distinct as having a p< 0.05. Results Effect of Milk Thistle and Royal Jelly on Malondialdehyde (MDA) in Serum Against Hepatotoxicity of Imipramine According Table 1. There is a significant reduction (p value<0.05) in serum MDA level in group B (367.43 ±129.2 nmol/g) compared to group A192.83 ± 12.15 nmol/g. All of the groups (C, D, E, and F) had a statical significant decrease in MDA compared to the B group. Group D had a greater reduction than Group E relative to Group B, while Group F had the greatest decrease among D and E, in comparison to Group B as Figure 1 The Effect of milk thistle and royal jelly on glutathione (gsh)in serum in opposition to the hepatotoxicity of imipramine As from Figure 2, group (B) showed a statistically significant decrease (p < 0.05) in GSH levels (7.71 ± 0.973 µmol/g) as compared to Group (A) GSH content in group (D)(13.08 ± 1.76 µmol/g) and in the group (E)(12.20 ± 0.989 µmol/g) were considerably higher compared to the group (B) (Both p value0.05 which is statistically nonsignificant as in (Table 1). The group (F) had higher GSH levels (11.44 ± 0.828 µmol/g) than the group (B), with a significant p value<0.05.but lower than the groups (D) and (E). Also, the GSH content of the group(C) is (14.39 ± 1.07 µmol/g), hence statistically significant with group (B) and not significant with other groups. Effect of milk thistle and royal jelly on tumor necrosis factor (tnf)alpha in serum against hepatotoxicity of imipramine. In the recent study, the (B) group possessed a sufficient statistical significance (p<0.05) for serum concentration of proinflammatory cytokine TNF alpha when compared with the group (A)(4.93±0.307and2.004±0.377) (mean ±S.E.), respectively, as shown in Figure 3. The group(C) revealed a fall in the level of TNF alpha when compared to the group (B), with statistically significant differences (p < 0.05), found as (2.88±0.335) and (4.93±0.307), respectively. Both the group (D) and the group (E) experienced a statistically significant reduction in TNF alpha from their corresponding group(B). The reduction in the group (E) was greater compared to the reduction found in the group (D), as reflected in Table 1. Maximum decrease in blood TNF alpha concentrations (1.71±0.294) was seen in (F) group versus the group (B) (4.93±0.307). Effect of milk thistle and royal jelly on IL-10 in serum against the imipramine-induced hepatotoxicity. Table 1 showed there was a reduction in IL-10 level of group (B) when compared to group (A), but the reduction was insignificant (p-value>0.05) from 32.69 ± 4.44 to 27.74 ± 2.49 pg/ml mean±SE. Group C exhibited the highest IL-10 level, 35.62 ± 4.75 in comparison to Group B 27.74 ± 2.49 pg/ml (mean± SE) but still nonsignificant For (D group), the difference was insignificant with the group (B).The group (E) and the group (F) both exhibited an increase in IL-10 levels compared to (B) group, but there was a nonsignificant p-value <0.05 (the values are shown in Figure 4) Table (1): Serum levels of MDA, GSH, TNF alpha, and IL-10 in all rat groups. Group name MDA serum level (mean±SE) GSH serum level (mean±SE) TNF alpha serum level (mean±SE) IL10 serum level (mean± SE) Group(A) negative control 192.83 ± 12.15 15.29±0.568# 2.004±0.377# 32.69± 4.44 Group(B) imipramine 367.43 ±12.92 7.71±0.973* 4.93±0.307* 27.74±2.49 Group(C)milk thistle +royal jelly 172.79±19.11 14.39±1.07# 2.88±0.335# 35.62±4.75 Group(D)milk thistle+imipramine 218.85±28.54 13.08±1.76# 3.30±0.407# 24.03±3.12 Group(E)royal jelly+imipramine 241.26 ±5.23 12.20 ±0.989 2.84 ±0.497# 34.41 ±4.38 Group(F)milk thistle, royal jelly, and imipramine 195.45 ±13.57 11.44 ± 0.828 1.71 ±0.294# 28.41 ±3.51 Symbol* = Statistically significant differences (p_ p-value <0.05) with respect to negative control (con), while the symbol# is for statistically significant differences (p_ p-value <0.05) with respect to the Imipramine group. Discussion Imipramine, a tricyclic antidepressant, is utilized for depression treatment and other off-label indications, but one of its side effects is the ability to induce hepatotoxicity, especially when used chronically. Oxidative stress and inflammation might play roles in imipramine-induced hepatotoxicity. Low GSH levels, high MDA and TNF-α levels indicate liver damage caused by redox imbalance and activation of the immune system [ 23 ]. Administration of imipramine may require the use of hepatoprotective agents to prevent or reduce the hepatotoxicity. Milk thistle has liver-protective antioxidants; it defends against free radicals, increases levels of GSH, and strengthens cell membranes to protect liver tissues against oxidative injury. Antioxidants are of utmost significance in defense against drug-induced hepatotoxicity and the re-establishment of liver function. [ 24 ] Royal jelly has antioxidant activities that act to protect the liver. The substance has various bioactive compounds, including peptides, flavonoids, and fatty acids, which enhance GSH levels and reduce lipid peroxidation, it helps to safeguard the liver cells from oxidative stress and toxins [ 25 ]. Imipramine significantly increases serum MDA levels, reflecting the promotion of lipid peroxidation and oxidative stress. This elevation is due to reactive oxygen species generated during imipramine's hepatic metabolism. The rise in MDA across other experimental research emphasizes lipid peroxidation as a main mechanism in imipramine-induced hepatotoxicity [ 1 ]، which is consistent with the current study results that confirm significant enhancement in MDA in the imipramine group rats relative to the control group rats Fig. 1. From table 1 showing that MDA serum level in groups that were given milk thistle was significantly reduced compared to imipramine treated rats (group B), which is consistent with the result of Faryadi, S., et al. (2024) that confirmed milk thistle markedly reduced hepatic MDA levels compared with untreated hepatotoxic groups [ 26 ] pointing out its protective