Assessment of the biosafety profile of Galium verum in vitro on myoblasts and in ovo on chorioallantoic membrane

Abstract

BACKGROUND

Cardiovascular diseases are the first cause of death in the world. Ischemic heart disease is the main cause of heart failure. New approaches are continuously sought to identify better therapeutic success. Thereby, current research has been drawn to identifying and completing the therapeutic profile of natural sources. Galium species are representatives exhibiting diuretic and antibacterial potential in living organisms and can treat burns, wounds, and skin diseases. Moreover, it was also observed that these plants manifest cardioprotective effects as well as having antihemolytic, antioxidant, antibacterial, anti-inflammatory, immunomodulatory, and antiproliferative potential. In ischemic heart disease, Galium verum (G. verum) extract manifested preservative properties in terms of contractility, systolic and diastolic function maintenance, and reduced damage to the heart after ischemia. In addition, G. verum extract upregulated the activity of antioxidant enzymes alleviating the production of pro-oxidants.

AIM

To test the ethanolic extract of G. verum on the H9C2(2-1) cell line by evaluating the in vitro biosafety profile and in ovo irritative potential.

METHODS

Cells were tested in vitro for viability (using the MTT test), cellular morphology, cell number, confluence, nuclear morphology (by immunofluorescence staining of cell nuclei and F-actin assay) and in ovo by the hen’s egg chorioallantoic membrane (CAM) test and CAM anti-irritant methods to study the irritation potential on the CAM.

RESULTS

The extract demonstrated a dose-dependent stimulatory activity. The viability increased to 170% for the dose of 55 µg/mL and decreased to 135% at 200 µg/mL. The results of cell number, confluence, and morphological analysis did not present significant changes compared with control untreated cells. The immunofluorescence assay showed insignificant apoptotic potential, and the hen’s egg CAM test revealed that the extract was in the weak to moderately irritating category with an irritation score of 5.3. When applying the sample to the CAM, only slight coagulation was observed (128 s). The anti-irritant test revealed the protective potential of the extract in the vascular plexus.

CONCLUSION

The ethanolic extract of G. verum manifests a stimulating effect on cardiomyocytes, enhancing cell viability, and maintaining a normal elongated shape, cell number, and confluence, without significant signs of apoptosis and with a weak irritative effect in ovo. In addition, the extract demonstrated a protective effect against hemorrhage, lysis, and coagulation of blood vessels induced by sodium dodecyl sulfate on the CAM.

Key Words: Galium verum L; Ethanolic extract; Cardiomyocytes; Ischemic heard; Cellular viability stimulation; In vitro; In ovo

Core Tip: Ischemic heart disease is a condition that affects a growing number of patients every day despite advances in the medical field. This phenomenon implies the need to identify new therapeutic approaches. Galium verum is a plant with cardioprotective effects, including maintenance of cardiac contractility. However, studies on Galium verum extract on cardiomyoblasts and vascular plexus have not been performed. We observed a stimulating effect of the plant on the H9C2(2-1) cell line, without signs of apoptosis or irritating effects in ovo and with a protective effect on the chorioallantoic membrane.

INTRODUCTION

Cardiovascular diseases (CVD) are the first cause of death in the European region and the United States, despite continued progress in the medical field[1,2]. Premature death due to CVD refers to subjects aged below 70 years. Over 60 million eventual years of life are lost in Europe annually[1]. In 2020, the American Heart Association published the Heart Disease and Stroke Statistics and specified that in 2016 the prevalence of stroke in the United States was 2.5%. In addition, 7 million people > 20 years of age had endured a stroke, and there were nearly 150000 deaths[3]. Using the available reports, it was observed that CVD caused around 4 million deaths/year across Europe, accounting for > 1.8 million deaths in males and > 2 million deaths in females, corresponding to 39% of all deaths in males and 46% of all deaths in females[1].

