Effects of vitexin derived from Prosopis farcta in a rat model of experimental heart failure

Abstract

Background:

Heart failure (HF) is a chronic cardiovascular condition and remains a leading cause of morbidity and mortality worldwide despite advances in pharmacological and non-pharmacological interventions. Vitexin, a natural flavonoid derived from Prosopis farcta, has demonstrated cardioprotective effects in several cardiovascular conditions; however, its efficacy in HF remains unclear. This study aimed to examine the effects of vitexin in a rat model of HF.

Method:

Thirty male albino rats were randomly assigned to five groups (n = 6 per group). Group I (negative control) received intraperitoneal isotonic saline for 7 days, followed by oral placebo for 14 days. Group II (positive control) received intraperitoneal isoprenaline (5 mg/kg/day) for 7 days to induce HF, followed by placebo for 14 days. The remaining 18 HF-induced rats also received isoprenaline for 7 days and were subsequently randomized into three treatment subgroups: vitexin (30 mg/kg/day, orally), digoxin (7 µg/kg/day, oral gavage), or carvedilol (5 mg/kg/day, oral gavage) for 14 days. The CODA tail-cuff system was used to measure blood pressure and heart rate on days 1, 7, and 14. At the end of the experiment, rats were anesthetized, and blood and 24-hour urine samples were collected to assess cardiac biomarkers (troponin-I, B-type natriuretic peptide, N-terminal pro-B-type natriuretic peptide, matrix metalloproteinase-9, alkaline phosphatase, and creatine phosphokinase) and renal function parameters.

Results:

Isoprenaline-induced HF significantly increased cardiac biomarkers, impaired renal function, and reduced systolic, diastolic, and mean arterial pressures. Vitexin significantly improved cardiac biomarkers, renal function parameters, and blood pressure, while heart rate remained unchanged. Digoxin and carvedilol also improved these parameters.

Conclusion:

Vitexin derived from P. farcta exhibited cardioprotective and renoprotective characteristics in a rat model of HF, and the observed therapeutic effects were comparable to those of the reference treatments evaluated in this study; these findings support further preclinical and clinical investigations to evaluate the therapeutic potential of vitexin.

Introduction

Heart failure (HF) remains the leading cause of mortality, morbidity, hospital admissions, and reduced quality of life worldwide, causing significant health and financial costs^[1^]. Its prevalence rises markedly with increasing age. Worldwide, it affects about 1.5–2% of the population^[2^], and its prevalence is expected to continue rising due to factors such as an aging population and the rising prevalence of risk factors^[3^].

HIGHLIGHTS

• Vitexin, derived from Prosopis farcta, demonstrated significant cardioprotective effects in an isoprenaline-induced rat model of heart failure, reducing troponin-I, creatine phosphokinase, alkaline phosphatase, B-type natriuretic peptide, and N-terminal pro-B-type natriuretic peptide.

• Vitexin improved renal function parameters, including creatinine, blood urea nitrogen, urine output, and estimated glomerular filtration rate, demonstrating dual cardio-renal protective actions.

• Vitexin markedly attenuated inflammatory and remodeling markers, most notably matrix metalloproteinase-9, thereby indicating a potential role in preventing myocardial fibrosis.

• The efficacy of vitexin was comparable to, or exceeded, that of carvedilol and digoxin in several measured biochemical and hemodynamic endpoints.

• The study provides the first direct comparative evaluation of vitexin versus standard heart failure drugs within a unified experimental model.

• Findings support vitexin as a promising plant-derived therapeutic candidate for future pharmacological development in cardiovascular disease.

In HF, the body compensates for the reduced cardiac output by stimulating the sympathetic nervous system and activating different neurohormonal pathways^[4^] such as the renin-angiotensin-aldosterone system (RAAS), which promotes fluid retention, vasoconstriction, and increased cardiac muscle contractility^[5^]. These compensatory mechanisms, however, can worsen HF over time^[6^]. Cardiac biomarkers represent a significant advancement in the diagnosis of HF^[7,8^], especially B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP), which promote natriuresis, diuresis, and vasodilation^[9,10^]. Other important HF biomarkers include alkaline phosphatase (ALP), creatine kinase, troponin-I, and matrix metalloproteinases (MMPs)^[11^].

