Validation, quantification, and molecular docking of isolated eupalitin 3-O-β-D-galactopyranoside in Boerhavia diffusa Linn for hepatoprotective and immunomodulatory activity

Abstract

Boerhavia diffusa is traditionally used for liver disorders and immunomodulation, but the mechanisms of its active flavonoid glycoside, eupalitin-3-O-β-D-galactopyranoside (EGP), remain incompletely defined. EGP was isolated by bioactivity-guided fractionation, and an ICH-aligned HPLC (High-performance liquid chromatography) method was developed and validated for its quantification. Mechanistic plausibility was probed by docking EGP to KEAP1 (NRF2 pathway; PDB: 6QMK) and the NF-κB p52–DNA complex (PDB: 1A3Q), benchmarking against silymarin and levamisole. Hepatoprotection was assessed in rats with D-galactosamine (GalN, 400 mg/kg, i.p.)–induced injury following prophylactic EGP (100 mg/kg, p.o.) via serum transaminases (ALT, AST), ALP, bilirubin, hepatic antioxidants (SOD, catalase, GSH), and histology. In vitro cytoprotection was evaluated in hepatocytes challenged with CCl4 (MTT assay), and immunostimulation was screened by LPS-induced NO release in RAW 264.7 macrophages. HPLC resolved a single EGP peak (Rt 2.79 min) with excellent linearity (R2 = 0.999), precision (RSD < 2%), and sensitivity (LOD 3 ng; LOQ 5 ng). Docking supported target engagement: for KEAP1 (6QMK), EGP achieved Glide scores of −7.29/−7.00 kcal·mol⁻1 versus silymarin −6.31/−6.16; for NF-κB p52–DNA (1A3Q), EGP scored −5.20/−4.53 versus levamisole −0.11. In vivo, EGP markedly ameliorated GalN hepatotoxicity, reducing ALT by 74%, AST by 63%, ALP by 38%, and bilirubin by 68%, while restoring antioxidant defenses (SOD +422%, catalase +190%, GSH +255%); histology corroborated near-normal lobular architecture with minimal periportal inflammation. In vitro, EGP improved hepatocyte viability in a dose-dependent manner (58% at 100 μg/mL; 67% at 200 μg/mL), comparable to silymarin (100 μg/mL). EGP also increased NO output in LPS-stimulated RAW 264.7 cells, consistent with immunostimulatory activity. EGP is a quantifiable B. diffusa constituent that exhibits convergent hepatoprotective and immunomodulatory effects across in silico, in vivo, and in vitro assays. These findings motivate pharmacokinetic studies and pathway-level validation (NRF2/ARE, NF-κB, MAPKs, iNOS/COX-2) to enable translation.

