In Silico, In Vitro, and In Vivo Evaluation of Woodfordia Fruticosa for Cardio Protection against Doxorubicin‐Induced Cardiomyopathy

ABSTRACT

This study aimed to evaluate the therapeutic potential of Woodfordia fruticosa, known for its cardioprotective properties, using in silico, in vitro, and in vivo methods. Fourier‐transform infrared spectroscopy (FTIR) and gas chromatography–mass spectrometry (GC–MS) analyses were used to identify and characterize the bioactive compounds in W. fruticosa. Molecular docking with dock score assessed the binding affinities of hecogenin, β‐sitosterol, and pulmatin. Antioxidant potential was evaluated using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. In vivo experiments involved inducing cardiomyopathy in Wistar albino rats with doxorubicin (16 mg/kg, ip) and treating them with W. fruticosa flower extract at doses of 100, 200, and 400 mg/kg for 16 days. Molecular docking studies revealed that hecogenin, β‐sitosterol, and pulmatin exhibited strong binding affinities against human protein kinase ZAK, with Dock scores of −12.07, −11.42, and −11.04, respectively, surpassing the standard drug ramipril (−9.32). FTIR and GC–MS confirmed the presence of key phytoconstituents, including β‐sitosterol, hecogenin, ellagic acid, kaempferol, and 2‐hydroxy‐1,4‐naphthoquinone (Lawsone). In vitro antioxidant assays demonstrated significant free radical scavenging activity comparable to ascorbic acid. In vivo studies in Wistar albino rats showed that W. fruticosa flower extract significantly (p < 0.001) improved electrocardiogram (ECG) parameters, and significantly (p < 0.001) reduced cardiac biomarkers, including creatine kinase myocardial band (CK‐MB), lactate dehydrogenase (LDH), and cardiac troponin I, and mitigated histopathological damage in a dose‐dependent manner caused by doxorubicin. The 400 mg/kg dose exhibited the most pronounced cardioprotective effects, normalizing ECG patterns, decreasing myocardial injury, and reducing fibrosis. W. fruticosa flower extract exhibited significant cardioprotective potential via the human protein kinase ZAK pathway against doxorubicin‐induced cardiomyopathy through in silico, in vitro, and in vivo evaluations. These findings suggest W. fruticosa as a promising natural agent for cardiomyopathy management, warranting further studies to elucidate its mechanisms, optimize dosing, and validate long‐term safety and efficacy.

1. Introduction

Cardiomyopathy encompasses a spectrum of heart muscle disorders characterized by structural and functional abnormalities, posing significant challenges to cardiovascular health globally. These disorders impair the heart's ability to efficiently pump blood, leading to complications such as heart failure, arrhythmias, and an increased risk of sudden cardiac death [1, 2]. Cardiomyopathies are classified into primary forms, including genetic, mixed, or acquired conditions, and secondary forms such as infiltrative, toxic, or inflammatory causes, presenting diverse clinical manifestations and epidemiological profiles [1].

Dilated cardiomyopathy (DCM), marked by enlarged ventricles and reduced pumping efficiency, is one of the most prevalent types, often linked to genetic predispositions and environmental factors [3]. Hypertrophic cardiomyopathy (HCM), characterized by thickened heart muscle, particularly affecting the left ventricle, is a prominent genetic disorder associated with an increased risk of sudden cardiac death [4, 5]. Restrictive cardiomyopathy (RCM), although rare, involves the stiffening of the heart muscle, impairing diastolic filling, often secondary to conditions like amyloidosis [6]. Arrhythmogenic right ventricular cardiomyopathy (ARVC), primarily affecting the right ventricle with genetic underpinnings, predisposes individuals to arrhythmias and cardiac dysfunction [7].

The pathophysiology of cardiomyopathy involves intricate molecular mechanisms contributing to myocardial dysfunction and remodeling, including abnormal calcium handling, mitochondrial dysfunction, oxidative stress, and genetic mutations affecting structural proteins like titin [8, 9]. Molecular targets such as human protein kinase ZAK (sterile alpha motif and leucine zipper containing kinase AZK) involved in stress response pathways and cardiac remodeling, and sodium glucose co‐transporter 2 (SGLT2), impacting cardiac metabolism and oxidative stress, highlight potential avenues for therapeutic intervention [10, 11]. Recent research has underscored the significance of protein kinases in the pathophysiology of cardiomyopathies, emphasizing their regulatory roles in cellular processes like apoptosis, hypertrophy, and fibrosis [10, 12]. Recognizing human protein kinase ZAK as a target for cardiomyopathy represents a major breakthrough in understanding and potentially treating this disease. Targeting ZAK through molecular docking studies is expected to speed up the discovery of new therapeutic agents, providing hope for better clinical outcomes for patients. Future research should focus on developing and validating ZAK inhibitors, with an emphasis on their pharmacodynamics and pharmacokinetics to ensure clinical efficacy and safety [13].

Among the various etiologies contributing to cardiomyopathy, one notable cause is the cardiotoxic effects induced by chemotherapeutic agents like doxorubicin. Doxorubicin, an anthracycline antibiotic widely used in cancer treatment, is associated with dose‐dependent cardiomyopathy characterized by oxidative stress, mitochondrial dysfunction, and cardiomyocyte apoptosis [14, 15]. Understanding the molecular mechanisms underlying doxorubicin‐induced cardiotoxicity is crucial for developing effective cardioprotective strategies.