Multiple traditional medicines have been used over the years to treat HF, including beta-blockers, inotropic agents, aldosterone antagonists, and diuretics. These drugs are costly and often cause side effects, which lead to nonadherence to treatment ^[12^]. Because of this, alternative therapies that are safer and more cost-effective are promoted, especially those derived from botanical sources. Keihanian et al (2023)^[13^] found that several plants showed great potential to significantly improve cardiac function by enhancing left ventricular remodeling and reducing hs-CRP and NT-proBNP levels, without causing liver or kidney damage. Zhang et al (2023)^[14^] identified additional plants that promote heart health by modulating the NF-κB and RAAS pathways.

Prosopis farcta (Syrian Mosquito) is a native species in the Fabaceae family. Due to the benefits of this plant, which have been reported earlier, it was chosen for its medicinal properties linked to phytochemicals such as alkaloids, glycosides, flavonoids, tannins, quinones, and phenolic compounds with antioxidant activities^[15,16^]. It contains vitexin, a natural flavonoid with therapeutic benefits. In a study by Al-Jeboory and Dizaye (2006)^[17^], it was reported that vitexin produced a positive inotropic effect and decreased mean arterial blood pressure (BP) through diuretic actions; however, its role in HF remains unclear. Thus, this study aims to evaluate the potential of vitexin extracted from P. farcta for the treatment of HF, in comparison with digoxin and carvedilol.

This article has been prepared in accordance with TITAN 2025 guidelines^[18^].

Materials and methods

Animals

In this study, 30 male albino rats weighing 200–250 g were included. The animals were obtained from the Animal House at Hawler Medical University’s College of Pharmacy. The sample size (n = 6 per group) was determined based on previous experimental pharmacology studies that used isoprenaline-induced HF models. In these studies, similar group sizes demonstrated measurable biochemical and hemodynamic differences. This number was considered sufficient to detect effects that are biologically relevant while adhering to ethical principles aimed at minimizing animal use in preclinical research. All animals that were included were male albino rats weighing ≤300 g, clinically healthy, and with no known history of cardiovascular disease or prior experimental manipulation. Rats were excluded if they exhibited signs of illness, infection, injury, or abnormal behavior during the acclimatization period; if body weight exceeded 300 g; or if congenital or suspected cardiac abnormalities were identified. Animals that experienced unexpected morbidity or mortality during HF induction, or from which adequate biological samples could not be obtained, were also excluded. No animals met the exclusion criteria, and all were included in the final analysis. They were housed in clean, well-organized polypropylene cages under standard laboratory conditions, with a 12-hour light-dark cycle and a controlled temperature of 22 ± 2°C in the animal house. Rats had free access to water and a solid pellet diet, except during specific experimental protocols. All animals were acclimatized for 2 weeks to reduce stress and ensure physiological stability. All procedures followed institutional ethics guidelines, and reporting complied with the ARRIVE guidelines. This study was approved by the Research Ethics Committee of the College of Medicine at Hawler Medical University, with approval document code 17 dated 27 May 2024.

Extraction, purification, and HPLC identification of Vitexin

Dried fruits of P. farcta were collected from the Mergasor region of Erbil, Iraq, and taxonomically identified by the Department of Botany, College of Education, Salahaddin University. Extraction, purification, and chromatographic characterization of vitexin were performed in a specialized phytochemistry laboratory. The fruits were air-dried and ground into a fine powder. Vitexin was extracted from the powdered fruit using ultrasonic-assisted extraction, a widely used technique for improving the recovery of plant bioactive compounds, as reported by Kumar et al (2021)^[19^] and Zafari and Sharifi (2020)^[20^], who described the optimized extraction conditions.

