Vitex Simplicifolia Abates Cadmium‐Induced Cardiotoxicity Through Antioxidant Activity and Keap1 Targeting

TL;DR

This study shows that Vitex simplicifolia extract protects the heart from cadmium toxicity by acting as an antioxidant and interacting with a key protein called Keap1.

Contribution

The novel contribution is the identification of Vitex simplicifolia's cardioprotective effects through antioxidant activity and Keap1 targeting, supported by molecular docking.

Findings

VSME reversed cadmium-induced oxidative stress and cardiac damage in rats.

VSME phytochemicals showed strong binding to Keap1 with better drug-likeness than standard inhibitors.

VSME improved lipid profiles and restored antioxidant enzyme levels in cadmium-exposed rats.

Abstract

This study evaluated the cardioprotective potential of Vitex simplicifolia methanol extract (VSME) and explored its underlying mechanisms of action. Twenty‐five male rats were assigned to five groups (n = 5). Group 1 served as the normal control, while groups 2–5 were exposed to 5 mg/kg body weight of cadmium chloride (CdCl2) daily. Group 2 received no treatment, whereas groups 3 and 4 were treated with 200 and 400 mg/kg VSME, respectively, and group 5 received 10 mg/kg propranolol (standard drug), all for 21 days via oral administration. Biochemical and histopathological analyses of the heart were conducted post‐treatment. Cadmium exposure significantly elevated cardiac malondialdehyde, triglycerides, cholesterol, low‐density lipoprotein, creatine kinase, lactate dehydrogenase, and C‐reactive protein, while reducing high‐density lipoprotein, superoxide dismutase (SOD), catalase, and glutathione peroxidase. VSME treatment reversed these changes, restoring antioxidant status, lipid profile, cardiac biomarkers, and myocardial architecture. Molecular docking revealed strong binding affinities of VSME phytochemicals (kaempferol, isorhamnetin, luteolin) with Kelch‐like ECH‐associated protein 1 (Keap1), primarily through hydrogen bonds. These compounds exhibited superior drug‐likeness, pharmacokinetics, and safety profiles compared to the standard Keap1 inhibitor, CPUY192018. These findings suggest that VSME may exert cardioprotective effects partly through antioxidant activity and potential interference with the Keap1‐Nrf2 interaction, as supported by molecular docking.

Figures

Effect of VSME on antioxidant enzymes (SOD, CAT, and GPx) and lipid peroxidation markers (MDA) of CdCl2‐intoxicated Wistar rats. Bar charts depict the mean ± SD (n = 5). The significance thresholds were set at *p < 0.05, p < 0.01, *p < 0.001, and p < 0.0001, with ns indicating nonsignificance compared with the untreated group.

Effects of VSME on Lipid Profile of CdCl2 Intoxicated Wistar Rats. Bar charts depict the mean ± SD (n = 5). The significance thresholds were set at *p < 0.05, p < 0.01, *p < 0.001, and p < 0.0001compared with the untreated group.

Effect of VSME on cardiac biomarkers of CdCl2‐intoxicated Wistar rats. Results are displayed as mean ± SD (n = 5). The significance thresholds were set at *p < 0.05, p < 0.01, *p < 0.001, and p < 0.0001 compared with the untreated group.

2D and 3D complexes of kaempferol (A), isorhamnetin (B), luteolin (C), and CPUY192018 (D) with Keap1.

Representative histological sections of heart tissue from experimental groups. Figure legends: White arrow (myocyte nuclei), black arrow (pericyte), yellow arrow (myocardial necrosis), and red arrow (capillaries). (A) Group 1: the normal rats, no intoxication and treatment; (B) Group 2: intoxicated only (untreated); (C) Group 3: intoxicated and treated with 200 mg/kg of VSME; (D) Group 4: intoxicated and treated with 400 mg/kg of VSME; and (E) Group 5: intoxicated and treated with 10 mg/kg of propranolol.

Tables

TABLE 1. Compounds identified in VSME using HPLC.

Peak no.	Identified compound	RT	MW	Area	Class of compounds
1	Caryophyllene	1.266	33.553	1181.3855	Sesquitepenoid
2	Beta‐phellandrene	2.516	24.849	2658.3150	Monoterpenoid
3	Eucalyptol	4.450	12.809	744.0795	Monoterpenoid
4	Butein	5.466	7.886	312.4090	Flavanoid
5	Epigallocatechin‐3‐gallate	6.483	6.040	199.6535	Flavanoid
6	Beta‐sitosterol	7.333	4.121	61.0590	Phytosterol
7	Stigmasterol	7.950	3.412	72.2160	Phytosterol
8	Campesterol	8.750	4.510	50.2930	Phytosterol
9	Alpha‐Amyrin	9.350	3.468	67.5390	Triterpenenoid
10	Quercetin	11.050	160.005	9371.7180	Flavanoid
11	Kaempferol	12.166	66.168	3730.7180	Flavanoid
12	Vitexin	13.700	43.616	3263.0900	Flavanoid
13	Casticin	16.250	5.374	103.2340	Flavanoid
14	Luteolin	17.616	6.575	496.2720	Flavanoid
15	Apigenin	18.750	4.117	55.8660	Flavanoid
16	Quercitrin	19.250	3.908	56.9525	Flavanoid
17	Isorhamnetin	19.683	4.332	96.9040	Flavanoid
18	Artemetin	20.500	3.529	55.5840	Flavanoid
19	Bicyclogermacrene	21.133	3.107	61.0860	Sesquiterpene

TABLE 2. Median lethal dose and acute toxicity study (LD50) of VSME.