Woodfordia fruticosa a medicinal plant traditionally used in Ayurvedic medicine, has garnered attention for its diverse pharmacological properties, including anti‐inflammatory, antimicrobial, and antioxidant effects [16, 17, 18]. Recent studies have highlighted its potential cardioprotective properties, suggesting it may mitigate cardiac damage induced by oxidative stress and inflammation [19, 20].

This study aims to evaluate the therapeutic potential of W. fruticosa in the management of cardiomyopathy induced by doxorubicin, employing a comprehensive approach encompassing in silico, in vitro, and in vivo methodologies. Molecular docking was conducted to identify potential bioactive compounds within W. fruticosa, while antioxidant assays assessed its ability to combat oxidative stress. In vivo studies using a doxorubicin‐induced cardiomyopathy model in Wistar albino rats further elucidated its efficacy in preserving cardiac function and morphology.

2. Materials and Methods

2.1. Drugs and Chemicals

W. fruticosa dry extract (flower) was sourced from Kaiwal Biotech, Gujarat. Doxorubicin was sourced from United Biotech Pvt. Ltd., Baddi, known for its cardiomyopathy‐inducing properties, and ramipril from Sanofi India Ltd., Goa, recognized for its cardioprotective effects. Diagnostic kits used included the creatine kinase‐MB (CK‐MB) kit (Coral Clinical Systems; Catalog no. 1102070210), the lactate dehydrogenase (LDH) kit (Q‐line Biotech Pvt. Ltd.), Catalog no. (30293SLB) and the Rat Cardiac Troponin I (cTn‐I) ELISA kit (Abbkine; Catalog no. KTE101019). All chemicals and reagents utilized were of analytical grade.

2.2. In Silico Studies

2.2.1. Preparation of Target Protein

The three‐dimensional crystal structure of human protein kinase ZAK was retrieved from the RCSB‐PDB database (Research Collaboratory for Structural Bioinformatics‐Protein Data Bank) with PDB ID 5HES [10]. The receptor structure was prepared by removing water molecules, cofactors, and other molecules not involved in the binding site.

2.2.2. Retrieval of Compounds by Molecular Docking

The three‐dimensional structures of three compounds, namely Hecogenin (CID 91453), β‐sitosterol (CID 222284), and pulmatin (CID 442731), as well as the reference standard drug ramipril (CID 5362129), were retrieved from the NCBI‐PubChem database [21]. The ligand structures were initially downloaded in two‐dimensional file formats and then converted into three‐dimensional file formats for docking. Molecular docking was performed on the ZAK receptor (PDB ID 5HES) [22, 23].

2.2.3. Absorption, Distribution, Metabolism, and Excretion Analysis and Toxicity Prediction

ADME analysis evaluates a drug candidate's absorption, distribution, metabolism, and excretion properties, which is crucial for understanding its pharmacokinetic profile and guiding drug discovery and development decisions [24]. SwissADME (http://www.swissadme.ch/) was used for predicting these properties in small molecules. By entering the compound's chemical structure in SMILES format or using the drawing tool on the website, a detailed report is generated that includes physicochemical properties, lipophilicity, solubility, pharmacokinetics, drug‐likeness, and medicinal chemistry friendliness [25]. Although β‐sitosterol exhibited one violation of Lipinski's rule of five due to a higher lipophilicity value (MLOGP > 4.15), and pulmatin approached the upper limit for molecular weight, such deviations do not necessarily preclude biological activity or drug‐likeness, particularly for natural compounds. Many phytoconstituents with minor rule violations have demonstrated excellent pharmacological efficacy and bioavailability through alternative mechanisms such as active transport or metabolic conversion. Therefore, these compounds were retained for in silico molecular docking to explore their potential interactions with the human protein kinase ZAK and to assess their comparative binding affinities and inhibitory profiles.

ProTox‐II (https://tox.charite.de/protox3/) is a powerful tool in computational toxicology, offering reliable and quick predictive toxicological analysis [26]. By inputting the chemical structure in SMILES, InChI, or using the drawing tool, the software generates a detailed report with predicted toxicological endpoints, LD₅₀ values, and potential toxicological alerts. ProTox‐II helps to provide early toxicity forecasts, eliminate the need for animal testing, and promote regulatory compliance, making it essential for the safe and effective development of new chemical entities [27].

2.3. Qualitative Analysis

2.3.1. Fourier‐Transform Infrared Spectroscopy

Fourier‐transform infrared spectroscopy (FTIR) was used to analyze the chemical composition and functional groups in plant extracts [28, 29]. For preparing the KBr pellet with W. fruticosa flower extract, 1–2 mg of dried plant residue was mixed with 100–200 mg of dry potassium bromide (KBr) in a 99:1 ratio, ground into a fine powder, and pressed into a transparent pellet [30]. The pellet was analyzed using an FTIR spectrometer (Shimadzu IR Affinity 1) across a wavenumber range of 4000–400 cm⁻¹. The obtained FTIR spectrum reveals characteristic absorption bands corresponding to various functional groups present in the extract [31].