Specifically, 50 g of powdered fruit material was extracted with 500 mL of 80% ethanol at 40°C for an hour using an ultrasonic bath (Labtech Power Sonic 405, South Korea). Later, the extract was filtered through Whatman filter paper (Whatman Limited, England) and concentrated under reduced pressure at 40°C using a rotary evaporator to obtain a dry crude extract. The dried extract was subsequently dissolved in absolute ethanol and purified by preparative reversed-phase high-performance liquid chromatography (HPLC) using a C18 column. The mobile phase consisted of 0.1% formic acid in distilled water (solvent A) and methanol (solvent B), with a flow rate of 1.0 mL/min and ultraviolet detection at 330–340 nm. After establishing the retention time using an authentic vitexin reference standard, the dissolved extract was repeatedly injected into the HPLC system, and fractions corresponding to the vitexin peak (approximately 16 minutes) were collected until sufficient purified vitexin was obtained for the experimental study.

Analytical HPLC confirmed the identity of the isolated vitexin through comparison with an authentic vitexin reference standard, which showed a retention time of 16.547 minutes, whereas the purified sample exhibited a major chromatographic peak at 16.036 minutes, confirming the identity of the isolated compound. Quantification was performed by comparing the sample peak area with that of the reference standard, and the corresponding chromatograms for the reference and the purified sample are provided as Supplemental Digital Content Figures S1 and S2, available at: https://links.lww.com/MS9/B418, respectively.

Experimental design

The experimental unit was composed of thirty individual albino rats, which were randomly divided into three groups. Randomization was performed using a computer-generated random number sequence in Microsoft Excel. Each animal had an equal chance of being assigned to any group. The first and second groups each consisted of six rats, while the third group was divided into three subgroups (n = 6 each). Group I (Control) received an intraperitoneal injection of isotonic saline for 7 days, followed by 2 weeks of oral placebo administration. Group II served as a positive control, receiving intraperitoneal injections of isoprenaline at a dose of 5 mg/kg for 7 days^[21^]. The rats were then given placebos for 2 weeks. Group III included 18 rats injected with isoprenaline using the same procedure as mentioned earlier and was then subdivided into three subgroups: Group IIIa received 30 mg/kg/day of vitexin orally for 14 days^[17^]. The dose of vitexin (30 mg/kg/day) was chosen based on pharmacological studies previously demonstrating cardioprotective, antioxidant, and anti-inflammatory effects of vitexin in experimental cardiovascular injury models^[21^]. Earlier investigations have shown that vitexin reduces myocardial injury by modulation of oxidative stress pathways, apoptosis, and inflammatory signaling. Although some studies administered vitexin intraperitoneally, the oral route was selected in the present study to better reflect potential translational therapeutic use and to reduce stress associated with repeated injections. Furthermore, earlier pharmacological investigations of vitexin isolated from P. farcta demonstrated positive inotropic and diuretic effects, providing mechanistic support for its evaluation in HF models^[17^]. Group IIIb was administered a daily dose of digoxin (7 μg/kg/day) via oral gavage for 14 days^[21^], and Group IIIc received a daily dose of carvedilol (5 mg/kg/day) by oral gavage for 14 days^[22^].

Isoprenaline-induced HF was produced by intraperitoneal injection of isoprenaline at 5 mg/kg/day for 7 days. The isoprenaline dosage (5 mg/kg/day for 7 days) was selected based on validated experimental HF models described in previous studies. These studies demonstrated that repeated administration of isoprenaline at similar doses induces myocardial injury, neurohormonal activation, and ventricular remodeling consistent with HF pathology. The pathophysiology of this outcome can be attributed to sustained β-adrenergic overstimulation, oxidative stress, and cardiac dysfunction^[21^]. Isoprenaline HCl (Mediver Ltd., London, UK) was weighed using an analytical balance (Sartorius, Germany) and dissolved in sterile water. Vitexin was administered orally as a freshly prepared suspension made daily. The required dose was measured precisely and suspended in an appropriate volume of distilled water to ensure uniform dispersion prior to administration.

