The Effectiveness of Hydroalcoholic Extract and Fraction of Cucumis melo on Amelioration of High Fat Diet Induced hepatotoxicity: A Comprehensive Approach Integrating Computational and Preclinical Validation

Non-Alcoholic Fatty Liver Disease (NAFLD) presents a significant worldwide health concern characterized by hepatic dysfunction and persistent dyslipidemia.^1,2 This condition results in the abnormal accumulation of fat and cholesterol within liver tissue, devoid of inflammatory processes and is driven by a complex interplay of signaling mechanisms. Metabolic disorders such as obesity, type 2 diabetes, dyslipidemia and hepatic steatosis are major precursors for the pathogenesis of the liver.^1,2 Studies suggest that mitochondrial dysfunction is a major precursor pathogenesis of the liver. As per previous reports, prolonged consumption of HFD is responsible for abnormal lipid accumulation in the form of Free Fatty Acid (FFA) and Triglyceride (TG) in the serum/ liver, increasing the weight of the liver, oxidative stress liable to steatohepatitis, cirrhosis, and Hepatocellular Carcinoma. (HCC).^1,2 Cholesterol major precursor of the hepatic cells, impairment in the regulation of cholesterol level, metabolism of lipoprotein, dietary uptake, endogenous synthesis excretion,³ elevated plasma levels of Total Cholesterol (TC), Low-Density Lipoprotein Cholesterol (LDL-C), Triglyceride Concentration (TG), decrease in plasma High-Density Lipoprotein cholesterol level (HDL-C) are highly manifested leading to dyslipidemia.

The Nuclear Receptor (NR) is the biggest family ligand-modulated of transcriptional regulators, which has 49 members in mice and 48 in humans and provides a framework for a better knowledge of liver physiology and pathobiology. Endogenous and exogenous substances such as hormones, Fatty Acids (FAs), Bile Acids (BAs), medicines, toxins and intermediary molecules in metabolism are examples of ligands for NRs.⁴ These occurrences play a pivotal role in governing signal transduction pathways, both in normal physiological states and during pathological conditions.⁵ Over time, Numerous steroid hormone Receptors (NRs) have been identified, including the Farnesoid X Receptor (FXR), Liver X Receptor (LXR), Pregnane X Receptor (PXR), Peroxisome Proliferator-Activated Receptor (PPAR) and Retinoid X Receptor (RXR). Prior to their discovery, the natural ligands and functions of these receptors remained elusive. However, their regulation has been extensively researched and identified to regulate lipid glucose metabolism, BA homeostasis, drug disposition, reproduction, inflammation, cell differentiation and various aspects of tissue repair including liver regeneration, fibrosis and, finally, tumour formation.⁶

The current investigation aims to evaluate the potential beneficial effects of Cucumis melo Linn in managing the HFD- induced hepatotoxicity in mice. C. melo is known for its traditional medicinal consideration in India, exhibits protection against hepatic damage, dyslipidaemia and inflammation. The seeds and fruit used traditionally for multiple purpose such as obesity, cough, flatulence, jaundice. The flesh of the fruit is a considerable source of protein, fatty acids, ascorbic acid, folic acid, carotenoids, terpenes and steroidal glycosides.⁷

The fresh fruits of C. melo were harvested in the Belagavi region of Karnataka. By comparing morphological traits, the Central Research Facility B.M.K. Ayurveda Mahavidyalaya, Belagavi, authenticated with accession number CRF/Auth./2020/1. Initially, Soxhlet extraction technique was used for 100 g of C. melo seeds with solvent as 95% v/v ethanol and the obtained extract was subjected to bio-guided fractionation as shown in Figure 1.

The DPPH assay of C. melo extract and Ascorbic acid was performed as per the method of Tubachi et al.,⁸ and the IC₅₀ was determined by,

HepG2 cells were seeded in 96-well plates at a density of 20000 cells/well and were incubated at 37°C for 24 hr with 95% humidity in a CO₂ incubator. After 24 hr, the cells were treated with test substance (C. melo extract and fraction), standard (Silymarin) and HFD and was incubated at 37°C and 5% CO₂. Cytotoxicity was evaluated after 24 hr by adding 20 μL of MTT solution (5 mg/mL in phosphate buffer saline) for 4 hr incubation followed by 100 μL DMSO for dissolving formazan crystals. The intensity of color was measured at 570 nm using an ELISA plate reader and CC₅₀ was determined.⁹

Healthy Swiss albino mice (15-20 g) were purchased from Shri Venkateshwara Enterprises. The ethical clearance was obtained at KLE College of Pharmacy, Belagavi IAEC (Reg No. 221/Po/ Re/S/2000/CPCSEA).