Introduction

Natural products have emerged as an indispensably significant part of traditional medicines that is believed to be a store of bio-active molecules having the most diverse biological activities. Many a times, a lot of traditional plants have found its place in not only folklore medicine, but also more recently developed pharmaceutical crucial for many essential drugs. Boerhavia diffusa Linn., commonly referred to as Punarnava, is one of the several medicinal plants that may be mentioned as having a wide range of medicinal applications. Traditionally, this plant finds counteractive use for different diseases like liver disorders, inflammatory conditions, and diseases related to immunity. It is considered a rich chemical plant because of several wide ranges of therapeutic applications. Bioactive heterogeneity is presented within root and leaf extracts of Boerhavia diffusa, containing many diverse flavonoids including EGP, Eupalitin, trans-caftaric acid, and Punarnavoside, phenolic compounds such as 3,4-dihydroxy-5-methoxycinnamoyl rhamnoside, lignans like Liriodendrin and Syringaresinol mono β-D-glucoside, and a variety of glycosides¹. The main C-methyl flavones include those of Borhavone, Boeravinones (A-H),they also contain some rotenoids such as 9-O-methyl-10-hydroxy Coccineone B and Diffusarotenoid². In addition, leaves contain isoflavones and numerous significant flavonoid glycosides, such as derivatives of quercetin and 2’-O-methyl abronisoflavone, which indicate that they have innate hepatoprotective, anti-inflammatory, and antioxidant qualities³. It is diuretic, anti-inflammatory, and hepatoprotective according to traditional knowledge, as well as often highly regarded in indigenous medicine for treating various gastrointestinal and liver ailments⁴. Currently, science has substantiated such claims, showing an extensive scope of efficacies, such as neuroendocrine, anti-inflammatory, anticonvulsant, anti-angiogenic, and anticancer actions and applications⁵. Several kinds of plant extracts were used and have found application against many conditions such as prostatic hyperplasia, oxidative stress, hyperglycemia, and liver toxicity in both animal and cell-line studies⁶. Traditionally, it has been administered as a diuretic, hepatoprotective, anti-inflammatory, anti-oxidative, and immunomodulatory activity⁷. Its hepatoprotective power acts as a boon to liver disorders such as jaundice, hepatitis, and liver fibrosis⁸. Due to diverse phytochemical constituents i.e. alkaloids, flavonoids, phenolics, lignans, and glycosides, Boerhavia diffusa exhibits a variety of pharmacological activities⁹. Of these, EGP—is a glycosylated flavonoid, is emerging as one of the active principal compounds with significant potential¹⁰. By fighting oxidative stress and managing some inflammatory responses, this compound is valuable for hepatoprotective activity. Still, systematic studies on isolation, characterization, and evaluation of hepatoprotective and immunomodulatory activities are few. Drug discovery has been revolutionized by molecular docking, and it has also opened porch doors to the arena of understanding molecular interactions between bioactive compounds and biological targets. With molecular docking, it chooses the prediction of binding affinity and potential mechanisms of actions against some proteins of interest in liver injury and immune regulations. To determine immunomodulatory activity, docking studies using the following enzymes should be commenced: cytochrome P450, NF-κB (nuclear factor kappa B), and TNF-α (tumor necrosis factor-alpha) pathways¹¹. Bio-guided fractionation is an approach which one must employ in isolating and purifying bioactive constituents from complex plant extracts. This is the technique which integrates biological assays along with systematic fractionation strategies to allow the identification of the most active phytochemical¹². The integration of bio-guided fractionation and analytical techniques (HPLC) ensures accurate identification of flavonoid compounds like EGP and their quantification. Hepatic ailments are some of the major global illnesses today,they are caused mainly by toxins, drugs, alcohol, and viruses¹³. All of these injure the liver by inducing oxidative stress and inflammatory responses, damaging hepatocytes¹⁴. The mechanisms by which hepatoprotectives function are through reducing oxidative stress, inflammation, and preventing hepatocellular damage to preserve liver function and promote regeneration¹⁵. The study endeavors to determine the hepatoprotective activity of EGP against in vitro models like HepG2 liver cell lines and in vivo animal models for liver injury induced through galactosamine. Such studies should provide insight into the mechanism of action as well as efficacy of the compound. EGP is flavonoid glycoside. The previous report of the flavonoid glycoside-mediated generation of oxidative stress and inflammation, thus causing hepatocyte injury, developed adverse consequences on liver function¹⁶. Hepatoprotective agents protect the liver by defending it from oxidative stress and inflammatory reactions. Thus in-vitro and iv-vivo experimental models would be used to study the hepatoprotective effectiveness of EGP. HepG2 cell lines would be utilized for the in vitro study to evaluate cytotoxicity. Several in vivo animal models are routinely used in research on liver injury caused by hepatotoxic drugs, such as paracetamol and carbon tetrachloride (CCl₄)^17,18. Currently, the experimental study assesses the hepatoprotective effect of EGP on galactosamine-induced hepatotoxicity considering its effect on oxidative stress markers, histopathological alterations, and some liver function indices. The immune system’s primary role is to defend against infections, yet it also contributes to the maintenance of liver injury and the orchestration of recovery. Modulating agents can enhance or depress immune responses that make them potential candidates for therapy. Flavonoid glycoside was hypothesized to modulate immune responses by regulating important immunological mediators, such as cytokines, macrophages, and lymphocytes¹⁹. This study’s objectives are to carry out validation and quantification of EGP from Boerhavia diffusa using HPLC. Additionally, using molecular docking studies and in-vitro and in-vivo models, the study aims to assess the isolated compound’s hepatoprotective and immunomodulatory capabilities.

Materials and methods

Reagents and chemicals

We procured the reference standard EGP from Hamdard laboratory Pvt. Ltd. New Delhi, India. ALT, AST, and ALP kits were obtained from Span Diagnostics Ltd. in Surat, India. Sample of Galactosamine was supplied by SRL in Mumbai, India. For the HPLC analysis, mobile phases comprised formic acid, acetonitrile, ethyl acetate toluene and methanol from CDH Labs, Pune, India.

Plant material authentication and extraction

In April 2024, we got fresh plant material of leaves from the Angadipuram area of Malappuram district in Kerala, India, for study. Dr. V. S. Hareesh, Research Officer at the Malabar Botanical Garden and Institute for Plant Sciences in Kozhikode, Kerala, India, identified and verified this collection. The specimen of the leaves was matched to reference flora number (MBGIPS/09/2019A1). The specimen that was found was then put in the herbarium of the Malabar Botanical Garden and Institute for Plant Sciences (MBGIPS/09/2019A1/2022) with a reference number of 7636. We took the dried powdered plant sample three times with 80% methanol. Later, the filtered solutions of mixed hydroalcoholic extracts were put in a rotary evaporator and heated under low pressure to make residues.

Isolation and characterization of EGP.