2.3.2. Gas Chromatography–Mass Spectrometry

Gas chromatography–mass spectrometry (GC–MS) analysis of W. fruticosa flower extract revealed the complex chemical composition of this medicinal plant [32]. The analysis was performed using an Agilent 5977B EI/CI MSD system at the Central Research Facility, Indian Institute of Technology, Delhi. One microliter sample was injected, with the injector temperature set at 250°C. The GC program started at 60°C for 1 min, ramped to 180°C, over 3 min, and then reached 240°C, over 12 min, with a ramp rate of 10°C/min, resulting in a total analysis time of 34 min. Helium was used as the carrier gas at a flow rate of 0.7 mL/min. The ion source temperature was 230°C, and the transfer line was maintained at 250°C. Electron impact ionization at 70 eV was used for component fragmentation, and the mass filter scanned m/z 45–450. Data processing was conducted using Agilent Technologies, Inc.'s Mass Hunter GC–MS Acquisition software version 10.1.49 (November 2, 2020).

2.4. In Vitro Antioxidant Activity

2.4.1. DPPH Radical Scavenging Activity

The DPPH assay evaluates antioxidant activity in vitro by measuring the reduction of DPPH, a stable free radical, indicated by the discoloration of the DPPH solution monitored spectrophotometrically. The assay involves preparing a 4 mg/100 mL DPPH solution in ethanol, mixing 1 mL of W. fruticosa flower extract or ascorbic acid (50–250 µg/mL) with 3 mL DPPH solution, homogenizing, and incubating in the dark at 25°C for 30 min. Absorbance was measured at 517 nm using a UV–visible spectrophotometer. The percentage of scavenging activity (%RSA) was calculated using %RSA = [(Ac − As)/Ac] × 100, where Ac and As are the absorbance of the control and sample. IC₅₀ values are determined by plotting %RSA against concentration [33, 34, 35, 36].

2.4.2. Ferric Reducing Antioxidant Power Assay

The ferric reducing antioxidant power (FRAP) assay assesses the antioxidant activity of W. fruticosa in vitro by quantifying its electron‐donating capacity, crucial for reducing ferric (Fe³⁺) to ferrous (Fe²⁺) ions. This assay supports the potential of W. fruticosa to mitigate oxidative stress, beneficial for conditions like cardiomyopathy, and validates its traditional use in Indian medicine [37]. Dilutions of W. fruticosa flower extract and ascorbic acid (50–250 µg/mL) were prepared. Each sample was mixed with 2.5 mL of phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide solution, then incubated at 50°C for 20 min. The reaction was stopped with 2.5 mL of 10% trichloroacetic acid (TCA) solution, followed by centrifugation at 3000 rpm for 10 min. The supernatant (2.5 mL) was mixed with 2.5 mL of deionized water and 0.5 mL of 0.1% ferric chloride solution, and absorbance was measured at 593 nm. The results were analyzed to report the FRAP values, ensuring the standardization and quality of the extracts [25, 38].

2.5. In Vivo Studies

2.5.1. Experimental Animals

Fifty‐four male Wistar albino rats (10–12 weeks old, weighing 180–200 g) were obtained from the Animal House Facility of KIET School of Pharmacy, Ghaziabad (UP), with CPCSEA Reg No. 1099/PO/RE/S/07/CPCSEA, sourced from the National Institute of Biologicals (NIB), Noida, India. The Institutional Animal Ethics Committee (IAEC) of KIET School of Pharmacy approved the protocol (IAEC/KSOP/2023‐24/04). Rats underwent 7–10 days of quarantine and 5 days of acclimatization before experiments, with an additional 10‐day acclimatization period for blood pressure recordings. Housing conditions followed CCSEA guidelines, maintaining 22 ± 2°C temperature, 50%–70% relative humidity, and a 12‐h light‐dark cycle, with rats provided ad libitum access to water and a balanced diet of dry pellets.

2.5.2. Treatment Schedule

In this study, 54 Wistar albino rats were divided into six groups comprising 9 animals per group. The normal control group received daily doses of 0.5% carboxymethyl cellulose (CMC) over a period of 16 days. The negative control group was administered a cumulative dose of doxorubicin totaling 16 mg/kg (2 mg/kg every other day) throughout the 16‐day period. The standard drug group received a daily dose of 10 mg/kg of ramipril along with doxorubicin, following the same dosing schedule as the negative control group. The low‐dose treatment group received a daily dose of 100 mg/kg of W. fruticosa extract alongside doxorubicin. The medium‐dose treatment group received 200 mg/kg/day of W. fruticosa extract, while the high‐dose treatment group received 400 mg/kg/day of W. fruticosa extract, both administered alongside doxorubicin. The dose levels were selected based on previous literature. Furthermore, the median lethal dose (LD₅₀) of W. fruticosa was greater than 2000 mg/kg [39, 40].

2.5.3. Experimental Model

Cardiomyopathy was induced by intraperitoneal (ip) administration of doxorubicin, with a cumulative dose of 16 mg/kg (administered as 2 mg/kg on alternate days) [41, 42]. Before administration on Day 0 and on the last day of the induction period, body weight, electrocardiogram (ECG), heart rate, and systolic blood pressure were measured [43, 44]. After a 24‐h period, all biochemical parameters were assessed, and subsequently, the animals were euthanized for histopathological analysis [43, 45].

2.5.4. Measurement of Body Weight

Body weight measurements were recorded at both the start and conclusion of the animal study. The changes in overall body weight were determined relative to the initial body weight of the rats on the first day of the experiment [46, 47].