Digoxin (Accord Healthcare, Intas Pharmaceuticals Ltd., Durham, USA) and carvedilol (Itqan Pharma, Amman, Jordan) tablets were freshly prepared every morning by dissolving the required dose in distilled water at room temperature. The validation of successful model induction was based on previously established protocols in the literature, supported by the biochemical and hemodynamic findings observed in this study. Additional evidence included clinical observations of reduced physical activity and fatigue, as well as histopathological evaluation of cardiac tissue. A CODA tail-cuff system (specifically designed for rats) was used to measure BP (systolic, diastolic, and mean arterial BP) and heart rate (HR) non-invasively. The rats were introduced to the restraint device gradually so they could become accustomed to it prior to starting the experiment. This was done to reduce stress, since stress can affect BP readings. At each measurement session, a minimum of five consecutive readings were taken for every rat, and the average of these readings was used in the final analysis. Baseline BP was recorded on day 1 for all groups. In the HF groups, additional measurements were taken on day 7 of isoprenaline administration to assess the early effects. The final measurements were recorded on day 14 of isoprenaline administration to evaluate the effect of the treatment. A warming pad was placed under the rats during the procedure to keep the tail warm and improve the accuracy and stability of the signal. Although the tail-cuff method is commonly used in animal studies and is considered practical and non-invasive, it can still be affected by stress-related changes in BP, which is one of the limitations of the study.

After the experiment, rats were anesthetized with ketamine HCl (75 mg/kg; Alfasan Woerden, Holland) and xylazine (10 mg/kg; OVER Medicina Veterinaria, Argentina)^[23^]. Blood was collected via cardiac puncture for analysis of cardiac biomarkers using rat enzyme-linked immunosorbent assay (ELISA) kits (Sunlong Biotech, China) and measurement of other biochemical parameters. Biomarker concentrations were measured using ELISA kits that quantify protein levels rather than enzymatic activity; therefore, results are expressed as mass concentrations (pg/mL or ng/mL). Twenty-four-hour urine samples were also collected to assess renal function. Because the investigator responsible for HF induction, group allocation, hemodynamic measurements, and treatment administration was required to know the assigned interventions, complete blinding of the personnel was not feasible. Therefore, these procedures were performed in an open-label manner. However, laboratory investigators responsible for biomarker assessment, the pathologist responsible for histopathological evaluation, and the statistician conducting data analysis were blinded to treatment allocation. Serum and urine samples were coded by the researcher before submission to the laboratory, and the biochemical analysis was performed by a laboratory investigator who was unaware of the treatment allocation. Histopathological evaluation was performed by a pathologist who was also blinded to the treatment groups. Group identities were not disclosed until all outcome assessments and data analyses had been completed.

Statistical analysis

Data were analyzed using the Statistical Package for the Social Sciences (SPSS), version 26, for Windows. Results are presented as mean ± standard error of the mean (SEM). The Shapiro–Wilk test was used to assess the normality of the data distribution. Group differences for normally distributed data were evaluated using one-way analysis of variance, followed by Tukey’s post hoc test for multiple pairwise comparisons. For non-normally distributed data, the Kruskal–Wallis test was used. A P-value < 0.05 was considered statistically significant.

Declaration of AI usage

No artificial intelligence tools were used in the writing, generation, and analysis of this article.

Results

Validation of isoprenaline-induced HF

Representative hematoxylin and eosin (H&E)-stained myocardial sections are shown in Figure 1. In the isoprenaline-treated rats, myocardial tissue displayed inflammatory cell infiltration, vascular congestion, degenerative myocardial cells, and interstitial edema (Fig. 1B), which are all indicative of successful HF modeling when compared with the normal myocardial tissue of the negative control rats (Fig. 1A). These representative images provide qualitative confirmation of model induction.

Figure 1.

Representative H&E-stained myocardial sections of the negative control and isoprenaline-induced heart failure groups. (A) Negative control myocardium showing (1) normal myocytes and (2) intact nuclei. (B) Heart failure myocardium showing (1) degenerative myocardial cells, (2) inflammatory cell infiltration, (3) interstitial edema, and (4) vascular congestion. Original magnification, ×400.

Effects of vitexin, digoxin, and carvedilol on different biomarkers in rats with HF

Table 1 and Figures 2 and 3 show that HF induction in rats significantly increased cardiac biomarker levels compared to controls (P < 0.05). Vitexin, digoxin, and carvedilol significantly reduced these biomarkers. However, BNP levels decreased in the digoxin (183.64 ± 17.47) and carvedilol (187.05 ± 7.55) groups but remained slightly above control levels. Vitexin had the greatest effect, lowering BNP to 177.04 ± 9.25, which was closer to the normal range (170.22 ± 5.75).

