Basic Study
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World J Cardiol. Feb 26, 2025; 17(2): 102310
Published online Feb 26, 2025. doi: 10.4330/wjc.v17.i2.102310
Assessment of the biosafety profile of Galium verum in vitro on myoblasts and in ovo on chorioallantoic membrane
Diana Maria Morariu-Briciu, Sorin Lucian Bolintineanu, Andreea Rata, Alexandra Denisa Semenescu, Alina Anton, Robert Jijie, Andreea Kis, Ingrid Hrubaru, Alina Heghes
Diana Maria Morariu-Briciu, Sorin Lucian Bolintineanu, Department of Anatomy and Embryology, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania
Diana Maria Morariu-Briciu, Doctoral School, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania
Andreea Rata, Department of Vascular Surgery, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania
Alexandra Denisa Semenescu, Alina Anton, Robert Jijie, Andreea Kis, Department of Toxicology, Drug Industry, Management and Legislation, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania
Alexandra Denisa Semenescu, Alina Anton, Robert Jijie, Andreea Kis, Research Center for Pharmaco-Toxicological Evaluations, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania
Ingrid Hrubaru, Department of Obstetrics and Gynecology, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania
Alina Heghes, Department of Pharmaceutical Technology, “Victor Babes” University of Medicine and Pharmacy, Timisoara 300041, Romania
Author contributions: Morariu-Briciu DM, Bolintineanu SL, Rata A, Anton A, and Heghes A designed and coordinated the study; Morariu-Briciu DM, Semenescu AD, Jijie R, Kis A, and Hrubaru I performed the experiments and acquired and analyzed data; Morariu-Briciu DM, Anton A, Heghes A, Rata A, Bolintineanu SL, Semenescu AD, Jijie R, Kis A, and Hrubaru I interpreted the data; Morariu-Briciu DM, Bolintineanu SL, Heghes A, Anton A, Hrubaru I, Semenescu A, Rata A, Jijie R, and Kis A wrote the manuscript; All authors approved the final version of the article.
Institutional review board statement: The current study was conducted within the research center for Pharmacotoxicological Evaluations, “Victor Babes” University of Medicine and Pharmacy, Eftimie Murgu Sq. No. 2, 300041 Timisoara, Romania. In vitro and in ovo studies did not require any special Institutional Review Board Approval.
Institutional animal care and use committee statement: The study presented in manuscript 102310 was performed using two types of experimental models, namely in vitro (e.g., cell line) and in ovo (e.g., fertilized chicken eggs). An ethics committee approval was not necessary for the present in vitro study, as the cell line used for the conducted experiments was commercially obtained from licensed manufacturers and cultured according to their recommendations (H9-C2(2-1), from ATCC CRL-1446™, https://www.atcc.org/products/crl-1446). In addition, regarding the experiments performed in ovo, the Institutional Animal Care and Use Committee, the National Institutes of Health United States, and the DIRECTIVE 2010/63/EU state that experiments on fertilized chicken eggs can be conducted without ethical approval if they are carried out up to the 14th day of development (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4427727/; https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100700/). Our experiments were performed in compliance with these conditions, on the 9th day of development.
Conflict-of-interest statement: The authors declare no conflicts of interest.
Data sharing statement: No additional data are available.
Open Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Alina Anton, Assistant Professor, Department of Toxicology, Drug Industry, Management and Legislation, “Victor Babes” University of Medicine and Pharmacy, Eftimie Murgu square nr 2, Timisoara 300041, Romania. dolghi.alina@umft.ro
Received: October 14, 2024
Revised: December 9, 2024
Accepted: January 3, 2025
Published online: February 26, 2025
Processing time: 133 Days and 16.8 Hours
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.

Keywords: 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.