Isolation of Volatile Compounds by Microwave-Assisted Extraction from Six Veronica Species and Testing of Their Antiproliferative and Apoptotic Activities

This study was conducted to determine the differences in the chemical composition of the essential oils and hydrosols of six different Veronica species (V. agrestis, V. anagalloides, V. austriaca ssp. jacquinii, V. beccabunga, Veronica cymbalaria, and V. officinalis) and to test their antiproliferative and apoptotic activities, according to the authors’ knowledge, because of insufficient research and lack of information. Also, the goal was to determine which obtained samples were better in achieving antiproliferative and apoptotic activities and due to which volatile components. Therefore, essential oils (EOs) and hydrosols (HYs) were isolated from the above-mentioned Veronica species by microwave-assisted extraction (MAE). Phytochemical identification of the free volatile compounds was performed using a GC equipped with a flame ionization detector and a mass spectrometer. Their antiproliferative and apoptotic activities against two human cancer cell lines, breast cancer cell line MDA-MB-231 and bladder cancer cell line T24, were determined. The main compounds identified in the studied Veronica EOs and HYs were terpinen-4-ol (0.34–6.49%), linalool (0.34–6.61%), (E)-caryophyllene (0.97–7.55%), allo-aromadendrene (0.18–2.21%), caryophyllene oxide (1.42–23.83%), benzene acetaldehyde (0.26–13.34%), and β-ionone (1.08–16.53%). In general, HYs of the tested Veronica species showed higher antiproliferative activity (IC50 13.41–42.05%) compared to EOs (IC50 158.1–970.4 µg/mL) on MDA-MB-231 and T24 cancer cell lines after 48 and 72 h. V. agrestis EO showed the best apoptotic effect among the EOs on the MDA-MB-231 cancer cell line (10.47 ± 0.53% and 9.06 ± 0.74% of early/late apoptosis, compared with control 3.61 ± 0.62% and 0.80 ± 0.17% of early/late apoptosis, respectively) and among the HYs V. cymbalaria showed 9.95 ± 1.05% and 3.06 ± 0.28% of early/late apoptosis and V. anagalloides 8.29 ± 1.09% and 1.95 ± 0.36% of early/late apoptosis compared with control (for EO was 7.45 ± 1.01% and 0.54 ± 0.25%, and for HY was 4.91 ± 1.97% and 0.70 ± 0.09% of early/late apoptosis, respectively) on the T24 cancer cell line. Future research will include other Croatian species of the genus Veronica to gain a more complete insight into the biological activity of the volatile products of this genus for potential discovery of drugs based on natural plant extracts.


Introduction
Throughout the long development of civilization, people have selected plants as food or/and medicine, primarily based on organoleptic evaluations.Scientific confirmation of the use of medicinal plants began with the development of analytical methods, especially chromatography.Chromatographic techniques enabled the identification and quantification of components of specialized metabolites from plant extracts and stimulated interest in studying the effects of these natural products on long-term health and preventive treatment [1].Prior to the identification and quantification of interesting and potentially biologically active compounds, they must first be isolated from plant material.In their study, Dunkić et al. [2] reported the results of isolation of volatile compounds (VCs) by classical hydrodistillation and microwave-assisted extraction (MAE).Some VCs were only isolated with either hydrodistillation or MAE.The results obtained can be explained by the fact that hydrodistillation method is known to have some disadvantages, since the same compounds are decomposed because of the high temperatures and long extraction times.On the other hand, microwave distillation can sometimes lead to the isolation of few components, as stated in the study of Wu et al. [3].However, modern techniques such as MAE are much faster, easier to use, and environmentally acceptable and enable extraction of bioactive components with less energy than conventional extraction methods [4,5].Two types of samples can be obtained using the MAE technique, essential oil (EO), and hydrosol (HY).Essential oils are lipophilic, aromatized liquids with volatile constituents, obtained from plant material by steam distillation and named after the plant from which they originate [6].Hydrosols are actually flavored waters obtained by condensation of water vapor in distillation processes [7] and contain a small quantity of water-soluble volatiles.Therefore, unlike EOs, HYs are safer