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Abstract

Background

Cardiovascular diseases represent 32% of global fatalities. A novel treatment is imperative to prevent heart failure (HF). This study investigates Allium cepa L. oil (ACO) as a prospective therapy for HF while improving HF treatments. ACO exhibited an abundant amount of organosulfides and antioxidants during GC-MS analysis.

Method

In vivo trial was conducted in male Wistar albino (n = 30), six were kept as NC, while 24 were injected with isoproterenol (ip; 5 mg/kgb.w) for seven days to induce heart failure and divided into positive control (PC), standard (digitalis 0.0225 mg/kgb.w), T1 (ACO; 40 µmol/ kgb.w), and T2 (co-treated; ACO 40 µmol/kgb.w + digitalis 0.01125 mg/kgb.w). Treatments were administered orally for 28 days, afterwards, rats were decapitated to collect serum and tissues.

Result

T2 groups showed a significant increase (P ≤ .05) in serum and urine H₂S; 85.7 ± 1.4 µmol/ml, 59.2 ± 0.7 µmol/ml respectively. The mRNA expression of CBS; 2.2 ± 0.24, CSE; 1.9 ± 0.5, 3-MPTS; 1.48 ± 0.4, eNOS; 2.9 ± 0.6 were significantly (P ≤ .05) upregulated in T2 group. Myocardial profiles, and oxidative stress markers Pro-BNP; 138.5 ± 1.7 pg/ml, Trop-1; 1.4 ± 0.2 µg/l, CK-MB; 48.7 ± 1.1 µg/ml, LDH; 48.9 ± 1.02 µg/dl, TOS; 35.8 ± 1.4 mmol/L, MDA; 0.3 ± 0.1 µmol/l, and R-R interval; 0.74 ± 0.50 ms were significantly (P ≤ .05) decreased in T2 as compared to PC. Reduced collagen and increased elastic fiber contents were observed in ventricular and aortic tissue of T2 group suggest ventricular remodelling.

Conclusion

Co-treated group (ACO 40 µmol/kgb.w + digitalis 0.01125 mg/kgb.w) exhibited notable cardioprotective effects, thereby improving myocardial profile and antioxidant status, as well as elevated serum hydrogen sulfide (H₂S) levels in HF.

Keywords

heart failure oxidative stress ventricular remodelling hydrogen sulfide

Introduction

Heart failure is caused by a loss of the critical quantity of functional myocardial cells after injury to the heart from several causes. Heart failure is a clinical illness characterized by the heart's inability to deliver adequate blood flow to satisfy metabolic demands or manage systemic venous return. This prevalent ailment impacts more than 5 million individuals in the United States, incurring an annual expense of $10-38 billion.¹ The World Health Organization (WHO) estimates that there were 17.9 million deaths worldwide in 2019. The situation in the Mediterranean region is increasingly concerning, with 54% of fatalities attributed to cardiovascular problems.² Approximately 80% of these deaths occur in low- and middle-income countries.³ The mortality rate among heart failure patients exceeded that of infectious and nutritional diseases.⁴

Over sixty-four million individuals worldwide suffer from heart failure (HF). In Pakistan 28.9% experienced de novo heart failure. Recurrence of heart failure (HF) was noted in 57.7%, while the death rate in the heart failure cohort was 24%.⁵ A cross-sectional study conducted in 2018 revealed that 17.5% (1109/6351) of the population exhibited cardiovascular anomalies.⁶ Thus, efforts to alleviate its financial and social impacts have emerged as a critical priority for global public health.⁷

Heart Failure (HF) results from circulatory abnormalities and is characterized by significant costs, impaired cardiac function, morbidity, diminished quality of life, and mortality.⁸ HF is typically affected by ventricular remodeling during its initiation and progression.⁹ The American Heart Association recommends that treatment for heart failure (HF) includes combination therapy utilizing many pharmaceutical categories, including angiotensin-converting enzyme (ACE) inhibitors or angiotensin receptor antagonists (ARBs), diuretics, and beta-blockers etc.¹⁰

Despite the availability of guideline-based medications, their limited efficacy highlights the urgent need for innovative therapies. A comprehensive approach to re-examining the pathways of heart disease as a progressive condition could significantly benefit public health and medical research. Investigating plants as a rich source of medicinal compounds, such as organosulfides, presents promising therapeutic possibilities for cardiovascular health. Preliminary studies indicate that organosulfides may aid in developing sustainable, natural therapies that enhance patient outcomes and offer protection against cardiovascular disease.¹¹

Hydrogen sulfide (H₂S) is a third endogenous gasotransmitter that has been known after nitric oxide and carbon monoxide.¹² It is endogenously produced by cystathionine-alpha lyase (CSE), cystathionine beta-synthase (CBS), and 3- mercapto pyruvate sulfurtransferase (3-MPST).¹³ Organosulfides act as exogenous sources of hydrogen sulfide (H₂S), which anticipated cardiac defense by opening K^ATP channels in cardiomyocytes.¹⁴ Similarly, exogenous hydrogen sulfide (H₂S) administration reduces the severity of myocardial infarction and heart failure among rodents following ischemia or reperfusion injury in vivo.¹⁵

Phytochemicals, with their diverse bioactive properties, offer significant potential for novel therapeutic strategies, particularly in personalized medicine, emerging diseases, and combination therapies. Phytochemicals like polyphenols, isothiocyanates, and curcumin can modulate the gut microbiota, influencing various health outcomes.^16,17 Allium cepa L., commonly known as onion, is the most grown and consumed crop worldwide as well as an important source in folk medicines used traditionally as an antioxidant, antimicrobial, anti-inflammatory, antihypertensive, and hypolipidemic due to the presence of a variety of bioactive compounds.¹⁸ The diverse bioactive constituents of Allium cepa L., such as polysaccharides, saponins, phenolic molecules, and organosulfur compounds, are chiefly accountable for their health benefits.¹⁹ The young leaves and their flower stalk are also eaten and used as edible. Moreover, the plant has a strong background in traditional medicinal systems to cure multiple diseases.²⁰ However, very little scientific research has been conducted to evaluate the cardioprotective effect of ACO as a source of condensed organosulfides, which release H₂S during its colonal enzymatic degradation. However, lower levels of plasma H₂S increase the progression of heart failure.²¹ Therefore, in the current study, we aimed to evaluate the ACO to promote reversal of ventricular remodelling by administering ACO orally as a rich source of organosulfides in isoproterenol-induced heart failure in rat model.

