Exploring Hepatoprotective Shielding action of Glycitein in alleviating Paracetamol- Induced Liver Damage in Albino Mice

Exploring Hepatoprotective Shielding action of Glycitein in alleviating Paracetamol- Induced Liver Damage in Albino Mice | 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 Exploring Hepatoprotective Shielding action of Glycitein in alleviating Paracetamol- Induced Liver Damage in Albino Mice Payal Mittal, Aritri Chowdhury, Vaneet Kumar, Navneet Sharma This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6876928/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 The liver is a vital organ involved in numerous metabolic and detoxification processes, but it is susceptible to damage from hepatotoxic substances. Natural remedies, such as essential oils and plant-derived compounds, have shown promise in supporting liver health. Isoflavone-containing essential oils, in particular, offer potential hepatoprotective effects. These effects are mediated through multiple mechanisms, including modulation of MAP kinase pathways, liver enzymes like SGOT and SGPT, and reactive oxidative species (ROS). Objective This study aimed to evaluate the hepatoprotective potential of Glycitein, an isoflavone compound, against paracetamol-induced hepatic damage in mice. Additionally, it investigated the in-silico interaction of Glycitein with key inflammatory and hormonal targets, namely IL-6, IL-1β, TNF-α, and Estrogen Receptors, which are implicated in various hepatological disorders. Methods Hepatotoxicity was induced in albino mice (either sex) via paracetamol (500 mg/kg) administration. Glycitein was administered intraperitoneally at doses of 3, 6, and 12 mg/kg. Hepatic function was assessed by evaluating changes in biochemical markers such as SGPT, SGOT, and bilirubin, along with oxidative stress markers including SOD and GSH-Px. Histopathological examinations were performed to observe liver tissue morphology. Molecular docking studies were conducted to assess Glycitein's binding affinity towards IL-6, IL-1β, TNF-α, and Estrogen Receptors. Results Paracetamol administration led to significant elevations in SGPT, SGOT, bilirubin levels, and depletion of antioxidant enzymes SOD and GSH-Px, indicating hepatic injury and oxidative stress. Histopathological analysis showed marked liver cell damage. Among the tested doses, Glycitein at 6 mg/kg demonstrated the most robust hepatoprotective effect, with notable restoration of enzyme levels and improved liver histology. Docking results indicated Glycitein exhibited moderate binding affinity with IL-6: –6.4 kcal/mol, IL-1β: –6.4 kcal/mol, TNF-α: –6.5 kcal/mol, Estrogen receptor: –6.8 kcal/mol. Conclusion Glycitein effectively alleviates paracetamol-induced liver injury by reducing oxidative stress, restoring the levels of key antioxidant enzymes, and normalizing liver histology. The in-silico findings suggest moderate binding affinity to inflammatory and hormonal targets, supporting its potential as a natural hepatoprotective agent. Glycitein Hepatotoxicity Paracetamol Liver function tests Molecular Docking ADMET Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction As a key organ in human body, liver is responsible for a wide range of essential function such as major detoxification and biological functions in the body(1). A proper examination of more harmless and effectual medications with the hepatoprotective nature has been the important research areas. After the long use of herbal and synthetic remedies which are currently present do not prove to be effective for the cure of liver illness. Hence, the procurement of new therapy from the nature with the least complexity is gaining more attention now. This is essential as the frequent cause of hepatic dysfunction would include Drug Induced Liver Injury (DILI)(2). Paracetamol (PCM) is an efficient and safe medication utilized as analgesic and antipyretic drug. The acute overdosing of PCM happen to produce some adverse effects (such as centrilobular hepatic necrosis) besides its effectiveness. This also involves generation of Reactive Oxidative Species (ROS) along pathway to liver toxicity. Currently, the liver damage production by PCM is very common mechanistic model for the evaluation of hepatoprotective interventions (3). Isoflavones are naturally occurring nonsteroidal phenolic compounds which are that are typically present in the plants of the Fabaceae family (4). Expanding evidence reveals that abnormally low estrogen levels have been associated with the emergence of liver disorders. As a hepatoprotective agent, estradiol has an indispensable impact. It has a greater affinity for Estrogen (5). Glycitein is found in soy supplements made from soy germ along with other organisms such as Psidium guajava, Ammopiptanthis mongolocus (6). It works as a fungal metabolite, a phytoestrogen and a plant metabolite. It serves as a phytoestrogen, a fungal metabolite, and a plant metabolite. Molecular Docking is a computational technique used to predict the interaction between ligand molecules and target proteins at the molecular level (7,8). Ligands are prepared using ChemDraw and saved in SDF format, while protein structures are obtained from the Protein Data Bank (PDB) and processed using BIOVIA Discovery Studio to optimize their geometry and eliminate unnecessary elements. Docking is performed using AutoDock Vina to calculate binding affinities (in kcal/mol) and analyze interactions, including hydrogen bonding, hydrophobic interactions, and pi-pi stacking. These analyses help evaluate the ligand's potential to bind effectively at receptor sites. Materials and Methods 2.1. Chemical used: Test drug Camphene was procured from TCI Chemicals Hyderabad. Silymarin and paracetamol were obtained from Oyster Labs, Ambala. Other chemicals such as carboxy methyl cellulose, NBT, DTNB, EDTA, Riboflavin, methionine, sodium dodecyl sulfate, thiobarbituric acid, DMSO, saline, phosphate buffer of analytical grade and were obtained from Chandigarh University. 2.2. Requisition of Animals 36 Albino mice (either sex), age 4 weeks (26-30g), were attained from the Chandigarh University. The mice of either sex weighed around 25–30 gm. Animals were kept under standard laboratory conditions in the animal house, where they had access to ad libitum standard commercial rat feed and pure drinking water. The experiment duration was 5 days and prior permission was obtained from IAEC (Institutional Animal Ethics Committee) to conduct experiments by Protocol No. CU/2022/IAEC/7/03. 2.3 Animal Procurement: After procuring the mice, they were maintained under standard laboratory conditions for a specific period that was around 15 days for acclimatization in the animal house. The mice were then divided depending on their body weight into 6 groups containing 6 mice in each categorise. 2.4. Experimental Blueprint For the induction of hepatotoxicity Swiss Albino Mice of (either sex) having mass around 26–36 gram, involved for study. Animals were housed inside cages with polycarbonate material, under standard laboratory conditions (27+-2 C) 12- hour Light-Dark Cycle along period of acclimatization and experimentation. Animals were sustained with a pallet meal along with water ad libitum. Each mice were grouped as 6 mice in 6 groups. In Group I (Control) 1% CMC was administered for 5 days and on the 5th day the mice sacrificed after the time period of 24 hours. Thereafter, in Group II (Positive Control) 1% CMC p.o. was given orally till day 5 with treatment with paracetamol (500mg/kg p.o.). After 24 hours, animals were sacrificed. In the case of Group III standard (Silymarin, 100mg/kg) administered orally for 5 days with treatment of Paracetamol on the 5th day (500mg/kg). Animals were then sacrificed after 24 hours. Moreover, in Group IV test (Glycitein, 3mg/kg) was dispensed orally for 5 days with treatment with Paracetamol (500 mg/kg) on the 5th day. After 24 hours, animals were sacrificed. Then in Group V test (Glycitein, 6mg/kg) was applied orally for 5 days with the treatment of Paracetamol 9500 mg/kg) on the 5th days. Animals were sacrificed after 24 hours. At the last, the Group VI test (Glycitein, 12mg/kg) was given orally for 5 days with treatment of Paracetamol (500mg/kg) on 5th day. Animals were abdicated after 24 hours. Groups Treatment No. of animals Group I Control (CMC (0.1% w/v); oral gavage 6 Group II Paracetamol (Positive Control) 500 mg/kg 6 Group III Silymarin (Standard) 6 mg/kg, oral + PCM (500 mg/kg, oral). 6 Group IV Glycitein (Test Low Dose)(3 mg/kg, oral) + PCM (500 mg/kg, oral). 6 Group V Glycitein (Test Medium Dose) (6 mg/kg, oral) + PCM (500 mg/kg, oral). 6 Group VI Glycitein (Test High Dose) (12 mg/kg, oral) + PCM (500 mg/kg, oral). 6 Total 36 The doses for the treatments and references were selected based on previously published studies. This experimental design targets to analyse impact of camphene at different dosages on a paracetamol-induced model. 2.5. Collection of sample and scarification of Animal : The retro-orbital plexus of the mice was used to extract blood (9). This collection was done under light anaesthesia of ketamine on the 6th day. About 0.5ml blood was collected in clean test tubes and was allowable to clot for half an hour & later was centrifuged for around 8–10 minutes at 3000rpm. The serum separated was collected in 1ml micro-centrifuged tubes. After that, these were subjected to SGOT, SGPT, and bilirubin estimation (10). Six animals from each group were put to death that same day following ketamine anaesthesia. Liver was stored in 10% neutral buffered formalin and saline for histopathology and oxidative parameter examination respectively. 