Key findings:

Key findings include: the ethanolic extract of Verbena officinalis L. exhibited potent antioxidant activity with an IC50 of 94.12 μg/ml in DPPH radical scavenging; significant cytotoxic effects against MCF-7 breast carcinoma cells with an IC50 of 47.78 μg/ml in MTT assay, indicating its potential as a natural source of antioxidants and anti-cancer agents.

What is known and what is new?

The known aspect is the traditional medicinal use of Verbena officinalis L. (V. officinalis) in treating various diseases. The new contribution is the study demonstrating the strong antioxidant and cytotoxic effects of ethanolic V. officinalis extract on breast carcinoma cell lines (MCF7), with significant DPPH radical scavenging and anti-cancer activity, highlighting its potential as a therapeutic agent for breast cancer treatment.

What is the implication, and what should change now?

The implication of this study is the potential of Verbena officinalis L. extract as a valuable source of antioxidants and anti-cancer agents for breast carcinoma treatment. Changes needed include further research to elucidate the specific bioactive compounds responsible for these effects, clinical trials to validate its efficacy, and exploration of potential formulations for therapeutic applications in oncology.

Breast cancer is a type of malignant neoplasm that is caused when cells in the breast grow out of control and become tumorous. This type of cancer affects more women than any other type of cancer in the world, Moreover, metastasis is the major cause of death for cancer patients [1]. Chemotherapy and radiotherapy are both clinically expensive and can have side effects. In terms of side effects and cost-effectiveness, Medicinal plants may be the most effective alternative to chemotherapy drugs in addressing these concerns. Secondary metabolites with diverse biological activities are abundant in plants [2, 3]. Therefore, they have identified numerous bioactive compounds in plants that have significant therapeutic potential. Antitumor activity is just one of the many medicinal uses discovered for plant chemicals like flavonoids, carotenoids, alkaloids, and phenolics[4,5]. Verbena Officinalis L. is one of these plants. It is regarded as one of the medicinal traditional plants found in various countries around the world.  Verbena officinalis, a plant from the Verbenaceae family, is commonly called pigeons’ grass, herb of grace, or vervain. It is a perennial erect herb, which grows to a height of about 25–100 cm, having toothed and lobed leaves. It is a flowing species that belongs to the Verbenaceae family, has medium-sized leaves, and is deciduous [6]. V. Officinalis contains glycosides, flavonoids, sterols, saponins, terpenoids phenolic acids,iridoids , and ursolic, according to the phytochemical qualitative analysis [7]. In vitro studies have shown that this plant's extracts have been employed for the treatment of many ailments such as gastric diseases, abrasion, skin burns,[8] wounds, thyroid problems, rheumatic pain,[9] asthma and cough,[10] amenorrhea, enteritis, acute dysentery,[11] expectorant and diuretic [12]. V. officinalis studied for its new important bioactivities including analgesic and anti-inflammatory,[13,14] antioxidant,[15] antifungal,[16] antibacterial,[17,18] anticancer,[19,20] neuroprotective,[11] antidepressant,[21] antiproliferative,[22] and, urolithiasis.[23] properties. V. Officinalis extract showed selective anticancer activity. The purpose of this study was to investigate the anticancer and antioxidant activities of an ethanol extract of V. O fficinalis grown in Iraq in vitro using the human breast cancer MCF-7 cell line.

Experimental Section

Plant Collection and Extraction

Leaf of V. Officinalis was collected from Baghdad, Iraq. The collected leaves were thoroughly washed using tap water. Following a thorough washing, the V. Officinalis leaves were dried in an incubator at 37 °C for one full night. A WileyMill grinder was used to grind the leaves into a fine powder (Standard Model 3). Employing the Soxhlet device and the procedure outlined in, [24] An ethanol extract with a purity level of 99.9% was produced. The round bottom flask was filled with 150 mL of ethanol and (50 g) fine powder. After 8 hours of extraction at 70 °C, the solvent extract was concentrated in a rotary evaporator at 40 °C. After collecting the dry extracts, they were weighed and placed in a refrigerator at 4 °C for long-term storage.