Materials and Methods

Collection of Plant Material

Allium cepa. (L) was collected from the local market of Faisalabad for oil extraction. The bulbs were washed with water to remove dust and foreign material and subjected to shade drying under subdued light and good ventilation for 6 weeks. After drying, the plant material was powdered using a mechanical grinder (INTSUPERMAI, US) to obtain coarse powder, and subjected to steam distillation.²² The oil was preserved in an airtight container with proper labeling and stored at 4 °C in a refrigerator for further use.

In-Vitro Studies

GC-MS of Allium cepa Bulbs Oil

Allium cepa. (L) bulb oil was analyzed using GC-MS Agilent, Model 7890B (Santa Clara, CA, USA) linked with Mass hunter acquisition as software. The instrument consisted of an ultra-inert capillary non-polar column (HP-5MS) with various proportions of 30 mm × 0.25 mm ID ×0.25 µm specification film. Helium, at a flow of 1.0 mL/min was used as a carrier gas while the oven was adjusted at 50 °C for 5 min, then the temperature was gradually raised to 250 °C at a rate of 100 °C/min, and finally to 300 °C for 10 min at a rate of 70 °C/min. The metabolites existing in the extracted sample were identified by the NIST library.^23,24

In Vivo Studies

Experimental Animals and Induction of Heart Failure

Normal, healthy male adult Wistar albino rats (n = 30) aged 8 to 10 weeks, having an average weight of 150 ± 20 grams, were selected for the study. All necessary approvals were sought from the Institutional Animal Ethical Committee at GCUF (Ref. No. GCUF/REC/316), ensuring adherence to ethical standards for animal research. Following a 7-day acclimatization period, the rats were randomly divided into five groups labeled (NC, PC, STD, T1, and T2), each with six rats. Rats (n = 24) were administered intraperitoneal injections of isoproterenol (5 mg/kg b.w) for 7 days to induce heart failure²⁵ except for negative control. All the rats were fed on a normal chow diet and water ad libitum for consecutive 28 days and Tween-80 was used as a vehicle for the oral administration of oil. The protocols for each group were as follows: Negative control (NC; received Tween-80 (0.5 ml), Positive control (PC; Tween-80; 0.5 ml), Standard group (STD; digitalis; 0.0225 mg/kgb.w), Treatment 1 (T1;40 µmol /kgb.w), Treatment 2 (T2; digitalis; 0.01125 mg/kg b.w along with Allium cepa oil; 40 µmol /kgb.w). At the end of the trial, a period of 28 days all the animals were decapitated for the collection of blood samples, and as well as organ collection the aorta and left ventricle were excised after dissection.

Physical Parameters

Body and Organ Weight

The weekly body weight of each rat was recorded during the experimental tenure and the animals followed ARRIVE guidelines²⁶ to harvest organs of rats, relative organ weight was determined by using the following formula.²⁷

Relative organ weight = \frac{Organ weight}{body weight} \times 100

Electrocardiogram (ECG)

An electrocardiogram (ECG) was recorded by using the Powerlab AD instruments (15 T, Model No. ML4818, New Zealand) data acquisition system.²⁸ Heart rate, RR interval, QRS complex, PQ interval, and QT intervals were recorded

Sample Collection

Blood samples were collected by providing gel tubes (clotting vials) for serum analysis. An inch of the left ventricle and upper aortic tissue was isolated, and stored in formalin (10%) for histological examination, while triazole was used to procure tissues of the heart and aorta to extract RNA (mRNA) that is then utilized for gene expression analysis.

Assessing Biochemical Parameters

Oxidative Stress Measurements

Oxidative stress estimation includes total oxidant status (TOS) and total antioxidant capacity (TAC). Total oxidative stress (TOS) in serum samples of different experimental groups was estimated because oxidants are present in the serum sample, and ferrous ion oxidizes to ferric ions. In an acidic solution, ferric ions and xylenol orange generate a color complex that is quantified spectrophotometrically as a change in color intensity using the colorimetric technique. The calibration curve was created by utilizing various H₂O₂ dilutions, and the outcomes were reported in units of μmol of H₂O₂ equivalent/L. The measurement yielded a sensitivity of 1.13 H₂O₂ equivalent/L and a precision of less than 3% with a linearity of 200 μmol H₂O₂ equivalent/L. Using Trolox as a reference, the total antioxidant capacity (TAC) was determined and expressed in milligrams of Trolox equivalent/L. The assay demonstrated linearity up to 6 mmol Trolox equivalent/L and accuracy within 3%. The activity of the arylesterase and PON1 enzymes was assessed using accepted techniques. Malondialdehyde (MDA) in serum samples was determined by spectrophotometric protocols 25 μl serum, 25 μl sodium dodecyl sulfate, 190 μl acetic acid, 190 μl TBA, and 75 μl distilled water were put into labeled tubes. After an hour of incubation at 95 °C, the tubes were cooled down and 625 μl of butanol was added. Absorbance was measured at 535 nm following ten minutes of centrifugation at 4000 rpm.²⁹ In serum samples level of catalase was measured spectrophotometrically (BIOLAB-310, URIT, China) using a novel automated colorimetric method, using dichromate in acetic acid as the substrate, and the amount of catalase in serum sample was determined spectrophotometrically. This process involves heating dichromate in acetic acid in the presence of hydrogen peroxide (H₂O₂) that hasn't broken down to producechromic acetate, with perchromic acid acting as an unstable intermediary. The quantity of chromic acetate generated by the reaction is directly correlated with the concentration of hydrogen peroxide. Calorimetric measurement of the generated chromic acetate is done at 570 nm.³⁰

Liver Profile

Total cholesterol, triglycerides, and high-density lipoprotein cholesterol (mg/dl) were calculated using the HDL-Cholesterol phosphotungstic precipitation method (Bioresearch catalog number CS009 1100), which has a CV of less than 10%. The kit's instructions were followed to process the samples. The components were carefully mixed in a test tube according to the specifications. The mixture was then incubated at 37 °C for five minutes. After incubation, the sample had an optical density of 546 nm compared to the blank reagent. Serum LDL cholesterol levels were calculated using the Friedrick equation as mentioned below.

