Hypophyllanthin Protects Against Alcohol-Induced Hepatotoxicity in Rats: Involvement of Oxidative Stress, Inflammatory Cytokines, Apoptosis, and Autophagy

Abstract

Introduction

Ethanol consumption is a primary global health concern, frequently leading to severe hepatic injury. The present study assessed the hepatoprotective effects of hypophyllanthin against ethanol-induced hepatotoxicity in rats.

Methods

A Wistar rats were randomly assigned into five groups (n=6/group): Group I (normal control), Group II (ethanol control), Group III-IV (ethanol + low-high-doses hypophyllanthin [5 and 10 mg/kg]), and Group V (high-dose hypophyllanthin per se [10 mg/kg]). At the end of the study, the biochemical assessment, including serum biomarkers (ALT, AST, ALP, TG, TBA, TC), serum alcohol, oxidative stress, inducible nitric oxide synthase (iNOS), tumor necrosis factor-alpha (TNF-α), interleukins (IL-1β and IL-6), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), caspase-3, Beclin-1, and LC3B, complex II, and complex IV, were determined. Additionally, a histological evaluation was also performed.

Results

Hypophyllanthin significantly attenuated ethanol-induced oxidative stress, as evidenced by decreased mitochondrial enzymes such as complex II and complex IV, as well as increased activity of antioxidant enzymes (SOD, CAT, GSH) and decreased lipid peroxidation (MDA). Hypophyllanthin mitigated inflammation by reducing cytokines (IL-1β, IL-6, TNF-α, NF-κB, iNOS, and COX-2) and serum biomarkers (ALT, AST, ALP, TG, TBA, and TC), as well as serum alcohol levels. Hypophyllanthin promoted apoptosis by modulating caspase-3 activity, facilitating the elimination of damaged cells. It also inhibited autophagy by suppressing the levels of Beclin-1 and LC3B, potentially limiting excessive cellular degradation. Histopathological analysis confirmed the protective effects of hypophyllanthin, demonstrating reduced hepatic necrosis, steatosis, and inflammatory cell infiltration. A dose-dependent effect was observed between the treated groups.

Discussion

These findings suggested a strong physiological basis for ethanol-induced hepatotoxicity and the hepatoprotective effects of hypophyllanthin.

Graphical Abstract

Introduction

The liver serves as a master metabolic regulator, orchestrating many physiological processes essential to homeostasis. This multifunctional organ primarily metabolizes carbohydrates, proteins, and lipids, ensuring efficient nutrient utilization for energy production and storage. The liver is critical in detoxification by processing and eliminating xenobiotics and foreign compounds, including drugs.¹ Consequently, hepatic dysfunction manifests as diverse pathologies, including degenerative cirrhosis, acute/chronic hepatitis, and non-inflammatory steatosis.² Cholestasis often results from the inhibition of hepatobiliary transporters, crucial for bile flow and xenobiotic elimination, by drug metabolites. Hepatocellular injury involves diverse pathways, including direct toxicity and immune responses. Drug-induced liver injury can be dose-dependent (eg, acetaminophen) or idiosyncratic.³

Ethanol exposure can lead to hepatotoxicity, impairing normal liver function. This is primarily attributed to the generation of toxic metabolites during ethanol metabolism, which induces oxidative stress.^4,5 Ethanol exerts hepatotoxicity by altering redox state via NADH-mediated alcohol dehydrogenase metabolism and by disrupting mitochondrial fatty acid β-oxidation by elevating the NADH/NAD+ ratio, thereby promoting steatosis. Additionally, it enhances intestinal lipid uptake and adipose tissue mobilization, further burdening the liver.⁶ Functionally, ethanol disrupts hepatocyte membranes, elevating serum transaminases, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are indicative of cellular injury. Leakage of alkaline phosphatase (ALP) from damaged hepatocytes further confirms hepatic dysfunction. Gamma-glutamyl transferase (GGT), a marker of oxidative stress protection, also increases upon membrane damage. Alcohol depletes glutathione (GSH), a crucial antioxidant, while promoting the accumulation of oxidative stress markers glutathione reductase (GR) and malondialdehyde (MDA), perpetuating hepatocellular damage.^7–9 As a key preclinical model, ethanol-induced hepatotoxicity in rats faithfully recapitulates human alcoholic liver disease (ALD), allowing for comprehensive exploration of disease mechanisms through various induction protocols, including chronic feeding, intragastric gavage, and “second-hit” paradigms.¹⁰

Despite advancements in allopathic medicine, effective treatment for hepatotoxicity remains elusive. While traditional medicine utilizes plant-derived phytoconstituents, highlighting the potential of unexplored plant-based antioxidants for hepatoprotective effects.²

Hypophyllanthin, a naturally occurring lignan, is synthesized in plants of the genus Phyllanthus through the dimerization of cinnamic acid via the shikimic acid and phenylpropanoid pathways. Hypophyllanthin demonstrates potent immunomodulatory properties, including anti-tumor, anti-allergic, anti-inflammatory, anti-hypertensive, and phytoestrogenic activities.¹¹ However, the precise mechanistic role of hypophyllanthin in ethanol-induced hepatotoxicity, particularly its modulation of oxidative stress, inflammatory cytokines, apoptosis, and autophagy, remains insufficiently explored. This study employed a rat model of ethanol-induced hepatotoxicity, as it has established capacity to recapitulate key pathophysiological features of ALD. This established model provides a relevant in vivo system for assessing the effects of new hepatoprotective agents. Therefore, this study aimed to assess the hepatoprotective effects of hypophyllanthin in rats with ethanol-induced liver damage.

Materials and Methods

Drugs and Solutions

Hypophyllanthin (≥95.0%) Was Procured from MSW, Maharashtra, India. It was dissolved in minimum concentration of dimethyl sulfoxide (DMSO) and diluted with saline, prior to administration to rats. TNFα (TNFα, Cat. No.: MBS2507393), interleukins (IL-1β, Cat. No.: MBS2023030 and IL-6, Cat. No.: MBS355410), NF-κB (NF-κB p65, Cat. No.: MBS2505513), and caspase-3 (Casp-3, Cat. No.: MBS763727) were commercially available enzyme-linked immunosorbent assay (ELISA) kits from MyBioSource, USA.

Animals

Male Wistar rats (10–12 weeks old, 180 ± 20 g) were procured for the investigation. All animals were housed in polycarbonate cages within a controlled environment. Standardized housing conditions included a 12-hour light-dark cycle, a constant temperature of 23 ± 2°C, and a relative humidity of 55 ± 5%. Following seven days of acclimatization to the laboratory environment, the rats, which were provided with standard rodent chow and water ad libitum, were used in the experimental procedures. All experiments were conducted in accordance with the CCSEA (Committee for the Control and Supervision of Experiments on Animals), Government of India, guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Ethics Committee, Transgenica, M.S., India (IAEC/TRS/PT/024/024). The study was also performed and reported in accordance with the ARRIVE guidelines.