Phases	Doses of VSME (mg/kg)	Mortality
Phase 1	10	nil
	100	nil
	1000	nil
Phase 2	1600	nil
	2900	nil
	5000	nil

TABLE 3. Effects of VSME and standard drug on atherogenic indices in CdCl2‐intoxicated rats.

Groups	Atherogenic/dyslipidemia indices in rats
	CRR	AC	CR		AIP
1	2.79 ± 0.13 ****	1.79 ± 0.13 ***		1.15 ± 0.01 ****	−0.09 ± 0.01 ***
2	7.23 ± 0.44	6.23 ± 0.44		4.02 ± 0.23	0.60 ± 0.09
3	2.22 ± 0.54 ****	1.22 ± 0.54 ****		0.92 ± 0.212 ****	−0.19 ± 0.05 ****
4	2.51 ± 0.31 ****	0.81 ± 0.69 ****		1.14 ± 0.26 ****	−0.08 ± 0.12 ***
5	2.29 ± 0.52 ****	1.29 ± 0.52 ****		0.93 ± 0.19 ****	−0.14 ± 0.07 ****

TABLE 4. Molecular interactions of VSME top‐scoring compounds with Keap1.

Compounds	Interacting residue	Types of interaction	Distance (À)	Binding energy (kcal/mol)
Kaempferol	GLY 367	H. bond	1.97	−10.190
	VAL 606	2 H. bonds	1.75, 1.92
	ALA 510	H. bond	1.61
	VAL 512	H. bond	2.28
Isorhamnetin	GLY 367	H. bond	1.96	−9.413
	VAL 606	2 H. bonds	1.62, 2.33
	ALA 510	H. bond	1.82
Luteolin	GLY 367	H. bond	1.83	−9.211
	VAL 606	H. bond	2.10
	ALA 510	H. bond	1.86
	THR 560	H. bond	2.37
CPUY192018	VAL 418	H. bond	1.83	−8.149
standard drug	VAL 465	H. bond	2.36
	VAL 467	H. bond	2.36
	ILE 559	H. bond	2.08
	VAL 561	H. bond	2.19
	VAL 606	H. bond	1.70

TABLE 5. Drug‐likeness, pharmacokinetics, and toxicity properties of VSME ligands.

Properties	Kaempferol	Isorhamnetin	Luteolin	CPUY192018	References
PubChem CID	5280863	5281654	5280445	73330369	—
Formula	C15H10O6	C16H12O7	C15H10O6	C28H26N2O10S2	—
MW (g/mol)	286.24	316.26	286.24	614.64	150–500
R. bond	1	2	1	12	0–9
HB donor	4	4	4	2	01–5
HB acceptor	6	7	6	10	0–10
MR	76.01	82.50	76.01	154.12	40–130
TPSA (A2)	111.13	120.36	111.13	184.58	20–130
LogPo/w (XLOGP3)	1.90	1.87	2.53	3.72	−0.7–5
Consensus Log P	1.58	1.65	1.73	2.85	≤ 3.5
Fraction Csp3	0.00	1.65	0.00	0.14	−0.25 – < 1
Druglikeness (Lipinski rule)	Yes 0 violation	Yes (0 violation)	Yes (0 violation)	No (2 violation, MW > 500, No R > 10)	MLog p ≤ 4.15, MW≤ 500, HBA ≤ 10, HBD ≤ 5
Lead‐likeness	Yes	Yes	Yes	No (3 violation, MW > 350, Rotors > 7, XLOGP3 > 3.5)	250 ≤ M.W ≤ 350, XLOGP3 ≤ 3.5, R‐bonds ≤ 7
BA score	0.55	0.55	0.55	0.11	> 0.1 (10 %)
BBB	No	No	No	No	BBB+ ≥ 0.30, BBB− < −1
GIA	High	High	High	Low	—
Synthetic Accessibility	3.14	3.26	3.02	3.83	1‐10 (very easy –very difficult)
Log kp (cm/s)	−6.70	−6.90	−6.25	−7.41	−8.0 to −1.0
Predicted LD50 (mg/kg)	3919	5000	3919	2000	—
Toxicity class	5	5	5	4	—