effect. Hence, the reduction of MDA acts as a reliable indicator of the hepatoprotective effect of milk thistle. Royal jelly groups showed a significant reduction in MDA levels compared to imipramine groups, Table 1, which proved its hepatoprotective potential in reducing lipid peroxidation and its antioxidant effects, Karadeniz, A., et al. (2011) demonstrated that royal jelly effectively reduces MDA levels, indicating inhibition of lipid peroxidation in hepatotoxicity models. In cisplatin-treated rats, royal jelly supplementation significantly decreased hepatic MDA and restored antioxidant balance, showing protection against oxidative stress [ 27 ]. The (F) group rats, which were given milk thistle and royal jelly in combination with imipramine, had the greatest reduction in MDA, proving a synergistic effect. In the present research, imipramine (B group) significantly reduced hepatic GSH levels compared to the control group (A group), Fig. 2 , reflecting an impaired redox balance. The observed decline aligns with a previous study of Chang et al. (2021), that tricyclic antidepressants diminish endogenous antioxidants [ 28 ]. Their experimental results in obese mice found that imipramine alone diminished antioxidant reserves and aggravated liver injury These findings emphasize that monitoring GSH depletion can serve as a sensitive indicator of oxidative damage during imipramine exposure. Milk thistle with imipramine (group D) had increased levels of GSH compared to imipramine alone (group B), reflecting against oxidative stress. According to Surai, P. F. (2015). Silymarin in milk thistle improves antioxidant defenses by enhancing GSH content, restoring redox balance, and then reducing hepatic injury [ 29 ]. The Royal Jelly with Imipramine (group E) contains higher levels of GSH compared to the Imipramine group. Erzincan et al. (2025) observed that in rats exposed to vincristine, administration of royal jelly significantly enhanced hepatic GSH. [ 30 ], indicating that royal jelly strengthens endogenous antioxidant capacity. These findings support the hepatoprotective role of royal jelly, with GSH elevation serving as a main mechanism of its protective activity The triple regimen of milk thistle, royal jelly, and imipramine (F) contained reduced levels of GSH than the double regimens (D & E). This difference may not reflect decreased efficacy, but rather elevated GSH consumption. A more effective therapeutic response may trigger cellular repair processes and detoxification that depend on GSH as a cofactor, resulting in its faster depletion. furthermore, milk thistle and royal jelly may stimulate phase II detoxifying enzymes (glutathione S-transferases), which expend GSH in conjugation reactions. so, the lower GSH might represent increased antioxidant turnover rather than reduced protection [ 31 ] Figure 3 shows that imipramine alone treatment caused a marked boost in the level of TNF-α, indicating a strong inflammatory reaction underlying its hepatotoxicity compared with the negative control group This TNF-α increase is in agreement with findings of Mu et al. (2023), which reported that imipramine causes hepatic inflammation through oxidative stress and cytokine dysregulation, which caused an increase in TNF-α [ 32 ]. Co-treatment of imipramine with royal jelly and/or milk thistle reduced TNF-α levels compared to imipramine alone. (D, E, F)-treatment groups (Table 1) had significantly reduced levels of TNF-α (p < 0.05) relative to (B). These findings evidenced anti-inflammatory activity of milk thistle and royal jelly. The (F) treated group with milk thistle and royal jelly in combination with imipramine had the highest reduction in TNF-α, showing a synergistic effect Pourhanifeh et al . (2022) summarized experimental studies in which silymarin administration to rats exposed to hepatotoxins such as doxorubicin, carbon tetrachloride, and paracetamol reported that the hepatoprotective activities of milk thistle are a result of the anti-inflammatory and antioxidant properties of silymarin [ 33 ]. The honeybee salivary secretion, royal jelly, with its bioactive fatty acids and peptides, is hepatoprotective and immunomodulatory. Anti-inflammatory cytokine IL-10 is typically elevated following oxidative or immunologic stress [ 34 ]. Figure 4 demonstrates minor differences in mean blood levels of IL-10 in imipramine, milk thistle, royal jelly, or their combinations. None were statistically significant (p > 0.05) compared to the untreated control; these alterations appear to be natural biological variation rather than effects of therapy. IL-10 levels were not significantly declined in imipramine group compared to the control may be due to mild liver injury that does not result in suppression of IL-10 synthesis, IL-10’s compensatory mechanism, which keeps its levels stable for controlling inflammation, or the time of sampling might not detect transient IL-10 changes Duda et al. (2016) found that chronic imipramine administration in rats changed oxidative stress markers without consistent decline of IL-10, suggesting that its modulation is limited in early or moderate stages of hepatotoxicity [ 1 ]. For milk thistle, it may be because IL-10 modulation by silymarin is apparently model-dependent and might require more severe inflammatory stimuli to present significantly Abenavoli et al . (2018) showed experimental and clinical data that in rodent models of toxin-induced hepatotoxicity (paracetamol, CCl₄, and alcohol), silymarin consistently decreased oxidative stress and pro-inflammatory cytokines such as IL-6 and TNF-α, but reports for IL-10 elevation were inconsistent. This proposes that while milk thistle exerts antioxidant and anti-inflammatory effects, its influence on IL-10 may not be strong enough to manifest in models of milder injury [ 24 ]. The primary action of royal jelly is on cellular antioxidant defense, which strongly enhances GSH, superoxide dismutase, catalase, etc., that