Acute Toxicological Study

An acute oral toxicity study was conducted in accordance with OECD Guideline 423. Rodent subjects were monitored for 14 days post-administration to evaluate potential toxicological effects. Hypophyllanthin was administered orally at 5 and 10 mg/kg, consistent with previously reported.¹¹ Observations included the assessment of clinical signs, specifically behavioral modifications, ophthalmological abnormalities, alterations in body weight, and changes in dermal and fur observations.

Prediction of ADMET by Computational Analysis

The absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile of Hypophyllanthin was computationally predicted using the web-based tool pkCSM (https://biosig.lab.uq.edu.au/pkcsm/). The pkCSM platform was used to estimate key pharmacokinetic parameters and potential toxicity endpoints, providing a rapid in silico evaluation of hypophyllanthin’s ADMET properties.

Experimental Design

This study aimed to assess the hepatoprotective efficacy of hypophyllanthin against ethanol-induced liver damage in a rodent model. Thirty male Wistar rats were randomly taken and allocated to four groups of six animals each to receive treatments (Figure 1).

Figure 1.

Experimental design.

Group I: Normal control (received saline) for 28 days.

Group II: Ethanol Control (ETOH-5 mL/kg/p.o) for 28 days.

Group III: Hypophyllanthin low dose (Ethanol + Hypophyllanthin 5 mg/kg) for 28 days.

Group IV: Hypophyllanthin high dose (Ethanol + Hypophyllanthin 10 mg/kg) for 28 days.

Group V: Hypophyllanthin 10mg per se group.

Hypophyllanthin 5 and 10 mg/kg were administered orally to the rats during the morning session.^7,11

Sample Collection and Preservation

Twenty-four hours after performing the last treatment, the fasted rats were anesthetized with an intraperitoneal administration of 80 mg/kg ketamine (Ciron Drugs and Pharmaceuticals Pvt Ltd., M.S., India) and 10 mg/kg xylazine (PCI, Karnataka, India). The rats were sacrificed as per the guidelines, and blood was collected from each rat via the abdominal aorta using a 3 mL syringe and transferred to sterile tubes. After 10 min at room temperature (RT) for coagulation, samples were centrifuged at 1200 g for 5 min (RT). The clots were incubated at 4°C for 1.5–2 hr to stabilize fibrin, then serum was extracted using forceps with gentle compression. Following two additional centrifugations (1200 g, 5 min, RT) to remove residual cells, the serum was incubated at 56°C for 30 min, cooled to RT, aliquoted (10 mL), and stored at –80°C until use.

After dissection, the liver of each rat was removed, weighed, and the liver tissue was quickly washed with cooled saline, then frozen at −80°C until biochemical analyses. For histological inspection, liver sections were excised and fixed in a 10% buffered formalin solution.

Body Weight and Liver Mass

All rats were weighed on a calibrated scale at two time points: before the start of intervention and after the end of the experiments. We recorded the weight changes for each rat based on these measurements. After dissection, the liver of each rat was removed and weighed.

Determination of Serum Biomarkers

Clinical biochemical tests were conducted to determine liver enzymatic activities in the normal control, ETOH-induced, and treatment groups based on levels of AST, ALT, ALP, triglycerides (TG), total bile acids (TBA), and serum total cholesterol (TC). Using commercially available kits, all serum enzymes were quantified according to the manufacturer’s instructions (Modern Lab, Wani, M.S., India).

Serum Alcohol

Serum alcohol levels were determined at 0, 1, 3, and 5 h after the oral administration of ethanol to rats. Serum alcohol concentrations were measured according to the manufacturer’s protocol using enzyme-based spectrophotometric (absorbance at 570 nm) kits.

Preparation of Tissue Homogenate

Liver tissues were homogenized in 1.15% potassium chloride buffer using a homogenizer (REMI RQ-127 A/D, Instruments, M.S., India), then centrifuged at 1000 x g for 15 min at 4°C. The obtained supernatant was used for further experimentation.

Determination of Complex II (Succinate Dehydrogenase) and Complex IV (Cytochrome c Oxidase)

Complex II (succinate dehydrogenase) and Complex IV (cytochrome c oxidase) activity were measured spectrophotometrically based on the previously reported method.¹² SDH activity was expressed as the amount of enzyme required to degrade 1 nmol of succinic acid/min/mg of protein.

Determination of Antioxidant Defenses

Glutathione (GSH) Activity

The concentration of GSH was quantified using the Ellman method.¹³ This method involves the addition of Ellman reagent and 10% trichloroacetic acid (TCA) to the homogenate in a 1:1 ratio. The Ellman reagent reacts with the sulfhydryl group of glutathione (GSH), producing a colored product measurable by its absorbance at 412 nm. The rate of this reaction, reflected by the absorbance change, is directly proportional to GSH activity in the homogenate, which is measured in mmol/mg.

Superoxide Dismutase (SOD) Activity

SOD was quantified according to the Misra and Fridovich method.¹⁴ In this assay, tissue homogenate supernatant was incubated with glycine buffer (50 mM, pH 10.4) and 0.02 mL of epinephrine. The change in optical density of the reaction mixture at 480 nm, monitored over a five-minute interval, reflects the rate of epinephrine autoxidation mediated by superoxide radicals. SOD activity is quantified by its ability to inhibit this reaction, normalized to a blank control lacking enzyme. The final result is expressed in units per milliliter (U/mg).

Catalase (CAT) Activity

CAT activity was quantified using the hydrogen peroxide (H₂O₂) decomposition assay. A solution containing 0.1 mL of tissue homogenate and 1.9 mL of 50 mM phosphate buffer (pH 7.0) was prepared. The reaction was initiated by adding 1.0 mL of a freshly prepared 30 mM H₂O₂ solution, and the mixture was incubated at 25°C. The reduction in absorbance at 240 nm, indicative of H₂O₂ consumption, was monitored every 10 seconds for 1 minute.¹⁵

Lipid Peroxidation (MDA Activity)

The analysis of lipid peroxidation employed the Wills technique.¹⁶ This method leverages the reactivity of Sigma-Aldrich thiobarbituric acid with thiobarbituric acid-reactive substances. The formation of the thiobarbituric acid-MDA metabolite was measured spectrophotometrically (Shimadzu Analytical Pvt. Ltd., M.S., India) by monitoring the peak absorbance at 532 nm.

Determination of Pro-Inflammatory Cytokines

The levels of IL-1β, IL-6, and TNF-α in liver tissue homogenates were determined utilizing an ELISA kit. The estimation method followed the manufacturer’s instructions. Standard curves were used to assess marker concentrations, which were reported as picograms per gram (pg/g) of tissue.

Determination of NF-κB and COX-2

NF-κB and COX-2 (MSW-COX-2, MSW Pharma, M.S., India), key regulators of inflammation, were quantified using an ELISA according to the manufacturer’s protocol. NF-κB levels were quantified in pg/mL, while COX-2 concentrations were determined in ng/g tissue.

Determination of Inducible Nitric Oxide Synthase (iNOS)

iNOS is a pivotal enzyme that generates nitric oxide (NO). A commercially available ELISA kit (MSW Pharma, M.S., India) was used to measure iNOS protein concentration in liver tissue homogenate samples. The procedure adhered strictly to the manufacturer’s protocol.