Full text

Introduction

1

Cadmium (Cd), a highly ubiquitous and toxic heavy metal, is a major environmental pollutant released through cigarette smoke, fuel oil combustion, phosphate fertilizer production, and the smelting of nickel and copper [1]. Humans are constantly exposed to Cd through inhalation of airborne particles, consumption of contaminated food and water, and occupational contact [2, 3]. Cadmium, owing to its long biological half‐life of 20–40 years [3] and inadequate excretion, accumulates in essential organs, leading to various cancers, hepatotoxicity, nephrotoxicity, genotoxicity, and making the heart especially susceptible [4, 5]. To this effect, the correlation between Cd exposure and the incidence of cardiovascular diseases (CVDs), such as heart failure, myocardial infarction, stroke, and coronary and peripheral artery diseases, has been demonstrated in prior epidemiological and animal research [2, 6]. For instance, the detrimental effects of Cd on smooth muscle cells, vascular endothelial cells, and cardiomyocytes have been documented in both in vitro and in vivo studies [2]. According to Karadas et al. ([7], CVDs are a global public health concern that accounts to 35% of deaths, and they are expected to continue to be the leading cause of global health burdens in the years to come [8]. This burden is exacerbated by rising Cd intake, which often exceeds the (FAO/WHO) and the European Food Safety Authority (EFSA) permissible tolerable Weekly Intake (TWI) of 2.5 µg/kg of Cd per body weight [9] and the FAO permissible daily intake of 1 µg/kg of body weight [4]. These observations highlight an urgent need for effective interventions against Cd‐induced cardiotoxicity.

Cadmium exerts cardiotoxic effects via multiple mechanisms, including dysregulated lipid metabolism, mitochondrial dysfunction, apoptosis, and oxidative stress—all of which culminate in myocardial injury and dyslipidaemia [10]. Research indicates that cadmium‐mediated deregulation of lipid levels in the heart may compromise myocardial integrity, resulting in myocardial fibrosis, cardiomyocyte apoptosis, and diminished contractility due to mitochondrial dysfunction, ultimately leading to cell death [6, 11]. Furthermore, Cd exposure alters cellular signaling pathways and the structural integrity of lipids. It impacts membrane‐bound enzymes by enhancing the formation of reactive oxygen species (ROS) and disrupting the antioxidant defense system, thereby undermining cellular protection against oxidative damage and exacerbating the detrimental effects of cellular oxidative stress [12]. Consequently, the impairment of antioxidant defense mechanisms has been identified as the primary and most deleterious mechanism of cadmium‐induced toxicity [13].

A promising therapeutic strategy for oxidative damage comprises inhibiting the nuclear factor erythroid 2–related factor 2 (Nrf2) signaling pathway, a negative regulator, by targeting Kelch‐like ECH‐associated protein 1 (Keap1). This enables Nrf2 to migrate to the nucleus, leading to the activation of antioxidant response element (ARE)‐dependent genes that shield against oxidative damage [14]. Additionally, cardiac biomarkers including creatine kinase (CK), troponins, lactate dehydrogenase (LDH), and C‐reactive protein (CRP) are critical for diagnosing and monitoring myocardial injury. Elevated levels of these biomarkers reflect myocardial damage, inflammation, and metabolic dysfunction [15]. Therefore, restoring antioxidant capacity and normalizing these biomarkers could offer a meaningful strategy to mitigate Cd‐induced cardiotoxicity.

Despite extensive research, therapeutic options for CVDs remain limited. Medicinal plants, which serve as valuable sources of bioactive compounds, have been explored for drug development, especially in resource‐limited settings [16, 17]. Vitex simplicifolia is a flowering plant widely distributed from the Ivory Coast to Africa and China [18]. The roots are woody and fibrous, brown to dark brown in color, and vary in size depending on the age and growth stage of the tree. Information from native traditional healers in Nigeria shows that a decoction of chopped stem barks, roots, and leaves of V. simplicifolia is consumed locally to cure many medical ailments such as hypertension, diabetes, infertility, cancer, anodyne, stiffness, liver disease, kidney diseases, and eye problems [19, 20]. Although V. simplicifolia has demonstrated strong antioxidant potentials, the cardioprotective role of Vitex simplicifolia and the molecular mechanisms through which its bioactive compounds may modulate antioxidant defenses, such as the Keap1–Nrf2 pathway, remain largely unexplored. In this context, this research sought to assess the cardioprotective properties of V. simplicifolia methanol extract (VSME) in a rat model of cadmium‐induced cardiotoxicity, with a particular focus on oxidative stress parameters, cardiac biomarkers, lipid profiles, histological features, and molecular interactions with Keap 1.

Results and Discussion

2

Cadmium exposure is strongly associated with oxidative stress–mediated tissue injury, and the heart is particularly susceptible due to its high metabolic demand. Therefore, identifying safe phytochemical‐rich interventions that can restore redox balance and reduce myocardial injury is of therapeutic interest. The following sections discuss our results sequentially in the order obtained.

HPLC Profiling of Compounds in VSME

2.1

The HPLC screening of VSME identified 19 phytochemical constituents belonging to five major classes: flavonoids (e.g., quercetin, kaempferol, luteolin, apigenin), monoterpenoids (eucalyptol, β‐phellandrene), sesquiterpenoids (caryophyllene), phytosterols (β‐sitosterol, campesterol, stigmasterol), and triterpenoids (α‐amyrin). Flavonoids were the most abundant, with quercetin registering the highest peak area. Detailed retention times, molecular weights, and peak areas are presented in Table 1, and the chromatographic profile is shown in Figure S1.