2.6. Assessment of Biochemical Parameters 2.6.2 Estimation of SGOT: The SGOT concentration was estimated by the IFCC method using the commercially available kit. The working reagent (1.0ml) and the sample (0.05ml) were mixed. At a wavelength of 340 nm, the reaction was tracked by detecting the drop in NADH absorption. The deduction rate in absorbance is related to the activity of SGOT in the sample. The mean change was determined in absorbance per minute that is ∆A/min and the test result was calculated (11). 2.6.3 Estimation of SGPT: The SGPT concentration was estimated by the IFCC method using the commercially available kit(12)The Working Reagent (1.0ml) and the sample (0.05ml) were mixed and the first absorbance was read of the test at 1 minute and then 30nm, 60nm, 90nm, 120nm, and 340nm. The mean change was determined in absorbance per minute that is ∆A/min and the test result was calculated (13). 2.6.4 Estimation of Serum Bilirubin: The concentration of bilirubin was estimated by the J&G method using the commercially available kit. Preparation of the working reagent was done by adding the sample as Standard (0.05mL) and Test (0.05mL), working reagent as Standard and Test (0.01mL), 2 Bilirubin Standard (0.1mL) and Test and 3 Bilirubin Standard (0.1mL) and Test (1.0mL). All the chemicals given in the table were mixed and then cultivated for 5 minutes. At R.T. and read the concentration against the sample blank at 546nm. Estimation of Antioxidant Parameters 3.1 Superoxide dismutase (SOD) The parameter was established by using (14)approach(15,16). 0.25 mL of ethanol, 0.15 mL of chloroform & 0.5 mL of tissue homogenate combined, & combination was manually mixed for 15 mins. Samples were then centrifuged at 13000 g for 15 min at 40°C & supernatant obtained utilized for further procedure. To 0.5 mL of the homogenate, 2 mL of 0.1 M Tris-HCl (pH 8.2) and 1.5 mL distilled water was mixed, together with 0.5 mL 2 mM pyrogallol, mixed, and optical density value was measured spectrophotometrically at 420 nm in 0-, 1, 2 and 3-minutes. 3.2 Estimation of Glutathione Peroxidase (GPx) Rotruch et al. (1973) approach GPx’s activity. The reaction combination included 0.5 mL of 0.2 mM hydrogen peroxide (H2O₂), 2.0 mL of 0.4 M Tris-HCl buffer (pH 7.0), 0.2 mL of tissue homogenate & 0.2 mL of 10 mM glutathione. Mixture was incubated at 37°C for 10 minutes to facilitate enzymatic action (17). The reaction then terminated by adding 10% TCA (0.4 mL). After five minutes of centrifuging the sample at 5000 rpm, absorbance of supernatant was evaluated at 430 nm to ascertain GPx parameter. 3.3 Estimation of Malondialdehyde (MDA) Malondialdehyde was determined by the process described by (18). The TBA reactive substance test was utilized to determine the MDA levels in the rat liver and serum, which was previously described. In brief, plasma or supernatant samples were mixed with a TBA/buffer solution and 8.1% sodium dodecyl sulfate. TBA solution was prepared by dissolving 0.53% thiobarbituric acid (TBA) 20% acetic acid, with pH adjusted to 3.5 using NaOH. The incubation time for combination of process is 60 min at 95°C to terminate reaction, tubes were immediately chilled on ice and centrifuged at 4000 rpm for 10 minutes. Optical concentration of supernatant (pink) was then analysed at 532 nm to evaluate reaction outcome (19). 3.4 Histopathological evaluations : Sections from each liver lobe were taken right away for histopathological examination(20). After that, a 10% formalin solution was preserved, dehydrated using alcohol, subsequently dissolved inside paraffin. They were then divided into 4–5 µm thick segments, stained with Haematoxylin and Eosin dyes, which were tested further. 3.5. Molecular Docking Docking was performed utilizing Autodock-vina, and energy was minimized for all ligand molecules. Whereas, command prop was used to perform docking on selected molecules and docking scores were estimated in kcal/mol. Various interactions (pi-pi stacking, hydrophobic interaction, hydrogen bonding & Vander wall interaction) were analysed. Whereas, Bio-Discovery Studio software was used to evaluate the 2D and 3D interaction of selected ligand molecules against receptor sites. 3.5.1 Ligand Preparation : The ligand molecule was drawn using ChemDraw and was downloaded in SDF format. The docking of ligands against possible therapeutic sites has been done using the auto dock Vina tools. 3.5.2 Protein Preparation : The protein database (PDB) was used to download 3D formation of protein from protein data bank PDB ID:6D6U. The proteins were then processed by eliminating ligand molecules, excess water, and het-atoms with the help of the BIOVIA-discovery studio software, hydrogen atoms were added, minimal bonds were generated, charges were fixed, as well as any poor bonds were made were correct. It was subsequently imported into the Autodock Vina software. 3.6 Statistical Analysis: GraphPad Prism9 was used to do a statistical analysis of the observations. A mean ± SEM error was used to express each outcome. The results were analyzed using Tukey's multiple comparison test after performing ANOVA (with p < 0.0001) being statistically noticeable. Results and Discussion 4.2.1. Effects of Glycitein on SGOT in Paracetamol-Induced Hepatotoxicity: Paracetamol (500mg/kg) administration significantly induced hepatic damage as observed by elevated SGOT levels 156.8 ± 10.53 (mg/dl) as compared to vehicle control group 22.8 ± 1.56 (mg/dl). However, it was seen that the standard drug Silymarin drastically reduced these level 50.2 ± 1.88 (mg/dl) as compared to vehicle control group 22.8 ± 1.56 (mg/dl) and paracetamol treated group 156.8 ± 10.53 (mg/dl) pre-treatment with test drug Glycitein in 5 days in different doses (3mg/kg: 116.8 ± 4.45 mg/dl, 6mg/kg: 104.5 ± 3.51mg/dl and 12mg/kg: 81.6 ± 2.50mg/dl) significantly decreased SGOT levels as compared to disease control group (156.8 ± 10.53) as shown in Fig. 1. 4.2.2. Glycitein’s impact on SGPT in Paracetamol-Induced Hepatotoxicity: Paracetamol (500mg/kg) administration significantly induced hepatic damage as observed by elevated SGOT levels 109 ± 2.39 (mg/dl) as contrast to vehicle control group 21.4 ± 1.07 (mg/dl). However, it was seen that the standard drug Silymarin drastically reduced these level 36.4 ± 1.91 (mg/dl) as collated with vehicle control group 21.4 ± 1.07 (mg/dl) and paracetamol treated group 109 ± 2.39 (mg/dl). Pre-treatment with test drug Glycitein in 5 days in different doses (3mg/kg: 85.2 ± 1.56 mg/dl, 6mg/kg: 61.6 ± 1.57 mg/dl and 12mg/kg: 57.8 ± 1.48 mg/dl) significantly decreased SGOT levels as compared to disease control group (156.8 ± 10.53) as shown in Figure No 2. 4.2.3. Effects of Glycitein on Bilirubin Level in Paracetamol-Induced Hepatotoxicity: Effects of Glycitein on SGOT amount in Paracetamol-Induced Liver Toxicity One-way analysis of variance (ANOVA) followed by Tukey’s test was utilized to develop statistical significance. a = p < 0.05 vs Paracetamol, b = p < 0.05 vs Silymarin, c = p < 0.05 vs Glycitein (3mg/kg), d = p < 0.05 vs Glycitein (6mg/kg), e = p < 0.05 vs Glycitein (12mg/kg). * Represents the p < 0.01; ** Represents the p < 0.05. Glycitein’s Influence on Oxidative Stress Biomarkers in Paracetamol-Induced Hepatotoxicity a. Effects of Glycitein on SOD Level in Paracetamol-Induced Hepatotoxicity : Paracetamol (500mg/kg) administration significantly induced hepatic damage as observed by elevated SGOT levels 6.58 ± 0.07 (mg/dl) as compared to vehicle control group 10.70 ± 0.15 (mg/dl). However, it was seen that the standard drug silymarin drastically increased these level 12.75 ± 0.14 (mg/dl) as compared to vehicle control group 10.70 ± 0.15 (mg/dl) and paracetamol treated group 6.58 ± 0.07 (mg/dl) pretreatment with test drug Glycitein in 5 days in different doses (3mg/kg: 8.48 ± 0.17 mg/dl, 6mg/kg: 8.87 ± 0.17 mg/dl and 12mg/kg: 9.07 ± 0.09 mg/dl) significantly decreased SGOT levels as compared to disease control group (3.57 ± 0.03) as shown in Figure No 4. b. Effects of Glycitein on GSH-Px Level in Paracetamol-Induced Hepatotoxicity : Paracetamol developed a certain lowering in the GSH level from 5.26 ± 0.04 to 2.07 ± 0.07 when compared with vehicle control group. In addition, amount of SOD significantly decreased from 5.26 ± 0.04 to 4.96 ± 0.03 in standard control group (Silymarin) when it was compared to vehicle control group. Glycitein at 3mg/kg, 6mg/kg, and 12mg/kg for 5 days with Paracetamol exhibited a considerable increase in Serum GSH quantities 2.51 ± 0.14, 3.67 ± 0.17 and 4.07 ± 0.09 when it was compared with control group (5.26 ± 0.04) as depicted in Table 10. Treatment with Glycitein significantly produced the hepatoprotective effect in Paracetamol-treated mice as shown in Figure No 5. c. Effects of Glycitein on MDA/TBARS Level in Paracetamol-Induced Hepatotoxicity : Paracetamol produced significant elevation in levels of MDA from 0.68 ± 0.37 to 4.24 ± 1.08 when compared with vehicle control group. In addition, standard control group (Silymarin) depicts considerable elevation inside the level of MDA from 0.68 ± 0.37 to 0.95 ± 0.03 when it was compared to the vehicle control group. On other hand, Glycitein at 3mg/kg, 6mg/kg, and 12mg/kg for 5 days with Paracetamol exhibited lowering of MDA from 4.24 ± 1.08 to 3.00 ± 1.26, 2.46 ± 1.37 and 1.48 ± 1.08 respectively when compared with treated control group. Treatment with Glycitein significantly produced the hepatoprotective effect in the mice as shown in Fig. 6. 