reduces triggers of inflammation, possibly lowering the need for a marked IL-10 response as mentioned by Tavares et al. (2019), experimental evidence [ 35 ] Conclusion This study verifies that imipramine causes hepatotoxicity through oxidative stress and inflammation, as signified by elevated MDA and TNF-α and reduced GSH. Milk thistle and royal jelly significantly reduced the levels of MDA and TNF-α and replenished GSH, indicating their anti-inflammatory and antioxidant activity. Milk thistle possessed greater MDA reduction and GSH elevation than royal jelly, whereas the latter possessed more TNF alpha reduction, thereby indicating their promising potential towards the alleviation of drug-induced liver injury. The combination of milk thistle and royal jelly with imipramine results in the greatest reduction in MDA and TNF alpha than each one alone with imipramine Limits of the study 1. The Sample size limit: The experiment was led on a small sample of experiments, and this constrain the generalizability of the results. 2. Short interval: The experimental duration not be capable of reflecting long-term conclusions or late hepatotoxic responses then protecting effects 3. Absence of human statistics: The study has been shown the usage of animal models, in addition, results will not be easily translatable to human physiology otherwise clinical practice without further studies. 4. No functional or behavioral outcomes assessed: The study focused predominantly on biochemical parameters without monitoring any change in behavior or liver function completed time. Declarations Acknowledgements The present study is based upon a master's project offered to Al-Basrah university college of pharmacy department of pharmacology & toxicology. In recognition of their caring assistance and support in conducting this study, the authors thank and acknowledge the Department of Pharmacology and Toxicology. Contribution of the Author: Daniah Ahmed Altuma, first author, idea, proposal, also preparation of manuscript. Ass. 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The Potential Hepatoprotective Effect of Vinpocetine against Lead-Induced Inflammatory and Apoptotic Cytokines in Rats. Iraqi Journal of Pharmaceutical Sciences.2024 Sep. 15 [cited 2025 Aug. 21];33(3):56–62. Available from: https://bijps.uobaghdad.edu.iq/index.php/bijps/article/view/2622 Tavares, W. R., Gonçalves, M. C. R., Campos, M. G., & Ribeiro, C. A. (2019). Royal jelly: A review of chemical composition and biological activity. Journal of Functional Foods, 58, 655–664. https://doi.org/10.1016/j.jff.2019.02.018 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7442584","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":508820112,"identity":"87c1e976-f39f-444b-ae7f-a0224c2a8d5e","order_by":0,"name":"Daniah Ahmed Altuma","email":"","orcid":"","institution":"Ministry of Health, Basrah Health Directorate, Al Shifaa general Hospital","correspondingAuthor":false,"prefix":"","firstName":"Daniah","middleName":"Ahmed","lastName":"Altuma","suffix":""},{"id":508820113,"identity":"0d93c606-3cf5-44b0-9b94-122cc7546dc4","order_by":1,"name":"Manal Abdul Khaliq Ibrahim","email":"","orcid":"","institution":"University of Basrah","correspondingAuthor":false,"prefix":"","firstName":"Manal","middleName":"Abdul Khaliq","lastName":"Ibrahim","suffix":""},{"id":508820114,"identity":"addb78a7-122b-40ec-96e7-7b591f001d6b","order_by":2,"name":"Manal N Alhayder","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABUklEQVRIie2RT0vDMBiHUwrbpZvXhqn7CimF6kDdV0koeCp1MBBBqQUhnvQ8mX6HePSWUVgv1V4jetjYtcJOMrCIWd2ltSh4EulDEsILz+/NHwAqKv4giMtlAgAGXKWfpboP1Gyjy6llI0dnqeBMUVaKxr9Xul8VHeeVIlZ4P5pjsOc2wxGdLFKvvQUTY9Y78Q7Whr4yeaFgvcnzSuTaMtbuw4icGxc0MO6GjmkOxkFn8MxV44YCDRYU7qDlSQjjhOoNnyvsybFaWo0jIHCt1ZAKyisoTswFBqeExVMK09TrssdIKu8eagtcfytThGPJLgFhglAZLtsJzZLhKkKyi1qqJNY2RmEfiiltbdDAZtF+37y6DJAhyBm8ftCLd0GxY4r50bHbjO0xTFJvl4XB7az36qFNYY/myeFOt/BiK7GkJlF8kP1OmfIDv1AqKioq/hMft3uCEfkpr80AAAAASUVORK5CYII=","orcid":"","institution":"University of Basrah","correspondingAuthor":true,"prefix":"","firstName":"Manal","middleName":"N","lastName":"Alhayder","suffix":""}],"badges":[],"createdAt":"2025-08-23 17:08:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7442584/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7442584/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90605520,"identity":"1f2a3d92-d828-4f23-aa2e-4487485b872c","added_by":"auto","created_at":"2025-09-04 15:26:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":42785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum Level of MDA in Treated Groups of Rats.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7442584/v1/14be26232c8e49c64af850d6.png"},{"id":90604474,"identity":"378e9d42-05e7-47c1-bbfd-631b7c83581d","added_by":"auto","created_at":"2025-09-04 15:18:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum Level of GSH in Treated Groups of Rats.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7442584/v1/18914762d90ea898942f1356.png"},{"id":90604478,"identity":"9f5c863c-1b9e-4526-904e-197a425d4858","added_by":"auto","created_at":"2025-09-04 15:18:59","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38639,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSerum Concentration of TNF alpha in Treated Rat Groups.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7442584/v1/10112c45315a31fbc69f5885.png"},{"id":90605522,"identity":"9a7a52af-edc8-4ee5-87ae-a5440774e1db","added_by":"auto","created_at":"2025-09-04 15:26:59","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":39765,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-10 \u0026nbsp;\u0026nbsp;Serum Level in Treated Groups of Rats.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7442584/v1/0aaf0b1edd51f66ccc778b44.png"},{"id":92607911,"identity":"5aba3c83-001a-44f5-b128-6ddc9d0841f1","added_by":"auto","created_at":"2025-10-01 15:32:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":930410,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7442584/v1/b351cbd0-c7ee-4668-8cbb-6f46754585aa.