Overall, the flavonoid‐rich profile revealed by HPLC supports a plausible biochemical basis for the antioxidant and cardioprotective effects observed in vivo. Flavonoids such as quercetin, kaempferol, luteolin, apigenin, and related polyphenols are widely reported to mitigate oxidative stress and inflammatory signaling, preserve membrane integrity, and improve cardiovascular risk markers [21]. For instance, Srivastava et al. [22] reported that quercetin protected rats against Cd‐induced damage, while Ginwala et al. [23] suggested anti‐atherosclerotic actions via inhibition of matrix metalloproteinases. Similarly, EGCG has been shown to mitigate metal toxicity by protecting mitochondria, scavenging ROS, enhancing metal excretion, and upregulating Nrf2 [24]. More importantly, the cardioprotective effects of kaempferol and apigenin identified in the extract have been extensively studied [25]. Thus, the HPLC‐identified constituents provide biological plausibility for the protective effects of VSME observed in the subsequent in vivo endpoints.

Acute Toxicity Studies (LD 50) of VSME

2.2

Acute toxicity assessment revealed no mortality or observable signs of toxicity such as salivation, tremors, convulsions, or behavioral abnormalities up to the highest tested dose of 5000 mg/kg b.w. This indicates a high safety margin for VSME (Table 2).

The absence of mortality or overt toxicity up to 5000 mg/kg suggests a wide safety margin for VSME. It supports the selection of 200 and 400 mg/kg as moderate and high nontoxic doses for efficacy assessment. These doses represent approximately 1/25 and 1/12.5 of the highest tested dose, consistent with standard practice for in vivo pharmacological evaluation.

Effects of VSME on Oxidative Stress in the Heart

2.3

A significant reduction in the activities of SOD and CAT, accompanied by an elevation in MDA levels, was observed in the cardiac homogenate of untreated rats following CdCl_2_ intoxication, in comparison to normal rats. Notably, treatment with 200 and 400 mg/kg b.w. of VSME markedly enhanced the activities of SOD, CAT, and GPx compared to the untreated rats. Moreover, treatment with both graded doses of the extract and the standard drug, propranolol, reduced MDA levels compared to untreated rats (Figure 1).

*Effect of VSME on antioxidant enzymes (SOD, CAT, and GPx) and lipid peroxidation markers (MDA) of CdCl2‐intoxicated Wistar rats. Bar charts depict the mean ± SD (n = 5). The significance thresholds were set at *p < 0.05, **p < 0.01, ***p < 0.001, and ***p < 0.0001, with ns indicating nonsignificance compared with the untreated group.

Emerging research outcomes have established that oxidative stress plays a pivotal role in cadmium‐induced toxicity [12]. Remarkably, studies have shown that the heart is particularly susceptible to oxidative stress due to its relatively low antioxidant capacity, high mitochondrial content, and metabolic demands [26, 27]. Cd‐induced oxidative stress can cause cardiac damage by depleting endogenous antioxidant defenses required to maintain redox homeostasis [5, 28]. In this study, the elevated MDA and reduced SOD, CAT, and GPx in the untreated group indicate enhanced lipid peroxidation and impaired antioxidant defense following Cd exposure. Hussein et al. [1] similarly reported increased MDA with decreased SOD and CAT after CdCl_2_ intoxication. VSME significantly restored antioxidant enzyme activities and reduced MDA in Cd‐intoxicated rats, indicating mitigation of Cd‐driven oxidative stress. Phenolic compounds can neutralize oxidants by donating protons/electrons [29, 30], while flavonoids stabilize radicals via electron delocalization [31] and may chelate metals, reducing absorption and enhancing elimination. Taken together, the restoration of cardiac antioxidant defenses by VSME strongly supports its role in mitigating Cd‐driven oxidative stress, which is a central trigger of downstream lipid dysregulation and myocardial injury.

Effects of VSME on Lipid Profile

2.4

Intoxication of the rats with CdCl_2_ elicited a significant increase in the heart levels of TAG, CHOL, and LDL‐C compared with the normal rats. In contrast, untreated rats showed a decrease in HDL‐C levels. Oral administration of 200 and 400 mg/kg b.w. of VSME and 10 mg/kg b.w. of propranolol in the CdCl_2_‐intoxicated rats significantly reduced the levels of TAG, CHOL, and LDL‐C, together with an increase in HDL‐C levels compared with the untreated rats (Figure 2).

*Effects of VSME on Lipid Profile of CdCl2 Intoxicated Wistar Rats. Bar charts depict the mean ± SD (n = 5). The significance thresholds were set at *p < 0.05, **p < 0.01, ***p < 0.001, and ***p < 0.0001compared with the untreated group.

Alterations in lipid metabolism, characterized by elevated cholesterol and LDL‐C and reduced HDL‐C, contribute significantly to cardiovascular disease risk [32]. The dyslipidemia observed in Cd‐intoxicated untreated rats suggests Cd‐mediated impairment of lipid homeostasis, which may be driven by oxidative stress and metabolic dysfunction. Cd exposure has been linked to lipid metabolism disorders across organs, including the heart [6]. Notably, VSME restored lipid fractions toward normal levels, consistent with improved redox balance and reduced lipid peroxidation observed in this study. Thus, VSME may mitigate Cd‐mediated membrane damage, thiol depletion, and protein oxidation, as evidenced by reduced lipid peroxidation and restored antioxidant defenses [33].