L D L - C h o l e s t e r o l = T o t a l C h o l e s t e r o l - \frac{T r i g l y c e r i d e s}{5} - (H D L - C h o l e s t r o l)

A calorimetric technique was used to measure the amount of aspartate aminotransferase (AST; U/L) in the serum using a commercially available kit from Innoline (catalog number A220074). A similar approach was used to assess the serum alanine aminotransferase (ALT; U/L) concentration using an Innoline kit (catalog number A220433). To quantify ALT; U/L and AST; U/L mixtures were prepared by combining ten parts of the working solution (1 ml) with one part of serum (100 μl) in each Eppendorf tube respectively. All Eppendorf vials were assigned sample numbers. The mixture was allowed to settle at room temperature for one minute. Measured the optical density at 546 nm.

Serum Renal Function Test

Urea/BUN concentration was measured by the (Urea /BUN- UV ultimate single reagent catalog number 320 002) reagent kit, urease–UV fixed-rate (enzymatic method), and serum uric acid level was measured by the (Uric acid – liquefy, Uricase- PAP (single Reagent, Spectrum, catalog number 323000) reagent kit method kit have CV of less than 10%.

Myocardial Profile

Levels of Trop-I and proBNP were analyzed by using immunoassay kits by Elecsys proBNP II Ref (04842464-190) cobas^®.³¹ Levels of creatine kinase (CK-MB) and lactate dehydrogenase (LDH) were estimated spectrophotometrically. CK-MB (myocardial band; µg/mL) levels were determined with a Randox catalog number 498392 kit. Using Lab kit catalog number T-7751, the serum lactate dehydrogenase (LDH; mg/dL) was measured spectrophotometrically these kits have a CV of less than 10%.

Serum Electrolytes

Serum electrolytes (Sodium, Potassium, Calcium Magnesium) were analyzed by using Electrolytes Fully automated AU 700 Beckman coulter.³²

Serum and Urine Sulfide Quantification

Urine and plasma concentrations were measured using a spectrophotometric technique (methyl blue). Samples (100 µl) were mixed with 300 μL zinc acetate (1%w/v) to acquire H₂S, after five minutes, 200 μL of N, N-2 dimethyl-p-phenylenediamine sulfate (20 millimolar combined with 7.2 M HCl) was added to stop the reaction. Following this, add 200 μL of FeCl3 thirty millimolar in combination with 1.2 M HCl). After that, the mixture was left in the dark for twenty minutes. Subsequently, 150 microliters of 10% (w/v) trichloroacetic acid were introduced to the sample in order to precipitate the relevant material. The mixture was then separated from the supernatant after being spun 40 times at 10,000 rpm for 10 min in a temperature-controlled centrifuge (centrifuge5810R, Eppendorf, Germany). The concentration of H₂S in the supernatant was measured at 670 nanometer.³³

Histopathology of Left Ventricular Muscle and Aorta

Tissues from the left ventricular muscle and aorta were removed from 10% formalin and then embedded in to paraffin to create peraffin blocks for tissue slicing. Using a microtome, 4-5 μm slices were cut for histological investigations (Bk-Mt268m; Biobase Biodustry, Shandong Co. Ltd China). Tissue sections were fixed an albumin-coated glass slide. Hematoxylin and eosin was used to stain the tissues after the removal of paraffin and rehydration. On stained slides, a cover slip was applied following DPX mounting. Slides were later examined under a light microscope.

Gene Expression Studies

An inch of left ventricular muscle tissue was taken in Triazole (Therma Fisher Scientific) and kept at −40 °C until further processing. By using Nanodrop, isolated RNA was quantified. The cDNA synthesis was done by using the RevertAid cDNA synthesis kit (Therma Fisher Scientific) by using equal concentrations of RNA from each sample according to the manufacturer's instructions. Finally, gene expression of CSE, CBS, eNOS₁, rNOS₁, ACE₂_, and NOD₂ genes were done using qRT-PCR (Biorad; BM10-QPCR96 RT 2).

Statistical Analyses

All dataset collected for various parameters as Mean ± SEM. one-way and Two-way analyses of variance ANOVA were used to rule out the statistical significance of data among treated and untreated groups Significant difference among different group by sitting significance level (P ≤ .05) while post significance test Ducky test was applied as posthoc significance test (GraphPad Prism 9.1.2; California) software.³⁴

Results

Allium cepa L. oil (ACO) is a substantial source of organosulfur compounds (H₂S donor), in addition to containing anti-inflammatory, antioxidant, and antidyslipidemic phytochemical components. Organosulfides serve as a substrate for hydrogen sulfide generation. Restoring normal plasma levels of H₂S may reduce heart hypertrophy via modulating vascular resistance and myocardial dysfunction.

Identification of Compound by GCMS Analysis

Gas chromatography mass spectrometry analysis confirmed the presence of total 12 compounds, three major compounds; Methyl 10, 11-tetradecadienoate, 3-methyl-2-(2-oxopropyl)furan, and 3-n-hexylthiolane, s,s-dioxide having concentration (≥1%) while all other compounds were present in trace amounts (≤ 1%) displayed the most significant peaks with maximum area in the GCMS analysis of Allium cepa (L) bulb oil at elevated quantities, as illustrated in Figure 1.

Figure 1.

GC-MS Chromatogram Displaying the Peaks of Several Chemicals in Alium cepa. L Oil.

GC-MS analysis of Allium cepa. L showed a total of 12 bioactive compounds with a high concentration of Methyl 10, 11-tetradecadienoate (77.09), 3-methyl-2-(2-oxopropyl)furan (14.06), 3-n-hexylthiolane, s,s-dioxide (18.15), and Eucalyptol (3.9) while except are less than one percent (Table 1).

Table 1.

Area (%) of Different Compounds Assessed in Allium cepa. L GC-MS Anal.