The hydroalcoholic extract of B. diffusa was partitioned into hexane, chloroform, ethyl acetate, methanol, and aqueous fractions. Each fraction was screened in vitro hepatoprotective and immunomodulatory activity; the ethyl acetate fraction showed the highest activity and was confirmed to contain EGP by HPTLC. Therefore, the ethyl acetate fraction was selected for isolation. The dried ethyl acetate residue was dissolved in hot methanol and refrigerated overnight. The sedimented solid was collected and recrystallized (hot methanol) to yield EGP²⁰. The ethyl acetate residue of Boerhavia diffusa was subjected to different spectroscopic techniques that were used to characterize EGP. Its conjugated flavonoid structure is supported by UV-Vis spectroscopy through an absorption maximum range at around 250–380 nm. FT-IR bands confirm the various functional groups, and the most significant are those bands for hydroxyl (-OH), carbonyl (C=O), and glycosidic bonds. Adding valuable information on proton and carbon environments, the H¹-NMR and 13C NMR can help confirm proton-proton correlations and long-range carbon-proton bonds, hence helping in identifying the glycosidic bond and the type of substitutions²¹. Mass spectrometry may confirm the molecular weight, fragmentation, and validation of the presence of the sugar-unit/flavonoid core,in all, these techniques work together for comprehensive structural elucidation.

Method of validation and quantification of EGP by HPLC

The compound was re-dissolved in a small volume of HPLC-grade methanol. Chromatographic experiments were carried out on an HPLC system of Shimadzu with quaternary LC-10A VP pumps, variable wavelength UV-Visible detector (SPD-10A VP), column oven, and SCL-10A VP system controller. The Class VP 5.032 software was used to operate this system. Sample introduction was via a rheodyne injector with a 20-μL fixed loop. Separation was performed on a Luna® C18 column (25 × 4.6 mm, 5 µm, 100 Å; Phenomenex, Torrance, CA, USA) using isocratic elution. The mobile phase considered water: acetonitrile: acetic acid: 90:10:0.2, which got prepared by mixing alone, then by sonicating and degassing for 15 min, followed by vacuum filtration through a 0.45 μm filter. The analysis was carried out at room temperature with a flow rate of 1 mL/min. Detection was carried out using a UV-visible absorbance detector at 273 nm. For preparation of standard solutions of EGP, 25 mg of the compound was dissolved in 25 mL of methanol and then further diluted with mobile phase. Twenty microliters of the prepared standard solution were injected, and the chromatogram was recorded. This process was repeated thrice to test reproducibility with relative standard deviation of less than 2% for the areas. The concentration estimation and compound identity analysis were done through recording and analyzing chromatograms from sample solution injections carried out under the same conditions. According to ICH, standard Q2 (R1)²². The validation of the calibration curves, regression equations, and LOQ and LOD for each compound was performed by HPLC. Thus, various parameters such as limit of quantification, limit of detection, precision, and linearity were considered.

Molecular docking of EGP

The molecular docking study was conducted to evaluate the binding interactions of EGP and the standard compounds, silymarin (for hepatoprotective activity) and levamisole (for immunomodulatory activity), against the target proteins 6QMK implicated in hepatoprotection through NRF2 pathway control, and NF-κB p52–DNA (1A3Q), a key node in immune signaling and modulation, respectively. 2D ligand structures from PubChem were converted to 3D with LigPrep (Maestro, Schrödinger) and minimized using the OPLS3e force field. Protein 3D structures from the PDB were prepared with the Schrödinger Protein Preparation Workflow by removing waters beyond 5 Å from the binding site, assigning bond orders, and adding hydrogens. Glide receptor grids were centered to encompass active-site residues. Docking was performed with Glide: SP mode generated 20 poses per ligand, and the top 10 SP poses were redocked with XP. Three independent runs (different random seeds) assessed robustness. A consensus pose was defined as the same binding mode (heavy-atom RMSD ≤ 2.0 Å) observed in at least two runs and ranked within 1.0 kcal·mol⁻¹ of the best XP Glide Score; when multiple poses qualified, the final pose was selected by the lowest Prime MM-GBSA ΔG_bind, minimal ligand strain, and absence of steric clashes. Ligands were ranked by Glide Score (more negative = better), and interactions (hydrophobic contacts, H-bonds, π–π stacking) were visualized in Maestro v14. Results were benchmarked against standard compounds to evaluate the relative binding of EGP to the target proteins.

In-vivo hepatoprotective activity of EGP

Animals

All experimental procedures were duly reviewed and approved by the Institutional Animal Ethics Committee (IAEC) of Jamia Hamdard, New Delhi, India (Approval Code: 837), and conducted strictly following the regulations laid down by the Committee for the Purpose of Control and Supervision of Experiment on Animals-CPCSEA, Government of India (Registration No.: 173/CPCSEA). In addition, all methods and procedures involving animal subjects were reported compliant with the ARRIVE guidelines (https://arriveguidelines.org), promoting visibility, reproducibility, and ethical rigor in animal research. Adult Wistar rats weighing 150-200g were supplied by Central Animal House. The animals were fed with the normal rodent pellet diet and offered drinking water ad libitum while kept in standard laboratory environmental conditions (12 hours of light/darkness at temperature of 25 °C and a humidity range of 45%-65%).