Table 1.

Effects of administration of vitexin, digoxin, and carvedilol on the serum biomarkers in rats with isoprenaline-induced HF (n = 6).

Parameters	Negative control	Heart failure	Vitexin 30	Digoxin 7	Carvedilol 5
			mg/kg/day	μg/kg/day	mg/kg/day
Troponin-I	22.64 ± 2.31	32.86 ± 2.78	24.82 ± 3.36	20. 66 ± 2.54	21.25 ± 2.32
pg/mL	a	b	a	a	a
BNP	170.22 ± 5.75	217.34 ± 13.80	177.04 ± 9.25	183.64 ± 17.47	187.05 ± 7.55
pg/mL	a	b	a	ab	ab
NT-Pro BNP	136.19 ± 5.52	213.44 ± 12.30	168.85 ± 7.62	165.06 ± 21.60	169.88 ± 6.52
pg/mL	a	b	a	a	a
ALP	1.80 ± 0.06	2.18 ± 0.12	1.79 ± 0.05	1.79 ± 0.09	1.60 ± 0.14
ng/mL	a	b	a	a	a
MMP-9	27.55 ± 0.45	40.10 ± 2.75	25.19 ± 1.82	25.87 ± 2.20	29.49 ± 1.49
pg/mL	a	b	a	a	a
CPK	197.58 ± 11.56	281.06 ± 36.54	215.69 ± 9.90	169.58 ± 7.95	167.64 ± 18.07
pg/mL	a	b	a	a	a

CPK, Creatine phosphokinase.

Data are expressed as mean values ± SEM.

Different letters indicate the significance of the result (P < 0.05).

Figure 2.

Serum troponin levels (pg/mL) in all study groups, including the negative control and the positive control (HF), as well as the treatment groups. Data are expressed as mean ± standard error (n = 6).

Figure 3.

Serum BNP (pg/mL) across all study groups, including the negative control and the positive control (HF), as well as the treatment groups. Data are expressed as mean ± standard error (n = 6).

Effects of vitexin, digoxin, and carvedilol on renal function in rats with heart

Table 2 summarizes the renal function parameters. The HF group had a significantly lower urine flow rate (0.77 ± 0.05) compared to the negative control group (1.22 ± 0.09) (P < 0.05). However, after vitexin treatment, urine output (1.23 ± 0.10) returned to control levels. Both digoxin (1.08 ± 0.13) and carvedilol (0.94 ± 0.15) also increased urine flow, though to a lesser extent. HF rats had significantly lower GFR (4.61 ± 0.27) than controls (3.08 ± 0.35) (P < 0.05). Digoxin (3.35 ± 0.36) and carvedilol (3.18 ± 0.41) modestly improved GFR, while vitexin (3.76 ± 0.38) nearly restored it.

Table 3.

Effects of vitexin, digoxin, and carvedilol on systolic, diastolic, and mean BP and HR in rats with isoprenaline-induced HF (n = 6).

Parameters	Negative control	Heart failure	Vitexin	Digoxin	Carvedilol
			30 mg/kg/day	7 μg/kg/day	5 mg/kg/day
Systolic BP	118.70 ± 4.02	98.02 ± 3.85	114.37 ± 3.48	116.65 ± 6.20	110.38 ± 6.13
mm Hg	a	b	a	a	ab
Diastolic BP	82.93 ± 2.98	67.47 ± 2.80	75.87 ± 5.22	82.37 ± 5.52	75.25 ± 4.83
mm Hg	a	b	ab	a	ab
Mean BP	94.69 ± 3.41	77.65 ± 3.05	88.70 ± 4.51	93.79 ± 5.56	86.66 ± 4.86
mm Hg	a	b	ab	a	ab
HR	294.33 ± 9.62	249.22 ± 20.56	292.60 ± 22.25	263.87 ± 23.53	234.32 ± 21.49
pulse/minute	a	a	a	a	a

Data are expressed as mean values ± SEM.

Different letters indicate the significance of the result (P < 0.05).

Table 2.

Effects of administration of vitexin, digoxin, and carvedilol on the renal function parameters in rats with isoprenaline-induced HF (n = 6).