Sr. no	% Area	Compound Name	M.F	M.W. (g/mol)	Pharmacological Activities
1	14.06	3-methyl-2-(2-oxopropyl)furan	C₈H₁₀O₂	138.16	Antioxidant, antimicrobial and anti-inflammatory activity35
2	77.07	Methyl 10,11-tetradecadienoate	C₁₅H₂₆O₂	238.37	Not reported
3	18.15	3-n-hexylthiolane, s,s-dioxide	C₁₀H₂₀O₂S	204.33	Anti-tumor36, Antimicrobial, Antiviral, Antihypertensive, Antidiabetic, Anticancer37
4	0.94	1,10-hexadecanediol	C₁₆H₃₄O₂	258.44	Antioxidant, Hypocholesterolemia, Hemolytic, 5- Alpha reductase inhibitor, Antialopecic38
5	0.57	1-nonylcycloheptane	C₁₆H₃₂	224.42	Anti-inflammatory, analgesic, anticonvulsant39
6	0.0017	Oleic acid	C₁₈H₃₄O₂	282.5	Antiatherosclerosis, Anti-inflammatory, Antiobesity, Antioxidant, Antihepatotoxic, Vasodilatory40
7	0.065	Z,z-6,28-heptatriactontadien-2	C₃₇H₇₀O	530.9	Vasodilator 41
8	0.43	Z, z, z-8,9-epoxyeicosa-5,11,14-	C₂₁H₃₄O₃	334.5	inhibitors of soluble epoxide hydrolase, vasodilatory42
9	3.9	Eucalyptol	C₁₀H₁₈O	154.25	substrates for CYP3A enzymes43
10	0.22	5,5-dimethyl-cyclohex-3-en-1-ol	C₈H₁₂O₂	140.18	antitumor, anticonvulsant and anti-inflammatory44.
11	0.13	3-n-hexylthiane, s, s-dioxide	C₁₁H₂₂O₂S	218.36	Thianes
12	0.31	2-ethylthiolane, s, s-dioxide	C₆H₁₂O₂S	148.23	Thiolanes (Volatile compound)

Body Weight and Relative Organ Weight

Overall mean body weight (gm ± SEM) was significantly (P ≤ .05) decreased in all groups during the induction period of heart failure for 7 days except NC. At day 14th to 28th, body weight was significantly (P ≤ .05) increased in STD, T1, and T2 as compared to the PC group. However, body weight continued to reduce significantly (P ≤ .05) in PC, compared to the negative control from day seventh to day 28th as shown in Figure 2(A). Relative organ weight was significantly (P ≤ .05) increased in positive control (PC) as compared to negative control at the end of the treatment period of 28 days as shown in Figure 2(B).

Figure 2.

Body Weight and Relative Organ Weight in Normal Control (NC), Positive Control (PC; Isoproterenol 5 mg/kg b.w), and Standard (STD; Digitalis 0.225 mg/kgb.w), T1; Allium cepa Linn. Bulb Oil 40 µg/kgb.w), and Co- Treated Group (T2 ; Allium cepa Linn. Bulb Oil 40 µg/kgb.w + Digitalis 0.01125 mg/kgb.w) in Isoproterenol-Induced Heart Failure Rat Model. Data are Mean ± SEM, N = 6. **** P < .0001, *** P < .001, ** P < .01, * P < .05.

Myocardial Profile

Heart failure was followed by a significant increase (P ≤ .05) in overall levels of markers of myocardial damage, including Pro-B-type natriuretic peptide (A), Troponin-I (B), creatine kinase-MB (C), and lactate dehydrogenase (D) in PC group as compared to STD, T1 group, and T2 as seen in Figure 3. In T2 level of Pro-BNP levels (pg/mL; 137 ± 1.5), Trop-I (µg/L; 1.2 ± .07), CK-MB (µg/ml; 45.1 ± 1.2) and, LDH (mg/dl; 45.1 ± 1.2) were significantly (P ≤ .05) decreased as compared to PC.

Figure 3.

Cardiac Profile A; Pro-BNP (pg/ml), B; Trop-I (µl/l), C; Ckmb (µg/ml), D; LDH (mg/dl) in Normal Control (NC), Positive Control (PC; Isoproterenol. 5 mg/kgb.w), and Standard (STD; digitalis 0.225 mg/kgb.w), T1; (Allium cepa Linn. Bulb oil 40 µg/kgb.w), and Co- Treated Group (T2; Allium cepa Linn. oil 40 µg/kgb.w + Digitalis 0.01125 mg/kgb.w) in Isoproterenol-Induced Heart Failure Rat Model. Data are Mean ± SEM, N = 6. **** P < .0001, *** P < .001, ** P < .01, * P < .05.

Electrocardiogram Analysis

The present investigation of electrocardiogram revealed a substantial (P ≤ .05) prolongation (ms ± SEM) R-R interval (ms) (1.45 ± 0.70^a), QRS complex (ms) (0.19 ± 0.43^a), PQ-Interval (ms) (0.17 ± 0.43^a), QT interval (ms) (0.24 ± 0.28^a) in the PC group relative to the NC group, indicating the presence of cardiac arrhythmia. As demonstrated in Figure 4 and Table 2, however, the R-R interval (ms) in T2 (0.74 ± 0.50^d) indicates a significant (P ≤ .05) decrease as compared to PC (1.45 ± 0.70^a) which helps to improve heart rate in T2 (242.58 ± 1.14^a). QRS- complexes, PQ- interval, and QT interval were significantly (P ≤ .05) shortened in T2 (0.04 ± 0.018^c), (0.040 ± 0.017^c), and (0.043 ± 0.21^b) respectively.

Figure 4.

Representative ECG Leads A (Negative Control), B (Positive Control), C (STD Group; Digitalis 0.225 mg/kgb.w), D (T1; ACO 40 µmol/kgb.w), E (T2; Allium cepa Linn. Oil 40 µmol/kgb.w + Digitalis 0.01125 mg/kgb.w).

Table 2.