High fat diet was prepared by mixing the powder pallet diet+5g of Ghee (34%)+1 g of Vanaspati (18%)+5 g of yeast powder (8%)+5 g of lard (12%)+1 mL of Coconut oil (6%), totally 78% of calories from fat and make up the volume up to 10 mL/kg. The percentage of fat is conformed according to the RM value.¹⁰ Albino mice were acclimatized for 7 days, then divided into six groups (n=6). Except for group I, all group animals received HFD daily for 28 days while Silymarin (50 mg/kg p.o) served as the standard. Group I: food and water. Group II: HFD (10 mL/kg).¹¹ Group III: Silymarin 50 mg/kg and HFD (10 mL/kg). Group IV: 50mg/kg of C. melo fraction and HFD (10 mL/kg), and lastly Group V: 500mg/kg of C. melo extract and HFD (10 mL/kg). 24 hr after the last dosing, a blood sample was collected through the retro-orbital puncture and animals were sacrificed by cervical dislocation under general anaesthesia. The liver was collected for estimation of biochemical parameters and liver histopathological examination.

Phytocompounds of C. melo. were retrieved from reported literature and open database viz., Dr. Dukes DB (https://www.nal.usda.gov/dr-dukes-database), IMMPAT DB (https://cb.imsc.re s.in/imppat/), Phytochemical integration DB (https://www.genome.jp/db/pcidb/) and their structures with PubChem CID and SMILES were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/).

Molsoft tool was used to predict the Druglikeness score for each phytocompound based upon Lipinski’s rule of 5. Moreover, Molecular Weight (MW), Formula (MF), number of Hydrogen Bond Acceptors (HBA), Donor (HBD). Surface area (MolPSA) and lipophilicity (MolLogP) of screened phytocompounds were obtained. The adverPred database¹² (https://www.way2drug.com/adverpred/) was used to predict the side effects for the screened phytocompounds.

We used ADMETlab 2.0 (https://admetmesh.scbdd.com/)¹³ to predict the ADME of the selected phytocompounds which is been widely recognized for their pharmacokinetics properties i.e., absorption, distribution, metabolism, and excretion.

SuperPred (https://prediction.charite.de/subpages/targetprediction.php)¹⁴ server, was used to predict targets of each phytocompounds with a p-value of ?0.7 and their respective Gene ID were obtained from UniProtKB server (https://www.uniprot.org/).

A set of predicted protein targets modulated by the phytocompounds targeting FXR were submitted to STRING¹⁵ ver 11.0 (https://stringdb.org/) and were used to identify the modulated pathways by a set of queried gene targets. Additionally, KEGG,¹⁶ genomes pathway database and published literature were also used to compile modulated genes and their related pathways in liver cirrhosis. Finally, using Cytoscape¹⁷ ver 3.6.1 a network was created between phytocompounds, targets and pathways using edge count as a topological parameter. For the network representation, the map node was set from low to high and small to big.

3D structure of each phytocompounds and known standard inhibitor Silymarin were retrieved from PubChem database (https://pubchem.ncbi.nlm.nih.gov/) in “sdf” file format and converted into “pdb” format using Discovery Studio Visualizer (https://discover.3ds.com/discoverstudio-visualizer-download) ver 17.2. Similarly, FXR (PDB Id: 6HL1) was retrieved from Protein Data Bank¹⁸ (RCSB; www.rcsb.org). Molecular docking was performed using AutoDock Vina via executed through the POAP pipeline.¹⁹ Standard docking was performed and the grid was set around the active site with the box dimensions center x=9.070, y=17.169, z=13.316 and size x=27.408, y=29.430 and z=23.349 and exhaustiveness was set to 50. The binding results were further, analyzed for interaction between ligand and target and were visualized using ‘BIOVIA Discovery studio visualizer 2019’.