Ischemic heart disease (IHD) and stroke account for 50% and 35% of CVD deaths, respectively, and represent the main cause of death[4]. Thus, in developed countries, IHD is considered the dominant cause of heart failure and represents a significant influential factor in morbidity and mortality at the global level[5]. Ischemia is an irreversible self-propagating process that negatively impacts cardiac function. To avoid myocardial ischemia, it is important to maintain a perfect match between consumption and delivery of myocardial oxygen. An almost exclusively aerobic mechanism represents myocardial metabolism, as cardiomyocytes contain limited glycogen stocks, differentiating them from other tissues and muscles[6]. The main physiopathological factors involved in IHD are presented in Figure 1. The degree of the effect on the ischemic myocardium, the extent of coronary occlusion, the involvement of collateral circulation, genetic predisposition, pre-existent myocardial metabolic rate, and the intrinsic survival potential of the myocytes can collectively determine the impact and stage of myocardial ischemia[7].

Figure 1  Physiopathological implications in ischemic heart disease.

To improve prognosis and therapeutic success, new approaches are being studied continuously. Lately, attention has been focused the identification and completion of the therapeutic profile of natural sources. A recent review highlighted that the majority of studies about medicinal plants of interest in the field of heart diseases were focused on the effects of herbs on different factors associated with heart failure, following myofibroblast activation, proinflammatory mediators, oxidative stress, energy metabolism, and apoptosis[8]. Thus, the most frequently used herbs studied for their antifibrotic mechanisms were Salvia miltiorrhiza, Carthamus tinctorius, and Panax ginseng[9,10]. Moreover, Salvia miltiorrhiza, Astragalus membranaceus, and Schisandra chinensis were tested for anti-apoptotic potential[11].

Due to abundant active ingredients, traditional Chinese herbs greatly affected cardiac metabolism reorganization in IHD. This disorder displays a significant association with metabolism disturbances, including oxidative phosphorylation, glycolysis, the citric acid cycle interaction, fatty acid β-oxidation, ketone body metabolism, branched chain amino acids, glycerol-phospholipid, and sphingolipid metabolism. Chinese medicinal plants have depicted their therapeutic potential and ability to regulate the dysfunctional cardiac metabolism[12]. Another review highlighted the ability of rosmarinic acid to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells and to activate peroxisome proliferator-activated receptor γ, thus alleviating reperfusion/ischemia and inducing cardioprotective effects[13].

Galium species exhibit cardioprotective effects as well as having antihemolytic, antioxidant, antibacterial, anti-inflammatory, immunomodulatory, and antiproliferative potential[14-18]. The genus Galium includes over 670 species worldwide, most of them in Europe but also in Asia, North Africa, and the United States[19]. It was also observed that Galium species manifest diuretic, inflammatory, and antibacterial potential in living organisms and can treat burns, wounds, and skin diseases[15]. A recent study highlighted that Galium verum (G. verum) extract (also known as lady’s bedstraw) manifested preservative properties in terms of contractility, systolic and diastolic function maintenance, and reduced damage to the heart after ischemia. In addition, G. verum extract upregulated the activity of antioxidant enzymes alleviating the production of pro-oxidants[17]. Milevic et al[20] observed that treatment with methanol extracts of G. verum may improve the post-ischemic prognosis and modulate myocardial redox signaling, thus significantly reducing cardiac oxidative stress in a dose-dependent manner.

Death of cardiomyocytes is the essential driver of ischemic heart failure. Emerging from this, the present study aimed to test the ethanolic extract of G. verum (Gv_EtOH) on the H9C2(2-1) cardiomyocyte cells and the chorioallantoic membrane (CAM) to evaluate the in vitro and in ovo biosafety profile and complete the knowledge about the biological potential of the herb in heart disorders. To the best of our knowledge, this is the first study in the literature that examines the in vitro effect of G. verum extract on cardiomyocytes.

MATERIALS AND METHODS

Reagents and equipment

To obtain the Gv_EtOH extract from the aerial parts of G. verum L., ethanol 95% (v/v) acquired from Girelli alcool SRL (Milano MI, Italy) was used. The preparation method is detailed in the article by Semenescu et al[21].

Several reagents were used for the in vitro evaluation of the Gv_EtOH extract on the myoblast cell line. High glucose DMEM and fetal bovine serum were purchased from PAN-Biotech GmbH (Aidenbach, Germany). Trypsin-EDTA solution, penicillin/streptomycin (10000 IU/mL), DMSO, PBS, and the MTT kit were procured from Sigma Aldrich (Steinheim, Germany). The DAPI and Texas Red^™-X Phalloidin were acquired from Thermo Fisher Scientific (Waltham, MA, United States).