2.5.5. Measurement of Blood Pressure and Heart Rate Using the Tail Cuff Method

In this study, blood pressure and heart rate were measured utilizing the tail cuff method, a widely employed technique for assessing cardiovascular function in rodents. The noninvasive blood pressure (NIBP) instrument (AD Instrument) was utilized alongside LabChart‐8 software for measuring systolic blood pressure and heart rate. This powerful combination enabled precise and real‐time monitoring and analysis of blood pressure parameters [48, 49]. Rats were acclimatized in a quiet, temperature‐controlled environment for 15–30 min. After cleaning the tail, an appropriately sized cuff was placed at its base, and the animal was positioned in a restrainer. Blood pressure was recorded over multiple cycles to ensure accuracy, and average values were used for analysis [50].

2.5.6. Measurement of Electrocardiography

ECG is an important diagnostic technology that measures the electrical activity of the heart. In the case of doxorubicin‐induced cardiomyopathy, an ECG can reveal many distinctive changes that represent the doxorubicin's negative effects on heart function. In this research work, ECG was measured on Day 0 and the last day of the animal study to evaluate the effects of the drug on the heart [51, 52]. Rats were placed in a quiet, temperature‐controlled environment for 15–30 min to minimize stress. Anesthesia was induced using a ketamine–xylazine mixture (87 and 13 mg/kg, respectively; 10:1 ratio, intraperitoneally). Once anesthetized, electrodes were positioned as follows: positive (red) on the left thoracic region, negative (black) on the right thoracic region, and reference (green) on the right thigh. ECG signals were then recorded for analysis [53, 54, 55].

2.5.7. Biochemical Estimation

The obtained serum samples were analyzed for cardiac injury markers, including CK‐MB, LDH, and cTn‐I, using commercially available diagnostic kits as per the manufacturer's instructions [56, 57, 58, 59, 60].

2.5.8. Histopathological Analysis of Heart

At the conclusion of the research, the experimental animals were euthanized through cervical decapitation. The heart with the aorta was collected and firmly preserved in 10% formalin solution. The slides were prepared with 5 µm sections of cardiac tissue and stained using hematoxylin and eosin (H&E) before being observed under a fluorescent microscope. All slides were examined to observe morphological changes in the heart [61].

2.6. Statistical Analysis

All data were presented as mean ± standard error of the mean (SEM) and analyzed using GraphPad Prism 9 software. One‐way analysis of variance (ANOVA) followed by Tukey's post hoc test (IBM SPSS Statistics 20) was conducted to compare groups. A significance level of p < 0.05 was used.

3. Results

3.1. In Silico Studies

3.1.1. Molecular Docking of W. fruticosa with Human Protein Kinase ZAK

The molecular docking analysis evaluated the interaction between compounds in the flower extract of W. fruticosa and the target human protein kinase ZAK. The results indicated a significant binding affinity of the active constituents of W. fruticosa with the kinase domain of ZAK (Table 1).

TABLE 1.

Molecular docking of phytoconstituents from Woodfordia fruticosa with target human protein kinase ZAK.

S. no.	Name	nConfs	Dock score (kcal/mol)	RF score (pKd)
1	Hecogenin	9	−12.07	8.16
2	β‐Sitosterol	9	−11.42	7.24
3	Pulmatin	9	−11.04	6.47
4	Prunin	9	−10.31	6.19
5	Quercitrin	9	−10.07	6.15
6	Epigallocatechin gallate	9	−9.87	6.43
7	Myricetin 3‐galactoside	9	−9.85	6.17
8	Avicularin	9	−9.81	6.06
9	Ellagic acid	9	−9.72	5.81
10	Kaempferol	9	−8.99	5.95
11	Cyanin	9	−8.35	6.55
12	Pelargonidin 3,5‐diglucoside	9	−7.87	6.76
13	2‐Hydroxy‐1,4‐naphthoquinone	9	−7.51	4.13
14	1‐Octacosanol	9	−7.47	6.87
15	β‐Sitosterol‐β‐d‐glucoside	9	−7.13	7.9
16	Ursolic acid	9	−5.35	7.64
17	Asiatic acid	9	−3.82	7.77
18	Maslinic acid	9	−3.28	7.76
19	Heterophylliin A	9	−2.97	6.75
20	Ramipril	9	−9.32	7.39

Docking simulations revealed that the phytochemicals from W. fruticosa interact with key residues in the active site of ZAK, suggesting potential inhibitory effects. These interactions were primarily stabilized through hydrophobic interactions, hydrogen bonds, and van der Waals forces, all of which are crucial for binding efficacy. The binding energies of the top compounds were compared to those of the standard drug ramipril, demonstrating promising potential for these natural compounds as competitive inhibitors of ZAK (Figure 1).

FIGURE 1.

3D Visualization and 2D interactions of the Woodfordia fruticosa inhibitors (A) hecogenin, (B) β‐sitosterol, (C) pulmatin and standard drug, (D) ramipril with human protein kinase ZAK protein.

The molecular docking analysis supports the hypothesis that W. fruticosa contains bioactive compounds capable of inhibiting human protein kinase ZAK, which could be beneficial for developing new treatments for cardiomyopathy induced by doxorubicin.

Furthermore, the three compounds with the highest docking scores were selected for further analysis. These compounds, namely hecogenin, β‐sitosterol, and pulmatin, exhibited promising scores of −12.07, −11.42, and −11.04, respectively. Ramipril, which is a standard drug, has a dock score of −9.32, which proves the phytochemicals in molecular docking show better binding affinity with the target than the standard drug. Subsequent investigations involved ADME analysis conducted using SwissADME, and toxicity prediction utilizing ProTox‐II.