Effect of VSME on Atherogenic/Dyslipidemia Indices

2.5

The levels of CRR, AC, CR, and AIP in the untreated group were significantly higher than those in group 1 (normal rats). Interestingly, these anomalies were markedly restored in all the extract‐treated groups (3 and 4) compared with the untreated group (2). The therapeutic activity of the standard drug was comparable to that of groups receiving graded doses of VSME (Table 3).

Atherogenic indices (AC, CRR, CR, and AIP) provide an integrated assessment of cardiovascular risk and may predict atherosclerosis more robustly than individual lipid fractions. The significant increases in these indices in Cd‐intoxicated untreated rats indicate elevated cardiovascular risk. The normalization of these indices following VSME treatment suggests that VSME may reduce overall cardiovascular risk by improving the balance of atherogenic and protective lipoproteins. Therefore, beyond improving lipid fractions, VSME may reduce overall cardiovascular risk by normalizing atherogenic indices, consistent with its antioxidant and protective biochemical effects.

Effect of VSME on Cardiac Biomarkers

2.6

There was a significant increase in CRP and troponin‐1 levels, together with increased activities of creatine kinase and lactate dehydrogenase, in the untreated group compared with normal rats. However, treatment with VSME significantly reduced these parameters compared with the untreated group, except for LDH activity, for which a nonsignificant decrease was observed in rats treated with 200 mg/kg b.w. of VSME. Interestingly, the restorative effects of the extract on CRP and troponin were comparable to those of the group treated with the standard drug, propranolol (Figure 3).

*Effect of VSME on cardiac biomarkers of CdCl2‐intoxicated Wistar rats. Results are displayed as mean ± SD (n = 5). The significance thresholds were set at *p < 0.05, **p < 0.01, ***p < 0.001, and ***p < 0.0001 compared with the untreated group.

Cardiac biomarkers such as troponin I, creatine kinase, LDH, and CRP are essential indicators of myocardial injury and systemic inflammation. The elevated levels observed in Cd‐intoxicated untreated rats indicate cardiomyocyte damage, which is consistent with cadmium's documented cardiotoxicity [4]. Troponin I is particularly valuable due to its high specificity for myocardial injury [34], while CRP reflects inflammatory activation [35]. In this study, VSME reduced the elevated cardiac biomarkers in Cd‐intoxicated rats, indicating preservation of myocardial membrane integrity and attenuation of inflammatory responses, which aligns with the observed restoration of antioxidant defenses and improved lipid status

Histological Assessment of the Heart Tissues

2.7

The heart section of the untreated group was characterized by multifocal areas of myocardial necrosis (black arrow) with infiltration of mononuclear leukocytes, compared with the normal myocardial histomorphology showing myocyte nuclei (white arrow), pericytes (black arrow), and capillaries (red arrow) in normal rats. On the other hand, CdCl_2_‐induced cardiac lesions were reduced by treatments with VSME and the standard drug, as shown in the observed myocyte nuclei (white arrow) and pericyte (black arrow) (Plate 1).

2D and 3D complexes of kaempferol (A), isorhamnetin (B), luteolin (C), and CPUY192018 (D) with Keap1.

Histopathological analysis is commonly used to validate biochemical findings [5]. The myocardial necrosis and inflammatory infiltration observed in Cd‐intoxicated untreated rats confirm structural injury. Oxidative stress and inflammation can provoke necrosis/apoptosis and activate pathways such as NF‐κB [12]. The improved myocardial architecture in VSME‐treated groups corroborates the biochemical restoration of antioxidant defenses and normalization of cardiac biomarkers. Overall, the histological recovery provides structural confirmation of the biochemical cardioprotection conferred by VSME.

Molecular Interaction of VSME With Keap1

2.8

Three of the phytochemicals identified from VSME, kaempferol, isorhamnetin, and luteolin, had significant binding interaction with Keap1. The molecular docking results revealed that kaempferol, isorhamnetin, and luteolin registered docking scores of −10.190, −9.413, and −9.211 kcal/mol, respectively, compared with −8.149 kcal/mol for the standard inhibitor, CPUY192018. The binding was achieved via hydrogen bonds with key amino acids, including GLY 367, VAL 606, ALA 510, VAL 512, and THR 560, as shown in Table 4 and Figure 4.

Representative histological sections of heart tissue from experimental groups. Figure legends: White arrow (myocyte nuclei), black arrow (pericyte), yellow arrow (myocardial necrosis), and red arrow (capillaries). (A) Group 1: the normal rats, no intoxication and treatment; (B) Group 2: intoxicated only (untreated); (C) Group 3: intoxicated and treated with 200 mg/kg of VSME; (D) Group 4: intoxicated and treated with 400 mg/kg of VSME; and (E) Group 5: intoxicated and treated with 10 mg/kg of propranolol.