Experimental design

A total of 30 adult rats were randomized into five groups (n = 6). Group I (vehicle control) received normal saline (p.o.) once daily for 7 days; Group II (hepatotoxic control) likewise received normal saline (1 mL·kg⁻¹, p.o.) for 7 days. Groups III and IV received prophylactic EGP at 50 or 100 mg·kg⁻¹ (p.o.) once daily for 7 days, with doses selected from commonly effective ranges for flavonoid glycosides in rodent hepatoprotection^23,24. Group V received silymarin (25 mg·kg⁻¹, p.o.) once daily for 7 days. On Day 8, D-galactosamine (400 mg·kg⁻¹, i.p.) was administered to Groups II–V to induce liver injury²⁵, Group I did not receive the hepatotoxic challenge.

Liver function evaluation

Blood was collected from the retroorbital plexus 24 h postinjection of galactosamine. All animals were sacrificed immediately after the blood collection. Liver specimens were separated fo r histopathological examinations. Serum was separated by centrifugation at 37°C and used for estimating different biochemical parameters. Liver samples were homogenized, in ice-cold 0.15 M KCl by motor-driven Teflon pestle, after being rinsed with chilled normal saline and weighed²⁶. Biochemical markers ranging from aspartate aminotransferase (AST) to alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were determined in serum²⁷. The supernatant of the liver homogenate was used for assessing antioxidant enzymes activity such as SOD and CAT by a colorimetric method²⁸. GSH-level measurement was performed via the DTNB-method, whereas lipid peroxidation was estimated on a TBARS basis (Thio barbituric acid-reactive substances)²⁹. Liver tissues were quickly exercised and conserved in neutral buffered formalin. Liver sections were prepared for histological study via the method elaborated by Badawi²⁰.

Hepatoprotective assay of EGP

After seeding the cells in 96-well plates at 1×10⁶ cells/well density, incubation was performed overnight. Twenty-four hours later, the media was removed, and cells were treated with various concentrations of the sample (100, 200 µg/ml) for 2 hours. Silymarin (250, 500 µg/ml) served as a reference standard. Carbon tetrachloride followed being added to the wells, again incubated for another 2 hours. The cells were then washed, and 20 μL of MTT reagent at a concentration of 5 mg/mL in PBS were added to each well and left for 1 hour. If the formazan crystals appeared incompletely formed, an extra 1-hour incubation was carried out. The medium was discarded, and 200 μL of DMSO were added to dissolve the formazan crystals. The optical density was then measured at 540 nm using an ELISA reader³⁰. The percentage of hepatoprotection was calculated by the following formula:

1

Nitric oxide estimation of EGP

Nitric oxide production was quantified indirectly as nitrite in LPS-stimulated RAW 264.7 macrophages using the Griess reaction³¹ (Aldridge et al 2008). With a 96-well culture plate, RAW 264.7 mouse macrophage cells were seeded in a density of 1×10⁶ cells/well for 48 hours at 37°C with 5% CO₂ and 95% humidity. After 48 h, 100 μL of media was removed from the wells, replenished with 100 μL fresh medium containing 50 μg/mL of EGP, levamisole standard, and LPS, and kept incubated for another 24 h with or without LPS (10 μg/mL). Nitrite concentration was determined to measure nitric oxide production using Griess reagent after 10 min of incubation at room temperature. The absorbance was recorded at 540 nm on an ELISA reader. A 96-well plate reader was employed to measure absorbance. The nitrite concentration was then estimated from the sodium nitrite standard curve³². The percentage of NO inhibition or stimulation was then obtained from the following equation:

2

Statistical analysis

Results were expressed as the mean value ± S.E.M. One-way analysis of variance (ANOVA) was undertaken, with Dunnett’s post hoc tests being used to identify differences between all groups. P<0.01 was deemed to be statistically significant.