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eComparative Anti-inflammatory and Oxidative Effects of Milk Thistle and Royal Jelly Against Hepatotoxicity of Imipramine in Rats\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImipramine is a tricyclic antidepressant, a dibenzoazepine derivative, FDA approved imipramine as an antidepressant drug in the 1950s [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Imipramine is a second-line drug primarily used for severe depression with atypical features [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition to its antidepressant effect, imipramine has proven to be a good off-label medication for the management of nocturnal enuresis in children over six years of age [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Imipramine has also been prescribed by doctors as an off-label medication for the management of chronic neuropathic pain [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs for other tricyclic drugs, toxicity with imipramine is associated with serious side effects like critical hypotension, cardiac dysrhythmia, convulsions, respiratory depression, hepatotoxicity, and coma [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eImipramine causes hepatotoxicity primarily through oxidative stress and inflammatory processes, the therapy with this drug significantly enhances pro-inflammatory cytokines such as TNF-α, which is held accountable for inducing liver injury. Hepatotoxicity is also linked with loss of antioxidant defense strategies, e.g., decreased reduced glutathione (GSH), these changes reflect oxidative damage to the liver and redox homeostasis imbalance. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eSilybum marianum L. (Milk thistle), a plant of the Carduus marianum group, is an earliest medicine plant has been used for times to treatment diseases for example gallbladder and liver disorders, protecting the liver from mushroom toxins, insect bites, snake bites, and alcoholism [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThis is found in Kashmir, Mexico, Canada, and the United States, it has big leaves and purple-red thorny flowers, also the drug parts of the plant are seeds or fruits [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Milk thistle also has antifibrotic, anti-inflammatory, and immunomodulatory effects in addition to its antioxidant effect by increasing the glutathione content by neutralizing free radicals, so it can be used in the management of hepatic cirrhosis and hepatitis, as well as mushroom poisoning [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Royal jelly (RJ) a whitish natural fluid secreted by bees (by hypopharyngeal glands of bees) has been shown to reduce nephrotoxicity induced by most drugs, such as cisplatin [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], RJ comprises fatty acids (such as 10-HDA), minerals, proteins (such as royalactin), free amino acids, monosaccharides, lipids, and trace vitamins. The major activities of RJ are credited to its anti-inflammatory activity, strong antioxidants, and free radical scavenging activity [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSo far, there is no study that has assessed the hepatoprotective action of milk thistle and royal jelly on imipramine -induced toxicity. Additionally, long-term use of imipramine in depression has been linked to liver damage. As such, this study sought to demonstrate the comparative protective action of milk thistle and royal jelly against imipramine -induced hepatotoxicity, through an assessment of inflammatory cytokines and oxidative parameters in female albino Wistar rats.\u003c/p\u003e"},{"header":"Methods and Materials","content":"\u003cp\u003eImipramine tablets (Accord, UK), milk thistle capsules (Mason Natural, USA), royal jelly capsules (Basic Nutrition, UK). distilled water and normal saline 0.9% (Pioneer, Iraq). diethyl ether (Thomas Baker, India), and formaldehyde solution (SDFC Limited, India) \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThirty healthy albinos female Wistar rats were used in the current study, that were within the age of 10-12 weeks and weighed between 180-250 grams. They were shipped from Iraq, Babylon, AL Raqeem office, and housed in polypropylene cages with sawdust at Pharmacy College, University of Basrah animal home. In order to acclimatize them to the environment, they kept the room temperature at 21±4°C and light/dark photoperiods (12L-12D) for two weeks. This possibly avoided making them unnecessarily stressed out, commercial pellet diet and water were offered to trial rats.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExperimental Method:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGroup A: (Negative Control) The control rats were given distilled water (1 ml).\u003c/p\u003e\n\u003cp\u003eGroup B: (Positive control) animals were treated with imipramine (10 mg/kg/day) [13]. \u003c/p\u003e\n\u003cp\u003eGroup C: Milk thistle was given 140 mg/kg daily [14]. Then, rats were treated by royal jelly (150 mg/kg/d) [15].\u003c/p\u003e\n\u003cp\u003eGroup D: Administration of milk thistle (140 mg/kg/day) to the rats as a pretreatment, imipramine (10 mg/kg/day) was then given to the rats.\u003c/p\u003e\n\u003cp\u003eGroup E: Pretreatment was given in the form of royal jelly (150 mg/kg/day), and then imipramine (10 mg/kg/day).\u003c/p\u003e\n\u003cp\u003eGroup F: Milk thistle was administered to the rats, followed by royal jelly, and then imipramine \u003c/p\u003e\n\u003cp\u003eAll of the milk thistle, royal jelly, and imipramine were dissolved in distilled water before administration.