Determination of Apoptosis and Autophagy

Apoptotic markers were assessed by quantifying caspase-3, a key regulator of apoptosis. Autophagic markers were evaluated by measuring Beclin-1 (MSW-Beclin-1) and light chain 3B (LC3B) levels (MSW-LC3B, MSW Pharma, M.S., India). Both sets of markers were determined by ELISA, following the manufacturer’s protocol. The concentration of these markers was measured and expressed in nanograms per gram of tissue.

Histopathology

The rats were euthanized at the end of the experiment. Liver tissues were promptly immersed in a 10% phosphate-buffered formalin solution for fixation. Subsequently, the tissues were meticulously embedded in paraffin wax using metallic molds. Molten paraffin was cast onto the embedded tissues and allowed to solidify under refrigerated plates. The fixed tissues were sectioned at a 3–5 µm thickness using a microtome, generating cross-sections. To evaluate liver histology, tissue sections were stained with hematoxylin and eosin (H&E) and subsequently examined under a light microscope (Magnus 030357, Olympus Opto Systems India Pvt. Ltd., Noida, India). All slides were assessed by a pathologist who was unaware of the experimental groups.

Statistical Analysis

An analysis of variance (ANOVA) was utilized to assess inter-group variations. Statistical analyses were performed using GraphPad Prism^® version 8.0.2 (GraphPad Software, San Diego, CA, USA). The Shapiro–Wilk test was used to assess the normality of the data. One-way ANOVA followed by Tukey’s multiple comparisons test was used for the remaining data analysis, with a significance level of p < 0.05.

Results

The present study provides novel insights by systematically investigating the hepatoprotective effects of hypophyllanthin in a well-established rat model of ethanol-induced liver injury. Importantly, this work extends beyond previous studies by comprehensively evaluating several parameters, including oxidative stress, inflammatory cytokines, apoptosis, and autophagy. By integrating these biochemical parameters, the study elucidates the multi-targeted mechanism by which hypophyllanthin mitigates ethanol-induced liver damage. Thus, the findings contribute new mechanistic evidence supporting hypophyllanthin as a promising therapeutic source for alcohol-related liver disorders.

Acute Toxicity

Hypophyllanthin was tested for acute oral toxicity (LD₅₀) as per the OECD’s 423 criteria. Rats are typically observed for 14 days following doses of 5 and 10 mg/kg administered within 24 hours. No mortality or adverse effects were recorded (Table 1).

Table 1.

Post-Treatment Behavioral Assessment Following Acute Oral Hypophyllanthin Administration

Observations	Acute Toxicity
Skin and Fur	Normal
Eyes	Normal
Respiration	Normal
Somatomotor activity	Normal
Tremors	Normal
Convulsions	Normal
Salivation	Not seen
Diarrhea	Not seen
Lethargy	Not seen
Sleep	Normal
Coma	Not seen
Mortality	Not seen

Predicted Pharmacokinetic (PK) Properties

The predicted ADMET profile of hypophyllanthin (Table 2). The acute oral median lethal dose (LD50) of hypophyllanthin was predicted to be 2.924 mol/kg.

Table 2.

Predicted ADMET Properties of Hypophyllanthin

Property	Model Name	Predicted Value	Unit
Absorption	Water solubility	−5.676	Numeric (log mol/L)
	Caco2 permeability	1.177	Numeric (log Papp in 10−6 cm/s)
	Intestinal absorption (human)	100	Numeric (% Absorbed)
	Skin Permeability	−2.711	Numeric (log Kp)
	P-glycoprotein substrate	No	Categorical (Yes/No)
	P-glycoprotein I inhibitor	Yes	Categorical (Yes/No)
	P-glycoprotein II inhibitor	Yes	Categorical (Yes/No)
Distribution	VDss (human)	−0.079	Numeric (log L/kg)
	Fraction unbound (human)	0	Numeric (Fu)
	BBB permeability	−1.694	Numeric (log BB)
	CNS permeability	−3.144	Numeric (log PS)
Metabolism	CYP2D6 substrate	No	Categorical (Yes/No)
	CYP3A4 substrate	Yes	Categorical (Yes/No)
	CYP1A2 inhibitor	No	Categorical (Yes/No)
	CYP2C19 inhibitor	Yes	Categorical (Yes/No)
	CYP2C9 inhibitor	Yes	Categorical (Yes/No)
	CYP2D6 inhibitor	No	Categorical (Yes/No)
	CYP3A4 inhibitor	Yes	Categorical (Yes/No)
Excretion	Total Clearance	0.358	Numeric (log mL/min/kg)
	Renal OCT2 substrate	No	Categorical (Yes/No)
Toxicity	AMES toxicity	No	Categorical (Yes/No)
	Max. tolerated dose (human)	−0.069	Numeric (log mg/kg/day)
	hERG I inhibitor	No	Categorical (Yes/No)
	hERG II inhibitor	No	Categorical (Yes/No)
	Oral Rat Acute Toxicity (LD50)	2.924	Numeric (mol/kg)
	Oral Rat Chronic Toxicity (LOAEL)	1.17	Numeric (log mg/kg bw/day)

Abbreviations: ADMET, absorption, distribution, metabolism, excretion, and toxicity; BBB, blood-brain barrier; CNS, central nervous system; CYP, Cytochrome; hERG, human ether-a-go-go gene; LD50, lethal dose 50; OCT2, organic cation transporter 2; VDss, volume of distribution.

Effect of Hypophyllanthin on Body Weight and Liver Mass

Figure 2 illustrates the outcome of hypophyllanthin on body weight and liver mass in ethanol-induced liver damage in rats. Ethanol administration caused a marked reduction in body weight and an elevation of liver index compared to the normal control (p < 0.01). In contrast, ethanol-induced rats treated with both doses of hypophyllanthin showed a notable increase in body weight (p < 0.05; p<0.001] and a reduction of liver index (4.938 ± 0.1106, p < 0.01; 4.135 ± 0.1898, p<0.001). No significant changes were found in the hypophyllanthin per se group.

Figure 2.

Effect of Hypophyllanthin on (A) Body weight and (B) Liver Index. Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: # p< 0.01 vs normal control; @p < 0.05, $p < 0.01, £p <0.001 vs ETOH-induced.

Effect of Hypophyllanthin on Serum Biomarkers

AST, ALT, ALP, TBA, TG, and TC concentrations were measured in the ethanol-induced liver damage group compared to the normal control group (Figure 3). The ethanol-induced group (177.7 ± 3.149) had significantly (p < 0.01) higher AST levels than the control group (64.18 ± 4.686). However, treatment with both doses of hypophyllanthin significantly reduced elevated AST levels (119.5 ± 3.838, p < 0.05; 106.1 ± 5.483, p < 0.01). No significant changes were found in the hypophyllanthin per se group.

Figure 3.

Effect of hypophyllanthin on Serum biomarkers: (A) ALT, (B) AST, (C) ALP, (D) TBA, (E) TG, and (F) TC. Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: #p < 0.01 vs normal control; @p < 0.05, $p < 0.01, £p <0.001 vs ETOH-induced.