Electrocardiogram; Heart Rate(bpm), R-R Interval (s), QRS Complex (ms), PQ Interval(ms), QT Intervals (ms) in Normal Control (NC), Positive Control (PC; Isoproterenol. 5 mg/kgb.w), and Standard (STD; digitalis 0.225 mg/kgb.w), T1; Allium cepa Linn. Bulb oil 40 µg/kgb.w), and co-Treated Group (T2 ; Allium cepa Linn. Bulb oil 40 µg/kg b.w + Digitalis 0.01125 mg/kgb.w) in Isoproterenol-Induced Heart Failure rat Model. Data are Mean ± SEM Different Superscripts (a, b, c, d, & e) Showing the Significant Difference Among Various Groups While Setting (P ≤ 0.05).

Groups	Heart Rate (BPM)	R-R Interval (Sec)	QRS- Complex (ms)	PQ Interval (ms)	QT- Interval (ms)
NC	248.97 ± 0.53^a	0.72 ± 0.49^d	0.04 ± 0.005^c	0.040 ± 0.005^c	0.045 ± 0.03^b
PC	124.01 ± 0.89^c	1.45 ± 0.70^a	0.06 ± 0.522^a	0.058 ± 0.020^a	0.081 ± 0.15^a
STD	225.71 ± 1.4^b	0.84 ± 0.53^c	0.04 ± 0.004^b	0.040 ± 0.005^c	0.044 ± 0.22^b
T1	214.33 ± 1.02^b	0.80 ± 0.52^c	0.04 ± 0.017^b	0.042 ± 0.018^c	0.045 ± 0.20^b
T2	242.58 ± 1.14^a	0.74 ± 0.50^d	0.04 ± 0.018^c	0.040 ± 0.017^c	0.043 ± 0.21^b

Renal Profile

After the administration of Isoproterenol, significantly (P ≤ .05) elevated levels of serum urea and creatinine level were evident, particularly in the PC group due to the damage of cardiac tissue which subsequently affects kidney function and ultimately leads to the disturbance of the electrolytes. The level of urea; mg/dl (A) and creatinine; mg/dl (B) significantly (P ≤ .05) decreased in T2 (23.7 ± 1.2), and (0.2 ± 0.2) as compared to PC. Levels of Na⁺ and Ca⁺⁺ (mmole/L) significantly (P ≤ .05) reduced were seen in T2 (124 ± 1.1, 1.8 ± 0.15). On the contrary, the levels of K⁺ and Mg⁺⁺ (mmole/L) were significantly (P ≤ .05) increased in T2 (8.2 ± 0.2, 3.4 ± 0.3) as shown in Figure 5.

Figure 5.

Renal Profile A; Urea, B; Creatinine, C; Sodium (Na⁺), D; Potassium (K⁺) E; Calcium (Ca⁺⁺) F; Magnesium (Mg⁺⁺) in Normal Control (NC), Positive Control (PC; Isoproterenol 5 mg/kgb.w), and Standard (STD; Digitalis 0.225 mg//kgb.w), T1 (Allium cepa Linn. Bulb Oil 40 µg/kgb.w), and Co-Treated Group T2 (Allium cepa Linn. Bulb oil 40 µg/kgb.w + digitalis 0.01125 mg/kgb.w) in Isoproterenol-Induced Heart Failure Rat Model. Data are Mean ± SEM, N = 6. **** P < .0001, *** P < .001, ** P < .01, * P < .05.

Liver Profile

Serum ALT and AST levels were, significantly (P ≤ .05) elevated in PC as compared with NC. The level of ALT (U/L) and AST (U/L) significantly (P ≤ .05) decreased in T2 (98.1 ± 1.5, 147 ± 2.3) as compared to PC. Fluctuations in liver enzymes also contribute to lipid metabolism, which could affect cellular activity. Accordingly, levels of TC, TG, and LDL (mg/dl) significantly (P ≤ .05) increased in PC however significant (P ≤ .05) reductions were seen in T2 (149.9 ± 1, 84.9 ± 0.9, F19.4 ± 1.1,). On the contrary, the levels of HDL (mg/dl) were significantly (P ≤ .05) reduced in PC as compared with T2 (45.5 ± 1.2) as shown in Figure 6.

Figure 6.

Liver Profile; A; TC (mg/dl), B; TG (mg/dl), C; HDL (mg/dl), D; LDL (mg/dl), E; AST (U/L), F; AST (U/L) in Normal Control (NC), Positive Control (PC; Isoproterenol. 5 mg/kg b.w), and Standard (STD; Digitalis 0.225 mg/kgb.w), T1 (Allium Cepa Linn. bulb oil 40 µg/kgb.w), and Co-Treated Group T2 (Allium Cepa Linn. Bulb Oil 40 µg/kgb.w + Digitalis 0.01125 mg/kgb.w) in Isoproterenol-Induced Heart Failure Rat Model. Data are Mean ± SEM, N = 6. **** P < .0001, *** P < .001, ** P < .01, * P < .05.

Oxidative Stress Markers

The overall mean of Total oxidative stress (µmol H₂O₂ equivalent/L), and Malondialdehyde (µmol/L) was significantly (P ≤ .05) reduced in T2 (40.6 ± 1.5, 0.27 ± 0.1) as compared to PC, as shown in Figure 7(A) and (C) respectively. Total antioxidant status (mmol Trolox equivalent/L) and catalase (kU/L) levels were significantly (P ≤ .05) increased in T2 (7.3 ± 0.5, 5.4 ± .02) as compared to PC, as shown in Figure 7(B) and (D) respectively.

Figure 7.

Oxidative Stress; A; Total Oxidative Stress (Mmole H₂O₂ Equivalent/L), B; Total Antioxcidant Capacity (Mmole Trolox Equivalent/L) C; Malondialdehyde (Mmole/L) D; Catalase (kU/L) in Normal Control (NC), Positive Control (PC; Isoproterenol. 5 mg/kg b.w), and Standard (STD; Digitalis 0.225 mg/kgb.w), T1 (Allium cepa Linn. Bulb oil 40 µg/kgb.w), and Co-Treated Group T2 (Allium cepa Linn. Bulb oil 40 µg/kgb.w + Digitalis 0.01125 mg/kgb.w) in Isoproterenol-Induced Heart Failure Rat Model. Data are Mean ± SEM, N = 6. **** P < .0001, *** P < .001, ** P < .01, * P < .05.