Inhibition of the Keap1–Nrf2 interaction is an emerging strategy to enhance antioxidant responses in oxidative stress‐related diseases [36, 37]. It is worth mentioning that several phytochemicals have been reported to protect against oxidative stress by modulating the Keap1–Nrf2 pathway [36]. The strong binding of kaempferol, isorhamnetin, and luteolin to key Keap1 residues within the Nrf2‐binding domain provides supportive (predictive) mechanistic evidence that these constituents may hinder Keap1‐mediated repression of Nrf2. These docking results, therefore, complement the in vivo antioxidant restoration, although experimental validation of Nrf2 pathway activation is required.

Drug‐Likeness, Pharmacokinetics, and Toxicity Properties

2.9

In this study, our hit compounds (kaempferol, isorhamnetin, and luteolin) were predicted to have excellent drug‐likeness properties by not violating the rule of five by Lipinski against the standard CPUY192018, which violated two rules: MW > 500 and several rotatable hydrogen bonds > 10. Interestingly, the hits had a favorable bioavailability score (BSA) of 0.55, good lead‐likeness properties, high gastrointestinal absorption (GIA), and LD 50 values. In contrast, CPUY192018 was predicted to have poor BAS, low GIA, and LD50 values, and 3 violations of lead‐likeness features (MW > 350, XLOGP3> 3.5, Rotors > 7), as shown in Table 5.

ADMET prediction supports early assessment of drug‐likeness and safety [30]. The favorable Lipinski compliance, bioavailability score, high GIA, and predicted safety class of the top‐hit flavonoids support their potential translational relevance as antioxidant‐oriented candidates. Collectively, these in silico properties support the potential translational relevance of the identified bioactives as candidates for cardiovascular protection against oxidative stress.

Conclusions

3

This study demonstrates that VSME exerts significant protective effects against cadmium‐induced cardiotoxicity in Wistar rats. The extract improved oxidative stress parameters by restoring antioxidant enzyme activities and reducing lipid peroxidation, as evidenced by decreased MDA levels. It also corrected dyslipidaemia and reduced atherogenic indices, thereby mitigating cardiovascular risk. Additionally, VSME reduced levels of critical cardiac biomarkers (troponin, CK, LDH, CRP), indicating a protective role against myocardial injury. Histopathological evaluation corroborated these findings, showing preserved myocardial architecture in extract‐treated rats.

HPLC profiling revealed the presence of cardioprotective flavonoids, including quercetin, kaempferol, luteolin, and apigenin. Molecular docking analysis supported their potential to disrupt Keap1–Nrf2 interactions, which will enhance antioxidant gene expression. Furthermore, ADMET predictions confirmed the drug‐likeness, safety, and favorable pharmacokinetic profiles of the top‐scoring compounds. Collectively, these findings highlight the therapeutic potential of Vitex simplicifolia as a source of bioactive compounds for the management of Cd‐induced oxidative stress and cardiovascular toxicity.

Limitations and Future Perspectives

3.1

Despite the promising cardioprotective effects of VSME observed in this study, several limitations should be acknowledged. First, the mechanistic insights regarding the involvement of the Keap1–Nrf2 signaling pathway were inferred primarily from molecular docking analyses, which provide predictive rather than confirmatory evidence of molecular interactions. Future studies should incorporate targeted molecular and protein expression analyses, such as quantitative PCR, Western blotting, or immunohistochemistry, to validate Nrf2 pathway activation and downstream antioxidant enzyme expression in cardiac tissue. Second, the study employed only two doses of VSME and a single exposure duration; dose–response relationships, long‐term safety, and chronic exposure models warrant further investigation. Third, the chemical constituents identified by HPLC were not individually isolated or tested in vivo; therefore, the relative contributions of specific bioactive compounds to the observed effects remain to be elucidated. Future research should focus on bioactivity‐guided fractionation, pharmacokinetic profiling, and evaluation of isolated compounds. Finally, although the rat model provides valuable preclinical insight, translation of these findings to humans will require additional validation in other experimental models and, ultimately, clinical studies.

Experimental Section

4

Chemicals

4.1

All the chemicals and reagents used for the research were of analytical grade and sourced from reputable companies. The dose of CdCl_2_ (5 mg/kg b.w.) used to induce cardiotoxicity was selected based on a previous study [38, 39], while propranolol (10 mg/kg) was selected based on preliminary studies.

Plant Collection and Identification

4.2

The Roots of V. simplicifolia were collected from February to March 2023 by botanist Mr. Alfred Ozioko, a staff member of the International Centre for Ethnomedicine and Drug Development (InterCEDD) Research Centre in Nsukka, Enugu State, Nigeria. The voucher specimens with reference No interCEDD/868 were catalogued in the taxonomist's herbarium (InterCEDD).

Preparation of V. simplicifolia Extract

4.3

The air‐dried and pulverized roots of V. simplicifolia (800 g) were macerated in a methanol‐distilled water mixture (80:20 v/v) for 72 h with continuous agitation to ensure thorough mixing and efficient extraction of bioactive compounds ([40]. The macerated solution was initially filtered through muslin cloth and subsequently filtered again using Whatman filter paper no. 1 to eliminate residual particles. The filtrate was evaporated using a rotary evaporator (R‐215, Buchi, Flawil, Switzerland) at 40 °C. The dried methanol extract of V. simplicifolia (VSME) with a yield of 124 g (16%) was preserved in an airtight container and refrigerated until required.