Parameters	Negative control	Heart failure	Vitexin	Digoxin	Carvedilol
			30 mg/kg/day	7 μg/kg/day	5 mg/kg/day
Urine flow rate	1.22 ± 0.09	0.77 ± 0.05	1.23 ± 0.10	1.08 ± 0.13	0.94 ± 0.15
(mL/h/kg)	a	b	a	ab	ab
GFR	4.61 ± 0.27	3.08 ± 0.35	3.76 ± 0.38	3.35 ± 0.36	3.18 ± 0.41
(mL/min/kg)	a	b	ab	b	b
Sodium	144.5 ± 2.17	153.50 ± 3.97	144.67 ± 0.96	144.17 ± 2.86	144.33 ± 1.56
(mmol/L)	a	b	a	a	a
Creatinine	0.38 ± 0.03	0.92 ± 0.05	0.36 ± 0.01	0.35 ± 0.02	0.34 ± 0.01
(mg/dL)	a	b	a	a	a
BUN	19.23 ± 1.07	52.18 ± 1.57	14.45 ± 0.51	18.68 ± 4.21	17.71 ± 1.69
(mg/dL)	a	b	a	a	a

BUN, Blood urea nitrogen.

Data are expressed as mean values ± SEM.

Different letters indicate the significance of the result (P < 0.05).

Additionally, sodium levels in rats with HF (153.50 ± 3.97 mmol/L) were significantly higher than those in the negative control group (144.5 ± 2.17 mmol/L) (Table 2 and Fig. 4). Vitexin, digoxin, and carvedilol significantly improved serum sodium levels compared with the untreated HF group, with no significant differences among the treatment groups, and restored values to levels comparable with those of the control group. Similarly, creatinine levels in the HF group (0.92 ± 0.05 mg/dL) and blood urea nitrogen (BUN) levels (52.18 ± 1.57 mg/dL) were significantly elevated. Treatment with vitexin, digoxin, and carvedilol significantly reduced serum creatinine and BUN levels (P < 0.05), with no significant differences relative to the control group.

Figure 4.

Serum sodium levels (mmol/L) in all study groups, including the negative and positive control (HF) groups, as well as the treatment groups. Data are expressed as mean ± standard error (n = 6).

Effects of vitexin, digoxin, and carvedilol treatments on different hemodynamic measures (systolic, diastolic, mean BP, and HR) in rats with HF

Isoprenaline significantly reduced BP measurements, dropping systolic BP from 118.70 ± 4.02 mmHg to 98.02 ± 3.85 mmHg; diastolic BP from 82.93 ± 2.98 mmHg to 67.47 ± 2.80 mmHg; and mean BP from 94.69 ± 3.41 mmHg to 77.65 ± 3.05 mmHg (P < 0.05) (Table 3). Treatment with vitexin, digoxin, or carvedilol improved BP. Vitexin significantly restored systolic BP to 114.37 ± 3.48 mmHg and raised diastolic BP and mean BP compared with HF rats.

In addition, digoxin significantly lowered systolic BP to 116.65 ± 6.2 mmHg, diastolic BP to 82.37 ± 5.52 mmHg, and mean BP to 93.79 ± 5.56 mmHg compared with HF rats. Carvedilol showed consistent effects, further reducing the HR to 234.32 ± 21.49 bpm, while vitexin (292.60 ± 22.25 bpm) and digoxin (263.87 ± 23.53 bpm) partially restored it. Additionally, carvedilol improved all three BP parameters, whereas HR remained unchanged across groups.

Discussion

The present study investigated the impact of vitexin, a flavonoid extracted from P. farcta, on cardiac biomarkers, renal function parameters, hemodynamics, and electrolyte balance in male albino rats with HF. HF was induced via an intraperitoneal injection of isoprenaline (5 mg/kg/day) for 7 days, which triggers HF through sustained beta-adrenergic receptor stimulation, promoting oxidative stress, cardiomyocyte apoptosis, and neurohormonal activation. These processes cause myocardial tissue changes, renal dysfunction, and disrupted BP regulation^[24,25^]. The representative H&E-stained myocardial sections provided qualitative confirmation of successful isoprenaline-induced HF. The observed structural myocardial alterations were consistent with the biochemical and hemodynamic abnormalities detected in the current study, supporting the validity of the experimental HF model.