All the data were expressed as mean ± SEM. The data was analyzed by One way ANOVA followed by Tukey’s multiple comparison tests using Graph Pad Prism version 8 with p<0.05 considered statistically significant and linear regression was used to analyze the inhibitory constant. Enrichment analyses were analyzed using the whole genome and edge count was defined to analyze the network representation, kcal/mol showed the least binding energy.

The hydroalcoholic extract yield of C. melo. seeds per 100 g w/w of powder was found to be 16%. Additionally, the C. melo fraction yield for 16 g w/w of hydroalcoholic extract was calculated as 6% respectively.

The IC₅₀ of ascorbic acid and C. melo was found to be 47.89 and 153.0 μg/mL, respectively as shown in (Figure 2).

C. melo extract and its fraction (Conc. 12.5-100 μg/mL) showed the significant cell viability after 24 hr (Figure 3).

Disease control group (HFD) exhibited remarkable raise in body weight and liver weight compared to normal group (p<0.001). Positive control groups, C. melo fraction and extract group exhibited decline in body weight (p<0.001) and liver weight (p<0.001) compared to disease control group (Figure 4).

Disease control group (HFD) exhibited remarkable decrease (p<0.001) in Catalase, Nitrate, SOD and GSH level and increase in the LPO level compared to normal group. Whereas, treatment with Silymarin, C. melo fraction and C. melo extract reversed (p<0.001) the same compared to disease control group. Among these, C. melo fraction exhibited potent antioxidant property (Figure 5).

Disease control group (HFD) exhibited remarkable increase (p<0.001) in ALP, ALT, AST, total and serum bilirubin compared to normal group. Whereas, treatment with Silymarin, C. melo fraction and C. melo extract showed significant decrease (p<0.001) in the liver enzymes compared to disease control group. Among these, C. melo fraction was found to be good hepatoprotective compared to extract (Figure 6).

Disease control group (HFD) exhibited remarkable increase (p<0.001) in LDL, VLDL, TC, TG and decrease (p<0.001) in the HDL and TP compared to normal group. Whereas, treatment with Silymarin, C. melo fraction and C. melo extract showed significant reverse in the parameters (p<0.001) compared to disease control group (Figure 7).

In mice receiving vehicle, liver tissue containing a Central Vein (CV) appeared unharmed. Histological abnormalities that suggested liver cirrhosis showed clogged blood vessels, disintegrating tissue, fatty degeneration and excessive inflammation were improved after HFD treatment. Pretreatment with Silymarin, extract and fraction of C. melo anomalies was brought back to normal as shown in Figure 8.

A total of 160 phytocompounds were reported to be present in the C. melo.

Out of 160 phytocompounds, 16 phytocompounds were predicted to have a positive drug likeness score in which 24-methylenecyloartanol showed the highest molecular weight with 1 HBD and HBA of each, followed by Euphol with 426.7, the least was observed in Avenasterol and Stigmasterol with molecular weight 412.7. Moreover, none of the phytocompounds were identified to cross the blood-brain barrier as shown in Table S1.

Figure 1:
Hydroalcoholic extraction and crude fractionation of Cucumis melo.

Among all the screened phytocompounds (as shown in Table S2 and Table S3), Avenasterol and Sitosterol were predicted to have toxicity, but were observed within the limits of Pa 0.246 and Pi 0.228.

Gene enrichment analysis showed that 16 phytocompounds were predicted to modulate the 27 targets. Additionally, these 27 targets interacted with each other to modulate the 65 pathways in the KEGG database. Among, them 7 pathways were identified to be associated with liver cirrhosis. As depicted in Table S4 and Table S5, the PPAR signaling pathway scored the lowest FDR of 1.95E-13 by modulating the 8 protein targets, followed by insulin resistance and AMPK pathway. Furthermore, out of 16 phytocompounds, Euphol was found to highly modulated compound by regulating a maximum number of proteins (Figure 9).