Serum and Urine Hydrogen Sulphide Quantification

Plasma and urinary H₂S levels had been significantly reduced (P ≤ .05) among the heart failure animals compared to normal control, which can be linked to endothelial dysfunction, mostly associated with ventricular dilation. In heart failure, significantly (P ≤ .05) reduced levels of serum hydrogen sulphide and urine hydrogen sulphide were evident, particularly in the PC group, due to the damage of the heart, which subsequently affects kidney function and ultimately leads to the reduced level of H₂S in urine. The level of serum H₂S(µmole/ml) and urine H₂S (µmole/ml) significantly (P ≤ .05) increased in T2 (85.7 ± 1.4, 59.2 ± 0.7) as compared to PC, as shown in Figure 8.

Figure 8.

Hydrogen Sulfide Level A; Serum H₂S level (µmole/ml) B; Urine H₂S level (µmole/ml), in Normal Control (NC), Positive Control (PC; Isoproterenol. 5 mg/kg b.w), and Standard (STD; Digitalis 0.225 mg/kg b.w), T1 (Allium cepa Linn. Bulb Oil 40 µg/kgb.w), and co-Treated Group T2 (Allium cepa Linn. Bulb Oil 40 µg/kgb.w + Digitalis 0.01125 mg/kgb.w) in Isoproterenol-Induced Heart Failure Rat Model. Data are Mean ± SEM, N = 6. **** P < .0001, *** P < .001, ** P < .01, * P < .05.

Transulfration Pathway and Cardioprotective mRNA Expression Level

Transulfration pathway genes and cardioprotective transcriptional factors are known to have a significant role in regeneration, proliferation of cardiomyocytes and regulating the endothelial vascular tone .The results show that the levels of CBS, CSE, 3-MPTS and eNOS were significantly down regulated (P ≤ .05) in PC versus healthy control, while in T2 (CBS; 2 ± 0.18, CSE; 1.8 ± 0.4, 3-MPTS; 1.5 ± 0.4, eNOS; 2.9 ± 0.6), shown in Figure 9 upregulated (P ≤ .05) expression levels which is consistent with improved cardiac functioning (Figure 3). Moreover ACE-2 and NOD-2 were significantly (P ≤ .05) upregulated in PC as compared to the NC. However the downregulation (P ≤ .05) were observed in T2 (ACE-2; 0.29 ± 0.17, NOD-2; 1.3 ± 0.4) as compared to the PC.

Figure 9.

mRNA Expression Level of A; CBS, B; CSE, C; 3-MPTS, D; ACE-2, E; NOD-2 F; eNOS in Normal Control (NC), Positive Control (PC; Isoproterenol. 5 mg/kg b.w), and Standard (STD; digitalis 0.225 mg/kg b.w), T1 (Allium cepa Linn. Bulb oil 40 µg/kgb.w), and co-Treated Group T2 (Allium cepa Linn. Bulb oil 40 µg/kgb.w + digitalis 0.01125 mg/kgb.w) in Isoproterenol-Induced Heart Failure rat Model. Data are Mean ± SEM, N = 6. **** P < .0001, *** P < .001, ** P < .01, * P < .05.

Histopathology of Left Ventricular Muscle

Histopathological investigation at 400X magnification revealed that the negative control group displayed normal characteristics in the left ventricular muscle. The left ventricular myocardium exhibits normal nuclei and symmetrical alignment of muscle fibers. In contrast, the positive control group exposed to isoproterenol exhibited muscle growth and cellular infiltration. The STD group demonstrated cellular edema and muscle disruption, accompanied by elevated connective tissue content in the heart tissue. In Figure 10, T1 exhibited reduced collagen deposition alongside augmented smooth muscle and elastic fiber content. T2 exhibited outcomes more comparable to the negative control in the isoproterenol-induced myocardial injury rat model. After 28 days, histological examination with HE staining (40×) revealed that the cardiac and aortic muscles had restored their smooth and elastic fibers, while eNOS levels had risen and ACE-2 and NOD-2 levels had diminished.

Figure 10.

Histopathology of Left Ventricular Muscle of Rat in Normal Control (NC), Positive Control (PC; Isoproterenol. 5 mg/kg b.w), and Standard (STD; Digitalis 0.225 mg/kg b.w), T1 (Allium cepa Linn. Bulb oil 40 µg/kgb.w), and Co-treated Group T2 (Allium cepa Linn. Bulb oil 40 µg/kgb.w + Digitalis 0.01125 mg/kgb.w) in Isoproterenol-induced Heart Failure Rat Model. HE Staining (400 x Magnification); Scale bar = 20 µm.

Histopathology of Aorta Tissue

The histological examination of aorta tissues in the normal control (NC) group, observed under a light microscope, reveals normal morphology and structure. The aortic muscle exhibited an increase in the thickness of the Tunica Media (TM) and Tunica Adventitia (TA), uneven degradation of the intimal surface of smooth muscle (SM), loss of elastic fibers, and accumulation of connective tissue. The positive control exhibited muscle growth and cellular infiltration. The STD group demonstrated an aorta with organized tunica media and tunica adventitia, characterized by smooth muscle content, arranged elastic fibers, and reduced connective tissue deposition. In Figure 11, T1 exhibited reduced connective tissue deposition alongside heightened smooth muscle and elastic fiber composition. T2 exhibited outcomes more comparable to the negative control in the rat model of isoproterenol-induced cardiac injury. After 28 days, histological examination with HE staining (40×) revealed that the aortic musculature had restored its smooth and elastic fibers, while eNOS levels had risen and ACE-2 and NOD-2 levels had diminished.

Figure 11.

Histopathology of Aorta of in Normal Control (NC), Positive Control (PC; Isoproterenol. 5 mg/kg b.w), and Standard (STD; Digitalis 0.225 mg/kg b.w), T1 (Allium cepa Linn. Bulb oil 40 µg/kgb.w), and co-Treated Group T2 (Allium cepa Linn. Bulb oil 40 µg/kgb.w + Digitalis 0.01125 mg/kgb.w) in Isoproterenol-Induced Heart Failure Rat Model. HE Staining (400 x Magnification); Scale bar = 20 µm.