\u003c/p\u003e\n\u003cp\u003eWithin treated groups, the dosing interval was 60 min. Rats were fasted for 24 h after the final dose(28days). Rats were killed with diethyl ether anesthesia, dissected, and blood drawn. Blood was taken from rats by cardiac puncture, put in tubes free of anticoagulant, and allowed to stand at area temperature for 30 min to permit clot retraction [16]. Serum was got from the blood samples via centrifugation for 15 min (Genex, FL, USA). Subsequently centrifugation, the serum was collected intended for biochemical study [17].\u003c/p\u003e\n\u003cp\u003eRat-specific ELISA kits (Reed Biotech Ltd, China) were employed for the determination of serum MDA levels that have sensitivity of 9.15 ng per mL, an average of 2000ng/mL and a detection range of (31.25-2000ng/mL).The GSH content was measured using the (Reed Biotech Ltd, China) kit through a detection limit of 0.48 ng/ml, a standard of 100 ng/ml, and a detection variety of (1.56–100 ng/ml).TNF alpha was measured using (Reed Biotech Ltd, China) with a detection limit of 0.15 pg./ml, a standard of 50 pg./ml, and a detection range of 0.78 - 50 pg/ml. IL-10 was measured by using the (Reed Biotech Ltd, China), with a sensitivity of 1.70 Pg/ml, a standard of 500 (Pg/ml) and an assay range of 7.81- 500pg/ml. All assays followed the sandwich ELISA principle and optical density at 450 nm (OD450). [18-21]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn average value ± SE of study data is provided. SPSS Statistics package was used in the statistical analysis of this study. In estimation of differences among test groups, a one-way ANOVA comparison was used [22]. The statistical significance was distinct as having a p\u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffect of Milk Thistle and Royal Jelly on Malondialdehyde (MDA) in Serum Against Hepatotoxicity of Imipramine\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording Table 1. There is a significant reduction (p value\u0026lt;0.05) in serum MDA level in group B (367.43 ±129.2 nmol/g) compared to group A192.83 ± 12.15 nmol/g. All of the groups (C, D, E, and F) had a statical significant decrease in MDA compared to the B group. Group D had a greater reduction than Group E relative to Group B, while Group F had the greatest decrease among D and E, in comparison to Group B as Figure 1 \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe Effect of milk thistle and royal jelly on glutathione (gsh)in serum in opposition to the hepatotoxicity of imipramine\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs from Figure 2, group (B) showed a statistically significant decrease (p \u0026lt; 0.05) in GSH levels (7.71 ± 0.973 µmol/g) as compared to Group (A) GSH content in group (D)(13.08 ± 1.76 µmol/g) and in the group (E)(12.20 ± 0.989 µmol/g) were considerably higher compared to the group (B) (Both p value\u0026lt;0.05 statistically significant) though, the group (D) had more GSH than the group (E) but the p value\u0026gt;0.05 which is statistically nonsignificant as in (Table 1).\u003c/p\u003e\n\u003cp\u003eThe group (F) had higher GSH levels (11.44 ± 0.828 µmol/g) than the group (B), with a significant p value\u0026lt;0.05.but lower than the groups (D) and (E). Also, the GSH content of the group(C) is (14.39 ± 1.07 µmol/g), hence statistically significant with group (B) and not significant with other groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of milk thistle and royal jelly on tumor necrosis factor (tnf)alpha in serum against hepatotoxicity of imipramine.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the recent study, the (B) group possessed a sufficient statistical significance (p\u0026lt;0.05) for serum concentration of proinflammatory cytokine TNF alpha when compared with the group (A)(4.93±0.307and2.004±0.377) (mean ±S.E.), respectively, as shown in Figure 3.\u003c/p\u003e\n\u003cp\u003eThe group(C) revealed a fall in the level of TNF alpha when compared to the group (B), with statistically significant differences (p \u0026lt; 0.05), found as (2.88±0.335) and (4.93±0.307), respectively. Both the group (D) and the group (E) experienced a statistically significant reduction in TNF alpha from their corresponding group(B). The reduction in the group (E) was greater compared to the reduction found in the group (D), as reflected in Table 1.\u003c/p\u003e\n\u003cp\u003eMaximum decrease in blood TNF alpha concentrations (1.71±0.294) was seen in (F) group versus the group (B) (4.93±0.307).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of milk thistle and royal jelly on IL-10 in serum against the imipramine-induced hepatotoxicity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 1 showed there was a reduction in IL-10 level of group (B) when compared to group (A), but the reduction was insignificant (p-value\u0026gt;0.05) from 32.69 ± 4.44 to 27.74 ± 2.49 pg/ml mean±SE. Group C exhibited the highest IL-10 level, 35.62 ± 4.75 in comparison to Group B 27.74 ± 2.49 pg/ml (mean± SE) but still nonsignificant For (D group), the difference was insignificant with the group (B).The group (E) and the group (F) both exhibited an increase in IL-10 levels compared to (B) group, but there was a nonsignificant p-value \u0026lt;0.05 (the values are shown in Figure 4)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable (1): Serum levels of MDA, GSH, TNF alpha, and IL-10 in all rat groups.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"678\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup name \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMDA serum level (mean±SE)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGSH serum level (mean±SE)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTNF alpha serum level (mean±SE)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eIL10 serum level (mean± SE)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup(A) negative control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e192.83 ± 12.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15.29±0.568#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.004±0.377#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e32.69± 4.44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup(B) imipramine \u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e367.43 ±12.