1. Effects of Glycitein on Histological Changes in Paracetamol-Induced Hepatotoxicity: Histopathological substitution of liver features were observed in the experimental protocol shown in Fig. 7. The liver cell sin vehicle control was seen to be normal with properly defined cells of liver surrounded main vein with clear cell membrane and nuclear architecture shown in Fig. 7-A. Treated control was seen with severe hepatic injury (degeneration changes including vacuolation of the cell cytoplasm and fatty changes in scattered cells) in the present study. Moreover, hepatocytes covering main vein showed extensive necrosis with nuclear psychosis and vacuolar cytoplasmic degeneration in Paracetamol-treated mice (Fig. 7-B). Standard control (Silymarin) showed a significant hepatoprotective effect (Fig. 7-C). The different doses of Glycitein were observed to reduce hepatocyte necrosis caused by Paracetamol and restored a normal morphological feature with dosage of 3mg/kg (Fig. 7-D), 6mg/kg (Fig. 7-E) and 12mg/kg (Fig. 7-F). Hence it has been seen that supplementation of different doses of Glycitein produced a hepatoprotective effect in paracetamol-treated mice. Effects of Various Treatments on Histology of Mice’s Liver: A) Vehicle Control (it shows normal anatomy of liver including central vein), B) Treated Control (central vein as well as normal anatomy of liver got affected due to pathogenic activity of paracetamol), C) Standard Control (Treatment of Silymarin reduces the pathogenic effect of paracetamol to a greater extent, clear image of central vein can be seen, hepatocytes are intact), D) Glycitein (3mg/kg) (3mg/kg of Glycitein treated the hepatocytes but the central vein is not clear), E) Glycitein (6mg/kg) (6mg/kg of Glycitein treated the hepatocytes but the central vein is still not clear), F) Glycitein (12mg/kg) (12mg/kg of Glycitein treated the hepatocytes very well and the central vein is clear) [White arrows indicate inflammation; Yellow arrows indicate condensed nuclei of cells; Black arrows indicate fat infiltration] 2. Docking Interaction of Glycitein against targets TNF-α (4M4E) Glycitein exhibits docking score of -6.5kcal/mol. Here in the interaction of Glycitein against 4M4E it shows van der walls interactions with various amino acid residues like (PHE457(A), HIS403(A), ARG459(A), ILE311(A), PRO396(A), ASP395(A), SER394(A)) and conventional hydrogen bond interaction with (ARG452 PHE457(A), HIS403(A), ARG459(A), ILE311(A), PRO396(A), ASP395(A), SER394(A)) amino acid residues. The ribbon structure is a representation of the structure of proteins. 1ALU: Glycitein exhibits docking score of -6.4kcal/mol. Here in the interaction of glycitein against 1ALU it shows van der walls interactions with various amino acid residues like (ASP160(A), MET49(A), THR43(A), THR163(A), LEU165(A), LEU167(A), SER169(A), PHE170(A)) and conventional hydrogen bond interactions with (SER47(A), GLU172(A)) amino acid residues. Besides this it shows one alkyl bond (ILE36(A)). It exhibits a Pi-Sigma bond (CYS44 SER47(A), GLU172(A)). The ribbon structure is a representation of the structure of proteins. 3LTQ: Glycitein exhibits docking score of -6.4kcal/mol. Here in the interaction of glycitein against 3LTQ it shows van der walls interactions with various amino acid residues like (GLN81(A), THR79(A), LYS77(A), PRO78(A), PHE133(A), LEU80(A), GLU25(A), TYR24(A)) and conventional hydrogen bond interactions with (VAL132(A), LYS74(A)) amino acid residues. The ribbon structure is a representation of the structure of proteins. 1A52: Glycitein exhibits docking score of -6.8kcal/mol. Here in the interaction of glycitein against 1A52 it shows van der walls interactions with various amino acid residues like (ILE326(A), LEU403(A)) and conventional hydrogen bond interactions with (ASN439(A), GLN441(A), LEU440(A)) amino acid residues. Besides this it shows two alkyl and Pi-alkyl bonds (ARG436(A), MET396(A)). It exhibits a Pi-Sigma bond (ARG394(A)) and a Pi-Pi T-shaped and Pi-Pi Stacked bond (TRP393(A)). It also shows a Pi-Anion bond (GLU397(A))). The ribbon structure is a representation of the structure of proteins. 3D and crystal structures, 2D interactions, and molecular docking outcomes of Glycitein with hepatotoxicity-related targets and receptors were analysed to evaluate its therapeutic potential have been mentioned in Table 1 , 2 and 3 respectively. Table 1 Docking outcome of ligand-target site interaction for hepatotoxicity Name of Compound Site of target Protein Data Bank ID Score of Docking (kcal/mol) Amino Acids Residual interaction Glycitein TNF-α 4M4E -6.5 PHE457, HIS403, ARG459, ILE311, PRO396, ASP395, SER394 Glycitein IL-6 1ALU -6.4 ASP160, MET49, THR43, THR163, LEU165, LEU167, SER169, PHE170. Glycitein IL-1β 3LTQ -6.4 GLN81(A), THR79(A), LYS77(A), PRO78(A), PHE133(A), LEU80(A), GLU25(A), TYR24(A). Glycitein Estrogen Receptor 1A52 -6.8 ILE326,ASN439,LEU403, GLN441, LEU440(A). Conclusion The current work was selected to assess beneficial properties of Glycitein on paracetamol-induced liver injury in mice along with, to find out employment of SGOT, SGPT, Serum bilirubin, GPx, MDA, and SOD in liver injury. Paracetamol was administered to mice at dose of 500 mg/kg p.o for 5 days for the induction of hepatotoxicity. Index of hepatocyte damage was assessed by estimating amounts of SGPT, Serum bilirubin and SGOT. Furthermore, impact of Oxidative Stress was assessed by estimating stages of Anti-oxidant enzymes that are GPx, SOD, and MDA. To check the extent of liver tissue damage histopathological studies were performed which showed inflammation and necrosis in paracetamol-induced hepatotoxicity whereas, Glycitein 6 mg/kg successfully reverted action of paracetamol by reducing inflammation and bringing the hepatocytes to normal. The result of the present research suggested that Glycitein has both preventative and curative regimens which considerably diminished liver dysfunction and Oxidative Stress by Paracetamol. Moreover, the pathological alterations were evaluated by microscopic evaluations of the liver. Glycitein demonstrates binding affinity to TNF-α receptor with docking score of -6.4 kcal/mol, indicating moderate interaction strength. Key amino acid residues involved in the interaction include PHE457, HIS403, ARG459, ILE311, PRO396 and ASP395 suggesting potential stabilization within the receptor's active site. This highlights its potential as a ligand for TNF-α modulation. The finding concluded that paracetamol causes Severe Liver Damage characterized by an increase in Oxidative Stress. However, Glycitein was found to be a potent Anti-oxidant and hence helped in the prevention and treatment of liver cell damage upon paracetamol exposure. Abbreviations Drug induced liver injury (DILI) paracetamol (PCM) acetaminophen (APAP) Interleukin 6 receptor (IL 6) serum glutamate oxaloacetate transaminase (SGOT) Nuclear factor erythroid 2 related factor 2 (Nrf2) Reactive oxygen species (ROS) Alanine transaminase (ALT) activated protein kinase (AMPK) Nitro blue tetrazolium (NBT) Institutional Animal Ethics Committee (IAEC) serum glutamate pyruvate transaminase (SGPT) Tumor Necrosis Factor alpha (TNF-α) International Federation of Clinical Chemistry (IFCC) Superoxide dismutase (SOD) Aspartate transaminase (AST) room temperature (R.T.) Mitogen activated protein kinase (MAP) High Fat Diet (HFD) Nuclear Factor kappa light-chain-enhancer of activated B cells (NfkB). Declarations Acknowledgment The authors are thankful to the Chandigarh University, for providing the necessary facilities. Funding Declaration No funding Clinical Trail Protocol No. CU/2022/IAEC/7/03. Supplementary file Not required Conflict of interest The author declares that there is no conflict of interest in this article. Author Contributions All authors have an equal contribution. PM: Conceptualization, final check ; AC: Paper writing, experiment performed; VK: Data interpretation, experiments; NS: Software, data interpretation. The authors confirm that no paper mill and artificial intelligence was used Ethical Approval Not required References 1. Ray G. Management of liver diseases: Current perspectives. World J Gastroenterol [Internet]. 2022 Oct 28 [cited 2025 Apr 4];28(40):5818. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC9639658/ 2. Andrade RJ, Chalasani N, Björnsson ES, Suzuki A, Kullak-Ublick GA, Watkins PB, et al. Drug-induced liver injury. Nature Reviews Disease Primers 2019 5:1 [Internet]. 2019 Aug 22 [cited 2025 Apr 4];5(1):1–22. Available from: https://www.nature.com/articles/s41572-019-0105-0 3. 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An Evaluation of Hepato-protective Activity of Ethanolic Extract of Solanum nigrum with Varying Doses on CCL4 Induced Hepatic Injured Rat. Asian Journal of Advanced Research and Reports [Internet]. 2024 Mar 16 [cited 2025 Apr 4];18(4):75–80. Available from: https://journalajarr.com/index.php/AJARR/article/view/625 12. Singleton VL, Rossi JA. Colorimetry of Total Phenolics with Phosphomolybdic-Phosphotungstic Acid Reagents. Am J Enol Vitic. 1965;16(3):144–58. 13. Nuzul M, Siddiq AA, Marliyati A, Riyadi H, Winarsih W. Effects of Kersen leaves extract (Muntingia calabura L.) on SGOT and SGPT levels of soft drink induced mice. Jurnal Gizi dan Pangan [Internet]. 2019 Jul 30 [cited 2025 Apr 4];14(2):69–76. Available from: https://journal.ipb.ac.id/index.php/jgizipangan/article/view/22576 14. Grankvist K, Marklund S, Sehlin J, Taljedal IB. Superoxide dismutase, catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro. Biochemical Journal [Internet]. 1979 Jul 15 [cited 2025 Apr 4];182(1):17–25. Available from: /biochemj/article/182/1/17/4429/Superoxide-dismutase-catalase-and-scavengers-of 15. Thavasu PW, Longhurst S, Joel SP, Slevin ML, Balkwill FR. Measuring cytokine levels in blood. J Immunol Methods. 1992 Aug;153(1–2):115–24. 16. Reitman S, Frankel S. A Colorimetric Method for the Determination of Serum Glutamic Oxalacetic and Glutamic Pyruvic Transaminases. Am J Clin Pathol. 1957 Jul 1;28(1):56–63. 17. Gurudath S, Ganapathy KS, Sujatha D, Pai A, Ballal S, Ml A. Estimation of superoxide dismutase and glutathione peroxidase in oral submucous fibrosis, oral leukoplakia and oral cancer–a comparative study. Asian Pac J Cancer Prev [Internet]. 2012 [cited 2025 Apr 4];13(9):4409–12. Available from: https://pubmed.ncbi.nlm.nih.gov/23167351/ 18. Baliga S, Chaudhary M, Bhat S, Bhansali P, Agrawal A, Gundawar S. Estimation of malondialdehyde levels in serum and saliva of children affected with sickle cell anemia. Journal of Indian Society of Pedodontics and Preventive Dentistry [Internet]. 