Discussion

Heart failure is a common disorder in the elderly. However, recent studies have indicated that the heart failure burden in the young may be increasing.⁴⁵ Heart failure arises from myocardial injury due to several etiologies, including coronary artery disease, hypertension, and diabetes mellitus. Infrequent causes encompass cardiomyopathies, valvular disorders, myocarditis, infections, systemic poisons, and cardiotoxic pharmaceuticals.⁴⁶

Dietary phytonutrients help prevent and treat diabetes, obesity, hypertension, cardiovascular disease, cancer, neurological disorders, age-related disease, inflammatory disorders, and other biological activities. To properly use phytonutrients for disease prevention and therapy, more study and understanding of individual variations are needed.⁴⁷ In this regard, we investigated Allium. cepa Linn. oil (ACO) as a potential regulator of cardiovascular dysfunction in the experimental heart failure animal model (HF). In our study GC-MS of Allium. cepa Linn. oil (ACO) showed (Figure 1) the broad spectrum of organosulfides, antioxidant compounds, lipopolysaccharides, polyunsaturated fatty acid, and various substances that have a potent and synergetic effect in the regulation of cardiomyocyte and cardiac function. Organosulfides act as a substrate for the production of hydrogen sulfide and help regulate the physiological range of H₂S in the body. Hydrogen sulfide (H₂S) is an endogenous, gaseous signaling molecule that plays a critical role in cardiac and vascular biology. H₂S regulates vascular tone and oxidant defense and exerts cytoprotective effects in the heart and circulation. In our research, relative organ weight rose in the positive control (PC) relative to the negative control (NC), whereas body weight considerably reduced in the PC (Figure 2). Nonetheless, the standard (digitalis 0.225 mg/kgb.w), T1 (40 µmol/kgb.w ACO), and T2 (ACO + digitalis) groups all maintained body weight. In the treated groups, relative organ weight was also recovered.^48,49 An increase in the heart weight in ISO-induced rats might be due to the increased water content, oedematous intramuscular space, and extensive necrosis of cardiac muscle fibers followed by the invasion of damaged tissues by the inflammatory cells.⁵⁰ The weight restoration might be attributed to the cardioprotective and antioxidant properties of organosulfides.⁵¹

One of the important diagnostic tools thought to be involved in the pathophysiology of heart failure is H₂S quantification. According to the current investigation, sulfide levels and mRNA expression level of trans-sulfuration enzyme were significantly reduced in PC as compared to NC. In STD group (digitalis) and ACO-treated groups show significant increases in sulfide levels and mRNA expression levels of trans-sulfuration enzyme but the most significant upregulation was seen in T1 and T2 because of the availability of organosulfides which act as a substrate for the enzymes that are responsible for the production of H₂S lowest total plasma sulfide in patients of NYHA Class IV.^21,52 Furthermore, low total plasma sulfide also predicts higher mortality and rehospitalization rates because it negatively correlates with Pro-BNP, troponin-I, lactate dehydrogenase (LDH), and CK-MB levels, the primary biomarkers for myocardial necrosis. An elevated myocardial profile was found in both acute and chronic heart failure, with significant predictive value.^53,54 (Figure 3) Serum hydrogen sulfide levels were low in PC and showed a progressive hike in cardiac profile and oxidative stress marker (total oxidative stress and MDA) also increased as compared to NC. High levels of cardiac profile and oxidative stress markers seen in positive control may result from volume expansion and extreme pressure overload in ventricles that destroys cardiac muscle (necrosis). It exacerbates the cardiac profile and oxidative stress even further. The STD group has a noteworthy decline in T1 and T2.

In STD group digitalis helps to regulate sinus rhythm which ultimately regulate cardiac contractility, and T1 and T2 (ACO-treated and co-treated) groups showed reduced levels of overall cardiac profile as compared with PC and the increased level total antioxidant capacity (TAC) and catalase because of organosulfur compounds (H₂S donor) and synergetic effect with 3-Methyl-2-(2-oxopropyl)furan.^55,56 It has cardioprotective, anti-inflammatory, and antioxidant properties those found in onions prevent a decline in cardiac function and modulate the extracellular matrix, in part, by increasing ventricular H₂S production and ameliorating oxidative and proteolytic stress, and protecting the heart against adverse remodeling.⁵⁷

Despite some differences, there are essential similarities between rat and human ECG.⁵⁸ Therefore, ECG in rats is used in animal models of cardiovascular disease²⁴ Rapid onset of cardiac dysfunction can result from a decrease in H₂S, which can induce abnormal diastolic and systolic pressure control. This can involve the retention of sodium and calcium as well as the loss of potassium and magnesium, which can alter heart rhythm and cardiac events. Expansion of the QRS complex, extension of QRS complexes and ST elevation, and left ventricular hypertrophy due to hypokalaemia.⁵⁹

ACO significantly restored serum electrolytes and cardiac electrical events. Irregular hemodynamics play a part in ventricular dilation and fibrosis, leading to ECG abnormalities.²⁴ Treatment with ACO significantly reversed these changes, possibly due to the presence of z-6,28-heptatriactontadien-2 which act as vasodilatory molecules, and due to the presence of organosulfides that act as an exogenous source of H₂S which ultimately help in the regulation of contraction and relaxation that helps in the restoration of ejection fraction vasodilatory effect helps to decrease the vasoconstriction that eventually reduces the afterload by mitigating the production of renin and by the uptake of potassium and upregulates the Na⁺/K⁺ATPas activity and regulate the L-type calcium channels to prevent the cardiac arrythmia. additionally, serum K⁺ is negatively correlated with plasma renin activity and plasma noradrenaline.^60–62