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7.71±0.973*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4.93±0.307*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e27.74±2.49\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup(C)milk thistle +royal jelly\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e172.79±19.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e14.39±1.07#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.88±0.335#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e35.62±4.75\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup(D)milk thistle+imipramine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e218.85±28.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e13.08±1.76#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e3.30±0.407#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e24.03±3.12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup(E)royal jelly+imipramine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e241.26 ±5.23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e12.20 ±0.989\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2.84 ±0.497#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e34.41 ±4.38\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup(F)milk thistle, royal jelly, and imipramine\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e195.45 ±13.57\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e11.44 ± 0.828\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1.71 ±0.294#\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e28.41 ±3.51\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eSymbol* = Statistically significant differences (p_ p-value \u0026lt;0.05) with respect to negative control (con), while the symbol# is for statistically significant differences (p_ p-value \u0026lt;0.05) with respect to the Imipramine group.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eImipramine, a tricyclic antidepressant, is utilized for depression treatment and other off-label indications, but one of its side effects is the ability to induce hepatotoxicity, especially when used chronically. Oxidative stress and inflammation might play roles in imipramine-induced hepatotoxicity. Low GSH levels, high MDA and TNF-α levels indicate liver damage caused by redox imbalance and activation of the immune system [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Administration of imipramine may require the use of hepatoprotective agents to prevent or reduce the hepatotoxicity.\u003c/p\u003e\u003cp\u003eMilk thistle has liver-protective antioxidants; it defends against free radicals, increases levels of GSH, and strengthens cell membranes to protect liver tissues against oxidative injury. Antioxidants are of utmost significance in defense against drug-induced hepatotoxicity and the re-establishment of liver function. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eRoyal jelly has antioxidant activities that act to protect the liver. The substance has various bioactive compounds, including peptides, flavonoids, and fatty acids, which enhance GSH levels and reduce lipid peroxidation, it helps to safeguard the liver cells from oxidative stress and toxins [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Imipramine significantly increases serum MDA levels, reflecting the promotion of lipid peroxidation and oxidative stress. This elevation is due to reactive oxygen species generated during imipramine's hepatic metabolism. The rise in MDA across other experimental research emphasizes lipid peroxidation as a main mechanism in imipramine-induced hepatotoxicity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]، which is consistent with the current study results that confirm significant enhancement in MDA in the imipramine group rats relative to the control group rats Fig.\u0026nbsp;1. From table 1 showing that MDA serum level in groups that were given milk thistle was significantly reduced compared to imipramine treated rats (group B), which is consistent with the result of Faryadi, S., et al. (2024) that confirmed milk thistle markedly reduced hepatic MDA levels compared with untreated hepatotoxic groups [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] pointing out its protective effect. Hence, the reduction of MDA acts as a reliable indicator of the hepatoprotective effect of milk thistle.\u003c/p\u003e\u003cp\u003eRoyal jelly groups showed a significant reduction in MDA levels compared to imipramine groups, Table\u0026nbsp;1, which proved its hepatoprotective potential in reducing lipid peroxidation and its antioxidant effects, Karadeniz, A., et al. (2011) demonstrated that royal jelly effectively reduces MDA levels, indicating inhibition of lipid peroxidation in hepatotoxicity models. In cisplatin-treated rats, royal jelly supplementation significantly decreased hepatic MDA and restored antioxidant balance, showing protection against oxidative stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The (F) group rats, which were given milk thistle and royal jelly in combination with imipramine, had the greatest reduction in MDA, proving a synergistic effect.\u003c/p\u003e\u003cp\u003eIn the present research, imipramine (B group) significantly reduced hepatic GSH levels compared to the control group (A group), Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e, reflecting an impaired redox balance. The observed decline aligns with a previous study of Chang et al. (2021), that tricyclic antidepressants diminish endogenous antioxidants [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Their experimental results in obese mice found that imipramine alone diminished antioxidant reserves and aggravated liver injury These findings emphasize that monitoring GSH depletion can serve as a sensitive indicator of oxidative damage during imipramine exposure.