DPPH assays

The DPPH free radical scavenging test was used to evaluate the antioxidant activity of the ethanol extract. [25] Spectrophotometry (shimadzu) was used to evaluate the DPPH free radical scavenging activity. Colorimetric changes were measured at 517 nm. For each identified compound, a set of concentrations (25, 50, 100, and 200 μg ml-1) were used in this experiment. As a control, ascorbic acid was used. According to the percentage of free radicals.

Cell Culture

Michigan Cancer Foundation-7 (MCF-7) was derived from the pleural effusion of a year old female with carcinomas of the breast. ^[26] Dulbecco's Modified Eagle medium was used to culture the cells, which was supplemented with heat-inactivated foetal bovine serum (10%), streptomycin, penicillin (1%), and glutamine (2 mmol/L).

MTT cytotoxicity assay

V. Officinalis anticancer activity was determined using the MTT assay in the MCF7 cell line [27]. A stock solution of C. myxa was prepared by dissolving it in (DMSO), and then serial dilutions (25-200 μg/mL) were made from this solution. After 24 hours in a CO2 incubator, MCF7 cells were treated with extract (100 L) or doxorubicin (control). After 1–4 more hours of incubation, MTT (5 μg/mL) was added to each well. After discarding the media, DMSO was added to each well to dissolve the formazan crystals. Hidex Chameleon microplate reader absorbance was read at 575 nm (LabLogic Systems Ltd., Sheffield, United Kingdom)

Statistical analysis

To determine whether group variance was significant, a one-way analysis of variance (ANOVA) was used. The data were expressed as MeanStandard Deviation (SD), and statistical significance was determined using GraphPad Prism version 8.02.

Antioxidant Activity of V. Officinalis Crude Extract

V. Officinalis ethanol extract scavenging activity was determined using DPPH free radical scavenging assays. Results, in Figure (1), shows that the percentage of scavenged free radicals increase, with increasing concentrations of extract. The extract showed significantly high antioxidant activity (70.66% at 200 μg ml-1). (Figure1) shows that when the extract's free radical scavenging activity was compared to that of a positive control, ascorbic acid, the two showed nearly identical dose-dependent patterns, at 100 and 200 μg mL-1 concentrations. The basis of this method is the reduction of DPPH solution in the presence of a hydrogen-donating antioxidant. This ability to donate hydrogen atoms or electrons was determined spectro photochemically by bleaching the purple colored DPPH solution. [28] The findings show that V. Officinalis is a rich source of antioxidants due to its high flavonoid and phenolic compound content[29]. 

Figure 1: DPPH Scavenging Activity of Ethanolic V.Officinalis Extract on Concentrations Compared with Ascorbic Acid.

Viability Assay MTT of V. Officinalis.

To evaluate the cytotoxic effects of V. Officinalis extract on MCF-7 cells, was used a 3-(dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. For 24 hours at 37 °C., MCF7 cells were exposed to varying concentrations of V. Officinalis (200, 100, 50, and 25 μg ml^-1). (Figure 2) depicts the results of determining the viability of MCF7 cells after treatment with V. Officinalis; and it was found to be 42.32%, 54.13%, 65.27%,  and 87.43% respectively. V. Officinalis cytotoxicity thus increased with increasing concentration, IC₅₀ of 47.78 μg ml^-1. Several studies were conducted to determine the chemical content of V. Officinalis leaves extract, including qualitative tests for glycosides, tannins, flavonoids, and phenolics [30]. The extract's therapeutic value as an anti- inflammatory, antiparasitic, antioxidant, and anti-tumor agent was also evaluated [19,21]. The extract of V. Officinalis leaves contains a high concentration of flavonoids and other phenolic compounds, which are the main pharmacologically active ingredients [31].Based on the MTT assay results, V. Officinalis is the most potent cytotoxicity and antitumor activity extract against human breast cancer (MCF-7), which is used only in all of the following assay.

Figure 2: Cytotoxicity of Ethanol V. Officinalis extrac

Findings indicated that the ethanolic V. Officinalis extract effectively quenched DPPH free radicals. These results suggest that V. Officinalis extract may be useful as an anticancer agent because of its high cytotoxicity and antitumor activity against the MCF-7 breast cancer cell line.

Acknowledgement

The authors would like to express their appreciation to Al-Nahrain University's College of Science for providing the necessary facilities for this research.

No funding sources.

 None declared.

The study was approved by the Institutional Ethics Committee of Al-Nahrain University.

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