ALT levels substantially (p < 0.01) increased in the ethanol-induced group (57.57 ± 1.137) compared to the normal control group (22.35 ± 1.142). However, treatment with hypophyllanthin significantly reduced ALT levels (35.77 ± 1.258, p < 0.05; 30.58 ± 1.120, p < 0.01) compared to the ethanol-induced group. No significant changes were found in the hypophyllanthin per se group.

ALP levels were significantly (p < 0.01) increased in the ethanol-induced group (141.2 ± 5.674) compared to the normal control group (67.41 ± 5.147). However, treatment with hypophyllanthin significantly reduced ALP levels (115.4 ± 3.572, p < 0.01; 108.3 ± 4.637, p < 0.001) compared to the ethanol-induced group. No significant changes were found in the hypophyllanthin per se group.

Figure 3 showed that TBA (17.39 ± 2.248 vs 5.483 ± 0.8859; p < 0.01), TG (95.41±4.159 vs 44.99 ± 3.425; p < 0.01), and TC (146.9 ± 2.392 vs 77.23 ± 3.934; p < 0.01) in the ethanol-induced group were significantly increased compared with the normal control group. Treatment with both doses of hypophyllanthin showed a reduction of TBA levels (10.54 ± 1.424, p < 0.05; 8.282 ± 1.031, p < 0.01), TG levels (76.19 ± 3.236, p < 0.05; 71.43 ± 4.525, p < 0.01), and TC (123.8 ± 5.161, p < 0.01; 101.7 ± 5.295, p<0.001]. No significant changes were observed in the hypophyllanthin-per-se group. A comparison of the two hypophyllanthin-treated groups showed a dose-dependent reduction in ALT, AST, ALP, TBA, TG, and TC levels compared to the ethanol-induced group. These outcomes indicate that hypophyllanthin ameliorated serum biomarker levels and had hepatoprotective effects.

Effect of Hypophyllanthin on Serum Alcohol

Figure 4 illustrates the effect of hypophyllanthin on serum alcohol levels in ethanol-induced liver damage in rats. Ethanol administration caused a marked increase in serum alcohol levels compared to the normal control (92.02 ± 2.676 vs 2.517 ± 0.2845; p < 0.01). In contrast, ethanol-treated rats receiving both doses of hypophyllanthin showed a significant reduction in serum alcohol levels (79.05 ± 3.899, p < 0.01; 70.47 ± 4.563, p<0.001) compared with the ethanol group, indicating a dose-dependent effect. No significant changes were observed in the hypophyllanthin per se group.

Figure 4.

Effect of Hypophyllanthin on serum alcohol. Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: #p < 0.01 vs normal control; $p < 0.01, £p <0.001 vs ETOH-induced.

Effect of Hypophyllanthin on Complex II and Complex IV

Figure 5 illustrates the effects of hypophyllanthin on mitochondrial enzyme activities (Complex II and Complex IV) in ethanol-induced liver damage in rats. Ethanol administration significantly reduced the activities of Complex II (succinate dehydrogenase, 9.417 ± 0.3842 vs 18.97 ± 0.3930; p < 0.01) and Complex IV (cytochrome c oxidase, 17.03 ± 0.7710 vs 33.52 ± 0.5029; p<0.01) relative to the normal control, indicating mitochondrial dysfunction.

Figure 5.

Effect of Hypophyllanthin on (A) complex II and (B) complex IV SDH. Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: #p< 0.01 vs normal control; @p < 0.05, $p < 0.01 vs ETOH-induced.

In contrast, ethanol-induced rats treated with hypophyllanthin at both doses showed a significant restoration of Complex II (12.22 ± 0.7350, p < 0.05;12.98 ± 0.8905, p<0.01) and Complex IV activities (21.83 ± 1.168, p < 0.05; 23.30 ± 1.893, p<0.01) compared to the ethanol group, demonstrating a dose-dependent improvement in mitochondrial function. No significant changes were observed in the hypophyllanthin per se group.

Effect of Hypophyllanthin on Antioxidant Markers

Figure 6 illustrates the outcome of hypophyllanthin on antioxidant markers in ethanol-induced liver damage in rats. Liver damage induced by ethanol demonstrates a marked reduction in antioxidant markers compared to normal control [SOD (118.6 ± 5.66 vs 228.3 ± 5.17; p < 0.01), CAT (26.24 ± 2.96 vs 52.46 ± 3.27; p < 0.01), and GSH (8.94 vs 0.76 vs 18.91 ± 1.82; p < 0.01)]. In contrarily, ethanol-induced rats treated with both doses of hypophyllanthin showed a notable increase in antioxidant markers, including SOD (142.4 ± 5.63, p < 0.05; 151.6 ± 1.44, p < 0.01); GSH (15.36 ± 1.90, p < 0.05; 17.04 ± 4.67, p < 0.01), and CAT (38.95 ± 2.05, p < 0.05; 42.93 ± 2.84, p < 0.01). No significant changes were found in the hypophyllanthin per se group. Hypophyllanthin at 5 and 10 mg elicited a significant, dose-dependent increase in these antioxidant markers compared with the ethanol-induced group. These results indicate that hypophyllanthin could restore antioxidant levels.

Figure 6.

Effect of hypophyllanthin on antioxidant markers: (A) SOD, (B) CAT, and (C) GSH. Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: #p < 0.01 vs normal control; @p < 0.05, $p < 0.01 vs ETOH-induced.

Effect of Hypophyllanthin on Lipid Peroxidation

Figure 7 illustrates the level of lipid peroxidation in rats with ethanol-induced liver damage following treatment with hypophyllanthin. The ethanol-induced group exhibited a marked increase in MDA content compared with the normal control group (4.730 ± 0.628 vs 1.642 ± 0.352; p < 0.01). Hypophyllanthin at doses of 5 and 10mg/kg remarkably attenuated MDA (2.740 ± 0.50, p < 0.05; 2.247 ± 0.30, p < 0.01) content in the ethanol-induced group. No significant changes were found in the hypophyllanthin per se group. These changes showed a dose-dependent effect with respect to hypophyllanthin at doses of 5 and 10mg/kg. The findings suggest that elevated MDA levels indicate ethanol-induced lipid peroxidation in the liver; however, hypophyllanthin treatment reduced oxidative stress.

Figure 7.

Effect of hypophyllanthin on lipid peroxidation, MDA. Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: #p < 0.01 vs normal control; @p < 0.05, $p < 0.01 vs ETOH-induced.