Cytation 5 (plate reader), Lionheart FX (automated microscope), and Gen5™ Microplate Data Collection and Analysis Software (Version 3.14) were furnished by BioTek Instruments Inc. (Winooski, VT, United States) for the in vitro analyses, and Zeiss SteREO Discovery.V8 stereomicroscope coupled with a Zeiss Axiocam 105 color digital camera were used for the in ovo tests.

Cell culture requirements and treatment protocol

Myoblast immortalized cell line, H9C2(2-1), was obtained from the American Type Culture Collection (Manassas, VA, United States), as a frozen vial. The myocardium cells were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. During the tests, H9C2(2-1) cells were kept in an incubator under specific conditions (37 °C and 5% CO₂).

The Gv_EtOH extract was dissolved in 0.5% DMSO until a 1 mg/mL stock solution was obtained. Five other concentrations were prepared from the stock solution (15-200 μg/mL), which were applied to cells.

Cellular viability

The MTT technique evaluated the activity of the Gv_EtOH extract on the viability of myoblasts. Briefly, H9C2(2-1) cells were grown in 96-well plates (10⁴ cells/well) and stimulated for 24 h with Gv_EtOH (15, 35, 55, 100, and 200 µg/ mL). After 24 h of treatment, fresh medium and 10 µL of MTT reagent 1 were added to all wells, after which the plate was incubated for 3 h at 37 °C. Finally, 100 µL/well of solubilization buffer was added, and the plate was kept protected from light at room temperature for 30 min. Absorbances were read at a wavelength of 570 nm using Cytation 5 (BioTek Instruments Inc., Winooski, VT, United States).

Analysis of confluence and cell number

Confluence and cell number were assessed to determine the influence of Gv_EtOH treatment on H9C2(2-1) cells. Images of treated cells after 24 h were taken at × 4 magnification using the Lionheart FX automated microscope. Photographs were analyzed using the cell analysis tool offered by Gen5 Microplate Data Collection and Analysis Software, version 3.14 (BioTek Instruments Inc., Winooski, VT, United States), according to the standard protocol[22].

Cell morphology investigation

The cell morphology of H9C2(2-1) cells was evaluated after 24 h of treatment with Gv_EtOH (15, 35, 55, 100, and 200 µg/ mL) to underline the changes produced by the extract. Cells were examined microscopically under bright field illumination, and photographs were taken using Lionheart FX and Gen5^™ Microplate Data Collection and Analysis Software.

Immunofluorescence staining of cell nuclei and F-actin

An immunofluorescent assay was performed to analyze the cellular components including cell nuclei and F-actin filaments. H9C2(2-1) cells were seeded in 12-well plates (10⁵ cells/well). When 80% confluence was reached, cells were treated with the lowest (15 µg/mL) and the highest concentration (200 µg/mL). After 24 h of treatment, ice-cold PBS was used to wash the cells three times, paraformaldehyde (4%) fixed cells, and Triton X/PBS (2%) permeabilized membranes. A blocking step with 1% BSA in PBS was performed. DAPI counterstained the cell nuclei (10 min at room temperature). Texas Red^™-X Phalloidin was used to stain the F-actin fibers (20 min incubation at room temperature). The images were taken with the Lionheart FX automatic microscope.

Hen’s egg CAM test

The hen’s egg CAM test (HET-CAM) method was chosen to evaluate the irritant potential of the Gv_EtOH extract. To conduct the irritability test, we followed a protocol adapted from ICCVAM[23]. Fertile hen eggs, weighing 50-60 g, were selected and incubated at 37.5 °C and 60% humidity. On the fourth day of incubation, about 5-7 mL of egg white was removed with a syringe so that the developing CAM could be detached from the inner shell. On the fifth day of incubation, a window was cut in the top of the egg to observe the CAM, which was sealed with leucoplast to avoid dehydration. Eggs were placed back in the incubator until day 8 of incubation when the HET-CAM assay was performed. Distilled water was used as a negative control and 1% sodium dodecyl sulfate (SDS) was used as a positive control. The highest tested concentration(200 μg/mL) for the Gv_EtOH extract was chosen to be tested. We applied 0.6 mL of negative control, positive control, and sample to the CAM. Changes in the vascular plexus were monitored under a stereomicroscope for 300 s, looking for signs of vascular lysis (L = disintegration of the blood vessel), coagulation (C = protein denaturation), or hemorrhage (H = bleeding occurring from the vessels). The Zeiss SteREO Discovery.V8 stereomicroscope coupled with a Zeiss Axiocam 105 color digital camera was used for imaging and analysis. The irritability score (IS) was calculated according to the formula applied in our previous study[24].