The active site residues of FXR, namely Ser259, Tyr260, Gln263, Arg264, Met265, Thr270, Ile273, Leu287, Thr288, Met290, Ala291, His294, Val297, Leu298, Phe301, Asn327, Met328, Arg331, Ser332, Ile335, Phe336, Lys338, Ser345, Leu348, Ile352, Ile357, Tyr361, Ile362, Met365, Phe366, Tyr369, Thr386, His447, Met450, Trp454 and Trp469 were taken from the crystal structure “6HL1.pdb”. Among all the phytocompounds, Silymarin scored the lowest Binding Energy (BE) of -9.4kcal/mol via forming 3 hydrogen bonds and 5 non- hydrophobic interactions (Figure 10). Euphol scored the lowest binding energy among all others with a BE of -11.1kcal/mol via forming 2 hydrogen bonds and 23 non-hydrophobic interactions (Figure 11 and Table 1).

Figure 2:
Antioxidant activity C. melo by DPPH assay.

Figure 3:
MTT cytotoxicity activity.

Figure 4:
Effect of Cucumis melo extract and fraction on body weight and liver weight. ***p<0.001 compare to normal, ###p<0.001 when compared to disease control.

Figure 5:
Effect of Cucumis melo extract and fraction on catalase, GSH, Nitrate and LPO. ***p<0.001 compare to normal, ##p<0.01, ###p<0.001 when compared to disease control.

Figure 6:
Effect of Cucumis melo extract and fraction on AST, ALT, ALP, Serum bilirubin and Total bilirubin. ***p<0.001 compare to normal, ##p<0.01, ###p<0.001 when compared to disease control.

The present study was designed to explore the hepatoprotective and antioxidant effect of C. melo in HFD-induced hepatotoxicity in albino mice. In HFD animals, elevated hepatic fatty acid metabolism results in cholesterol and triglyceride accumulation as well as elevated liver inflammation and liver damage.²⁰ Firstly, both C. melo extract and fraction showed significant cell viability compared to HFD-treated groups in HepG2 cells. Secondly, the extract and a fraction were screened for its hepatoprotection in albino mice. In animal study, the significantly increased body weight (p<0.001) and liver weight (p<0.001) could be due to the accumulation of fat which was reversed in the treatment groups. This indicates the potential role of phytosterols in the regulation of lipid metabolism. In the present study, HFD-treated animals showed abnormal levels of antioxidant markers TP, GSH, LPO, SOD, nitrate and catalase, levels and were reversed in the C. melo extract and fraction groups. Similarly, lipid markers like HDL, LDL, VLDL, TG and TC were also abnormal in the HFD-treated animal and were found to be towards normal in C. melo extract and fraction groups. Serum ALP, AST, ALT and bilirubin are considered as a specific marker for hepatic damage and were significantly elevated in the HFD-treated animals and were found to be towards normal in C. melo extract and fraction groups. Histology of the liver showed remarkable histological changes after treatment. In HFD-treated groups, cirrhosis or liver injury was confirmed by clogged blood vessels, disintegrating tissue, fatty degeneration and excessive inflammation. Whereas, Silymarin, C. melo extract and fraction anomalies HFD diet mediated histological pattern of the liver.

Moreover, the current work traced the 160 reported phytocompounds of C. melo to propose a probable mechanism against the HFD-induced hepatotoxicity. Out of 160 phytocompounds, 16 were predicted to show a positive druglikeness and were further predicted to target 27 protein molecules and were involved in 65 pathways, in which 7 pathways were identified to be associated with liver cirrhosis. Among 27 predicted targets FXR, HMGCR was identified as a major modulated and therapeutic target involved in the pathogenesis of hepatotoxicity by modulating all 7 pathways within the network. FXR indirectly reduces lipid-induced hepatic inflammation by decreasing intracellular FA levels.²¹ It is highly expressed in the liver and intestine which further standalone in activating varieties of inflammatory markers causing hepatocellular or cholestatic conditions.²¹ Several tetracyclic triterpenes from fruits and vegetables have been reported to inhibit the production of FXR by blocking the inflammatory cellular signaling pathways primally MAPK, PI3-Akt and NF-kB.²² C. melo phytocompounds exhibit potent antioxidant, anti-inflammatory and anti-colitis activity by reducing the FXR expression in bile formation, TNF-α and IL-6 upregulations.²³ Furthermore, in vitro and in vivo studies demonstrated that euphol showed a both preventive treatments were effective in reducing the severity of inflammatory conditions and reducing intestinal smooth muscle with propulsion of faces by controlled acute inflammatory reactions. Also, it regulates nitric oxide production and mucosal damage and decreases in the component expression of inflammatory responses.²⁴ Therefore, the network analysis and predicted affinity of phytocompounds of C. melo. towards the active site residues of FXR seems to be concurrence with these findings and provide the possible molecular mechanism of action of C. melo. as a potent hepato-protective nutraceutical.