Effect of Hypophyllanthin on Pro-Inflammatory Cytokines

Figure 8 represents the pro-inflammatory cytokines substantially increased [IL-1β (151.7 ± 8.05 vs 75.86 ± 2.87; p < 0.01), IL-6 (108.0 ± 4.61 vs 61.96 ± 4.42; p < 0.01), and TNF-α (311.3 ± 8.26 vs 140.9 ± 8.08; p < 0.01)] levels in the ethanol-induced group compared with the normal control group. However, administration of hypophyllanthin (5 and 10 mg/kg) dose-dependently lowered this elevated concentration of IL-1β (119.3 ± 5.01, p < 0.05; 96.28 ± 4.33, p < 0.01), IL-6 (87.22 ± 2.99, p < 0.05; 76.90 ± 4.83, p < 0.01), and TNF-α (272.5 ± 8.52, p < 0.05; 252.0 ± 11.63, p < 0.01) compared to ethanol-induced group. No significant changes were found in the hypophyllanthin per se group. These outcomes indicate that hypophyllanthin mitigated the proinflammatory levels.

Figure 8.

Effect of hypophyllanthin on Pro-inflammatory cytokines: (A) IL-1β, (B) IL-6, and (C) TNF-α. Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: #p < 0.01 vs normal control; @p < 0.05, $p < 0.01 vs ETOH-induced.

Effect of Hypophyllanthin on NF-κB and COX-2

Figure 9 represents the outcome of hypophyllanthin on NF-κB and COX-2 in the ethanol-induced group. The disease control rats demonstrated higher levels of NF-κB (23.07 ± 2.34 vs 5.113 ± 0.72; p < 0.01) and COX-2 (29.78 ± 2.72 vs 10.77 ± 1.41; p < 0.01) than the normal control group. However, treatment with hypophyllanthin showed dose-dependent reductions in NF-κB (15.05 ± 1.83, p < 0.05; 13.19 ± 1.37, p < 0.01) and COX-2 (21.93 ± 1.21, p < 0.05; 19.12 ± 1.07, p < 0.001), compared to the ethanol-induced group. No significant changes were found in the hypophyllanthin per se group. These findings indicate its potent anti-inflammatory effect.

Figure 9.

Effect of hypophyllanthin on (A) NF-κB and (B) COX-2. Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: #p < 0.01 vs normal control; @p < 0.05, $p < 0.01, £p < 0.001 vs ETOH-induced.

Effect of Hypophyllanthin on iNOS

Figure 10 depicts the concentration of iNOS in rat liver tissue following ethanol-induced hepatic damage. The results demonstrate a marked elevation in iNOS (350.5 ± 11.22 vs 203.8 ± 8.52; p < 0.01) in the ethanol-induced group compared with the control group. However, hypophyllanthin at 5 and 10 mg/kg markedly attenuated iNOS levels (298.5 ± 12.96, p < 0.05; 279.1 ± 15.06, p < 0.01) in the ethanol-induced group. These changes showed a dose-dependent effect of hypophyllanthin treatment. No significant changes were found in the hypophyllanthin per se group. The findings suggest that ethanol is a potent inducer of iNOS in the liver, while hypophyllanthin exhibits a protective effect by suppressing iNOS levels.

Figure 10.

Determination of inducible nitric oxide synthase (iNOS). Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: #p < 0.01 vs normal control; @p < 0.05, $p < 0.01 vs ETOH-induced.

Effect of Hypophyllanthin on Apoptosis and Autophagy

Ethanol administration to rats significantly elevated the concentration of apoptotic markers, ie, caspase-3 (66.47 ± 3.461 vs 19.81 ± 2.129; p < 0.01), compared with the normal control group. However, hypophyllanthin at 5 and 10 mg/kg dose-dependently attenuated caspase-3 activity (52.84 ± 2.879, p < 0.05; 48.59 ± 2.738, p < 0.001) compared to the ethanol-induced group, suggesting its ability to prevent programmed cell death (Figure 11). The autophagic activity was also increased in the ethanol-induced group, as indicated by the high concentration of Beclin-1 (36.00 ± 3.346 vs 10.11 ± 1.470; p < 0.01) and LC3B (16.21 ± 1.547 vs 5.115 ± 0.6364; p < 0.01). These proteins are key components of the autophagy pathway, which degrades cellular components. Hypophyllanthin treatment dose-dependently suppressed the concentration of Beclin-1 (24.12 ± 2.612, p < 0.05; 20.57 ± 2.038, p < 0.001) and LC3B (10.66 ± 1.215, p < 0.05; 9.345 ± 0.9523, p < 0.01) compared to the ethanol-induced group, indicating its inhibitory effect on autophagy liver damage (Figure 11). No significant changes were found in the hypophyllanthin per se group.

Figure 11.

Determination of Apoptosis and autophagy: (A) Caspase-3, (B) Beclin-1 and (C) Light chain 3B (LC3B). Results are expressed as mean ± SEM (n = 6). A one-way ANOVA followed by Tukey’s post hoc analysis showed statistically significant findings compared to the ETOH-induced: #p < 0.01 vs normal control; @p < 0.05, $p < 0.01, £p <0.001 vs ETOH-induced.

Histopathology

Histopathological assessment (Figure 12) of the liver in the control and hypophyllanthin per se group demonstrates a normal hepatic architecture. Rats exposed to ethanol exhibited severe hepatic necrosis, steatosis, and inflammatory cell infiltration. Hypophyllanthin at 5 mg/kg resulted in moderate hepatic necrosis, degeneration, and inflammation. This pathological improvement was more pronounced than in the 10 mg/kg hypophyllanthin group, as evidenced by the reduced severity of hepatic lesions. There was a significant increase in hepatocellular necrosis in the ethanol-induced group, which was significantly (p < 0.05; p < 0.01) reduced by hypophyllanthin at both doses. Findings demonstrated a dose-dependent effect in the two treatment groups.

Figure 12.

The effect of hypophyllanthin on histological changes was evaluated across all groups. Liver sections were stained with hematoxylin and eosin (H&E, magnification X200, scale bar = 100 μm). (A) Normal control group: liver architecture remains intact. (B) ETOH control group: severe degenerative to necrotic lesions are observed (black arrow). (C) ETOH+Hypophyllanthin-5 mg/kg: Minor abnormalities are evident (yellow arrow). (D) ETOH+Hypophyllanthin-10 mg/kg: mild liver architecture (green arrow). (E) Hypophyllanthin per se group: normal hepatic architecture. (F) The percentage of necrotic areas in liver samples is quantified by using one-way ANOVA followed by Tukey’s post hoc analysis, which showed statistically significant findings compared to the ETOH-induced: #p < 0.01 vs the normal control; @p < 0.05, $p < 0.01 vs ETOH-induced.