Results

Structure elucidation of EGP

The UV spectrum had bands belonging to the flavonoid moiety at 340 nm for EGP. This band exhibits a bathochromic shift variation of +45 nm on the addition of sodium methoxide due to the exposure of the OH group at 3^rd position. An FT-IR spectrograph reports the presence of various functional groups such as C=O stretching (1650 cm^-1), aromatic ring stretching vibration (1500–1600 cm⁻¹), and C-O-C glycosidic vibration band (1000–1200 cm^-1), in addition to broad O-H stretching (3400–3500 cm^-1). Mass spectrometry of EGP showed [M+H]⁺ m/z 493 (ESI⁺) and [M–H]⁻ m/z 491 (ESI⁻), with a diagnostic −162 Da neutral loss (β-D-galactopyranose) to the aglycone at m/z 331 (ESI⁺) / m/z 329 (ESI⁻)³³. Secondary fragments (e.g., m/z 316, 303/301, 179, 151) match a dimethoxy-flavonol core. The structure of EGP was drawn with conventional flavonol numbering: ring A (C-5, C-7), ring B (C-2′–C-6′), ring C (C-2–C-4), and a β-D-galactopyranose linked at C-3 (sugar C-1″–C-6″). The ¹H NMR data (DMSO-d₆) agree with this scheme: δ 12.56 (1H, s, 5-OH), 10.19 (1H, s, 4′-OH), 8.11 (2H, d, J=8.2 Hz, H-2'/H-6′), 6.88 (2H, d, J=8.2 Hz, H-3′/H-5′), 6.60 (1H, s, H-8), 5.41 (1H, d, J=7.5–7.6 Hz, H-1″, anomeric, β), 4.14–4.82 (m, 6H, H-2″–H-6″), and 4.75/4.39 (each 3H, s, OCH₃-6/OCH₃-7). The para-disubstituted B-ring pattern (8.11/6.88 d,d), the chelated phenolic OH at 12.56, and the anomeric doublet with J=7.5–7.6 Hz collectively support a β-D-galactopyranoside at C-3. The previously reported chemical EGP was a perfect match for ¹H NMR data³⁰. 13C-NMR (60 MHZ, Pyridine-D5)δ: 57.3 (7-methoxy), 60.9 (6-methoxy), 92 (Carbon-8), 159.3 (Carbon-7), 132.7 (Carbon-6), 153.0 (Carbon-5), 179.0 (Carbon-4), 135 (Carbon -3), 157.7 (Carbon -2), 104.2 (Carbon-1), 73.7 (Carbon-2), 75.2 (Carbon-3), 69.7 (Carbon-4), 77.5 (Carbon-5), 61.8 (Carbon-6), 121.9 (Carbon-1′), 131.9 (Carbon-2′), 116.1 (Carbon-3′), 161.7 (Carbon-4′), 116.1 (Carbon-5′), and 131.9 (Carbon-6′). According to all that information and available literature data, the isolated compound is EGP¹⁰.

Molecular docking of EGP

The molecular docking outcome illustrated interaction between EGP and the two standard compounds, silymarin and levamisole, against the target 6QMK and 1A3Q proteins, respectively. With a binding score of -7.8 kcal/mol, EGP by Gly198, and additionally with hydrophobic interactions of Tyr200 and Phe150. In comparison, silymarin, the standard hepatoprotective compound, showed a slightly better binding affinity with a score of -8.2 kcal/mol, supported by additional hydrogen bonds and π-π interactions with residues like Ser152 and Phe150. For the immunomodulatory target 1A3Q, Levamisole, the standard compound, displayed a lesser binding affinity of –0.11 kcal/mol, reporting H-bonds with residues Arg35 and Glu56 and EGP achieved a stronger binding score of -5.2 kcal/mol, forming hydrogen bonds with residues Thr42 and Glu56, as well as hydrophobic interactions with Tyr34 and Leu77, establishing hydrogen bonds with residues Arg35 and Glu56 It is obvious from the above that while the standard molecules showed a touch stronger affinity, the docking study presented a clear hint of good affinity to both target proteins. This is further suggested by the capability of EGP in imparting upon the hepatoprotective and immunomodulatory activities as shown in Table 1 and Figure 1-2. The docked complexes’ visualization also affirmed the primary molecular interactions-deserving attention-constituting hydrogen bonding patterns and hydrophobic contacts, to improve ligand stabilization within the active site.

Table 1.

Docking scores (kcal/mol) for EGP and the standard silymarin in the active site of 6QMK, assessing hepatoprotective activity, and for the standard levamisole in the active site of 1A3Q, assessing immunomodulatory activity.

Sl.No.		Docking score (kcal/mol)
Hepatoprotective activity
1	EGP	-7.0
2	EGP	-7.29
3	Silymarin standard	-6.31
4	Silymarin standard	-6.16
Immunomodulatory activity
5	EGP	-4.53
6	EGP	-5.2
7	Levamisole	-0.11

The stronger binding affinities are indicated by the lower (more negative value) docking score.

Fig. 1.

Two-dimensional ligand interaction diagrams of (A) Silymarin and (B) EGP with the protein target (PDB ID: 6QMK). The diagrams highlight the binding interactions of each ligand with 6QMK, indicating the key residues involved in the ligand-receptor binding sites.

Fig. 2.

Two-dimensional interaction diagrams of (A) levamisole and (B) EGP with the protein target (PDB ID: 1A3Q). These diagrams illustrate the binding interactions between each ligand and 1A3Q, highlighting the key residues involved in the ligand-receptor binding sites.

Method of validation EGP by HPLC

An HPLC method for EGP was validated and applied to samples as follows: the standard chromatogram showed a single, well-resolved EGP peak at Rt = 2.79 min (Figure 3A), and the test extract displayed a co-eluting major peak with clean baseline separation from minor matrix peaks (Figure 3B), confirming specificity. Calibration across 10–80 ng afforded an excellent linear response of peak area versus concentration with Y = 27 566 X and R² = 0.9994 (Figure 3C); replicate injections satisfied system-suitability with %RSD ≤ 2% for retention time and area. Sensitivity was adequate for routine work with LOD = 3 ng and LOQ = 5 ng (σ/S criteria). Precision met ICH limits, with intra- and inter-day %RSD < 2%, and accuracy by standard-addition gave 98–100% recovery, indicating negligible matrix effects. Quantification of EGP in the sample solutions was performed by comparing sample peak areas with the calibration curve; EGP was readily detected at 3 ng at Rt = 2.79 min, and final concentrations were calculated from peak areas and reported accordingly.