In this study, isoprenaline, used to induce HF, resulted in significant increases in all cardiac biomarkers. Elevated troponin-I levels, a specific marker of cardiomyocyte necrosis, confirm structural damage resulting from oxidative stress and calcium overload^[26,27^]. A prior study showed similar results, indicating that BNP and NT-proBNP reflect ventricular wall stress and neurohormonal activation^[28^], which contribute to HF progression^[11,29^]. Chronic high levels of natriuretic peptides may indicate hemodynamic overload, RAAS, and sympathetic nervous system overactivation, which contribute to negative cardiac remodeling^[30,31^].

Furthermore, an increase in creatine phosphokinase (CPK) levels may indicate a loss of membrane integrity and leakage of intracellular enzymes into the circulation, a hallmark of cardiac injury in HF^[32^]. In addition, elevated levels of ALP could be a systemic indication of inflammation and endothelial dysfunction; both are associated with poor outcomes in HF^[33^]. Additionally, enhancement of extracellular matrix degradation and fibrotic remodeling might lead to an increase in MMP-9 activity, which contributes to ventricular dilation and impaired contractile function^[33^]. Together, these biomarkers may confirm that the isoprenaline model effectively represents HF pathology and provide a basis for assessing vitexin’s cardioprotective effects, including antioxidant, anti-inflammatory, and anti-apoptotic effects that may counteract HF pathological changes and serve as a comparison with conventional HF therapies.

Vitexin isolated from P. farcta lowered cardiac biomarkers, likely by reducing myocardial injury. Its antioxidant and anti-inflammatory properties may inhibit reactive oxygen species and pro-inflammatory cytokines, decreasing cardiomyocyte damage and troponin-I and CPK release. In addition, vitexin significantly reduced MMP-9 activity, which may suggest less cardiac fibrosis^[34^]. Babaei et al (2020)^[35^] showed that 30 mg/kg of vitexin administered intraperitoneally similarly reduced markers of cardiac hypertrophy, as well as BNP and pro-BNP levels. These findings are consistent with previous studies suggesting that vitexin may reduce myocardial stress. Although prior studies administered vitexin intraperitoneally, in this study, oral administration was used to better simulate potential clinical translation and to reduce stress during repeated procedural dosing protocols. Furthermore, earlier pharmacological investigations of vitexin isolated from P. farcta demonstrated positive inotropic and diuretic effects, providing mechanistic support for its evaluation in HF models^[17^].

Vitexin was compared with two established pharmacological agents used in the management of HF, namely digoxin and carvedilol. Digoxin is a cardiac glycoside that inhibits the Na +/K +-ATPase pump, increasing calcium and contractility, raising vagal tone, and indirectly reducing biomarker levels^[36^]. Carvedilol, a non-selective β-blocker with α1-adrenergic blocking properties, decreases HR, BP, sympathetic activity, and oxidative stress, thereby affecting biomarker levels^[37^]. Digoxin and carvedilol significantly reduced cardiac biomarkers; however, BNP levels remained numerically higher than those of the control group. Vitexin also reduced BNP levels and demonstrated a cardioprotective effect in this experimental model. Overall, the pattern of improvement was similar to that observed following treatment with digoxin and carvedilol.

In this study, HF induction impaired renal function, as shown by significant reductions in urine flow rate and glomerular filtration rate (GFR), along with increased BUN and serum creatinine levels, likely due to decreased cardiac output and renal hypoperfusion, affecting waste filtration and fluid regulation^[38,39^]. In this study, there was also significant hypernatremia in animals with HF, suggesting impaired renal sodium handling, worsened by RAAS and sympathetic activation. This imbalance might have led to fluid retention and cardiac congestion, suggesting that HF progression was accelerating. Vitexin treatment significantly improved the renal function parameters, which may be attributed to its antioxidant and anti-inflammatory properties^[40–44^].