CAM anti-irritant test

To evaluate the anti-irritative properties of Galium verum extract, a volume of 600 μL of the extract (200µg/mL) was administered to the CAM of chick embryos, maintaining contact for 1 h. Subsequently, an equivalent volume of the positive control, SDS, was applied to the membrane and observed for a duration of 5 min to assess any indications of hemorrhage, clotting, or lysis.

Statistical analysis

All results were presented as the mean ± SD, and the differences were compared by one-way analysis of variance followed by Dunnett’s multiple comparisons post-test. The utilized software was GraphPad Prism version 9.2.0 for Windows (GraphPad Software, San Diego, CA, United States, www.graphpad.com). The statistically significant differences among the results are marked with ^aP < 0.0001.

RESULTS

Gv_EtOH extract manifested a stimulatory effect on H9C2(2-1) cell viability

The potential impact of Gv_EtOH extract (15, 35, 55, 100, and 200 µg/mL) on the viability of H9C2(2-1) cells was investigated after 24 h of treatment. Figure 2 shows the viability percentages of the five concentrations tested. The extract produced a statistically significant increase in cell viability compared with the control. There was a slight decrease recorded at 100 µg/mL compared with the first three concentrations. The extract demonstrated a dose-dependent stimulatory activity, with viability increasing above 100%, with a percentage of 170% at 55 µg/mL and decreasing to 135% at 200 µg/mL.

Figure 2 In vitro cell viability assessment of the ethanolic extract of Galium verum (15, 35, 55, 100, and 200 µg/mL) in H9C2(2-1) cells after 24-h treatment. A one-way analysis of variance followed by Dunnett’s multiple comparisons post-test revealed the statistical differences between the untreated (control) group and the treated groups. ^aP < 0.0001. Gv_EtOH: Ethanolic extract of Galium verum.

Gv_EtOH increased cell number and confluence

Furthermore, the effect of the Gv_EtOH extract on the confluence and the number of cells was investigated. The results are in agreement with the data obtained regarding cell viability. Therefore, at the first three concentrations, an increase in both the number of cells and the confluence was observed (up to a percentage of 178.4% for cell number and 177.1% for cell confluence). While at the doses of 100 µg/mL (171.5% and 170%, respectively) and 200 µg/mL (145.6% and 142.0%, respectively), the percentages decreased slightly (Figure 3).

Figure 3 Confluence and cell number in H9C2(2-1) cells 24 h after treatment with the ethanolic extract of Galium verum (15, 35, 55, 100, and 200 µg/mL). A: Cell confluence; B: Cell number. Analyzed by one-way analysis of variance and Dunnett’s multiple comparisons post-test. ^aP < 0.0001. Gv_EtOH: Ethanolic extract of Galium verum.

Gv_EtOH did not affect normal cellular morphology

The next direction was the analysis of G. verum L. extract on the morphology of H9C2(2-1) cells after 24 h of treatment. The results confirmed that Gv_EtOH did not have a toxic effect on myoblasts. At all tested concentrations, no significant changes in the shape of the cells were observed. The cells kept their elongated shape, but in some places slight cellular remains were evident (Figure 4).

Figure 4 Morphology of H9C2(2-1) cells after 24 h of treatment with the ethanolic extract of Galium verum. The scale bars represent 100 µm.

Gv_EtOH extract did not induce apoptosis

To comprehensively outline the impact of the Gv_EtOH extract on cellular morphology, the aspects of nuclei and actin filaments were investigated after the application of the lowest and the highest tested concentration previously (15 µg/mL and 200 µg/mL), following the immunofluorescence method. Within H9C2(2-1) cells, Gv_EtOH induced no significant modifications in nuclear structure, resulting in a slight reduction in the number of nuclei compared with control untreated cells (Figure 5). The nuclei show an oval shape and uniformly distributed chromatin. The extract caused gentle reorganization and induced slight condensation of the F-actin filaments but maintained a uniform distribution in the cytoplasm, underlining the normal morphology of H9C2(2-1) cells.