for human use [8,9].The general study of specialized plant metabolites contributes to the development of different areas of phytochemistry [10].Specialized metabolites are very specific to certain plant families, genera, and species and contain an incredibly large library of bioactive compounds.Depending on the concentration, these compounds can also be toxic, and in adjusted doses, they represent a broad spectrum of phytochemical effects on human cells, bacteria, fungi, and parasites [10].Genus Veronica, which has recently been studied because of its characteristic specialized metabolites, formerly a member of the Scrophulariaceae, was subsequently transferred to the Plantaginaceae family after phylogenetic and chemotaxonomic studies [11].To date, research on specialized metabolites of the genus Veronica has mainly focused on iridoid glycosides, flavonoids, and saponins [12][13][14], while the free volatile compounds, which also constitute an important part of the specialized metabolites of this genus, have only recently begun to be studied in more detail [15][16][17].Some Veronica species are used in traditional medicine worldwide for the treatment of different disorders such as the following: as an antiscorbutic, for wound healing, in respiratory diseases for cough, or as an expectorant [18].Previous research on the Veronica species has shown that because of their specialized metabolites, they possess antioxidant, antimicrobial, cytotoxic, and antitumor activities [19][20][21].Harput et al. [22] demonstrated the antiproliferative activity of investigated methanolic extracts from five Veronica species against two tumor cell lines, KB and B16 cells.Their results showed that the MeOH extracts possessed anti-inflammatory and cytotoxic activities.EOs have been demonstrated to possess anticancer properties through different mechanisms, including cancer prevention mechanisms, as well as the impact of the established tumor cell itself and the interaction with the microenvironment [23].Key features of cancer include resistance to cell death.Therefore, therapeutic strategies are aimed to induce apoptosis [24].In medical treatment, EOs and raw natural extracts are generally well accepted by patients, although their good reputation (because of the widespread belief that their naturalness is a guarantee of safety) may hide occasional toxicity problems because of the presence of specific components [25].Despite different Veronica extracts having been used in traditional medicine for cancer treatment, only a few species have been studied for their cytotoxic and anticancer activity [14].Apoptosis, programmed cell death, is a process that includes cell changes such as cell contraction, blebbing, DNA fragmentation, nuclear fragmentation, chromatin condensation, and mRNA decay [26].Defects in apoptotic processes are associated with various diseases, including cancer.Uncontrolled cell proliferation is associated with insufficient apoptosis [26].Therefore, scientific research is increasingly focused on medicinal plant research and the elucidation of signaling pathways that control cell cycle arrest and apoptosis.Because of all mentioned above, six species of the genus Veronica were selected for this study.Consequently, the aim of this study was the phytochemical identification of FVCs in EOs and HYs isolated by microwave-assisted extraction (MAE) from six different Veronica species distributed in Croatia, V. agrestis L., V. anagalloides Guss., V. austriaca ssp.jacquinii L., V. beccabunga L., V. cymbalaria Bodard, and V. officinalis L., and determination of their antiproliferative and apoptotic activities against two human cancer cell lines: breast cancer cell line MDA-MB-231, and bladder cancer cell line T24.

Extraction of Volatile Components from Six Veronica Species
Extraction of volatiles from the six Croatian Veronica species collected in 2022 (Table 1) was performed by microwave-assisted extraction (MAE).Each extract consists of two parts, lipid and water, and both parts of the extracts of all studied species were analyzed by gas chromatography-mass spectrometry (GC-MS).The results of the composition of the lipid part (essential oil, EO) and the water part (hydrosol, HY) are presented in Tables 2 and 3.The compounds linalool, (E)-caryophyllene, allo-aromadendrene, caryophyllene oxide, hexahydrofarnesyl acetone, phytol, β-ionone, hexadecanoic acid, docosane, tricosane, tetracosane, and octacosane were detected in six studied Veronica EOs (Table 2).