Moreover, digoxin (7 µg/kg) and carvedilol (5 mg/kg) improved renal function in rats with HF, normalizing serum sodium, serum creatinine, and BUN levels compared to untreated HF animals. These effects may be attributed to increased cardiac output and neurohormonal modulation, particularly sympathetic and RAAS pathways^[45,46^].

Furthermore, hemodynamic measurements revealed that HF caused significant reductions in systolic, diastolic, and mean arterial BP compared with controls, aligning with prior research that HF significantly affects BP^[28^], reflecting compromised cardiac output and impaired vascular tone. HR, however, remained relatively constant across treatments.

Vitexin from P. farcta significantly improved BP after 14 days, potentially reflecting its cardioprotective properties, suppression of oxidative stress, and enhancement of overall cardiovascular function^[47–49^]. Digoxin improved BP possibly through its positive inotropic and vasoregulatory actions^[50,51^]. Carvedilol only partially improved BP readings, likely due to persistent structural and neurohormonal changes^[52^]. These findings suggest that vitexin improves hemodynamic parameters in this experimental model; however, further preclinical and clinical studies are required to establish its therapeutic role in HF.

This study has several limitations. Echocardiography was unavailable in our experimental setting. The relatively small sample size may have limited statistical power to detect subtle differences between treatment groups. In addition, although outcome assessment and data analysis were blinded, group allocation and treatment administration were not, potentially introducing bias. Another limitation is that complementary analytical techniques, such as ultraviolet-visible, mass spectrometry, or nuclear magnetic resonance spectroscopy, were not available to further characterize the isolated vitexin. Although identification was performed by comparison with an authentic reference standard using HPLC, these additional techniques would have provided further confirmation of its structural identity and purity. However, because complementary analytical characterization was unavailable, the purity of the isolated compound could not be experimentally verified. Therefore, no unsupported purity value is reported. Furthermore, representative histopathological images were included to qualitatively confirm successful establishment of the isoprenaline-induced HF model; however, quantitative histopathological scoring was not performed, and future studies incorporating standardized histopathological scoring would provide a more objective assessment of myocardial injury. Finally, a CODA tail-cuff system was used to measure BP, which is sensitive to stress and may introduce measurement variability.

Conclusion

To our knowledge, this is the first study to investigate the cardioprotective and renoprotective effects of vitexin derived from P. farcta in an isoprenaline-induced experimental model of HF. Vitexin significantly reduced cardiac biomarkers, including troponin-1, BNP, NT-proBNP, ALP, MMP-9, and CPK, which suggests preservation of cardiac performance. It improved renal function, with increased urine flow rate and GFR, and normalized serum sodium, creatinine, and BUN. Vitexin restored BP to normal levels and maintained a stable HR comparable to that of the control group, as shown by hemodynamic measurements. Notably, this may also be the first comparison of vitexin with HF drugs such as digoxin and carvedilol. These drugs produced the expected effects, and vitexin showed improvements in cardiac and renal parameters that were comparable to those observed following treatment with digoxin and carvedilol. These findings support further investigation of vitexin as a potential therapeutic candidate for HF. Further preclinical and clinical trials are needed to confirm these results and assess the translational potential of vitexin.

Ethical approval

This study received approval from the Research Ethics Committee of the College of Medicine at Hawler Medical University, with approval document code 17, dated 27 May 2024. All experimental procedures in this study followed the “Guide for Care and Use of Laboratory Animals” provided by the National Academy of Science and published by the National Institute of Health.

Consent

Not applicable.

Sources of funding

The authors did not receive any funding for this research.

Author contributions

S.R.A.: Conceptualization, study design, methodology, animal handling and experimental procedures, data collection, interpretation of results, writing – original draft preparation, manuscript revision, preparation of tables and figures, and submission responsibilities. K.D.: Supervision, project administration, validation of experimental methods, oversight of data accuracy, statistical analysis, interpretation of pharmacological findings, writing – review and editing, critical revision of the manuscript for intellectual content, and approval of the final version for publication.

Conflicts of interest disclosure

The authors declare no conflict of interest.

Research registration unique identifying number (UIN)

Not applicable.

Guarantor

Sundus Rashad Ahmed.

Provenance and peer review

Not commissioned, externally peer reviewed.

Data availability statement

All data are stored and secured in our database and are available upon request.