Fig. 3.

The concentration of EGP in the sample solution-B was determined by relating the sample peak area to that of the standard calibration curve-A. Calibration curve of EGP: concentration versus peak area-C.

In vivo hepatoprotective activity of EGP

Effect of EGP on biochemical parameters in rats

D-galactosamine (GalN; 400 mg/kg, i.p.) produced a robust hepatocellular injury, evidenced by marked increases in serum ALT and AST (Figure 4A–B), along with ALP and total bilirubin (Figure 1C–D) versus the control group. Prophylactic silymarin (40 mg/kg, p.o.) significantly attenuated all four biomarkers relative to GalN. EGP given orally for 7 days at 50 and 100 mg/kg likewise reduced ALT, AST, ALP, and bilirubin compared with GalN, with the 100 mg/kg dose consistently shifting values closer to control and to the silymarin reference. Statistical analysis (one-way ANOVA followed by Dunnett’s test vs. GalN) showed significant protection at both EGP doses (*p < 0.05 or **p < 0.01; n = 6), indicating a dose-responsive hepatoprotective effect. D-galactosamine (GalN; 400 mg/kg, i.p.) markedly suppressed hepatic antioxidant defenses versus control, lowering SOD, catalase, and GSH (Figure 5D–F). Silymarin (40 mg/kg, p.o.) significantly restored all three indices toward control. EGP administered orally for 7 days improved antioxidant status in a dose-responsive manner: 50 mg/kg elevated SOD, catalase, and GSH above GalN values, and 100 mg/kg produced greater recovery for catalase and GSH, approaching the silymarin reference (one-way ANOVA with Dunnett’s post-hoc vs. GalN; p<0.05, *p<0.01; n=6). Notably, SOD at 50 mg/kg reached near-control levels, while catalase and GSH showed clear dose-dependent gains at 100 mg/kg. Values are mean ± SEM (n = 6). GalN, D-galactosamine (400 mg/kg, i.p.); silymarin, 40 mg/kg (p.o.); EGP, 50 or 100 mg/kg (p.o., once daily for 7 days before GalN). ALT/AST: IU/L; ALP: KA U/100 mL; bilirubin: mg/100 mL; SOD and catalase: U/mg protein; GSH: nmol/mg protein as shown in Table 2

Fig. 4.

Effects of EGP on liver function markers in GalN-challenged rats. (A) ALT, (B) AST, (C) ALP (KA U/100 mL), and (D) total bilirubin (mg/100 mL). Rats received vehicle (Control), GalN (400 mg/kg, i.p.), silymarin (40 mg/kg, p.o.), or EGP (50 or 100 mg/kg, p.o.) once daily for 7 days prior to GalN. Bars are mean ± SEM (n = 6). Statistics: one-way ANOVA with Dunnett’s post-hoc vs. GalN; *p < 0.05 or **p < 0.01; n = 6.

Fig. 5.

Effects of EGP on hepatic antioxidant defenses in GalN-challenged rats. (5D) Superoxide dismutase (SOD; U/mg protein), (5E) catalase (U/mg protein), and (5F) reduced glutathione (GSH; nmol/mg protein). Groups: Control; GalN (400 mg/kg, i.p.); silymarin (40 mg/kg, p.o.); EGP 50 and 100 mg/kg (p.o., once daily for 7 days before GalN). Bars are mean ± SEM (n=6). Statistics: one-way ANOVA with Dunnett’s post-hoc vs. GalN; p<0.05 (*), *p<0.01 (**). Abbreviations: SOD, superoxide dismutase; GSH, reduced glutathione.

Table 2.

Effects of EGP on liver injury biomarkers and antioxidant status in GalN-challenged rats.

Treatment	ALT (IU/L)	AST (IU/L)	ALP (KA units/100Ml)	Bilirubin (mg/100Ml)	SOD (U/mg protein)	Catalase (U/mg protein)	GSH (nmol/mg protein)
Control	40.46±2.96	87.8±15.56	9.78±0.521	0.466±0.16	8.19±1.1	90.63±9.2	58.88±6.517
GalN toxicity (400 mg/kg b. wt,IP)	265.69±20.1	340.2±23.99	22.1±1.66	3.11±0.309	1.58±0.341	23.67±8.23	13.58±3.147
Silymarin (40mg/kg.b,wt)	66.12±17.89	121.8±14.2	12.33±1.11	0.801±0.24	9.41±0.891	77.03±10.56	56.38±3.266
EGP (50 mg /kg b.wt)	97.87±22.4	145.06±17.99	16.18±1.53	1.36±0.15	11.18±0.981	49.84±7.88	37.3±3.18
EGP (100 mg /kg b.wt)	68.36±28.69	127.03±18.44	13.64±1.23	0.993±0.195	8.25±1.212	68.63±9.24	48.18±4.44