3.1.2. ADME Analysis Using SwissADME

The SwissADME analysis of hecogenin, β‐sitosterol, pulmatin, and ramipril revealed that most of the compounds possess favorable pharmacokinetic properties. In addition, the analysis highlights the potential of hecogenin, β‐sitosterol, and pulmatin as promising candidates for therapeutic development. Their favorable pharmacokinetic profiles, characterized by efficient absorption rates and distribution patterns similar to ramipril, suggest robust bioavailability and tissue penetration. Detailed results for various parameters, including physicochemical properties, lipophilicity, water solubility, pharmacokinetic properties, and drug likeness aspects, are provided in Table 2 below.

TABLE 2.

Physicochemical, lipophilicity, pharmacokinetic, and drug‐likeness properties of hecogenin, β‐sitosterol, pulmatin, and ramipril analyzed using SwissADME.

	Hecogenin	β‐Sitosterol	Pulmatin	Ramipril
Physicochemical properties
Formula	C27H42O4	C29H50O	C21H20O9	C23H32N2O5
Molecular weight (MW) (g/mol)	430.62	414.71	416.38	416.51
Number of heavy atoms	31	30	30	30
Number of aromatic heavy atoms	0	0	12	6
Carbon bond saturation (fraction Csp3)	0.96	0.93	0.33	0.61
Number of rotatable bonds	0	6	3	11
Number of H‐bond acceptors	4	1	9	6
Number of H‐bond donors	1	1	5	2
Molar refractivity (MR)	122.27	133.23	100.88	116.96
Topological polar surface area (TPSA)	55.76 Å2	20.23 Å2	153.75 Å2	95.94 Å2
Lipophilicity
Log Po/w (iLOGP)	4.06	4.79	2.28	3.06
Log Po/w (XLOGP3)	4.83	9.34	1.27	1.43
Log Po/w (WLOGP)	4.97	8.02	−0.35	2.00
Log Po/w (MLOGP)	4.09	6.73	−1.26	1.98
Log Po/w (SILICOS‐IT)	3.99	7.04	0.92	2.57
Consensus Log Po/w	4.39	7.19	0.57	2.21
Pharmacokinetic properties
GI absorption	High	Low	Low	High
Blood–brain barrier (BBB) permeant	Yes	No	No	No
P‐gp substrate	Yes	No	Yes	Yes
CYP1A2 inhibitor	No	No	No	No
CYP2C19 inhibitor	No	No	No	No
CYP2C9 inhibitor	No	No	No	No
CYP2D6 inhibitor	No	No	No	Yes
CYP3A4 inhibitor	No	No	No	Yes
Log Kp (skin permeation)	−5.50 cm/s	−2.20 cm/s	−7.94 cm/s	−7.83 cm/s
Drug likeness
Lipinski rule	Yes; 0 violation	Yes; 1 violation: MLOGP > 4.15	Yes; 0 violation	Yes; 0 violation
Ghose rule	No; 1 violation: #atoms > 70	No; 3 violations: WLOGP > 5.6, MR > 130, #atoms > 70	Yes	Yes
Veber rule	Yes	Yes	No; 1 violation: TPSA > 140	No; 1 violation: Rotors > 10
Egan rule	Yes	No; 1 violation: WLOGP > 5.88	No; 1 violation: TPSA > 131.6	Yes
Muegge rule	Yes	No; 2 violations: XLOGP3 > 5, Heteroatoms < 2	No; 1 violation: TPSA > 150	Yes
Bioavailability score	0.55	0.55	0.55	0.55

3.1.3. Prediction of Toxicity Using ProTox‐II

The ProTox‐II tool was utilized to assess the toxicity profiles of hecogenin, β‐sitosterol, pulmatin, and ramipril, predicting various toxicological endpoints, including carcinogenicity, cytotoxicity, hepatotoxicity, immunotoxicity, and mutagenicity. The outcomes, presented in Table 3, detail the predicted toxicity class, LD₅₀ values, types of toxicity, prediction status, and corresponding probabilities for each compound.

TABLE 3.

Toxicity prediction of hecogenin, β‐sitosterol, pulmatin, and ramipril using ProTox‐II.

Compound name	Predicted toxicity class	Predicted LD50	Toxicity type	Prediction	Probability
Hecogenin	6	10 000 mg/kg	Hepatotoxicity	Active	0.50
			Carcinogenicity	Inactive	0.72
			Immunotoxicity	Active	0.97
			Mutagenicity	Inactive	0.87
			Cytotoxicity	Inactive	0.77
β‐Sitosterol	4	890 mg/kg	Hepatotoxicity	Inactive	0.87
			Carcinogenicity	Inactive	0.60
			Immunotoxicity	Active	0.99
			Mutagenicity	Inactive	0.98
			Cytotoxicity	Inactive	0.94
Pulmatin	5	3000 mg/kg	Hepatotoxicity	Inactive	0.85
			Carcinogenicity	Inactive	0.84
			Immunotoxicity	Active	0.98
			Mutagenicity	Active	0.91
			Cytotoxicity	Inactive	0.77
Ramipril	6	10 000 mg/kg	Hepatotoxicity	Inactive	0.92
			Carcinogenicity	Inactive	0.75
			Immunotoxicity	Inactive	0.99
			Mutagenicity	Inactive	0.89
			Cytotoxicity	Inactive	0.76

3.1.4. FTIR Analysis of W. fruticosa

Upon analyzing the FTIR spectrum (Figure 2) and examining the functional groups (Table 4), it was determined that the primary active component in W. fruticosa flower extract is β‐sitosterol (Figure 3).