2018 Jan 1 [cited 2025 Apr 4];36(1):43–7. Available from: https://journals.lww.com/jped/fulltext/2018/36010/estimation_of_malondialdehyde_levels_in_serum_and.9.aspx 19. Levy M, Kolodziejczyk AA, Thaiss CA, Elinav E. Dysbiosis and the immune system. Nat Rev Immunol. 2017 Apr 6;17(4):219–32. 20. Chowdhury AB, Mehta KJ. Liver biopsy for assessment of chronic liver diseases: a synopsis. Clin Exp Med [Internet]. 2023 Jun 1 [cited 2025 Apr 4];23(2):273–85. Available from: https://link.springer.com/article/10.1007/s10238-022-00799-z Tables Tables 2 and 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table23.docx 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-6876928","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":473162237,"identity":"26f2d3cd-438b-4167-a761-a59a7d8106ae","order_by":0,"name":"Payal Mittal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYLCCB0DMx8N8EETz8BGlJYHBgIGNhy3ZAKSFjQQtPGYSIA5BLebsZww/JDD8kWfjOWBW+TXHToaNgfnhoxt4tFj25BhLAG0xbONtSLstuy0Z6DA2Y+McPFoMDuQYgLQwtvEzHLstuY0ZqIWHTRqvlvNvjH8Atdi38TO2FUtuqydCy40cM5AtiW28zWyMH7cdJqzFcsazMosEA+PkNp5jzNKM247zsDET8Is5f/LmGx8q5Gz7efI/fvy5rdqen7354WO8DmPgMACRYMDMAybxKIdoYX8A5zD+IKB6FIyCUTAKRiYAACq7P7TMtg5AAAAAAElFTkSuQmCC","orcid":"","institution":"University Institute of Pharma Sciences (UIPS), Chandigarh University","correspondingAuthor":true,"prefix":"","firstName":"Payal","middleName":"","lastName":"Mittal","suffix":""},{"id":473162238,"identity":"5ba15551-a2fe-4076-b8ca-d49c13be1001","order_by":1,"name":"Aritri Chowdhury","email":"","orcid":"","institution":"University Institute of Pharma Sciences (UIPS), Chandigarh University","correspondingAuthor":false,"prefix":"","firstName":"Aritri","middleName":"","lastName":"Chowdhury","suffix":""},{"id":473162240,"identity":"b0750d0a-a422-4871-84f9-a23dca78fea1","order_by":2,"name":"Vaneet Kumar","email":"","orcid":"","institution":"Chandigarh University","correspondingAuthor":false,"prefix":"","firstName":"Vaneet","middleName":"","lastName":"Kumar","suffix":""},{"id":473162241,"identity":"3a239385-0a96-4d4a-97ff-ef054879559e","order_by":3,"name":"Navneet Sharma","email":"","orcid":"","institution":"University Institute of Pharma Sciences (UIPS), Chandigarh University","correspondingAuthor":false,"prefix":"","firstName":"Navneet","middleName":"","lastName":"Sharma","suffix":""}],"badges":[],"createdAt":"2025-06-12 06:08:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6876928/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6876928/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85056577,"identity":"8061cf78-f5c9-4167-8271-2721ea329025","added_by":"auto","created_at":"2025-06-20 13:01:16","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":18952,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlycitein mitigated SGOT levels in Paracetamol-induced hepatic Injury \u003c/strong\u003eSignificance was evaluated using\u003cstrong\u003e \u003c/strong\u003eOne-way analysis of variance (ANOVA) followed by Tukey’s test. a=p\u0026lt;0.05 vs Paracetamol, b=p\u0026lt;0.05 vs Silymarin, c=p\u0026lt;0.05 vs Glycitein (3mg/kg), d=p\u0026lt;0.05 vs Glycitein (6mg/kg), e=p\u0026lt;0.05 vs Glycitein (12mg/kg). *p\u0026lt;0.01; **p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6876928/v1/8ef4221c0a12a49a64ee4c39.jpg"},{"id":85057477,"identity":"82dfa29c-ec26-4e85-89e2-ef331a981b8e","added_by":"auto","created_at":"2025-06-20 13:09:16","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":43168,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e: Glycitein mitigated SGPT levels in Paracetamol-induced hepatic Injury\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificance was evaluated using\u003cstrong\u003e \u003c/strong\u003eOne-way analysis of variance (ANOVA) followed by Tukey’s test. a=p\u0026lt;0.05 vs Paracetamol, b=p\u0026lt;0.05 vs Silymarin, c=p\u0026lt;0.05 vs Glycitein (3mg/kg), d=p\u0026lt;0.05 vs Glycitein (6mg/kg), e=p\u0026lt;0.05 vs Glycitein (12mg/kg). *p\u0026lt;0.01; **p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6876928/v1/a29ccf652da663f1de7b6b29.jpg"},{"id":85056579,"identity":"2f2135ea-25b1-444b-9647-888ebd6d3485","added_by":"auto","created_at":"2025-06-20 13:01:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":321259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActions of Glycitein on Serum Bilirubin Stages in Paracetamol- Induced Liver Toxicity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificance was evaluated using\u003cstrong\u003e \u003c/strong\u003eTwo-way analysis of variance (ANOVA) later by Tukey’s test. a=p\u0026lt;0.05 vs Paracetamol, b=p\u0026lt;0.05 vs Silymarin, c=p\u0026lt;0.05 vs Glycitein (3mg/kg), d=p\u0026lt;0.05 vs Glycitein (6mg/kg), e=p\u0026lt;0.05 vs Glycitein (12mg/kg). *p\u0026lt;0.01; **p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6876928/v1/c0156e678d4c598e09616732.png"},{"id":85057478,"identity":"3b3a3cb6-8cc2-478b-ab7b-4e3be8425317","added_by":"auto","created_at":"2025-06-20 13:09:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":267002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAction of Glycitein on SOD Levels in Paracetamol-Induced Liver Toxicity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificance was evaluated using\u003cstrong\u003e \u003c/strong\u003eTwo-way analysis of variance (ANOVA) followed by Tukey’s test. a=p\u0026lt;0.05 vs Paracetamol, b=p\u0026lt;0.05 vs Silymarin, c=p\u0026lt;0.05 vs Glycitein (3mg/kg), d=p\u0026lt;0.05 vs Glycitein (6mg/kg), e=p\u0026lt;0.05 vs Glycitein (12mg/kg). *p\u0026lt;0.01; **p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6876928/v1/f89ed90adebbc6f070aff0b2.png"},{"id":85056586,"identity":"530a99bd-b508-4dd7-9725-abe9191d04cb","added_by":"auto","created_at":"2025-06-20 13:01:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":278276,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of Glycitein on GSH Levels in Paracetamol-Induced Liver Toxicity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSignificance was evaluated using\u003cstrong\u003e \u003c/strong\u003eTwo-way analysis of variance (ANOVA) followed by Tukey’s test utilized to state statistical significance. a=p\u0026lt;0.05 vs Paracetamol, b=p\u0026lt;0.05 vs Silymarin, c=p\u0026lt;0.05 vs Glycitein (3mg/kg), d=p\u0026lt;0.05 vs Glycitein (6mg/kg), e=p\u0026lt;0.05 vs Glycitein (12mg/kg). *p\u0026lt;0.01; ** p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6876928/v1/915d12eca3b6af050462642f.png"},{"id":85056590,"identity":"5684a120-361b-4bcb-9c86-756243e134d0","added_by":"auto","created_at":"2025-06-20 13:01:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":388504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of Glycitein on TBARS Levels in Paracetamol-Induced Liver Toxicity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwo-way analysis of variance (ANOVA) followed by Tukey’s test utilized to state statistical significance. a=p\u0026lt;0.05 vs Paracetamol, b=p\u0026lt;0.05 vs Silymarin, c=p\u0026lt;0.05 vs Glycitein (3mg/kg), d=p\u0026lt;0.05 vs Glycitein (6mg/kg), e=p\u0026lt;0.05 vs Glycitein (12mg/kg). * Represents the p\u0026lt;0.01; ** Represents the p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6876928/v1/3298c7d2222a581021d243d3.png"},{"id":85058122,"identity":"53190063-029b-4bfe-8314-0a34c07ade15","added_by":"auto","created_at":"2025-06-20 13:17:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5787129,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of Glycitein on histological changes (400x) in paracetamol-induced hepatotoxicity\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-6876928/v1/d38c0eb37351b8ced1d974d8.png"},{"id":86129750,"identity":"0bc6df37-4a76-4288-b28c-3cdc0785a138","added_by":"auto","created_at":"2025-07-07 06:32:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7891293,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6876928/v1/22f9e19b-197d-46ab-aafc-1fdd8deefdc8.pdf"},{"id":85056584,"identity":"fd6ffb82-7508-411a-87f0-eefa24e2bcd8","added_by":"auto","created_at":"2025-06-20 13:01:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1073358,"visible":true,"origin":"","legend":"","description":"","filename":"Table23.docx","url":"https://assets-eu.researchsquare.com/files/rs-6876928/v1/2e38436d77b507dfd0ec99c3.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploring Hepatoprotective Shielding action of Glycitein in alleviating Paracetamol- Induced Liver Damage in Albino Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs a key organ in human body, liver is responsible for a wide range of essential function such as major detoxification and biological functions in the body(1). A proper examination of more harmless and effectual medications with the hepatoprotective nature has been the important research areas. After the long use of herbal and synthetic remedies which are currently present do not prove to be effective for the cure of liver illness. Hence, the procurement of new therapy from the nature with the least complexity is gaining more attention now. This is essential as the frequent cause of hepatic dysfunction would include Drug Induced Liver Injury (DILI)(2).\u003c/p\u003e \u003cp\u003eParacetamol (PCM) is an efficient and safe medication utilized as analgesic and antipyretic drug. The acute overdosing of PCM happen to produce some adverse effects (such as centrilobular hepatic necrosis) besides its effectiveness. This also involves generation of Reactive Oxidative Species (ROS) along pathway to liver toxicity. Currently, the liver damage production by PCM is very common mechanistic model for the evaluation of hepatoprotective interventions (3).