High‐performance Liquid Chromatography Analysis (HPLC) Phytochemical Profiling

4.4

The phytochemicals present in the extract were characterized using HPLC (Shimadzu Co., Kyoto, Japan) coupled with a fluorescence detector. The extract and reference compounds were filtered with a syringe filter, and 50 mL of filtrate from each sample was injected at a flow rate of 1 mL/min for 50 min. The sample (10 g) was prepared by dissolving it in an amber bottle containing 20 mL of acetonitrile/methanol and shaking vigorously. Subsequently, the upper organic layer was carefully decanted into a standard flask (25 mL) and filled up to the mark. Calibration of the machine was done using standard analysts before inserting an aliquot of the sample. Finally, the retention times and peak areas of the samples were compared with those of the standard analytes to determine the concentration of each phytochemical.

Experimental Animals

4.5

A total of 25 mature male Wistar albino rats, weighing between 76.6 and 110.6 grammes, were acquired from the Animal House of the Department of Zoology at the University of Nigeria, Nsukka. The animals were housed in stainless steel cages under controlled laboratory conditions, with a 12‐hour light/dark cycle and an ambient temperature maintained between 23°C and 26°C. Rats were given unrestricted access to clean water and a conventional rodent diet. Before the investigation commenced, all animals underwent a 2‐week acclimatization phase to reduce stress and ensure physiological stability.

The guidelines of the National Institutes of Health Guide for the care and use of laboratory animals [41] were adhered to strictly, and official approval for the study with approval number: UNN/FBS/23/SS.16261was received from the Faculty of Biological Sciences Research Ethics Committee (FBSREC) of the University of Nigeria. Throughout the study, deliberate effort was made to minimize pain and distress, with animals handled gently by trained personnel and carefully observed for signs of discomfort or adverse effects. As the experimental procedures did not involve surgical interventions, routine analgesic administration was not required. Euthanasia after the study was done with intraperitoneal injection of thiopental (50 mg/kg) following guidelines set by the American Veterinary Medical Association (AVMA) for humane endpoints. Confirmation of death was established by evaluating respiratory cessation, fixed and dilated pupils, and absence of a heartbeat. This study was reported in accordance with the ARRIVE 2.0 guidelines, which promote transparent and reproducible reporting of animal research [42].

The Acute Toxicity and Tolerated Dose of the Extract

4.6

We assessed the safety profile and maximum tolerated dose of VSME using Lorke's [43] experimental protocols. Eighteen Wistar mice were randomized into six groups (n = 3) and orally administered VSME at doses of 10, 100, 1000, 1600, 2800, and 5000 mg/kg body weight. The animals were observed for 48 h for any signs of toxicity, behavioral changes, or mortality.

Induction and Treatment of Cardiotoxicity

4.7

The 25 study rats were randomly allocated to five groups, with five rats per group. Group 1 was not administered cadmium or experimental treatments. Groups 2 to 5 received CdCl_2_ at a dose of 5 mg/kg body weight orally daily for 21 consecutive days to induce cardiotoxicity [38, 39]. Group 2 underwent no therapy (Cd‐only control). Groups 3 and 4 were treated with 200 mg/kg and 400 mg/kg b. w. of VSME, respectively, through oral delivery. Group 5 received 10 mg/kg of propranolol, serving as the conventional pharmacological intervention. Based on the acute oral toxicity study, which revealed no mortality or observable toxic effects up to 5000 mg/kg body weight (LD_50_ > 5000 mg/kg), doses of 200 and 400 mg/kg were selected for the main experiment. These doses represent approximately 1/25 and 1/12.5 of the highest tested nontoxic dose and were chosen to ensure safety while allowing evaluation of potential cardioprotective and antioxidant effects. All treatments were delivered simultaneously with CdCl_2_ exposure during the 21‐day research period.

Sample Collection and Processing

4.8

On day 21 of the experiment, rats were fasted overnight and placed on a clean, sterile surgical surface under aseptic conditions. Animals were deeply anesthetized with thiopental sodium (50 mg/kg, intraperitoneal) in accordance with the AVMA Guidelines for the Euthanasia of Animals, which recognize barbiturates as acceptable agents for both anaesthesia and euthanasia when administered at appropriate doses [44]. Thiopental was selected due to its rapid onset of action and ability to induce stable deep anaesthesia, thereby minimizing stress and physiological alterations that could affect oxidative stress and cardiac biomarkers [45].

Along the sternum, a small midline incision was made in the chest area with a sharp sterile surgical blade. The heart was carefully located and isolated with a sterile spatula, then portioned into two segments. The first section was homogenized using a 0.1 M phosphate buffer solution prior to centrifugation for 10 min at 5000 rpm. The resultant supernatants were utilized immediately for biochemical analysis. The second section was preserved in a 10% neutral formalin fixative solution and utilized for histological analysis. An independent experimenter conducted sample decoding and coding in order to guarantee that the investigators were blinded to sample identity.

Biomarker Determination

4.9

Determination of Oxidative Stress Biomarkers

4.9.1

The extent of lipid peroxidation in the heart homogenate was assessed by quantifying the level of malondialdehyde (MDA), following the method outlined by Wallin et al. [46]. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were assayed by the protocols of Fridovich [47], Paglia and Valentine [48], and Aebi [49], respectively.