FIGURE 2.

FTIR spectrum of flower extract from Woodfordia fruticosa and identified peaks.

TABLE 4.

Prediction of compounds in Woodfordia fruticosa using FTIR analysis.

S. no.	Functional group present	Expected wavenumber (cm−1)	Observed wavenumber (cm−1)
1	Alcohol O─H stretch	3650–3200 cm−1	3361.93–3348.42 cm−1
	Alcohol O↓H bend	1440–1300 cm−1	1357.89 cm−1
	Alcohol C─O(H) stretch	1260–970 cm−1	1153.43 cm−1
2	Alkanes C─H stretch	3000–2840 cm−1	2929.87 cm−1
	Alkanes C↓H bend	1460 cm−1	1409.96 cm−1
	Alkanes CH3 Symmetric bend	1380 cm−1	1357.89 cm−1
	Alkanes CH2 vibration	770–720 cm−1	763.81 cm−1
3	Alkenes C═C stretch	1690–1635 cm−1	1627.92 cm−1
	Alkenes CH2─(C═C) stretch	1440 cm−1	1409. 96 cm−1
	Alkenes H─C(═C) stretch	1000–675 cm−1	856.39 cm−1

FIGURE 3.

Structure of β‐sitosterol (C₂₉H₅₀O).

3.2. GC–MS Analysis of W. fruticosa

The interpretation of GC–MS spectra (Figure 4) showed 05 prominent phytoconstituents present in W. fruticosa flower extract. These compounds were kaempferol, hecogenin, lawsone, β‐sitosterol, and ellagic acid (Figure 5) with retention times of 4.190, 6.341, 8.150, 8.419, and 17.683 min, respectively.

FIGURE 4.

GC–MS spectra of flower extract of Woodfordia fruticosa.

FIGURE 5.

Phytoconstituents present in the flower extract of Woodfordia fruticosa identified through GC–MS analysis.

3.3. In Vitro Antioxidant Activity of W. fruticosa

The DPPH radical scavenging activity of the W. fruticosa flower extract and ascorbic acid (serving as a standard reference) was determined at various concentrations (50, 100, 150, 200, and 250 µg/mL). The findings from the DPPH assay showed that W. fruticosa and ascorbic acid exhibit substantial antioxidant properties. W. fruticosa displayed a slightly lower IC₅₀ value, 149.05 µg/mL, in comparison to ascorbic acid, 152.47 µg/mL, suggesting a slightly stronger antioxidant efficacy. The FRAP assay results indicated that both W. fruticosa and ascorbic acid have strong antioxidant activities. W. fruticosa demonstrated a slightly lower IC₅₀ value, 176.40 µg/mL, compared to ascorbic acid, 181.52 µg/mL, indicating slightly higher potency. These findings showed its potential as a useful natural antioxidant. The results are outlined in Figure 6A,B.

FIGURE 6.

Antioxidant activity of Woodfordia fruticosa and ascorbic acid in DPPH assay (A) and FRAP assay (B).

3.4. In Vivo Studies

3.4.1. Effect of W. fruticosa on Body Weight of Wistar Albino Rats

The rats that received doxorubicin showed weight loss compared to the normal control group. An ip administration of doxorubicin caused a considerable (***p < 0.0001) decline in body weight as compared to rats that received 0.5% CMC. W. fruticosa exhibited an increase (^## p < 0.001) in body weight in a dose‐dependent manner against doxorubicin (Figure 7).

FIGURE 7.

Effect of Woodfordia fruticosa on changes in body weight induced by doxorubicin. Data represent mean ± SEM (n = 9). ***p < 0.0001 versus normal control group; ^# p < 0.05 versus DOX‐induced cardiomyopathy; ^## p < 0.001 versus DOX‐induced cardiomyopathy. Following the one‐way ANOVA, a Tukey's post hoc analysis was conducted.

3.4.2. Effect of W. fruticosa on Systolic Blood Pressure and Heart Rate of Wistar Albino Rats

Rats that received doxorubicin showed considerably (***p < 0.0001) high systolic blood pressure and heart rate as compared to normal control rats. W. fruticosa exhibited a dose‐dependent decrease (^## p < 0.001) in the systolic blood pressure and heart rate of the animals as compared to doxorubicin‐induced rats (Figure 8). In the study, the systolic blood pressure of animals approached that of the normal control group at the 400 mg/kg dose of W. fruticosa.

FIGURE 8.

Effect of Woodfordia fruticosa on changes in systolic blood pressure (A) and heart rate (B) induced by doxorubicin. Data represent mean ± SEM (n = 9). ***p < 0.0001 versus normal control group; ^# p < 0.05 versus DOX‐induced cardiomyopathy; ^## p < 0.001 versus DOX‐induced cardiomyopathy. Following the one‐way ANOVA, a Tukey's post hoc analysis was conducted.

3.4.3. Effect of W. fruticosa on ECG Measurements of Wistar Albino Rats

The normal control group receiving 0.5% CMC displayed normal ECG readings. In contrast, the group treated with DOX showed significant ST segment elevation, prolonged P wave and QRS complex, and changes in the R–R interval (p < 0.0001 vs. normal control group). The groups given 100 and 200 mg/kg of W. fruticosa extract demonstrated less pronounced changes in the ST segment, P wave prolongation, QRS complex, and R–R interval alterations in their ECG readings. The animals treated with 400 mg/kg of W. fruticosa extract exhibited an almost normal ECG pattern with reduced ST segment elevation, QRS complex, QT interval, and R–R interval (p < 0.001), whereas the cardiac cycle increased when compared to the normal control group (Figure 9).