\u003c/p\u003e \u003cp\u003eIsoflavones are naturally occurring nonsteroidal phenolic compounds which are that are typically present in the plants of the Fabaceae family (4). Expanding evidence reveals that abnormally low estrogen levels have been associated with the emergence of liver disorders. As a hepatoprotective agent, estradiol has an indispensable impact. It has a greater affinity for Estrogen (5). Glycitein is found in soy supplements made from soy germ along with other organisms such as Psidium guajava, Ammopiptanthis mongolocus (6). It works as a fungal metabolite, a phytoestrogen and a plant metabolite. It serves as a phytoestrogen, a fungal metabolite, and a plant metabolite. Molecular Docking is a computational technique used to predict the interaction between ligand molecules and target proteins at the molecular level (7,8). Ligands are prepared using ChemDraw and saved in SDF format, while protein structures are obtained from the Protein Data Bank (PDB) and processed using BIOVIA Discovery Studio to optimize their geometry and eliminate unnecessary elements. Docking is performed using AutoDock Vina to calculate binding affinities (in kcal/mol) and analyze interactions, including hydrogen bonding, hydrophobic interactions, and pi-pi stacking. These analyses help evaluate the ligand's potential to bind effectively at receptor sites.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemical used:\u003c/h2\u003e \u003cp\u003eTest drug Camphene was procured from TCI Chemicals Hyderabad. Silymarin and paracetamol were obtained from Oyster Labs, Ambala. Other chemicals such as carboxy methyl cellulose, NBT, DTNB, EDTA, Riboflavin, methionine, sodium dodecyl sulfate, thiobarbituric acid, DMSO, saline, phosphate buffer of analytical grade and were obtained from Chandigarh University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. \u003cb\u003eRequisition of Animals\u003c/b\u003e\u003c/h2\u003e \u003cp\u003e36 Albino mice (either sex), age 4 weeks (26-30g), were attained from the Chandigarh University. The mice of either sex weighed around 25\u0026ndash;30 gm. Animals were kept under standard laboratory conditions in the animal house, where they had access to ad libitum standard commercial rat feed and pure drinking water. The experiment duration was 5 days and prior permission was obtained from IAEC (Institutional Animal Ethics Committee) to conduct experiments by Protocol No. CU/2022/IAEC/7/03.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Animal Procurement:\u003c/h2\u003e \u003cp\u003eAfter procuring the mice, they were maintained under standard laboratory conditions for a specific period that was around 15 days for acclimatization in the animal house. The mice were then divided depending on their body weight into 6 groups containing 6 mice in each categorise.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Experimental Blueprint\u003c/h2\u003e \u003cp\u003eFor the induction of hepatotoxicity Swiss Albino Mice of (either sex) having mass around 26\u0026ndash;36 gram, involved for study. Animals were housed inside cages with polycarbonate material, under standard laboratory conditions (27+-2 C) 12- hour Light-Dark Cycle along period of acclimatization and experimentation. Animals were sustained with a pallet meal along with water ad libitum. Each mice were grouped as 6 mice in 6 groups.\u003c/p\u003e \u003cp\u003eIn Group I (Control) 1% CMC was administered for 5 days and on the 5th day the mice sacrificed after the time period of 24 hours. Thereafter, in Group II (Positive Control) 1% CMC p.o. was given orally till day 5 with treatment with paracetamol (500mg/kg p.o.). After 24 hours, animals were sacrificed. In the case of Group III standard (Silymarin, 100mg/kg) administered orally for 5 days with treatment of Paracetamol on the 5th day (500mg/kg). Animals were then sacrificed after 24 hours. Moreover, in Group IV test (Glycitein, 3mg/kg) was dispensed orally for 5 days with treatment with Paracetamol (500 mg/kg) on the 5th day. After 24 hours, animals were sacrificed. Then in Group V test (Glycitein, 6mg/kg) was applied orally for 5 days with the treatment of Paracetamol 9500 mg/kg) on the 5th days. Animals were sacrificed after 24 hours. At the last, the Group VI test (Glycitein, 12mg/kg) was given orally for 5 days with treatment of Paracetamol (500mg/kg) on 5th day. Animals were abdicated after 24 hours.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNo. of animals\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl (CMC (0.1% w/v); oral gavage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParacetamol (Positive Control) 500 mg/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup III\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSilymarin (Standard) 6 mg/kg, oral\u0026thinsp;+\u0026thinsp;PCM (500 mg/kg, oral).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup IV\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycitein (Test Low Dose)(3 mg/kg, oral)\u0026thinsp;+\u0026thinsp;PCM (500 mg/kg, oral).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycitein (Test Medium Dose) (6 mg/kg, oral)\u0026thinsp;+\u0026thinsp;PCM (500 mg/kg, oral).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup VI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGlycitein (Test High Dose) (12 mg/kg, oral)\u0026thinsp;+\u0026thinsp;PCM (500 mg/kg, oral).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe doses for the treatments and references were selected based on previously published studies. This experimental design targets to analyse impact of camphene at different dosages on a paracetamol-induced model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.5. Collection of sample and scarification of Animal\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe retro-orbital plexus of the mice was used to extract blood (9). This collection was done under light anaesthesia of ketamine on the 6th day. About 0.5ml blood was collected in clean test tubes and was allowable to clot for half an hour \u0026amp; later was centrifuged for around 8\u0026ndash;10 minutes at 3000rpm. The serum separated was collected in 1ml micro-centrifuged tubes. After that, these were subjected to SGOT, SGPT, and bilirubin estimation (10). Six animals from each group were put to death that same day following ketamine anaesthesia. Liver was stored in 10% neutral buffered formalin and saline for histopathology and oxidative parameter examination respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Assessment of Biochemical Parameters\u003c/h2\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Estimation of SGOT:\u003c/h2\u003e \u003cp\u003eThe SGOT concentration was estimated by the IFCC method using the commercially available kit. The working reagent (1.0ml) and the sample (0.05ml) were mixed. At a wavelength of 340 nm, the reaction was tracked by detecting the drop in NADH absorption. The deduction rate in absorbance is related to the activity of SGOT in the sample. The mean change was determined in absorbance per minute that is ∆A/min and the test result was calculated (11).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3 Estimation of SGPT:\u003c/h2\u003e \u003cp\u003eThe SGPT concentration was estimated by the IFCC method using the commercially available kit(12)The Working Reagent (1.0ml) and the sample (0.05ml) were mixed and the first absorbance was read of the test at 1 minute and then 30nm, 60nm, 90nm, 120nm, and 340nm. The mean change was determined in absorbance per minute that is ∆A/min and the test result was calculated (13).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.6.4 Estimation of Serum Bilirubin:\u003c/h2\u003e \u003cp\u003eThe concentration of bilirubin was estimated by the J\u0026amp;G method using the commercially available kit. Preparation of the working reagent was done by adding the sample as Standard (0.05mL) and Test (0.05mL), working reagent as Standard and Test (0.01mL), 2 Bilirubin Standard (0.1mL) and Test and 3 Bilirubin Standard (0.1mL) and Test (1.0mL). All the chemicals given in the table were mixed and then cultivated for 5 minutes. At R.T. and read the concentration against the sample blank at 546nm.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eEstimation of Antioxidant Parameters\u003c/h3\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.1 Superoxide dismutase (SOD)\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe parameter was established by using (14)approach(15,16). 0.25 mL of ethanol, 0.15 mL of chloroform \u0026amp; 0.5 mL of tissue homogenate combined, \u0026amp; combination was manually mixed for 15 mins. Samples were then centrifuged at 13000 g for 15 min at 40\u0026deg;C \u0026amp; supernatant obtained utilized for further procedure. To 0.5 mL of the homogenate, 2 mL of 0.1 M Tris-HCl (pH 8.2) and 1.5 mL distilled water was mixed, together with 0.5 mL 2 mM pyrogallol, mixed, and optical density value was measured spectrophotometrically at 420 nm in 0-, 1, 2 and 3-minutes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Estimation of Glutathione Peroxidase (GPx)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eRotruch et al. (1973) approach GPx\u0026rsquo;s activity. The reaction combination included 0.5 mL of 0.2 mM hydrogen peroxide (H2O₂), 2.0 mL of 0.4 M Tris-HCl buffer (pH 7.0), 0.2 mL of tissue homogenate \u0026amp; 0.2 mL of 10 mM glutathione. Mixture was incubated at 37\u0026deg;C for 10 minutes to facilitate enzymatic action (17). The reaction then terminated by adding 10% TCA (0.4 mL). After five minutes of centrifuging the sample at 5000 rpm, absorbance of supernatant was evaluated at 430 nm to ascertain GPx parameter.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Estimation of Malondialdehyde (MDA)\u003c/h2\u003e \u003cp\u003eMalondialdehyde was determined by the process described by (18). The TBA reactive substance test was utilized to determine the MDA levels in the rat liver and serum, which was previously described. In brief, plasma or supernatant samples were mixed with a TBA/buffer solution and 8.1% sodium dodecyl sulfate. TBA solution was prepared by dissolving 0.53% thiobarbituric acid (TBA) 20% acetic acid, with pH adjusted to 3.5 using NaOH. The incubation time for combination of process is 60 min at 95\u0026deg;C to terminate reaction, tubes were immediately chilled on ice and centrifuged at 4000 rpm for 10 minutes. Optical concentration of supernatant (pink) was then analysed at 532 nm to evaluate reaction outcome (19).