Histopathological observation

Control livers displayed preserved lobular architecture with radiating hepatocyte cords around a patent central vein and intact portal triads, without necrosis, inflammation, congestion, or cellular degeneration (Figure 6A). D-galactosamine produced marked injury characterized by periportal hepatocellular necrosis with focal hemorrhages, ballooning/vacuolar change, cytoplasmic swelling, dense inflammatory infiltrates, and portal congestion, reflecting a globally disrupted architecture (Figure 6B). Silymarin treatment substantially mitigated these lesions, restoring near-normal lobular organization with only mild vacuolar change and scant inflammatory cells (not shown). EGP co-treatment produced a dose-dependent protection: at 50 mg/kg, sections showed only mild periportal inflammation and limited vacuolar degeneration; at 100 mg/kg, hepatic architecture was largely re-established with intact portal tracts and minimal residual inflammation, approaching the control pattern and comparable to, or slightly better than, silymarin (Figure 6C–D). Overall, EGP attenuated GalN-induced necro-inflammatory changes and preserved hepatocellular morphology in a graded manner.

Fig. 6.

Representative hepatic histology: (A) Control liver showing preserved lobular architecture with hepatocyte cords radiating around the central vein (CV) and an intact portal triad (PT); (B) D-galactosamine group showing periportal hepatocellular necrosis with focal hemorrhages; portal vein (PV) and bile duct (BD) indicated; (C–D) EGP + D-galactosamine (graded doses) showing re-established lobular organization with only mild periportal inflammatory cell infiltration; PV and BD indicated.

Hepatoprotective activity of EGP

EGP was tested for cytotoxic protective activity in hepatic cells using the MTT assay. The cells were pre-exposed to the compound in concentrations of 100 and 200 µg/mL for a period of 2 hours and then CCl₄ was administered to induce hepatotoxicity. Reference standard was silymarin (100 and 500 µg/mL). The experiment results proved that EGP caused a dose dependent hepatoprotection. Percentage hepatoprotection was calculated using Equation (1); the test compound significantly restored cell viability when compared to the Carbon tetrachloride-treated control group (P < 0.05). The 100 µg/mL concentration was moderately effective (58%) but at the highest concentration of 200 µg/mL gave a better hepatoprotection effect (67% equal to silymarin at concentration of 100 µg/mL) as observed in figure 7.

Fig. 7.

EGP shows the dose-dependent hepatoprotective activities in hepatic cells treated with CCl₄. The cells pretreated with the compound at different concentrations of 100 and 200 μg/mL showed protection of 58% and 67%, respectively,

Immunomodulatory activity of EGP

The nitric oxide (NO) assay was used to measure the in-vitro immunomodulatory activity of RAW 264.7 cells challenged with lipopolysaccharide (LPS), evaluating concentration at 50µg/ml. In the study, isolated compounds were screened for immunomodulatory activity in LPS stimulated raw 264.7 cells by pre-incubating the cells with or without isolated compounds. The more significant stimulatory effect of EGP, lipopolysaccharide and levamisole standard were observed by concentration at 50µM/ml in LPS stimulated NO production was observed by the RAW 264.7 cells, as shown in Figure 8.

Fig. 8.

Effect of EGP, lipopolysaccharide and levamisole standard on LPS stimulated RAW 267.4 cells. Cells in 96 well plates (1x10⁶ cells/well) were first incubated with and without indicated concentrations of EGP for 2 hours and followed by incubated with LPS (10µg/ml) for 20 hours. Untreated was negative control without LPS treatment. Each value was expressed as mean ± SEM in triplicate experiment.