Figure 7:
Effect of Cucumis melo extract and fraction on HDL, LDL, VLDL, TC, TG, TP. ***p<0.001 compare to normal, ###p<0.001 when compared to disease control.

Figure 8:
Histopathological changes on HFD induced hepatotoxicity. I: Normal control, II: HFD, III: Silymarin+HFD, IV: C. melo extract [250 mg/kg+HFD], V: C. melo extract [500 mg/kg+HFD], VI: C. melo fraction [50 mg/kg+HFD].

Figure 9:
Network representation of Cucumis melo Linn phytocompounds with their probable protein molecules involved in Liver cirrhosis.

Figure 10:
Shows (a) the 2D interaction of the binding mode of silymarin with FXR, (b) 3D binding mode of silymarin with FXR.

Figure 11:
Shows (a) the 2D interaction of the binding mode of euphol with FXR, (b) 3D binding mode of euphol with FXR.

In network, non-alcoholic fatty liver diseases, AMPK, HIF-1, Insulin resistance, PPAR signalling and pathways in cancer were found to be highly modulated pathways via modulating multiple protein molecules. On looking into the Non-alcoholic fatty liver diseases pathway PPAR signaling pathway it includes 9 potential targets i.e., FABP4, PPARG, FABP1, FABP5, PPARD, FABP3, PPARA and, FXR. The current study identifies the tetracyclic triterpene and triterpenoids to majorly target PPAR signalling pathway by targeting the 9 protein targets, among which FXR is also present. Activation of the PPAR signalling pathway modulates lipid metabolism and energy homeostasis.^25,26 In some conditions the impaired activity of PPAR signalling pathway might contribute to Hepatocellular carcinoma.²⁷ Insulin resistance mechanism is a vital signaling pathway in which it binds to the FXR which further extends its role in regulating lipid and cholesterol homeostasis. Activation of FXR reverses the insulin resistance and lipid abnormalities gaining its protection against liver toxicity.²⁸ Further, it is well reported that, phytocompounds from C. melo act as an anti-inflammatory, anti-diabetic, diuretic, gastroprotective, anti-microbial and, hepatoprotective activity by inducing the antioxidant effect. Also, phytocompounds present in the C. melo are reported to target genes involved in hepatotoxicity.

Hence, the present investigation on hepato-protective activity of C. melo could be due to the modulation of PPAR, Insulin resistance, pathways in cancer, AMPK, non-alcoholic fatty liver disease and HIF-1 signaling pathways.

Thecurrentinvestigationemployed in vivo experimentalevaluation to explore the HFD-induced hepatoxicity of C. melo extract and fraction followed by computational approaches. Evaluation of the extract and fraction demonstrated an increase in antioxidant enzyme activity and reduced biochemical levels. Histopathology analysis revealed improved clogged vessels, disintegrating tissue, fatty degeneration and excessive inflammation. Further, we depicted the interaction of the phytocompounds from C. melo. with proteins involved in the etiology of hepatotoxicity. Gene enrichment analysis identified PPAR signalling pathway as a likely major pathway targeted by the phytocompounds to counter hepatotoxic activity. Key constituent like euphol suggested their potential role in inhibiting the FXR target. Further, the identified phytocompounds and enriched fractions need to be validated for clinical trials and their interaction studies with the standard anti-hepatotoxicity drugs to develop and formulate the adjunct therapies for effective management. On the other hand, it provides the critical suggestive possible mechanism of action that may be taken advantage of developing the C. melo. based research plans.

The authors are thankful to the Principal, KLE College of Pharmacy, Belagavi, Dept. of Pharmacology, KLE College of Pharmacy, Belagavi and to the Director, ICMR- National Institute of Traditional Medicine, Belagavi and to the Dept. of Pathology JNMC, Belagavi, Dr. Prabhakar Kore Basic Science Research Centre, for providing facilities and resources to conduct the research work.