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.4 Histopathological evaluations\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eSections from each liver lobe were taken right away for histopathological examination(20). After that, a 10% formalin solution was preserved, dehydrated using alcohol, subsequently dissolved inside paraffin. They were then divided into 4\u0026ndash;5 \u0026micro;m thick segments, stained with Haematoxylin and Eosin dyes, which were tested further.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Molecular Docking\u003c/h2\u003e \u003cp\u003eDocking was performed utilizing Autodock-vina, and energy was minimized for all ligand molecules. Whereas, command prop was used to perform docking on selected molecules and docking scores were estimated in kcal/mol. Various interactions (pi-pi stacking, hydrophobic interaction, hydrogen bonding \u0026amp; Vander wall interaction) were analysed. Whereas, Bio-Discovery Studio software was used to evaluate the 2D and 3D interaction of selected ligand molecules against receptor sites.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.5.1 Ligand Preparation\u003c/b\u003e: The ligand molecule was drawn using ChemDraw and was downloaded in SDF format. The docking of ligands against possible therapeutic sites has been done using the auto dock Vina tools.\u003c/p\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e\u003cb\u003e3.5.2 Protein Preparation\u003c/b\u003e:\u003c/h2\u003e \u003cp\u003eThe protein database (PDB) was used to download 3D formation of protein from protein data bank PDB ID:6D6U. The proteins were then processed by eliminating ligand molecules, excess water, and het-atoms with the help of the BIOVIA-discovery studio software, hydrogen atoms were added, minimal bonds were generated, charges were fixed, as well as any poor bonds were made were correct. It was subsequently imported into the Autodock Vina software.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Statistical Analysis:\u003c/h2\u003e \u003cp\u003eGraphPad Prism9 was used to do a statistical analysis of the observations. A mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM error was used to express each outcome. The results were analyzed using Tukey's multiple comparison test after performing ANOVA (with p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) being statistically noticeable.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e4.2.1. Effects of Glycitein on SGOT in Paracetamol-Induced Hepatotoxicity:\u003c/h2\u003e\n\u003cp\u003eParacetamol (500mg/kg) administration significantly induced hepatic damage as observed by elevated SGOT levels 156.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.53 (mg/dl) as compared to vehicle control group 22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56 (mg/dl). However, it was seen that the standard drug Silymarin drastically reduced these level 50.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 (mg/dl) as compared to vehicle control group 22.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56 (mg/dl) and paracetamol treated group 156.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.53 (mg/dl) pre-treatment with test drug Glycitein in 5 days in different doses (3mg/kg: 116.8\u0026thinsp;\u0026plusmn;\u0026thinsp;4.45 mg/dl, 6mg/kg: 104.5\u0026thinsp;\u0026plusmn;\u0026thinsp;3.51mg/dl and 12mg/kg: 81.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.50mg/dl) significantly decreased SGOT levels as compared to disease control group (156.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.53) as shown in Fig.\u0026nbsp;1.\u003c/p\u003e\n\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\n\u003ch2\u003e4.2.2. Glycitein\u0026rsquo;s impact on SGPT in Paracetamol-Induced Hepatotoxicity:\u003c/h2\u003e\n\u003cp\u003eParacetamol (500mg/kg) administration significantly induced hepatic damage as observed by elevated SGOT levels 109\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39 (mg/dl) as contrast to vehicle control group 21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 (mg/dl). However, it was seen that the standard drug Silymarin drastically reduced these level 36.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91 (mg/dl) as collated with vehicle control group 21.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07 (mg/dl) and paracetamol treated group 109\u0026thinsp;\u0026plusmn;\u0026thinsp;2.39 (mg/dl). Pre-treatment with test drug Glycitein in 5 days in different doses (3mg/kg: 85.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56 mg/dl, 6mg/kg: 61.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57 mg/dl and 12mg/kg: 57.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.48 mg/dl) significantly decreased SGOT levels as compared to disease control group (156.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.53) as shown in Figure No 2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n\u003ch2\u003e4.2.3. Effects of Glycitein on Bilirubin Level in Paracetamol-Induced Hepatotoxicity:\u003c/h2\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEffects of Glycitein on SGOT amount in Paracetamol-Induced Liver Toxicity One-way analysis of variance (ANOVA) followed by Tukey\u0026rsquo;s test was utilized to develop statistical significance. a\u0026thinsp;=\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs Paracetamol, b\u0026thinsp;=\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs Silymarin, c\u0026thinsp;=\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs Glycitein (3mg/kg), d\u0026thinsp;=\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs Glycitein (6mg/kg), e\u0026thinsp;=\u0026thinsp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 vs Glycitein (12mg/kg). * Represents the p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ** Represents the p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eGlycitein\u0026rsquo;s Influence on Oxidative Stress Biomarkers in Paracetamol-Induced Hepatotoxicity\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003ea. Effects of Glycitein on SOD Level in Paracetamol-Induced Hepatotoxicity\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eParacetamol (500mg/kg) administration significantly induced hepatic damage as observed by elevated SGOT levels 6.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (mg/dl) as compared to vehicle control group 10.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 (mg/dl). However, it was seen that the standard drug silymarin drastically increased these level 12.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 (mg/dl) as compared to vehicle control group 10.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 (mg/dl) and paracetamol treated group 6.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 (mg/dl) pretreatment with test drug Glycitein in 5 days in different doses (3mg/kg: 8.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 mg/dl, 6mg/kg: 8.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 mg/dl and 12mg/kg: 9.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 mg/dl) significantly decreased SGOT levels as compared to disease control group (3.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03) as shown in Figure No 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb. Effects of Glycitein on GSH-Px Level in Paracetamol-Induced Hepatotoxicity\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eParacetamol developed a certain lowering in the GSH level from 5.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 to 2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 when compared with vehicle control group. In addition, amount of SOD significantly decreased from 5.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 to 4.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 in standard control group (Silymarin) when it was compared to vehicle control group. Glycitein at 3mg/kg, 6mg/kg, and 12mg/kg for 5 days with Paracetamol exhibited a considerable increase in Serum GSH quantities 2.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, 3.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 and 4.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 when it was compared with control group (5.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04) as depicted in Table\u0026nbsp;10. Treatment with Glycitein significantly produced the hepatoprotective effect in Paracetamol-treated mice as shown in Figure No 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec. Effects of Glycitein on MDA/TBARS Level in Paracetamol-Induced Hepatotoxicity\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eParacetamol produced significant elevation in levels of MDA from 0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 to 4.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 when compared with vehicle control group. In addition, standard control group (Silymarin) depicts considerable elevation inside the level of MDA from 0.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 to 0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 when it was compared to the vehicle control group. On other hand, Glycitein at 3mg/kg, 6mg/kg, and 12mg/kg for 5 days with Paracetamol exhibited lowering of MDA from 4.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 to 3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26, 2.46\u0026thinsp;\u0026plusmn;\u0026thinsp;1.37 and 1.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.08 respectively when compared with treated control group. Treatment with Glycitein significantly produced the hepatoprotective effect in the mice as shown in Fig.