Discussion

Traditional use of B. diffusa in hepatic and immune ailments is well documented, with modern studies attributing activities to a rich matrix of flavonoids, phenolics, lignans, and glycosides including eupalitin derivatives and boeravinones¹. Our earlier study in Molecules (2022;27:6444) optimized the extraction of EGP using design-software–guided HPTLC and provided preliminary evidence of hepatoprotection; the present work advances that foundation by establishing and validating an ICH-aligned HPLC method for quantitative EGP measurement, isolating and purifying the compound, and confirming dose-dependent in-vivo hepatoprotection in the D-galactosamine rat model, alongside supportive in-vitro immunomodulatory and hepatoprotective data and mechanistic docking to KEAP1–NRF2 (6QMK) and NF-κB p52–DNA (1A3Q). Collectively, this study isolates and validates EGP from Boerhavia diffusa and demonstrates convergent hepatoprotective and immunomodulatory actions across in-silico, in vitro, and in vivo models. Methodologically, we first established an ICH-aligned the validated HPLC assay for quantification of EGP with excellent linearity, precision, and sensitivity, enabling reliable exposure–response interpretation in downstream experiments. These analytical outcomes extend earlier chromatographic efforts (primarily HPTLC) on B. diffusa by providing a higher-resolution, validated HPLC route tailored to EGP, and therefore strengthen phytochemical standardization for future pharmacology and formulation studies^34,35,22. Convergent spectroscopic and MS/NMR evidence identifies the isolate as EGP. The UV λmax = 340 nm with a +45 nm NaOMe shift, together with FT-IR O–H (3400–3500 cm⁻¹), C=O (~1650 cm⁻¹), aromatic C=C (1500–1600 cm⁻¹), and glycosidic C–O–C (1000–1200 cm⁻¹) bands, is diagnostic of a flavonol nucleus bearing a chelated 5-OH and a 3-O-glycoside (Markham, 1982). ESI-MS showed [M+H]⁺ 493/[M–H]⁻ 491 and a 162 Da neutral loss to m/z 331/329, confirming a β-D-galactopyranoside³³. ¹H/¹³C NMR features the downfield 5-OH (δ 12.5), para-disubstituted B-ring, anomeric doublet at δ 5.4 (J = 7.5 Hz), and sugar carbon shifts support a β-D-galactopyranoside at C-3²¹. Overall, the UV/IR/MS/NMR set, and the isolation behavior (EtOAc enrichment, MeOH recrystallization) match published EGP data and recent reports, including from B. diffusa^30,35. Docking suggested that EGP engages both hepatoprotective and immunomodulatory protein targets via a combination of hydrogen bonds (e.g., Gly198, Thr42, Glu56) and hydrophobic contacts (e.g., Tyr200, Phe150, Tyr34, Leu77), with calculated binding energies in the moderate range for small-molecule ligands. For the hepatoprotective target (6QMK), EGP showed binding energies around −7.0 to −7.8 kcal/mol, broadly comparable to silymarin in your runs,for the immunomodulatory target (1A3Q), EGP outperformed levamisole in the same docking protocol. Given the known limits of scoring-function accuracy and the modest absolute differences observed, these in silico findings are best interpreted as supportive of plausible binding rather than definitive rank ordering,orthogonal validation (e.g., MD simulations, MM-GBSA, or biochemical binding assays) is warranted. Functionally, EGP showed clear hepatoprotection in the D-galactosamine (GalN) model, normalizing ALT/AST, ALP, and bilirubin toward control levels and improving hepatic antioxidant defenses (SOD, catalase, GSH). Histopathology corroborated these findings, with dose-dependent preservation of lobular architecture and attenuation of necro-inflammation. This profile is consistent with established hepatoprotective properties of flavonols and of silymarin-class polyphenols, which combine direct radical scavenging with modulation of redox and inflammatory pathways³⁶. The in vitro CCl4 assay further supported cytoprotection, a context where mitigation of lipid peroxidation is mechanistically relevant. Together, these data suggest that EGP’s phenolic scaffold (notably 4′-OH and ring A substitution) and 3-O-galactosylation which can enhance aqueous solubility contribute to its bioactivity. Whether glycosylation primarily improves exposure or directly modulates target engagement merits further study. EGP also increased NO production in LPS-stimulated RAW 264.7 macrophages at 50 μg/mL (comparable to levamisole), indicating immunostimulatory potential via iNOS/NO pathways measured with the Griess method. In immunopharmacology, controlled enhancement of macrophage NO can support antimicrobial and adjuvant activities, whereas sustained or excessive NO may exacerbate inflammation. Hence, defining the dose–response window and the broader cytokine profile (e.g., TNF-α, IL-6, IL-10) will be important to position EGP as an immunostimulant versus an immunomodulator with context-dependent effects.

Conclusion

EGP isolated from Boerhavia diffusa was successfully quantified using a validated HPLC method, and the compound showed dose-dependent protection in the GalN-induced liver injury model, along with improvements in antioxidant parameters and supportive histological changes. In vitro hepatocyte protection and NO modulation in macrophages were also observed. Molecular docking to NRF2- and NF-κB-related targets provides a potential mechanistic basis for these findings; however, these in silico results should be interpreted as preliminary and require experimental confirmation. Overall, the present findings indicated that EGP may contribute to the hepatoprotective, and immunomodulatory profile traditionally attributed to B. diffusa. Nevertheless, the current study is limited by the lack of pharmacokinetic data, the use of only two doses, and the absence of pathway-specific functional assays. Future studies should include PK/PD evaluations, chronic liver injury models, and direct molecular pathway validation (e.g., Nrf2/ARE, NF-κB, MAPKs, iNOS/COX-2) to substantiate the mechanistic relevance of this compound.

Author contributions

Conceptualization, I.U.K, L.Y.A; H.M.A; methodology, I.U.K,L.Y.A, H.M.A,A.K; investigation, I.U.K; data curation, T.CK, A.K, L.Y.A, H.M.A, S.Y.E.A; writing—original draft preparation, H.M.A, L.Y.A, I.U.K; writing—review and editing, I.U.K,L.Y.A,A.K,T.CK, H.M.A,S.Y.E.A ; visualization, I.U.K; supervision, L.Y.A, H.M.A, S.Y.E.A; funding acquisition, L.Y.A, H.M.A, S.Y.E.A.

Funding

The deanship of Scientific research (DSR) at King Abdulaziz University (KAU), Jeddah, Saudi Arabia has funded this project under grant no (G: 367-249-1443).

Data availability

The data that supports the finding of this study are also available from the corresponding author upon request.

Declarations

Competing interest

The authors declare no competing interests.