\u003c/p\u003e\u003cp\u003eMilk thistle with imipramine (group D) had increased levels of GSH compared to imipramine alone (group B), reflecting against oxidative stress. According to Surai, P. F. (2015). Silymarin in milk thistle improves antioxidant defenses by enhancing GSH content, restoring redox balance, and then reducing hepatic injury [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe Royal Jelly with Imipramine (group E) contains higher levels of GSH compared to the Imipramine group. Erzincan et al. (2025) observed that in rats exposed to vincristine, administration of royal jelly significantly enhanced hepatic GSH. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], indicating that royal jelly strengthens endogenous antioxidant capacity. These findings support the hepatoprotective role of royal jelly, with GSH elevation serving as a main mechanism of its protective activity\u003c/p\u003e\u003cp\u003eThe triple regimen of milk thistle, royal jelly, and imipramine (F) contained reduced levels of GSH than the double regimens (D \u0026amp; E). This difference may not reflect decreased efficacy, but rather elevated GSH consumption. A more effective therapeutic response may trigger cellular repair processes and detoxification that depend on GSH as a cofactor, resulting in its faster depletion. furthermore, milk thistle and royal jelly may stimulate phase II detoxifying enzymes (glutathione S-transferases), which expend GSH in conjugation reactions. so, the lower GSH might represent increased antioxidant turnover rather than reduced protection [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e\u003cp\u003eFigure 3 shows that imipramine alone treatment caused a marked boost in the level of TNF-α, indicating a strong inflammatory reaction underlying its hepatotoxicity compared with the negative control group This TNF-α increase is in agreement with findings of Mu et al. (2023), which reported that imipramine causes hepatic inflammation through oxidative stress and cytokine dysregulation, which caused an increase in TNF-α [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Co-treatment of imipramine with royal jelly and/or milk thistle reduced TNF-α levels compared to imipramine alone. (D, E, F)-treatment groups (Table\u0026nbsp;1) had significantly reduced levels of TNF-α (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) relative to (B). These findings evidenced anti-inflammatory activity of milk thistle and royal jelly. The (F) treated group with milk thistle and royal jelly in combination with imipramine had the highest reduction in TNF-α, showing a synergistic effect\u003c/p\u003e\u003cp\u003ePourhanifeh \u003cem\u003eet al\u003c/em\u003e. (2022) summarized experimental studies in which silymarin administration to rats exposed to hepatotoxins such as doxorubicin, carbon tetrachloride, and paracetamol reported that the hepatoprotective activities of milk thistle are a result of the anti-inflammatory and antioxidant properties of silymarin [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The honeybee salivary secretion, royal jelly, with its bioactive fatty acids and peptides, is hepatoprotective and immunomodulatory.\u003c/p\u003e\u003cp\u003eAnti-inflammatory cytokine IL-10 is typically elevated following oxidative or immunologic stress [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Figure\u0026nbsp;4 demonstrates minor differences in mean blood levels of IL-10 in imipramine, milk thistle, royal jelly, or their combinations. None were statistically significant (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) compared to the untreated control; these alterations appear to be natural biological variation rather than effects of therapy. IL-10 levels were not significantly declined in imipramine group compared to the control may be due to mild liver injury that does not result in suppression of IL-10 synthesis, IL-10\u0026rsquo;s compensatory mechanism, which keeps its levels stable for controlling inflammation, or the time of sampling might not detect transient IL-10 changes Duda et al. (2016) found that chronic imipramine administration in rats changed oxidative stress markers without consistent decline of IL-10, suggesting that its modulation is limited in early or moderate stages of hepatotoxicity [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. For milk thistle, it may be because IL-10 modulation by silymarin is apparently model-dependent and might require more severe inflammatory stimuli to present significantly\u003c/p\u003e\u003cp\u003eAbenavoli \u003cem\u003eet al\u003c/em\u003e. (2018) showed experimental and clinical data that in rodent models of toxin-induced hepatotoxicity (paracetamol, CCl₄, and alcohol), silymarin consistently decreased oxidative stress and pro-inflammatory cytokines such as IL-6 and TNF-α, but reports for IL-10 elevation were inconsistent. This proposes that while milk thistle exerts antioxidant and anti-inflammatory effects, its influence on IL-10 may not be strong enough to manifest in models of milder injury [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe primary action of royal jelly is on cellular antioxidant defense, which strongly enhances GSH, superoxide dismutase, catalase, etc., that reduces triggers of inflammation, possibly lowering the need for a marked IL-10 response as mentioned by Tavares et al. (2019), experimental evidence [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study verifies that imipramine causes hepatotoxicity through oxidative stress and inflammation, as signified by elevated MDA and TNF-α and reduced GSH. Milk thistle and royal jelly significantly reduced the levels of MDA and TNF-α and replenished GSH, indicating their anti-inflammatory and antioxidant activity. Milk thistle possessed greater MDA reduction and GSH elevation than royal jelly, whereas the latter possessed more TNF alpha reduction, thereby indicating their promising potential towards the alleviation of drug-induced liver injury.