Figure 5 Immunofluorescence of nuclei and actin filament structures in H9C2(2-1) cells following 24 h of treatment with 15 µg/mL and 200 µg/mL of ethanolic extract of G. verum. Gv_EtOH: Ethanolic extract of Galium verum.

Gv_EtOH did not manifest as a potential irritant in ovo

To determine the potential irritant effect of the Gv_EtOH extract, the in ovo HET-CAM was chosen. This type of assay is used because of its capacity to produce a toxicity effect at a vascular level[25]. The European Union-approved test assesses the ability of a substance to cause changes in blood vessels leading to bleeding, clotting, hyperemia, or lysis[26]. Based on the observation of the occurrence of these processes, an IS is calculated, which helps to classify the test substances into one of the following categories: Non-irritant (0-0.9); weak irritant (1-4.9); moderate irritant (5-8.9); strong irritant (9-21)[27]. The positive control was 1% SDS, and the IS value was 18.501. Distilled water was selected as a negative control, and the IS value was 0.070. When applying 1% SDS to the CAM, the processes of hemorrhage (20 s), lysis (38 s), and coagulation (45 s) occurred, while for distilled water, none of these processes were observed. For 200 μg/mL G. verum extract, an irritation score of 5.230 was obtained, designating the extract as moderately irritating but close to the weakly irritating one (Table 1). When applying the sample to the CAM, only slight coagulation was observed (128 s) (Figure 6).

Figure 6 Anti-irritative hen’s egg chorioallantoic membrane test. Stereomicroscopic images of the chorioallantoic membrane treated with controls [H₂O used as negative control; sodium dodecyl sulfate (SDS) 1% used as positive control) and test samples [ethanolic extract of Galium verum (Gv_EtOH) 200 μg/mL] before treatment (t0) and 5 min post-treatment (t5). Scale bars represent 200 μm.

Table 1 Calculated irritation score for 200 μg/mL ethanolic extract of G. verum, H₂O (negative control), and 1% sodium dodecyl sulfate (positive control).

Sample	IS	Irritation category
H2O	0.070	Non-irritant
1% SDS 1%	18.501	Severe irritant
200 μg/mL Gallium verum	5.230	Irritant

IS: Irritation score; SDS: Sodium dodecyl sulfate.

CAM anti-irritant test

Stereomicroscopic imaging was employed to elucidate the morphological alterations observed on the CAM after the administration of SDS, which was used as a positive control. Additionally, the study examined the effects of SDS on the CAM that had been pretreated with G. verum extract at the highest concentration of 200 μg/mL. The comparative analysis of the stereomicroscopic images provided insight into the differential impacts of SDS on the CAM, both in the presence and absence of the G. verum extract, thereby contributing to the understanding of the potential protective or modulating effects of the extract against the cytotoxicity-induced by SDS. The application of SDS resulted in massive hemorrhage, accompanied by blood vessel lysis and blood clotting. Post-application of SDS on the CAM pretreated with G. verum extract, only minimal hemorrhage and lysis were noted (Figure 7), which implies that G. verum extract effectively mitigated the extensive bleeding, lysis, and coagulation processes typically induced by SDS. These findings suggest a potential protective role of G. verum extract against irritative responses in this in ovo experimental model.

Figure 7 Stereomicroscopic images of anti-irritant assay. The effects observed on the chorioallantoic membrane before and after the application of sodium dodecyl sulfate (SDS), which was used as a positive control, as well as the effects induced by SDS on the chorioallantoic membrane pretreated with the ethanolic extract of Galium verum (Gv_EtOH) extract at a concentration of 200 μg/mL demonstrated the cardioprotective effects of the Gv_EtOH.

DISCUSSION

Myocardial ischemia is a frequent condition found in failing hearts. It is an intricate clinical syndrome likely caused by alterations in functional or structural coronary circulation. Thereby, ischemia is a self-propagating trial that can negatively impact prognosis and irreversibly impair cardiac function[6]. Coronary arterial occlusion downregulates sudden myocardial contractility. Induced hypoxia is the main cause of the phenomenon, which is due to the decrease in aerobic ATP production by the ischemic myocardium. A downregulation in the influx of calcium ions is caused by sodium pump inhibition present in the ischemic heart. The coronary arterial occlusion shortens the potential plateau of the action, which is also associated with decreased systolic Ca²⁺ influx from the extracellular fluid. In addition, the ischemic myocardium induces a state of acidosis due to the lactate production that appears when shifting to anaerobic paths of energy production, which may disturb contractility by augmenting the tightness of Ca²⁺ binding to the sarcoplasmic reticulum of the heart. The upregulation of intracellular H+ concentration could also displace Ca²⁺ from its binding site on troponin, the sensitizing protein of the contractile apparatus, exerting a negative inotropic effect[28].