Peculiarities in the EO composition for each species were investigated.In the composition of V. agrestis, the dominant compound is phytol (56.57%); in V. anagalloides, the dominant compounds are hexahydrofarnesyl acetone (16.17%) and β-ionone (13.13%).In addition, in the composition of V. austriaca ssp.jacquinii EO, hexadecanoic acid (27.66%) and phytol (13.02%) are the predominant compounds.Phytol and hexadecanoic acid are also the predominant constituents in the species V. beccabunga, with 28.08% and 17.06%, respectively.In addition to these two compounds already mentioned, the compound caryophyllene oxide (23.83%) is significantly present in the species V. cymbalaria.Together with phytol and hexadecanoic acid, which are also significantly present in the EO composition of V. officinalis, heptacosane is the most abundant compound (17.21%)(Table 2).Retention indices (RIs) were determined relative to a series of n-alkanes (C8-C40) on capillary columns VF5-ms (RI a ) and CPWax 52 (RI b ); identification method: RI, comparison of RIs with those in a self-generated library reported in the literature [27] and/or with authentic samples; comparison of mass spectra with those in the NIST02 and Wiley 9 mass spectral libraries; * injection with reference compounds; -, not identified; SD, standard deviation of triplicate analysis.

Discussion
In this paper, the antiproliferative and apoptotic activities of free volatile compounds (FVCs) of six Croatian Veronica species, V. agrestis, V. anagalloides, V. austriaca ssp.jacquinii, V. beccabunga, V. cymbalaria, and V. officinalis, were investigated.The extraction of FVCs from all plant samples was performed by microwave-assisted extraction (MAE).Twelve samples were obtained-two samples for each species (essential oil (EO) and hydrosol (HY)).All samples were analyzed by gas chromatography-mass spectrometry (GC-MS), and the obtained data are presented in Tables 2 and 3.The plant material of the mentioned Veronica species was collected in 2022 (Table 1).
The FVCs of the other five Veronica species studied in this paper were compared with previously published data [2,7,28].In the composition of the EO extract of V. anagalloides, the dominant compounds are hexahydrofarnesyl acetone (16.17%) and β-ionone (13.13%).

Discussion
In this paper, the antiproliferative and apoptotic activities of free volatile compounds of six Croatian Veronica species, V. agrestis, V. anagalloides, V. austriaca ssp.jacquinii, V. beccabunga, V. cymbalaria, and V. officinalis, were investigated.The extraction of FVCs from all plant samples was performed by microwave-assisted extraction (MAE).Twelve samples were obtained-two samples for each species (essential oil (EO) and hydrosol (HY)).All samples were analyzed by gas chromatography-mass spectrometry (GC-MS), and the obtained data are presented in Tables 2 and 3.The plant material of the mentioned Veronica species was collected in 2022 (Table 1).
The FVCs of the other five Veronica species studied in this paper were compared with previously published data [2,7,28].In the composition of the EO extract of V. anagalloides, the dominant compounds are hexahydrofarnesyl acetone (16.17%) and β-ionone (13.13%).Comparing the composition of the oil components of this species with the data previously published in the article by Dunkić et al. [2], we note a similarity in the concentration value of hexahydrofarnesyl acetone.In that research, the composition of oil components obtained by classical (Clevenger apparatus, HD) and modern hydrodistillation (MAE) was compared, so the value of hexahydrofarnesyl acetone in the EO of V. anagalloides was 14.33% for HD and 19.12% for MAE [2].Moreover, in Nazlić et al. article [7], a much lower β-ionone relative percentage was found in this sample obtained by MAE compared to this study: only 4.22%.
In the composition of V. austriaca ssp.jacquinii EO, hexadecanoic acid (27.66%) and phytol (13.02%) were the predominant compounds (Table 2).Hexadecanoic acid was also the most abundant compound in a previously published study of V. austriaca ssp.jacquinii in MAE extract (22.17%) [2].Phytol and hexadecanoic acid are also the predominant EO constituents in the species V. beccabunga, with 28.08% and 17.06%, respectively.The dominant compound in V. beccabunga collected in 2021 is oxygenated diterpene phytol with 34.54% for MAE [2], and the biggest difference in the composition of EO compounds in this line comparing 2021 and 2022 is the identification of piperitone: 29.28% in 2021 [2] and only 2.46% in this research.