Determination of Lipid Profile Biomarkers

4.9.2

The following methods were used to determine the heart homogenate lipid profile using Randox commercial kits (Randox Lab. Ltd., UK, Antrim, UK): Cholesterol level was measured as described by Allain et al. [50]; triacylglycerol level was measured as described by Albers et al. [51]; HDL‐C was obtained after precipitating LDL‐C and VLDL as described in Albers et al. [51]. In contrast, LDL‐C was determined with the polyvinyl sulfate method as described by Assmann et al. [52].

Determination of Atherogenic/Dyslipidaemia Indices

4.9.3

The atherogenic/dyslipidaemia indices were computed from the formulae of Chukwuma et al. [32] as shown below:

• Atherogenic coefficient (AC) = Total cholesterol‐HDL‐CHDL‐C
• Cardiac risk ratio (CRR) = Total cholesterolHDL‐C
• Classical ratio (CR) = LDL‐CHDL‐C
• Atherogenic index of plasma (AIP) = log10 TriglycerideHDL‐C

Determination of Cardiac Biomarkers

4.9.4

The levels of C‐reactive protein (CRP) and troponin‐1 in the heart tissue homogenate were measured using an ELISA kit (Abcam, Cambridge, MA, USA), while the activities of creatine kinase and lactate dehydrogenase were investigated using the spectrometric assay by quantifying the change in absorbance at 340 nm due to NADH production and NAD^+^ reduction or NADH oxidation, respectively.

Cardiac Histopathology

4.9.5

The heart was processed in graded solutions of ethanol for histopathological examination as described in Onyesife et al. [29]. The tissue sections were stained with haematoxylin and eosin, and the photomicrographs were captured using a compound microscope (Olympus BX‐51, New York Microscope Company, USA) at magnifications of x100 and x400.

Molecular Docking Studies of Protein‐Ligand Interactions

4.10

The 3D x‐ray crystallographic structure of the target protein, Kelch‐like ECH‐associated protein 1 (PDB ID: 4l7b), was obtained from the Protein Data Bank (https://www.rcsb.org/). The SDF formats of VSME HPLC‐identified compounds and the standard Keap1 protein inhibitor, CPUY192018 (PubChem CID: 73330369), were retrieved from the PubChem chemical library (https://pubchem.ncbi.nlm.nih.gov/). The protein and ligands were prepared using the Protein Preparation Wizard and LigPrep panels of the Schrödinger Suite (version 2020–3), respectively. Subsequently, the prepared ligands were docked using the Schrödinger Ligand Docking program to the predicted binding site of Keap1, which was selected based on size, site score, Dvolume, and volume, as described in our previous studies [53, 54]. The docked complexes with the strongest binding interactions were further visualized to analyze protein–ligand interactions, and the results were presented as 2‐ and 3‐D conformations.

Evaluation of Drug‐Likeness and ADMET Properties of the HPLC‐Identified Compounds From VSME

4.11

The SwissADME algorithm was utilized to assess the drug‐likeness, pharmacokinetics (absorption, distribution, metabolism, and excretion (ADME)), and medicinal chemistry of the substances found using VSME. Furthermore, we used the ProTox‐II webserver (https://tox.charite.de/protox3/?site=compound_input) to assess the toxicity of the highest‐ranking compounds from VSME and the reference ligand.

Data Analysis

4.12

The data generated during this research were analyzed using GraphPad Prism version 9 software (GraphPad Software, Inc., California, USA) by one‐way analysis of variance (ANOVA). Differences within the experimental groups were assessed utilizing Tukey's post hoc multiple comparisons. The standard threshold for statistical significance was set at p < 0.05, p < 0.01, p < 0.001, or p < 0.0001. Results were displayed as mean ± standard deviation (SD).

Author Contributions

Ifeoma F. Chukwuma: conception, design of the experiment, experimental and in silico studies, data interpretation, supervision and critical review of the paper. Okechukwu Ignatius Eze: methodology, data analysis, writing of the original draft, Ogechukwu Colet Okeke: methodology, resources acquisition, and writing of the original draft. Victor O. Apeh: methodology, data curation, interpretation and writing of manuscript. Timothy Prince Chidike Ezeorba: methodology, data analysis, molecular docking, and writing of the manuscript. Chima Okafor: acquisition of the reagents and chemicals, methodology, drafted paper. All authors contributed to the reading of the final manuscript and approved it for publication.

Funding

The authors declare that they did not receive any financial support from any source. The study was funded solely by the authors.

Conflicts of Interest

There is no competing interest to declare.

Ethics Approval and Consent to Participate

The guidelines of the National Institutes of Health Guide for the care and use of laboratory animals [41] were adhered to strictly, and official approval for the study with approval number: UNN/FBS/23/SS.16261was received from the Faculty of Biological Sciences Research Ethics Committee (FBSREC) of the University of Nigeria.

Consent for publication

This does not apply to this paper.

Supporting information

Supporting File 1: cbdv71102‐sup‐0001‐SuppMat.pdf