The therapeutic management involves antiaggregants, anticoagulants, beta-blockers, high potency statins, angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, nitrates, hydralazine, angiotensin receptor neprilysin inhibitor, inhibitors of the cardiac late sodium current, spironolactone, and digoxin. The appropriate treatment is recommended by the specialist doctor depending on the patient’s needs[29]. Moreover, the toxicity of oral anticoagulant drugs has led to increased interest in the latest research [30] identifying various plants and natural compounds that have proven to be effective in the case of IHD, being mainly involved in anti-ischemic protection[31-34], apoptosis mechanisms[35-38], inflammatory processes[39-42], and anti-oxidative actions[43-54](Table 2).

Table 2 Effective natural sources in treating cardiac ischemia.

Natural source	Active compounds	Effect on the ischemic myocardium	Ref.
Ginkgo biloba L.	Flavonoids, terpenoids, ginkgolic acids	Reduce angina and myocardial ischemia symptoms	[46,47]
Vitis vinifera L.	Resveratrol	Defense against ischemia-reperfusion injury	[31,32]
		↓ H3K27me3 levels, ameliorating hypertension status
		Promoting mitochondrial biogenesis
Gynostemma pentaphyllum Thunb.	Gypenoside A	↓ apoptosis and ↑ cell viability and proliferation on human cardiomyocytes	[38]
Citrus x aurantium L.	Hesperidin	Anti-apoptotic effects by ↑ Bcl-2 protein and ↓ Bax protein expression	[41,42]
		Induction in the expression of peroxisome proliferatoractivated receptorγ
		↓ JNK and p38-MAPK
Herba Siegesbeckiae	Kirenol	Protection against the aggravation of cardiac function in myocardial ischemia/reperfusion injury rats	[33]
		↓ inflammation and oxidative stress process
		Anti-apoptotic effect
		Blocking the activation of the NF-κB pathway reducing inflammation and oxidative stress
Momordica charantia L.	Terpenoids, tannins, saponins, flavonoids, and steroids	↓ Creatine kinase MB, heme oxygenase 1, tumor necrosis factor α, C-reactive protein, interleukin-6, and cardiac troponin I levels	[34,35]
		Restoring isoprenaline-induced abnormal electrocardiogram changes to normal levels
Morus alba L. Psidium guajava L., Psidium guajava L. Maclura pomifera Raf., Maclura tinctoria L., Malus pumila Borkh., Allium cepa L., Prunus amygdalus Batsch	Morin	↓ inflammation (TNF-α/IL-6/NFκB/IKKβ) and apoptosis (increased Bcl-2, decreased Bax/caspase-3 and TUNEL positivity) in the myocardium	[39,40]
Vitis vinifera L.; Allium cepa L.; Fragaria x ananassa Duchesne	Fisetin	↓ RAGE/NF-κB mediated oxidative stress, apoptosis, and inflammation	[43]
Lagerstroemia speciose L.	Corosolic acid	Protection of the heart against ischemia-reperfusion injury	[36]
		↓ oxidative stress and improving mitochondrial structure and function in cardiomyocytes
		↑ The expression of the mitophagy-related proteins prohibitin 2, PTEN-induced putative kinase protein-1, and parkin
		Protective effect on hypoxia and reoxygenation in cardiomyocytes
Salvia miltiorrhiza Bunge	Salvianolic acid	Improving ST segment changes
		↑ antioxidant, anti-inflammatory, and anticoagulant potential	[37]
Glycine max (L.)	Genistein	Regulation of P2X7/NF-κB pathway	[44]
Scutellaria baicalensis Georgi	Baicalin	↓ oxidative stress, inflammation, and apoptosis by suppressing TLR4/MyD88/MAPKS/NF-κB pathway	[45]
Olea europaea L.	Apigenin and vitexin	↓ LDL cholesterol, lipid peroxidation	[48,49]
		↑ Antioxidant enzyme activity
Melissa officinalis L.		↓ peroxidation of lipids such as malondialdehyde	[50]
		Blood pressure, cardiac troponin I, and infarct size
Allium sativum L.	Hydrogen sulfide, apigenin, and vitexin	↓ lipid peroxidation and superoxide and hydroxyl radicals	[51-53]
		↑ antioxidative enzymes levels
		↓ rate of cellular metabolism
Avena sativa L.	Vitamin C	↓ blood cholesterol and arterial blockage	[54]
		Regulation of c-JNK and p38-MAPK pathways

↑: Upregulation; ↓: Downregulation.