Discussion

Excessive ethanol consumption constitutes a significant global public health concern. The liver serves as the primary site for alcohol metabolism. Alcohol consumption presents a unique epidemiological profile compared to other risk factors, as its attributable morbidity and mortality extend beyond the individual consumer, impacting non-consumers through secondary exposure mechanisms (eg, alcohol-impaired driving and fetal alcohol spectrum disorders).¹⁷ Moreover, the detrimental effects of alcohol transcend purely health-related outcomes, encompassing a broad spectrum of adverse consequences across multiple domains, including increased rates of criminal activity and diminished socioeconomic productivity.¹⁷ Alcohol consumption is associated with a spectrum of significant adverse health outcomes, encompassing hepatic pathologies such as ALD and hepatocellular carcinoma, extrahepatic malignancies, cardiovascular dysfunction, and neuropsychiatric sequelae, including depressive disorders and increased suicidality.¹⁸ Consequently, alcohol abuse triggers a spectrum of hepatic pathologies, encompassing alcoholic steatosis, alcoholic hepatitis, and alcoholic cirrhosis. This etiological factor contributes to the development of hepatocellular carcinoma.¹⁹ Steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma are well-established liver diseases caused by ethanol consumption. Hepatoprotective agents with minimal side effects are still scarce despite the availability of treatment options. Natural lignan bioactive compound hypophyllanthin has been found to have antioxidant and anti-inflammatory properties in Phyllanthus species, but its role in ethanol-induced hepatotoxicity has not been investigated in detail. This study evaluated hypophyllanthin in rats using an ethanol-induced liver injury model, focusing on liver damage markers. Liver function parameters, antioxidant enzyme activity, lipid peroxidation levels, cytokine levels, and histological evaluation were employed to assess the outcome of hypophyllanthin on the rat’s liver.

The pkCSM server was used as a reliable tool for ADMET prediction. The predicted results indicate hypophyllanthin has 100% intestinal absorbance and no hepatotoxicity.^11,20 Liver enzymes are crucial indicators of hepatocyte injury and liver damage. Among these, ALT, AST, and ALP are commonly used. Serum AST, ALT, and ALP serve as enzymatic biomarkers for assessing hepatic structural integrity and injury, thereby contributing to the clinical diagnosis of hepatotoxicity.²¹ ALT is considered the gold standard due to its sensitivity; even a small degree of hepatocyte necrosis can significantly elevate ALT levels. AST, primarily localized in hepatocyte mitochondria, is released into the bloodstream upon mitochondrial damage and cellular rupture. ALP, a marker of hepatobiliary function, is also elevated in cases of drug-induced liver injury. TBA is a unique marker reflecting liver metabolism, secretion, and cell damage. While rarely present in serum, TBA levels rise dramatically in acute hepatitis.^22,23 As a result of liver damage, hepatocytes were released into the stream, and serum biomarkers (AST, ALT, and ALP) were highly elevated in the intoxicated group. Hypophyllanthin suppressed ALT, AST, and ALP levels in a dose-dependent manner and restored them to normal ranges. Our results corroborate with previously reported studies.^21,23

A higher level of TBA, or cholemia, and TC can exacerbate alcohol-induced liver damage and steatosis (fatty liver). ALD is more common in rodents with pre-existing cholemia.²⁴ Elevated levels of TBA impaired liver function.

TGs are key mediators of hepatotoxicity, primarily due to their role in aberrant lipid metabolism, and are crucial for the pathogenesis of liver diseases. Intrahepatic triglyceride accumulation can result in conditions such as steatosis and steatohepatitis, which are often exacerbated by factors like alcohol abuse and metabolic syndrome, characterized by chronic inflammation and fibrosis.²⁵

This investigation found that ethanol-induced hepatic damage increased TBA and TG levels. Hypophyllanthin significantly attenuated this elevation in TBA, TG, and TC levels. Our results corroborate previous studies on the hepatoprotective effects of hypophyllanthin.²⁶

Consumption of alcohol stimulates the synthesis of fatty acids in the liver. Overconsumption of alcohol causes excessive fat accumulation in the liver cause steatosis. In the current study, ethanol treatment significantly increased serum alcohol levels compared to the normal control group. However, ethanol-induced rats treated with both doses of hypophyllanthin showed a marked reduction in serum alcohol level compared to ethanol-induced rats. These findings are consistent with the previous study.² Mitochondria are critical for hepatocyte survival, and impairment of the electron transport chain (ETC) is a hallmark of liver toxicity, particularly in conditions such as ethanol-induced injury. Complex II (succinate dehydrogenase) and Complex IV (cytochrome c oxidase) are crucial components of the ETC, each with distinct roles in energy production and redox regulation. Impairment of Complex II and Complex IV leads to disruption of oxidative phosphorylation, oxidative stress, and altered energy levels, which are crucial steps in the development of liver injury. In the present study, Ethanol administration significantly reduced the activities of Complex II and Complex IV relative to the normal control, indicating mitochondrial dysfunction. In contrast, ethanol-treated rats given hypophyllanthin at both doses showed a significant restoration of Complex II and Complex IV activities compared with the ethanol group, demonstrating a dose-dependent improvement in mitochondrial function. These results are in alignment with a previously reported study.¹²

Previous studies showed the role of oxidative stress in the development of alcohol-related liver disease (ARLD). This association is evidenced by elevated levels of reactive oxygen species (ROS) and a depletion of antioxidant defenses following ethanol exposure. Ethanol exposure disrupts mitochondrial function and upregulates CYP2E1 and NADPH oxidase expression, thereby enhancing ROS generation. Furthermore, research demonstrated reductions in both enzymatic antioxidants and non-enzymatic antioxidants, including SOD, CAT, and GSH.²⁷ However, administration of hypophyllanthin markedly increases the activity of SOD, CAT, and GSH in the liver, followed by a concurrent reduction in MDA levels.

Hepatic damage occurs primarily due to oxidative stress and abnormal antioxidant defense mechanisms. Several biomarkers, such as MDA, SOD, GSH, and CAT, are closely associated with the progression of oxidative stress-mediated hepatotoxicity.^28,29 MDA is an endogenous biomarker of lipid peroxidation that indicates oxidative stress-induced tissue damage. SOD is an essential antioxidant enzyme that scavenges ROS and protects against hepatic tissue injury. CAT is another key antioxidant enzyme that decomposes H₂O₂ and terminates free radical chain reactions, contributing to the body’s defense against oxidative stress. GSH is also a vital antioxidant that enhances the liver’s antioxidant defence system by neutralizing hydroxyl and superoxide radicals.^23,30

Ethanol metabolism, primarily mediated by alcohol dehydrogenase, generates ROS and depletes antioxidants, leading to the destruction of cellular components, including lipids, proteins, and DNA. Consequently, cellular death and tissue injury ensue, leading to ethanol-induced hepatotoxicity.³¹ The present study demonstrated that hypophyllanthin markedly increased the antioxidant enzymes, including SOD, GSH, and CAT, while reduced MDA levels. These findings suggest that hypophyllanthin possesses potent antioxidant properties. The elevation of SOD, GSH, and CAT indicates the ability of flavonoids to neutralize free radicals, whereas reduced MDA levels indicate their potential to suppress lipid peroxidation. These results are consistent with previous reports, including studies such as Astaxanthin attenuates 5-fluorouracil-induced liver toxicity and oxidative stress in male rats, where antioxidant compounds significantly restored enzymatic antioxidant levels and reduced lipid peroxidation under hepatotoxic conditions.³²

Hypophyllanthin is a lignan compound with the systematic chemical nomenclature (7R,8R,9S)-9-(3,4-dimethoxyphenyl)-4-methoxy-7,8-bis(methoxymethyl)-6,7,8,9 tetrahydrobenzo[g]^1,3 benzodioxole. It possesses a molecular formula of C₂₄H₃₀O₇ and a molecular weight of 430.5 g/mol. In conjunction with other prominent constituents of plant extracts, this compound, specifically geraniin, corilagin, and phyllanthin, is hypothesized to contribute significantly to the observed potent immunomodulatory effects. These compounds demonstrate the capacity to modulate innate immune responses in phagocytic cells at various stages. Collectively, these findings emphasize the promising antioxidant capabilities of hypophyllanthin.