Heart failure is a multifaceted pathophysiologic syndrome, with prevalent dysfunction of other vital organs and systems. The role of the liver in this disease has been little investigated, although up to 80% of patients with heart failure present with some form of liver dysfunction this current investigation reveals that the level of liver enzyme (ALT, AST) was (50%-60%) increased in PC as compared to NC which also affects the uptake of lipid. Lipid profile analyses revealed that elevated concentrations of serum total cholesterol, low-density lipoproteins and triglycerides, and decreased concentrations of high-density lipoproteins were remarkable in the positive control group suggestive of dyslipidemia that leads towards atherosclerosis which also contributes to the pathogenesis of heart failure and related complication such as nephropathy. The excessive lipids accumulate in the blood cause vascular stiffness that ultimately affects efferent arterioles which contribute to hypertensive nephropathy due to this reason urea and creatinine levels were also increased in PC and decreased levels of H₂S in urine also predicted the health of the kidney (Figure 5). Liver and renal profile disturbances in heart failure are often caused by factors such as inflammation, hormonal imbalances, impaired cardiac function, and decreased organ perfusion due to vasocontraction which leads to abnormal functioning.⁶³ The standard control group and onion oil-treated group showed significant restoration of the liver and renal profile which is comparable with negative control. However in ACO-treated groups (T1, T2) may be due to the presence of 1,10-hexadecane-diol, oleic acid, 3-methyl-2-(2-oxopropyl)furan that acts as hypercholesteraemic, antioxidant, Vasodilatory, Antihepatotoxic, and Antiatherosclerosis activity.^35,38,40

In a microscopic examination of a longitudinal section of the myocardium and aorta at 400X (Figures 10 and 11), the negative control group showed normal architecture of myofibers with a centrally located nucleus and intercalated discs while distracted in all other groups. Positive control left ventricle muscle showed immune cell infiltration and abundance of fibroblasts resulting in fibrosis of tissues due to sustained increase of afterload. It causes a disproportionate aggregation of fibroblasts leading to an increase in interstitial fibrillar collagen mass. Such fibroblast stimulation increases collagen synthesis and causes fibrosis resulting in a stiff myocardium, which interferes with ventricle filling. The stiffed myocardium and the reduced contractility are outcomes of pathological remodeling and serve as a marker of heart failure.⁶⁴ The aorta presents with increasing thickness of the Tunica Media (TM) and Tunica Adventitia (TA) and irregularity of the intimal surface destruction of smooth muscle, loss of elastic fibers, and deposition of connective tissue in PC. Upregulation of ACE-2, NOD-2 and downregulation of eNOS expression levels in PC are also in line with these histological findings of myocardium and aorta. In the treatment group, normal structural integrity is comparable with the negative control group whereas remarkably dissimilar to the positive control and standard control group and the downregulation of ACE-2, NOD-2 while upregulation of eNOS levels in these groups indicate the positive correlation of eNOS and ACE-2 and NOD-2 negative correlation with the structural remodelling of myofibers in heart and aorta that showed T1 (ACO; 40 µmole/kgb.w) and T2 (cotreated) has the potential to ameliorate fibrotic processes by downregulating the level of ACE-2 and NOD-2 in isoproterenol-induced cardiac failure because ACO contains organosulfides, which have been studied for their potential as ACE inhibitors, helping lower blood pressure.⁶⁵ Although Allium cepa Linn. oil (ACO) shows promising results in cardiovascular studies, the dosage must be done carefully because of its tight safety margin. Large-scale research is required to fully understand Allium cepa Linn. oil (ACO) safety profile and effectiveness in a system, more likely in primates and clinical studies in human models. Contemporary techniques like PET scans and echocardiography can enable more accurate evaluations of tissue-level effects and heart function, providing a clearer picture of the therapeutic potential and hazards and eventually assisting in the transfer to clinical application.

Conclusion

Phytonutrients are bioactive substances derived from plants found in dietary sources and utilized in the nutraceutical sector. The present research thoroughly examined Allium cepa L. oil (ACO) in vitro analysis as well as in vivo HF model. Our research demonstrates a beneficial impact of phytonutrients on cardiovascular illness, prevention, and management. Allium cepa L. oil (ACO) is a rich source of organosulfur compounds (H₂S donor) despite the presence of anti-inflammatory, antioxidant, and antidyslipidemic phytochemical constituents. Organosulfides act as a substrate for the production of H₂S. Restoration of the normal plasma levels of H₂S potentially inhibits cardiac hypertrophy by regulating the vascular resistance and myocardial dysfunction. As an add-on to existing therapies, ACO may halt or inhibit the progression of heart failure.

Supplemental Material

sj-docx-1-npx-10.1177_1934578X251349942 - Supplemental material for Cardioprotective Role of Allium cepa L. Bulb Oil in Isoproterenol-Induced Heart Failure in a Pre-clinical Trial

Supplemental material, sj-docx-1-npx-10.1177_1934578X251349942 for Cardioprotective Role of Allium cepa L. Bulb Oil in Isoproterenol-Induced Heart Failure in a Pre-clinical Trial by Sana Saleem, Haseeb Anwar, Arslan Iftikhar and Imran Mukhtar in Natural Product Communications

Footnotes

Acknowledgements

Staff of the animal experimental station of the Department of Physiology, GCUF are acknowledged for their efforts. We acknowledge Dr Humaira Muzaffar as head of the Animal Station running for pre-clinical trial as a part of a current research project.

ORCID iDs

Arslan Iftikhar

Imran Mukhtar

Ethical Considerations

Ethical approval to report this case was obtained from the Ethical Review Committee, Government College University, Faisalabad. Reference No. GCUF/ERC/316.

Author Contributions

Imran Mukhtar conceived the idea and supervised it. Sana Saleem is a PhD scholar and performed the in vivo experiment and prepared the original draft. Arslan Iftikhar helped with laboratory analysis. Haseeb Anwar helped write the manuscript, analyzed the data, edited the manuscript, and reviewed the final manuscript.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Statement of Human and Animal Rights

The ethical guidelines outlined in the Council for International Organizations of Medical Sciences (CIOMS) and the Declaration of Helsinki were adhered to in the study protocol approach for laboratory animals. According to the Algerian Executive Directive (No 10-90 JORA, dated 18 March 2004) and the regulations of Law No. 88 −08, issued on 26 January 1988, which deals with veterinary medicine activities and the protection of animal health (No JORA: 004 of 27-01-1988), these protocols were approved by ethical health research standards.

All procedures in this study were conducted in accordance with the Ethical Review Committee, Government College University, Faisalabad. Reference No. GCUF/ERC/316.

Statement of Informed Consent

There are no human subjects in this article, and informed consent is not applicable.

Supplemental Material

Supplemental material for this article is available online.

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