\u0026nbsp;6.\u003c/p\u003e\n\u003ch3\u003e1. Effects of Glycitein on Histological Changes in Paracetamol-Induced Hepatotoxicity:\u003c/h3\u003e\n\u003cp\u003eHistopathological substitution of liver features were observed in the experimental protocol shown in Fig.\u0026nbsp;7. The liver cell sin vehicle control was seen to be normal with properly defined cells of liver surrounded main vein with clear cell membrane and nuclear architecture shown in Fig.\u0026nbsp;7-A. Treated control was seen with severe hepatic injury (degeneration changes including vacuolation of the cell cytoplasm and fatty changes in scattered cells) in the present study. Moreover, hepatocytes covering main vein showed extensive necrosis with nuclear psychosis and vacuolar cytoplasmic degeneration in Paracetamol-treated mice (Fig.\u0026nbsp;7-B). Standard control (Silymarin) showed a significant hepatoprotective effect (Fig.\u0026nbsp;7-C). The different doses of Glycitein were observed to reduce hepatocyte necrosis caused by Paracetamol and restored a normal morphological feature with dosage of 3mg/kg (Fig.\u0026nbsp;7-D), 6mg/kg (Fig.\u0026nbsp;7-E) and 12mg/kg (Fig.\u0026nbsp;7-F). Hence it has been seen that supplementation of different doses of Glycitein produced a hepatoprotective effect in paracetamol-treated mice.\u003c/p\u003e\n\u003cp\u003eEffects of Various Treatments on Histology of Mice\u0026rsquo;s Liver: A) Vehicle Control (it shows normal anatomy of liver including central vein), B) Treated Control (central vein as well as normal anatomy of liver got affected due to pathogenic activity of paracetamol), C) Standard Control (Treatment of Silymarin reduces the pathogenic effect of paracetamol to a greater extent, clear image of central vein can be seen, hepatocytes are intact), D) Glycitein (3mg/kg) (3mg/kg of Glycitein treated the hepatocytes but the central vein is not clear), E) Glycitein (6mg/kg) (6mg/kg of Glycitein treated the hepatocytes but the central vein is still not clear), F) Glycitein (12mg/kg) (12mg/kg of Glycitein treated the hepatocytes very well and the central vein is clear) [White arrows indicate inflammation; Yellow arrows indicate condensed nuclei of cells; Black arrows indicate fat infiltration]\u003c/p\u003e\n\u003ch3\u003e2. Docking Interaction of Glycitein against targets\u003c/h3\u003e\n\u003cp\u003e\u003cstrong\u003eTNF-\u0026alpha; (4M4E)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGlycitein exhibits docking score of -6.5kcal/mol. Here in the interaction of Glycitein against 4M4E it shows van der walls interactions with various amino acid residues like (PHE457(A), HIS403(A), ARG459(A), ILE311(A), PRO396(A), ASP395(A), SER394(A)) and conventional hydrogen bond interaction with (ARG452 PHE457(A), HIS403(A), ARG459(A), ILE311(A), PRO396(A), ASP395(A), SER394(A)) amino acid residues. The ribbon structure is a representation of the structure of proteins.\u003c/p\u003e\n\u003ch3\u003e1ALU:\u003c/h3\u003e\n\u003cp\u003eGlycitein exhibits docking score of -6.4kcal/mol. Here in the interaction of glycitein against 1ALU it shows van der walls interactions with various amino acid residues like (ASP160(A), MET49(A), THR43(A), THR163(A), LEU165(A), LEU167(A), SER169(A), PHE170(A)) and conventional hydrogen bond interactions with (SER47(A), GLU172(A)) amino acid residues. Besides this it shows one alkyl bond (ILE36(A)). It exhibits a Pi-Sigma bond (CYS44 SER47(A), GLU172(A)). The ribbon structure is a representation of the structure of proteins.\u003c/p\u003e\n\u003ch3\u003e3LTQ:\u003c/h3\u003e\n\u003cp\u003eGlycitein exhibits docking score of -6.4kcal/mol. Here in the interaction of glycitein against 3LTQ it shows van der walls interactions with various amino acid residues like (GLN81(A), THR79(A), LYS77(A), PRO78(A), PHE133(A), LEU80(A), GLU25(A), TYR24(A)) and conventional hydrogen bond interactions with (VAL132(A), LYS74(A)) amino acid residues. The ribbon structure is a representation of the structure of proteins.\u003c/p\u003e\n\u003ch3\u003e1A52:\u003c/h3\u003e\n\u003cp\u003eGlycitein exhibits docking score of -6.8kcal/mol. Here in the interaction of glycitein against 1A52 it shows van der walls interactions with various amino acid residues like (ILE326(A), LEU403(A)) and conventional hydrogen bond interactions with (ASN439(A), GLN441(A), LEU440(A)) amino acid residues. Besides this it shows two alkyl and Pi-alkyl bonds (ARG436(A), MET396(A)). It exhibits a Pi-Sigma bond (ARG394(A)) and a Pi-Pi T-shaped and Pi-Pi Stacked bond (TRP393(A)). It also shows a Pi-Anion bond (GLU397(A))). The ribbon structure is a representation of the structure of proteins. 3D and crystal structures, 2D interactions, and molecular docking outcomes of Glycitein with hepatotoxicity-related targets and receptors were analysed to evaluate its therapeutic potential have been mentioned in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e respectively.\u0026nbsp;\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eDocking outcome of ligand-target site interaction for hepatotoxicity\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eName of Compound\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eSite of target\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eProtein Data Bank ID\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eScore of Docking (kcal/mol)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAmino Acids Residual interaction\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlycitein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eTNF-\u0026alpha;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4M4E\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\"\u003e\n\u003cp\u003e-6.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePHE457, HIS403, ARG459, ILE311, PRO396, ASP395, SER394\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlycitein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIL-6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1ALU\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\"\u003e\n\u003cp\u003e-6.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eASP160, MET49,\u003c/p\u003e\n\u003cp\u003eTHR43,\u003c/p\u003e\n\u003cp\u003eTHR163,\u003c/p\u003e\n\u003cp\u003eLEU165,\u003c/p\u003e\n\u003cp\u003eLEU167,\u003c/p\u003e\n\u003cp\u003eSER169,\u003c/p\u003e\n\u003cp\u003ePHE170.\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlycitein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIL-1\u0026beta;\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3LTQ\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\"\u003e\n\u003cp\u003e-6.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGLN81(A),\u003c/p\u003e\n\u003cp\u003eTHR79(A),\u003c/p\u003e\n\u003cp\u003eLYS77(A),\u003c/p\u003e\n\u003cp\u003ePRO78(A),\u003c/p\u003e\n\u003cp\u003ePHE133(A),\u003c/p\u003e\n\u003cp\u003eLEU80(A),\u003c/p\u003e\n\u003cp\u003eGLU25(A),\u003c/p\u003e\n\u003cp\u003eTYR24(A).\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eGlycitein\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003eEstrogen Receptor\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1A52\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\"\u003e\n\u003cp\u003e-6.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eILE326,ASN439,LEU403, GLN441, LEU440(A).\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003cdiv class=\"colspec\" align=\"left\"\u003e\u0026nbsp;\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe current work was selected to assess beneficial properties of Glycitein on paracetamol-induced liver injury in mice along with, to find out employment of SGOT, SGPT, Serum bilirubin, GPx, MDA, and SOD in liver injury. Paracetamol was administered to mice at dose of 500 mg/kg p.o for 5 days for the induction of hepatotoxicity.\u003c/p\u003e \u003cp\u003eIndex of hepatocyte damage was assessed by estimating amounts of SGPT, Serum bilirubin and SGOT. Furthermore, impact of Oxidative Stress was assessed by estimating stages of Anti-oxidant enzymes that are GPx, SOD, and MDA. To check the extent of liver tissue damage histopathological studies were performed which showed inflammation and necrosis in paracetamol-induced hepatotoxicity whereas, Glycitein 6 mg/kg successfully reverted action of paracetamol by reducing inflammation and bringing the hepatocytes to normal.\u003c/p\u003e \u003cp\u003eThe result of the present research suggested that Glycitein has both preventative and curative regimens which considerably diminished liver dysfunction and Oxidative Stress by Paracetamol. Moreover, the pathological alterations were evaluated by microscopic evaluations of the liver. Glycitein demonstrates binding affinity to TNF-α receptor with docking score of -6.4 kcal/mol, indicating moderate interaction strength. Key amino acid residues involved in the interaction include PHE457, HIS403, ARG459, ILE311, PRO396 and ASP395 suggesting potential stabilization within the receptor's active site. This highlights its potential as a ligand for TNF-α modulation.\u003c/p\u003e \u003cp\u003eThe finding concluded that paracetamol causes Severe Liver Damage characterized by an increase in Oxidative Stress. However, Glycitein was found to be a potent Anti-oxidant and hence helped in the prevention and treatment of liver cell damage upon paracetamol exposure.