Cytokines are small proteins that regulate various cellular activities, including cell survival, proliferation, differentiation, migration, immune cell activation, and death. These factors serve as transcription factors, modulating gene expression and stimulating cytokines such as TNF-α and IL-1β. TNF-α, a key mediator, initiates a cascade of events that leads to the upregulation of other proinflammatory cytokines, including IL-1β, which are essential for inflammatory responses. Increased levels of cytokines are often associated with high infiltration of inflammatory cells into hepatic tissues.^33,34 The NF-κB is a key regulator of various biological processes, including immune response, inflammation, and developmental pathways. Notably, NF-κB serves an essential role in upregulating death receptors like TNF-α. Upon activation, NF-κB promotes the transcription of pro-inflammatory genes, including TNF-α, IL-1β, COX-2, IL-6, and iNOS, which can contribute to hepatic tissue injury.³¹ Additionally, iNOS and COX-2 are key inflammatory mediators that catalyze the generation of NO and PGE2, respectively. The overproduction of NO can lead to oxidative stress by depleting intracellular GSH levels. Therefore, compounds suppressing iNOS and COX-2 levels could be promising candidates for managing chronic inflammation.³⁵ It is suggested that inhibition of these markers modulates ALD-associated sustained inflammatory cascades.

In the present study, ethanol-induced hepatotoxicity resulted in elevated levels of inflammatory cytokines, including IL-1β, IL-6, and TNF-α, as well as NF-κB, iNOS, and COX-2. However, the administration of hypophyllanthin restored these markers. These findings suggest a protective role for hypophyllanthin in mitigating inflammatory responses within the hepatotoxic rodent paradigm. This observation aligns with prior research.²³

Hepatocyte apoptosis is a critical, well-documented mechanism in liver toxicity. Apoptosis, a naturally occurring, tightly regulated program of cellular self-destruction, is activated by pro-apoptotic factors like caspases. In healthy hepatocytes, stress or injury triggers apoptosis. This controlled process effectively eliminates damaged cells, often caused by elevated free radical production. The mitochondrial efflux of cytochrome c initiates a cascade of events leading to apoptosis, characterized by the formation of apoptotic bodies and the sequential activation of caspase-3. Activated caspase-3 can cleave caspase-8, amplifying the pro-apoptotic mitochondrial signaling pathway. Additionally, the cleavage of downstream substrates by caspases ensures the perpetuation of the apoptotic cascade.³⁶ Autophagy, an evolutionarily conserved lysosomal degradative process, plays a crucial role in maintaining cellular integrity by eliminating compromised organelles and aggregated proteins. This degradative activity is upregulated under conditions of cellular duress, such as oxidative stress. Although critical for cellular adaptation and immunological competence, excessive or dysregulated autophagic activity may contribute to tissue pathology. Beclin 1 and LC3B levels serve as established biomarkers for monitoring autophagic activity. Ethanol-induced liver injury in rats led to upregulation of caspase-3, a marker of apoptosis. Beclin-1 and LC3B levels were elevated, suggesting activation of autophagic pathways as a cellular response to ethanol-induced stress and damage. Hypophyllanthin-5 and 10 mg/kg markedly attenuated these markers, indicating its protective effect against both apoptotic and autophagic processes in ethanol-induced liver damage. Notably, the observed decrease in LC3B and Beclin-1 does not necessarily indicate direct suppression of autophagy by hypophyllanthin; rather, it likely reflects a reduced need for autophagic activation due to alleviated cellular stress. These findings are consistent with previous research.³⁷

Histopathological analysis identified significant ethanol-induced hepatotoxicity in rats, marked by extensive tissue necrosis, severe steatosis, and pronounced inflammatory infiltrates. These structural pathologies provide compelling visual evidence of alcohol’s destructive impact on hepatic tissue, unlike transient biochemical fluctuations. Necrosis, steatosis, and inflammation collectively serve as definitive morphological markers of ethanol-induced liver damage, distinguishing actual injury from functional variations. Treatment with hypophyllanthin mitigated hepatic injury, as evidenced by decreased necrotic regions, reduced inflammatory cell infiltration, and diminished steatosis. Nevertheless, the protective effects of hypophyllanthin were not as pronounced as those observed in the control group. These findings corroborate previously reported studies and support the potential hepatoprotective properties of hypophyllanthin.^38,39

The immunosuppressive properties of an 80% ethanol extract of Phyllanthus amarus, and its constituent bioactive lignans, including hypophyllanthin, phyllanthin, and niranthin. These compounds have been shown to attenuate the release of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated U937 human macrophage cells. This attenuation is mediated through the modulation of key signaling pathways, namely NF-κB, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase/Akt (PI3K-Akt).¹¹ Hypophyllanthin’s hepatoprotective effects are consistent with similar findings using lignan-based compounds, such as silymarin, schizandrin, and arctigenin, which have shown liver protection.^40,41

Ethanol control rats showed clear signs of hepatic necrosis, steatosis, and inflammatory infiltration, hallmarks of sustained liver injury rather than transient stress. In the ethanol control group, liver enzymes and inflammatory cytokines significantly increased above normal physiological fluctuations, confirming liver injury. Based on these findings, hypophyllanthin exhibits potent hepatoprotective properties against ethanol-induced liver injury and can act by various mechanisms (Figure 13). Our study evaluated key biomarkers of liver injury and their clinical relevance. Ethanol-induced hepatic injury, or ALD, is characterized by a systemic inflammatory response, evidenced by elevated circulating cytokines (IL-1β, IL-6, TNF-α) and activation of downstream effectors (NF-κB, iNOS, COX-2). Concurrently, oxidative stress, indicated by increased lipid peroxidation (MDA) and impaired antioxidant capacity (SOD, CAT, GSH), contributes to hepatocyte damage, reflected by elevated transaminases and cholestatic markers. Dysregulated cell death mechanisms, particularly apoptosis and autophagy, further contribute to liver pathology, which is histologically defined by necrosis, steatosis, and fibrosis. Hypophyllanthin demonstrates a comprehensive hepatoprotective profile by normalizing hepatic function, restoring redox balance through antioxidant upregulation and lipid peroxidation suppression, inhibiting pro-inflammatory signaling, and modulating cell death pathways, ultimately leading to significant histopathological improvements and indicating its therapeutic relevance in ALD.

Figure 13.

Hypothetical mechanism of hypophyllanthin.

Hypophyllanthin exhibits potential hepatoprotective effects through antioxidant and immunomodulatory mechanisms, offering a natural therapeutic avenue. While it shares common hepatoprotective mechanisms with established agents such as silibinin, curcumin, and resveratrol, it provides unique advantages and certain limitations. Silibinin, derived from silymarin, is well-established for its efficacy against toxin-induced liver damage and boasts a robust safety profile, though its bioavailability is a concern.