FIGURE 9.

ECG readings across different experimental groups of rats.

3.4.4. Estimation of CK‐MB, LDH, and Cardiac Troponin I (cTnI) Levels

Serum levels of CK‐MB, LDH, and cTnI significantly increased (***p < 0.0001) in animals with doxorubicin‐induced cardiomyopathy compared to the normal control group. Treatment with W. fruticosa led to a dose‐dependent reduction in CK‐MB, LDH, and cTnI levels compared to the doxorubicin‐induced cardiomyopathy group. The administration of 100 and 200 mg/kg of W. fruticosa resulted in a slight decrease in these levels in animals (^# p < 0.05) compared to those with doxorubicin‐induced cardiomyopathy. Conversely, the administration of 400 mg/kg W. fruticosa (Table 5).

TABLE 5.

Estimation of CK‐MB, LDH, and cTnI of Wistar albino rats expressed in mean ± SEM.

Groups (n = 9)	CK‐MB (IU/L)	LDH (IU/L)	cTnI (ng/mL)
Normal control group (0.5% CMC)	64.31 ± 0.47	62.78 ± 0.57	0.087 ± 0.002
Negative control group (DOX)	186.96 ± 0.50 ***	207.40 ± 0.61 ***	0.788 ± 0.026 ***
Disease + standard drug group (DOX + ramipril)	77.90 ± 0.64 ##	77.52 ± 0.56 ##	0.322 ± 0.027 ##
Disease + low dose treatment group (DOX + WF 100 mg/kg)	147.37 ± 0.61 #	128.44 ± 0.53 #	0.6 ± 0.023 #
Disease + medium dose treatment group (DOX + WF 200 mg/kg)	107.45 ± 0.62 #	92.76 ± 0.63 #	0.344 ± 0.017 #
Disease + high dose treatment group (DOX + WF 400 mg/kg)	69.90 ± 0.44 ##	68.85 ± 0.44 ##	0.177 ± 0.022 ##

Note: Results are expressed as mean ± SEM; n = 9 animals per group. Following the one‐way ANOVA, a Tukey's post hoc analysis was conducted.

^*** p < 0.0001 versus normal control group.

^# p < 0.05 versus DOX‐induced cardiomyopathy.

^## p < 0.001 versus DOX‐induced cardiomyopathy.

3.5. Histopathological Findings in Cardiac Tissue

The H&E‐stained images of rat heart tissues depict the healthy architecture of cardiac tissue with uniform distribution of myocardium layers in normal control rats. In contrast, DOX‐induced cardiomyopathy in disease control rats is evident with the focal hemorrhagic patch (green arrow), infiltration of inflammatory cells (black arrowheads), necrotic patch (black arrow), and vacuolization (blue arrow). The ramipril‐10 mg/kg treatment image showed a marginal reversal of DOX‐induced cardiomyopathic features toward normal control. However, W. fruticosa extract treatment image showed dose‐dependent treatment of DOX‐induced cardiomyopathy, as evident with reversal of cardiomyopathic features toward normal control. Such findings are very well correlated via 3D‐interactive surface plots as presented in parallel along with H&E‐stained images (Figure 10).

FIGURE 10.

Effects of Woodfordia fruticosa on histopathological alterations induced by doxorubicin in the hearts of rats. Histopathological changes observed in the (A) normal control group, (B) doxorubicin‐induced cardiotoxicity, (C) doxorubicin + ramipril (10 mg/kg), (D) doxorubicin + low dose (100 mg/kg) W. fruticosa, (E) doxorubicin + medium dose (200 mg/kg) W. fruticosa, (F) doxorubicin + high dose (400 mg/kg) W. fruticosa. Images are taken at ×100 magnification with scale bar of 20 µm under Upright Research Microscope RXLr‐4NX, Radical Scientific Pvt. Ltd., Delhi, India.

4. Discussion

Cardiomyopathy is a multifactorial disorder affecting the heart muscle's structure and function, often leading to complications such as heart failure and arrhythmias [2]. Its complex etiology, involving genetic, biochemical, and environmental factors, underscores the pressing need for effective therapeutic options [62]. Among the known causes, doxorubicin—a potent chemotherapeutic agent—has been identified as a significant contributor to cardiomyopathy due to its cardiotoxic side effects. These effects are mediated by oxidative stress, mitochondrial dysfunction, and apoptosis of cardiac cells [63, 64]. The clinical presentations of doxorubicin‐induced cardiomyopathy vary from subclinical cardiac impairment to severe heart failure, emphasizing the necessity for cardioprotective agents [64, 65]. In this context, the present study evaluated the cardioprotective potential of W. fruticosa using an integrated approach comprising in silico, in vitro, and in vivo techniques. FTIR and GC–MS analyses were employed to corroborate the findings as well.

Molecular docking studies targeting human protein kinase ZAK, a key mediator in doxorubicin‐induced cardiac damage [10], revealed that phytoconstituents such as hecogenin, β‐sitosterol, and pulmatin exhibited stronger binding affinities than ramipril, a standard therapeutic agent. Their respective dock scores were −12.07, −11.42, and −11.04, in comparison to ramipril's −9.32, indicating promising ZAK inhibitory potential. SwissADME and ProTox‐II further validated the drug‐likeness and safety of these compounds, especially hecogenin and pulmatin, suggesting their suitability for drug development [21]. FTIR and GC–MS analyses supported these results by confirming the presence of β‐sitosterol and other bioactive molecules, including ellagic acid, kaempferol, and lawsone [29, 66]. Antioxidant studies using DPPH and FRAP assays demonstrated the extract's strong free radical scavenging and electron‐donating capacities, comparable or superior to those of ascorbic acid [25, 33].