Since cardiac contractility is directly involved in the ischemic process and natural products are hot research topics in cardiac disease research, the present work aimed to test G. verum ethanolic extract on the H9C2(2-1) cell line and to identify its potential on myoblasts and to analyze its anti-irritative and protective effect on the CAM in ovo.

The tested Gv_EtOH extract was previously analyzed by liquid chromatography coupled with mass spectrometry, and rutin was identified as the most abundant compound (14.811 µg/mL)[21]. Rutin is a flavonoid known to inhibit free radical-mediated cytotoxicity and lipid peroxidation[55]. The H9C2(2-1) cell line was selected due to its specificity for cardiac disorders, especially in the case of hypoxia/reoxygenation[56].

The first step of the present work was assessing cellular viability. Results highlighted significant growth stimulation (up to 170%) especially from 15 µg/mL to 55 µg/mL of Gv_EtOH (Figure 2). The obtained results are in accordance with another report, where flavonoids in seabuckthorn manifested a similar stimulatory effect on H9C2(2-1) cells[57] and were able to inhibit doxorubicin-induced cardiotoxicity. Moreover, Wang et al[58] using the same method to study the cytotoxicity of flavonoids observed no toxic effect of isoquercitrin on myoblasts from 5-500 μM concentrations tested from 6-36 h.

The next step of the study was the assessment of effect of Gv_EtOH extract on the confluence and cell number. Up to 55 µg/mL, an augmentation was observed in both, and a regressive tendency was noticed from 100 µg/mL (Figure 3). When analyzing cellular morphology, no significant changes were observed compared with control untreated cells (Figure 4). More than that, other in vitro studies confirm the protective properties of flavonoids on myoblasts, and in vivo studies empower the findings, highlighting the potential of flavonoids and polyphenols to alleviate the abnormal morphological alterations of the myocardium[59-61]. A recent study signaled the potential ability of G. verum extract to increase the activity of the defense system via antioxidants. The obtained results have shown that methanolic extract of G. verum was able to increase the level of reduced glutathione and the activity of superoxide dismutase and catalase while reducing thiobarbituric acid reactive substances levels compared with the control group. Thereby, the oxidative damage resulting from ischemia-reperfusion was significantly reduced by G. verum extract intake in the case of hypertensive rats[20]. The same findings were achieved by Bradic et al[18], who observed that G. verum extract preserved systolic and diastolic function, cardiac contractility, and structural cardiac damages after ischemia. They confirmed the ability of the plant to activate antioxidant enzymes and alleviate the production of pro-oxidants[62].

Another stage of the research examined possible modifications in nuclear morphology. It is well known that the augmentation in left ventricular filling pressures stretches myocytes, slows coronary perfusion, modifies arteriolar circulation by inducing external compression, and results in ischemia with related troponin release[63]. Myocyte necrosis may appear because of subendocardial ischemia, which arises in the subendocardium, both in systole and in diastole and is directly influenced by increased relative wall thickness[64]. Apoptosis is a phenomenon characterized by obvious morphological changes observed at the cellular level, including cellular detachment from the substrate, cellular contraction, nuclear chromatin condensation, nuclear membrane bleeding, and actin filament structure modification[65]. DAPI staining is a frequently used technique in analyzing cell biology for identifying nuclei modifications due to its strong binding to A-T-rich regions in DNA[66]. Additionally, staining with Texas Red^™-X Phalloidin was applied to understand the dynamic cytoskeletal changes in actin filaments[65]. Our results revealed only slow modification, a slight reduction in nuclei number, and a gentle actin fiber contraction (Figure 6). An in vivo study highlighted the anti-apoptotic potential of G. verum on male Wistar albino rats treated with doxorubicin. Thus, after succeeding in treatment with the extract, the assessment of cardiac function was performed to determine the redox state. Results showed that the consumption of G. verum extract suppressed the disturbed cardiac response to changes induced by the administration of doxorubicin. Intake of the extract was associated with a significant reduction in pro-oxidants, degenerative changes, and necrosis, which were observed in the group treated with the medicine[61].