Curcumin, known for its potent antioxidant and anti-inflammatory properties, shows promise for mitigating hepatic stress but also suffers from poor absorption. With its ability to improve metabolic markers and reduce oxidative stress, resveratrol presents another potential therapeutic option, albeit with ongoing investigations into long-term safety and drug interactions.⁴² Each compound presents unique advantages and limitations. Hypophyllanthin requires further research to validate its efficacy and safety. Extensive data support Silibinin’s clinical use, but formulation improvements are needed to enhance absorption. Curcumin and resveratrol, while promising, require strategies to address bioavailability issues and clarify their long-term effects. Consequently, continued research is crucial to optimize the therapeutic application of these compounds in liver health management.^42,43 The hepatoprotective effects of hypophyllanthin are comparable to those of silibinin, curcumin, and resveratrol, especially in the reduction of oxidative stress, modulation of cytokines, and adjustment of apoptosis/autophagy.^44,45

Limitations and Future Scope

The present study evaluated the potential hepatoprotective effect of hypophyllanthin in ethanol-induced liver injury in rats. While these findings are promising, they also have limitations due to interspecies variation, the complexity of human hepatotoxicity, the short duration of the study, and the use of a single hepatotoxicity model. Future research should employ additional hepatotoxicity models to enhance the robustness and clinical relevance of these findings. The primary objective of this pilot study was to generate preliminary data and initial insights to inform and support the design of future, larger-scale studies. This research provides evidence for the hepatoprotective potential of hypophyllanthin; however, it presents several methodological limitations. Specifically, the study did not elucidate the precise molecular mechanisms underlying this effect, including key cellular signaling cascades such as NF-κB, Nrf2, AMPK, and SIRT1. While ELISA assays provided valuable insights into the modulation of NF-κB and Nrf2 signaling pathways, these methods alone cannot conclusively demonstrate pathway activation. Specifically, for NF-κB, the assessment of the p-NF-κB/NF-κB ratio and for Nrf2, the demonstration of nuclear translocation via techniques such as Western blotting of nuclear extracts or immunofluorescence would be required for definitive confirmation, which was not performed due to resource constraints. The study did not evaluate lipogenic and bile acid synthesis pathways, restricting comprehensive mechanistic insight into hypophyllanthin’s hepatoprotective effects. Further investigations are required to characterize these pathways. The impact of oxidative stress on mitochondrial bioenergetics was not quantified. Future studies should evaluate mitochondrial membrane potential, ATP synthesis, and ROS production to assess mitochondrial function comprehensively. The long-term effects of hypophyllanthin, including the modulation of hepatic fibrosis and steatosis, were not examined. To address these aspects, subsequent studies should employ histological techniques, such as Masson’s trichrome, Sirius Red, and Oil Red O staining. Finally, a comparative efficacy analysis against established hepatoprotective agents, such as silymarin or N-acetylcysteine (NAC), is necessary to determine the relative potency of hypophyllanthin. Although ADMET predictions were conducted, experimental validation through animal studies is needed to confirm its systemic exposure, distribution, and therapeutic potential, which will be addressed in future investigations. The future should focus on direct comparisons and on the clinical application of these hepatoprotective agents for chronic liver disease, fibrosis, and metabolic dysfunction.

Conclusion

These findings demonstrate that hypophyllanthin exerts significant hepatoprotective effects against ethanol-induced liver injury in rats. Hypophyllanthin administration significantly attenuated oxidative stress, as evidenced by increased antioxidant enzyme activities and reduced lipid peroxidation. It also reduced pro-inflammatory cytokines and normalized liver function markers (ALT, AST, ALP, TBA, and TC) and serum alcohol levels, as supported by histopathological evidence of improved hepatic architecture. Histopathological study further corroborated the protective effects of hypophyllanthin on liver tissue. Overall, these findings highlight the antioxidant and anti-inflammatory potential of hypophyllanthin in mitigating ethanol-induced hepatotoxicity. Further studies are required to elucidate the underlying molecular mechanisms and to validate these findings in clinical settings.

Funding Statement

The researchers would like to thank the Deanship of Graduate Studies and Scientific Research at Qassim University for financial support (QU-APC-2026).

Abbreviations

ADMET, Absorption, Distribution, Metabolism, Excretion, and Toxicity; ALD, Alcoholic Liver Disease; ALP, Alkaline Phosphatase; ALT, Alanine Aminotransferase; ANOVA, Analysis of Variance; ARLD, Alcohol-Related Liver Disease; ARRIVE, Animal Research: Reporting of In Vivo Experiments; AST, Aspartate Aminotransferase; CAT, Catalase; COX-2, Cyclooxygenase-2; DMSO, Dimethyl Sulfoxide; DNA, Deoxyribonucleic Acid; ELISA, Enzyme-Linked Immunosorbent Assay; ETOH, Ethanol; GGT, Gamma-Glutamyl Transferase; GR, Glutathione Reductase; GSH, Glutathione; IL-1β, Interleukin-1 Beta; IL-6, Interleukin-6; iNOS, Inducible Nitric Oxide Synthase; LC3B, Microtubule-Associated Protein 1A/1B-Light Chain 3 Beta; LD50, Lethal Dose 50%; LPS, Lipopolysaccharide; MAPK, Mitogen-Activated Protein Kinase; MDA, Malondialdehyde; NAC, N-Acetylcysteine; NF-κB, Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells; OECD, Organisation for Economic Co-operation and Development; PI3K-Akt, Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway; PK, Pharmacokinetics; ROS, Reactive Oxygen Species; RT, Room Temperature; SOD, Superoxide Dismutase; TBA, Total Bile Acid; TCA, Trichloroacetic Acid; TG, Triglycerides; TNF-α, Tumor Necrosis Factor-Alpha.

Data Sharing Statement

All data generated or analyzed during this study are included in this published article. Additional information and supporting datasets related to the current study are available from the corresponding author upon reasonable request.

Ethical Approval

The study was approved by the Institutional Animal Ethics Committee, Transgenica, M.S., India (IAEC/TRS/PT/024/024).

Author Contributions

Muhammad Afzal: Conceptualization, Methodology, Investigation, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing. Sattam Khulaif Alenezi: Data curation, Formal analysis, Methodology, Validation, Writing – review & editing. Tariq G. Alsahli: Investigation, Project administration, Supervision, Validation, Writing – review & editing. Imran Kazmi: Formal analysis, Methodology, Supervision, Validation, Visualization, Writing – review & editing. Nadeem Sayyed: Methodology, Resources, Software, Writing – original draft, Writing – review & editing. Khalid Saad Alharbi: Conceptualization, Funding acquisition, Resources, Writing – review & editing. Reem Alqahtani: Validation, Visualization, Supervision, Writing – review & editing. All authors made a significant contribution to the work reported, whether in the conception, study design, execution, acquisition of data, analysis, and interpretation, or in all these areas; took part in drafting, revising, or critically reviewing the article; gave final approval of the version to be published; agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors report no conflicts of interest in this work.