\u0026nbsp;The combination of milk thistle and royal jelly with imipramine results in the greatest reduction in MDA and TNF alpha than each one alone with imipramine\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimits of the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. The Sample size limit: The experiment was led on a small sample of experiments, and this constrain the generalizability of the results.\u003c/p\u003e\n\u003cp\u003e2. Short interval: The experimental duration not be capable of reflecting long-term conclusions or late hepatotoxic responses then protecting effects\u003c/p\u003e\n\u003cp\u003e3. Absence of human statistics: The study has been shown the usage of animal models, in addition, results will not be easily translatable to human physiology otherwise clinical practice without further studies.\u003c/p\u003e\n\u003cp\u003e4. No functional or behavioral outcomes assessed: The study focused predominantly on biochemical parameters without monitoring any change in behavior or liver function completed time.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe present study is based upon a master\u0026apos;s project offered to Al-Basrah university college of pharmacy department of pharmacology \u0026amp; toxicology. In recognition of their caring assistance and support in conducting this study, the authors thank and acknowledge the Department of Pharmacology and Toxicology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContribution of the Author:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDaniah Ahmed Altuma, first author, idea, proposal, also preparation of manuscript. Ass. Prof. Manal Abdul Khaliq Ibrahim, search for literature and acquisition and definition of intellectual content. Ass. prof Manal N. Alhayder, statistical analysis and data analysis, and reviewing and editing manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were used otherwise analyzed throughout this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were approved by the Basrah College of Pharmacy ethical committee. Their judgment was based on the ethical terms mentioned in the \u0026quot;European Union Directive (86/609/EEC), 24 November 1986.\u0026quot;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDuda, W., Curzytek, K., Kubera, M., Iciek, M., Kowalczyk-Pachel, D., Bilska-Wilkosz, A., et al. (2016). The effect of chronic mild stress and imipramine on the markers of oxidative stress and antioxidant system in rat liver. Neurotoxicity Research, 30(2), 173\u0026ndash;184.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFayez, R., \u0026amp; Gupta, V. (2022). Imipramine. In StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing. [Updated 2021; cited 2022 Feb 9]. 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Journal of Functional Foods, 58, 655\u0026ndash;664. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jff.2019.02.018\u003c/span\u003e\u003cspan address=\"10.1016/j.jff.2019.02.018\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Milk Thistle, Imipramine, Hepatotoxicity, Hepatoprotective, Royal jelly, Comparative","lastPublishedDoi":"10.21203/rs.3.rs-7442584/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7442584/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eImipramine is an antidepressant drug that with repeated administration can cause hepatic toxicity, which results in hepatic injury; hence the need for liver protection from toxicity by the use of hepatoprotactants, like milk thistle and royal jelly.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eThis study aims to comparatively evaluate the antioxidant and anti-inflammatory effects of milk thistle and royal jelly, both as monotherapies and in combination, in a rat model of imipramine-induced hepatotoxicity.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e\u003cp\u003eThirty adult female Albino Wistar rats were divided into six groups (n\u0026thinsp;=\u0026thinsp;6).Group A: Negative control (1ml D.W.); Group B imipramine (10 mg/kg/day) ;Group C milk thistle (140 mg/kg/day ) and royal jelly (150 mg/kg/day); Group D milk thistle (140 mg/kg/day ) and imipramine (10 mg/kg); Group E royal jelly (150 mg/kg/day) and imipramine (10 mg/kg); Group F milk thistle (140 mg/kg/day ), royal jelly (150 mg/kg/day) and imipramine (10 mg/kg). All treatments were taken orally. At 4 weeks of treatment, rats will be sacrificed and anesthetized. Serum for the assessment of Tumor Necrosis Factor Alpha (TNF-α), Interleukin-10 (IL-10), Glutathione (GSH), and Malondialdehyde (MDA) will be collected. All the parameters were determined by ELISA.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eImipramine group exhibited hepatotoxicity as reflected by the elevation of MDA and TNF alpha and reduction of GSH compared to the control group. In contrast, the change in IL-10 was insignificant. Royal jelly and milk thistle had a remarkable antioxidant and anti-inflammatory effect by way of reduction of MDA and TNF alpha and elevation of GSH when compared to imipramine.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eBoth milk thistle and royal jelly have an anti-inflammatory and antioxidant action against imipramine hepatotoxicity.\u003c/p\u003e","manuscriptTitle":"Comparative Anti-inflammatory and Oxidative Effects of Milk Thistle and Royal Jelly Against Hepatotoxicity of Imipramine in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-04 15:18:55","doi":"10.21203/rs.3.rs-7442584/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5b3027a7-07e6-4b29-9b6d-5c2851fd0f4a","owner":[],"postedDate":"September 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-01T15:23:58+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-04 15:18:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7442584","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7442584","identity":"rs-7442584","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}