In addition to drug therapies, reperfusion therapies are successfully used in subjects with acute myocardial infarction with a significant reduction in ischemia-induced myocardial damage. Still, reperfusion-induced cardiac lesions have become more frequent. The characteristics of cardiac ischemia-reperfusion injury cover platelet activation, microvascular perfusion defects, and sequential cardiomyocyte death because of adjacent ischemic events during the reperfusion stage. Microvascular obstruction may occur with higher severity. Mitochondria in endothelial cells are reduced compared to other cardiac cells, and pathological events during cardiac microvascular injury can lead to disturbed mitochondrial dynamics and augmented mitochondrial reactive oxygen species levels (inducing oxidative stress and apoptosis). These modifications involve extracellular signals and intracellular processes such as migration, cell proliferation, permeability transition, adhesive molecule expression, metabolism, endothelial barrier function, angiogenesis, and anticoagulation[67,68].

For this reason, the next experiment performed in the present study was the analysis of the potential irritant potential of the Gv_EtOH extract in ovo. The highest tested in vitro concentration was applied on eggs. Thus, an irritation score of 5.230 (weak to moderate irritant) was obtained for 200 μg/mL G. verum extract (Table 1). When analyzing the coagulative potential, only slight coagulation events (128 s) were observed (Figure 6), highlighting the possible safety profile on ischemic cells. Another study also found that flavonoids could be introduced in the class of non-irritating active ingredients[69]. Moreover, flavonoids from ginkgo biloba were also found to be nontoxic in ovo[69].

The role of endothelial dysfunction in the pathophysiology of heart diseases is studied. It is well known that endothelial dysfunction may be instigated by oxidative stress, contributing to the development of heart failure. Downregulation in endothelial function and the nitric oxide-cyclic guanosine monophosphate pathway are frequently involved in the pathophysiological profile of heart failure with both preserved and reduced ejection fraction. Thus, an altered endothelium, favoring repeated episodes of ischemia/reperfusion, can determine a chronic stunned myocardium with increased diastolic stiffness with diastolic dysfunction and systolic disorders. Moreover, alterations induced in the nitric oxide-cyclic guanosine monophosphate pathway directly downregulates myocardial homeostasis.

Endothelial dysfunction is associated with a higher rate of cardiovascular events and a worse prognosis[70]. Thus, the last study followed the anti-irritant potential of the extract. The results of the anti-irritant assay conducted on the CAM indicated that pretreatment with G. verum extract at a concentration of 200 μg/mL effectively mitigates vascular damage induced by the positive control, SDS. Specifically, the extract demonstrated a protective effect against hemorrhage, lysis, and coagulation of blood vessels, which are critical indicators of vascular integrity. These findings suggest that G. verum may possess cardioprotective properties, potentially contributing to vascular health and offering therapeutic avenues in cardiology. Further investigation into the mechanisms underlying these protective effects is warranted to elucidate the role of the extract in cardiovascular protection.

CONCLUSION

Cardiovascular disorders have a significant influence on morbidity and mortality at the global level. IHD represents the main cause of death, accounting for half of heart failures. G. verum L. is a plant with important biological properties, including cardioprotective effects, with preservative potential in maintaining contractility function and reducing damage to the heart after ischemia. In vitro studies depicted the stimulatory effect on cellular viability, maintaining morphological characteristics, and increasing cell number. In addition, no significant apoptotic signs or irritating effects were observed, including the compound in the class of products of interest in the field of cardiology, especially in the case of IHD. The anti-irritant test revealed the protective potential on the vascular plexus, showing the effect against hemorrhage, lysis, and coagulation of blood vessels induced by SDS. Further investigation of the cellular mechanisms induced by G. verum extract in cardiomyocytes and on the CAM are necessary to elucidate the role of the extract in cardiovascular protection.

ACKNOWLEDGEMENTS

We would like to acknowledge “Victor Babes” University of Medicine and Pharmacy Timisoara for their support in covering the costs of publication for this research paper.