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDrug\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003einduced liver injury (DILI)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eparacetamol (PCM)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eacetaminophen (APAP)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eInterleukin 6 receptor (IL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e6)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eserum glutamate oxaloacetate transaminase (SGOT)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNuclear factor erythroid 2\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003erelated factor 2 (Nrf2)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eReactive oxygen species (ROS)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAlanine transaminase (ALT)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eactivated protein kinase (AMPK)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNitro blue tetrazolium (NBT)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eInstitutional Animal Ethics Committee (IAEC)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eserum glutamate pyruvate transaminase (SGPT)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTumor Necrosis Factor\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealpha (TNF-α)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eInternational Federation of Clinical Chemistry (IFCC)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eSuperoxide dismutase (SOD)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAspartate transaminase (AST)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eroom temperature (R.T.)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMitogen\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eactivated protein kinase (MAP)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHigh Fat Diet (HFD)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eNuclear Factor kappa\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003elight-chain-enhancer of activated B cells (NfkB).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to the Chandigarh University, for providing the necessary facilities.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical Trail\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtocol No. CU/2022/IAEC/7/03.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary file\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot required\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author declares that there is no conflict of interest in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have an equal contribution.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePM:\u003c/strong\u003e Conceptualization, final check\u003cstrong\u003e; AC:\u003c/strong\u003e Paper writing, experiment performed; \u003cstrong\u003eVK:\u003c/strong\u003e Data interpretation, experiments; \u003cstrong\u003eNS:\u003c/strong\u003e Software, data interpretation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors confirm that no paper mill and artificial intelligence was used\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot required\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e1. Ray G. Management of liver diseases: Current perspectives. World J Gastroenterol [Internet]. 2022 Oct 28 [cited 2025 Apr 4];28(40):5818. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC9639658/\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e2. Andrade RJ, Chalasani N, Bj\u0026ouml;rnsson ES, Suzuki A, Kullak-Ublick GA, Watkins PB, et al. Drug-induced liver injury. Nature Reviews Disease Primers 2019 5:1 [Internet]. 2019 Aug 22 [cited 2025 Apr 4];5(1):1\u0026ndash;22. Available from: https://www.nature.com/articles/s41572-019-0105-0\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e3. Wang X, Wu Q, Liu A, Anad\u0026oacute;n A, Rodr\u0026iacute;guez JL, Mart\u0026iacute;nez-Larra\u0026ntilde;aga MR, et al. Paracetamol: overdose-induced oxidative stress toxicity, metabolism, and protective effects of various compounds in vivo and in vitro. Drug Metab Rev [Internet]. 2017 Oct 2 [cited 2025 Apr 4];49(4):395\u0026ndash;437. 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Morris GM, Lim-Wilby M. Molecular Docking. Methods in Molecular Biology [Internet]. 2008 [cited 2025 Apr 4];443:365\u0026ndash;82. Available from: https://link.springer.com/protocol/10.1007/978-1-59745-177-2_19\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e8. Stanzione F, Giangreco I, Cole JC. Use of molecular docking computational tools in drug discovery. Prog Med Chem. 2021 Jan 1;60:273\u0026ndash;343.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e9. Vakkalagadda RK, Ravula P, Parameshwar K, Saraswathi K, Sindhuri P, Srikala R, et al. Protective Potential of Canthium dicoccum Methanolic Extract Against Hepatic Injury in Rats. Pharmacognosy Journal. 2021 Dec 1;13(6s):1648\u0026ndash;55.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e10. Khalil AW, Iqbal Z, Adhikari A, Khan H, Nishan U, Iqbal A, et al. 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Nuzul M, Siddiq AA, Marliyati A, Riyadi H, Winarsih W. Effects of Kersen leaves extract (Muntingia calabura L.) on SGOT and SGPT levels of soft drink induced mice. Jurnal Gizi dan Pangan [Internet]. 2019 Jul 30 [cited 2025 Apr 4];14(2):69\u0026ndash;76. Available from: https://journal.ipb.ac.id/index.php/jgizipangan/article/view/22576\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e14. Grankvist K, Marklund S, Sehlin J, Taljedal IB. Superoxide dismutase, catalase and scavengers of hydroxyl radical protect against the toxic action of alloxan on pancreatic islet cells in vitro. Biochemical Journal [Internet]. 1979 Jul 15 [cited 2025 Apr 4];182(1):17\u0026ndash;25. Available from: /biochemj/article/182/1/17/4429/Superoxide-dismutase-catalase-and-scavengers-of\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e15. Thavasu PW, Longhurst S, Joel SP, Slevin ML, Balkwill FR. Measuring cytokine levels in blood. 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Journal of Indian Society of Pedodontics and Preventive Dentistry [Internet]. 2018 Jan 1 [cited 2025 Apr 4];36(1):43\u0026ndash;7. Available from: https://journals.lww.com/jped/fulltext/2018/36010/estimation_of_malondialdehyde_levels_in_serum_and.9.aspx\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e19. Levy M, Kolodziejczyk AA, Thaiss CA, Elinav E. Dysbiosis and the immune system. Nat Rev Immunol. 2017 Apr 6;17(4):219\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e20. Chowdhury AB, Mehta KJ. Liver biopsy for assessment of chronic liver diseases: a synopsis. Clin Exp Med [Internet]. 2023 Jun 1 [cited 2025 Apr 4];23(2):273\u0026ndash;85. Available from: https://link.springer.com/article/10.1007/s10238-022-00799-z\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 2 and 3 are available in the Supplementary Files section.\u003c/p\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":"Glycitein, Hepatotoxicity, Paracetamol, Liver function tests, Molecular Docking, ADMET","lastPublishedDoi":"10.21203/rs.3.rs-6876928/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6876928/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe liver is a vital organ involved in numerous metabolic and detoxification processes, but it is susceptible to damage from hepatotoxic substances. Natural remedies, such as essential oils and plant-derived compounds, have shown promise in supporting liver health. Isoflavone-containing essential oils, in particular, offer potential hepatoprotective effects. These effects are mediated through multiple mechanisms, including modulation of MAP kinase pathways, liver enzymes like SGOT and SGPT, and reactive oxidative species (ROS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study aimed to evaluate the hepatoprotective potential of Glycitein, an isoflavone compound, against paracetamol-induced hepatic damage in mice. Additionally, it investigated the in-silico interaction of Glycitein with key inflammatory and hormonal targets, namely IL-6, IL-1β, TNF-α, and Estrogen Receptors, which are implicated in various hepatological disorders.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepatotoxicity was induced in albino mice (either sex) via paracetamol (500 mg/kg) administration. Glycitein was administered intraperitoneally at doses of 3, 6, and 12 mg/kg. Hepatic function was assessed by evaluating changes in biochemical markers such as SGPT, SGOT, and bilirubin, along with oxidative stress markers including SOD and GSH-Px. Histopathological examinations were performed to observe liver tissue morphology. Molecular docking studies were conducted to assess Glycitein's binding affinity towards IL-6, IL-1β, TNF-α, and Estrogen Receptors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParacetamol administration led to significant elevations in SGPT, SGOT, bilirubin levels, and depletion of antioxidant enzymes SOD and GSH-Px, indicating hepatic injury and oxidative stress. Histopathological analysis showed marked liver cell damage. Among the tested doses, Glycitein at 6 mg/kg demonstrated the most robust hepatoprotective effect, with notable restoration of enzyme levels and improved liver histology. Docking results indicated Glycitein exhibited moderate binding affinity with IL-6: –6.4 kcal/mol, IL-1β: –6.4 kcal/mol, TNF-α: –6.5 kcal/mol, Estrogen receptor: –6.8 kcal/mol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGlycitein effectively alleviates paracetamol-induced liver injury by reducing oxidative stress, restoring the levels of key antioxidant enzymes, and normalizing liver histology. The in-silico findings suggest moderate binding affinity to inflammatory and hormonal targets, supporting its potential as a natural hepatoprotective agent.\u003c/p\u003e","manuscriptTitle":"Exploring Hepatoprotective Shielding action of Glycitein in alleviating Paracetamol- Induced Liver Damage in Albino Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-20 13:01:11","doi":"10.21203/rs.3.rs-6876928/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":"1c3da8b2-972b-49ce-bb75-23323dac8d5a","owner":[],"postedDate":"June 20th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T06:23:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-20 13:01:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6876928","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6876928","identity":"rs-6876928","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}