In vivo studies in doxorubicin‐treated rats revealed elevated biomarkers (CK‐MB, LDH, and troponin I), ECG abnormalities, and increased systolic blood pressure—all of which were significantly improved following treatment with W. fruticosa, particularly at 400 mg/kg [67, 68]. Histopathological examination further demonstrated reduced myocardial damage and restored cardiac architecture in the treated groups [45, 56]. These findings collectively support the cardioprotective potential of W. fruticosa attributed to its antioxidant capacity and molecular interactions with ZAK, making it a promising candidate for mitigating doxorubicin‐induced cardiomyopathy.

In vivo studies confirmed these results, with animal models of cardiomyopathy demonstrating significant improvement in cardiac function after treatment with W. fruticosa extract. Treatment led to decreased levels of cardiac biomarkers such as CK‐MB, LDH, and cardiac troponin I, indicative of reduced myocardial damage. In addition, parameters such as body weight, systolic blood pressure, heart rate, and ECG readings showed favorable responses to the extract [67, 68]. Rats treated with doxorubicin experience cardiomyopathy associated with an elevation in systolic blood pressure. As the heart muscle weakens and loses its ability to pump effectively due to doxorubicin toxicity, systolic blood pressure may increase as a compensatory response to ensure sufficient blood flow. This higher blood pressure places additional stress on the heart, worsening cardiomyopathy. Thus, careful monitoring and management of systolic blood pressure in doxorubicin‐induced rats are essential to prevent cardiovascular complications and maintain heart function [48]. In this study, the normal control group treated with 0.5% CMC exhibited normal ECG patterns, whereas the group treated with doxorubicin showed significant deviations, including elevated ST segments, prolonged P wave and QRS complex, and altered R–R intervals, indicative of doxorubicin‐induced cardiomyopathy. Treatment with W. fruticosa extract at doses of 100 and 200 mg/kg partially mitigated these abnormalities, although some ECG changes persisted. The highest dose of 400 mg/kg demonstrated promising results, approaching normal ECG parameters with reduced ST segment elevation, shorter QRS complex duration, and improved QT and R–R intervals compared to controls. These findings suggest that W. fruticosa extract is showing potential in protecting against doxorubicin‐induced cardiac damage [51, 52]. Exploring biomarkers of cardiac injury, LDH, CK‐MB, and cardiac troponin I were scrutinized following treatments with doxorubicin and W. fruticosa. The administration of W. fruticosa, particularly at 400 mg/kg, demonstrated significant reductions in LDH, CK‐MB, and cardiac troponin I levels, suggesting a potential protective effect against doxorubicin‐induced cardiomyopathy. These findings underscore the potential of W. fruticosa in mitigating cardiac injury and warrant further exploration of its therapeutic mechanisms in cardiovascular health. The treatment not only reduced markers of oxidative stress and inflammation but also preserved myocardial tissue integrity, showcasing its cardioprotective effects [45, 56]. Histopathological analysis supported these findings by revealing decreased myocardial damage and improved cardiac tissue structure in animals treated with W. fruticosa flower extract. The normal control group exhibited normal cardiac tissue without signs of cardiomyocyte damage, whereas the doxorubicin‐treated group displayed perivascular fibrosis and vacuolated myocytes indicative of cardiac injury. W. fruticosa flower extract treatment at varying doses yielded promising results. Lower doses reduced interstitial fibrosis and inflammation, while the highest dose group showed evidence of healed cardiomyocytes. These findings highlight the potential cardioprotective effects of W. fruticosa flower extract against doxorubicin‐induced cardiac damage, suggesting its therapeutic significance in preserving cardiac function [45, 67, 68].

5. Conclusion

This study provides a strong basis for considering W. fruticosa as a promising adjuvant in the treatment of doxorubicin‐induced cardiomyopathy and as a valuable source of natural antioxidants. Through in silico, in vitro, and in vivo evaluations, the extract showed significant cardioprotective effects, likely linked to its ability to reduce oxidative stress and modulate human protein kinase ZAK‐related molecular pathways. The combined molecular, chemical, and biological findings suggest its potential as a safe and effective natural therapeutic option. Overall, the results add to the growing evidence supporting the role of medicinal plants in developing new treatments for complex diseases. Further work on isolating active phytoconstituents and studying their pharmacokinetics and optimal dosing will help in translating these findings into practical therapeutic use.

Author Contributions

Gauri Goyal: Conceptualization and Vinay Kumar: Conceptualization. Abhijeet Debnath: Methodology and Mohamad Taleuzzaman: Methodology. Ajay Kumar Pal: Validation and formal analysis and K. Nagarajan: Validation and formal analysis. Gauri Goyal: Statistical analysis and Vinay Kumar: Statistical analysis. Writing – original Gauri Goyal: draft preparation and Abhijeet Debnath: draft preparation. Vinay Kumar: Review and editing and Mohamad Taleuzzaman: Review and editing. All authors have read and agreed to the published version of the manuscript. The authors confirm that no paper mill and artificial intelligence was used.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding

The authors have nothing to report.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.