Phytol was identified in the EO of V. cymbalaria with 3.71% in 2021 and 16.66% in 2022 (Table 2), while caryophyllene oxide with 23.83% was identified as less compared to in 2021 when 32.72% was identified [2].Specifically, in the EO composition of V. officinalis, heptacosane is the most abundant compound (17.21%)(Table 2), while in the previous year, it was only identified as 5.52% [2].
The most significant differences in the proportions of the following compounds in the HY of V. anagalloides species are the following: in the extracts from 2022, the compound β-ionone was 16.53% and (E)-β-damascenone was 11.55% (Table 3), while in the material from 2021, these compounds were identified in significantly lower proportions, β-ionone with 6.07% and (E)-β-damascenone with 1.52% [7].The reported differences in the EOs' relative percentages of some compounds for the species, V. anagalloides, V. austriaca ssp.jacquinii, V. cymbalaria, and V. officinalis, could also be related to different locations of material collection, not just the difference in the year of collection.
Comparing the compositions of HYs from two years, the biggest discrepancy is the identification of methyl eugenol, which is 37.01% in the HY of V. austriaca ssp.jacquinii from 2022, while it was not identified at all in the year before; moreover, not a single phenolic component characteristic of species of the genus Veronica was identified [28].The composition of V. beccabunga HY is dominated by α-pinene and piperitone at 17.11% and 19.54% (Table 3), respectively, and the differences compared with published compositions are as follows: piperitone was identified at 79.86% and α-pinene was not identified [28].
The HY of V. cymbalaria in this study is rich in the phenolic constituents methyl eugenol (38.61%) and (Z)-methyl isoeugenol (31.32%), while oxygenated sesquiterpene caryophyllene oxide is 6.26% (Table 3).Caryophyllene oxide is the most abundant compound in the previously published manuscript for the species V. cymbalaria HY with 37.12% [7].The total phenolic components in V. cymbalaria in the research conducted on the material from 2021 are represented by only 5.51%, and the most abundant is thymol with 3.83%; methyl eugenol was not identified, and (Z)-methyl isoeugenol was identified with less than 1% [7].Methyl eugenol is the most represented phenolic component in HY of V. officinalis with 22.01%, while in the previously published composition, the hydrosols of phenolic compounds was the only one represented by 2-methoxy-4-vinylphenol with 11.12%.The compounds (E)-β-damascenone and β-ionone for V. officinalis were identified in a similar percentage in both years compared (Table 3) [7].From the comparison of the phytochemical profile of the Veronica species studied, it is evident that the composition of FVCs may vary within the same plant species, which is influenced by many factors such as abiotic and biotic factors, postharvest treatment, extraction methods, and storage conditions of the extract.Among the abiotic factors, microclimatic influence on plant growth is particularly important [29].This is precisely why it is important to determine the chemical composition of any extract before beginning research into its biological activity.So far, almost 300 natural compounds from species of the genus Veronica have been identified and their biological activity studied [12,14], confirming the importance of this genus as medicinal plants.
Only a few studies have shown that extracts that contain terpenoids can behave synergistically with conventional chemotherapy.In spite of the encouraging results obtained over more than 35 years on the beneficial effects of these components, only a few clinical studies on humans have been conducted in the field.The only existing studies were conducted on limonene and its derivatives with some promising results [25].
So far, only a few Veronica species have been studied for their cytotoxic activity in vitro and in vivo, mostly methanolic and aqueous extracts of various Veronica species.Also, in our review, just two studies regarding antiproliferative activity of EOs and HYs were conducted [19,30].In the present study, the antiproliferative activity of the EOs and HYs of the above-mentioned six Veronica species was tested on two cancer cell lines (MDA-MB-231 and T24).V. agrestis EO showed significant antiproliferative activity on MDA-MB-231 after 48 and 72 h (572.1 µg/mL and 586.9 µg/mL), and 340.6 µg/mL on T24 after 72 h.According to Nazlić et al. [30], similar activity was shown for V. saturejoides (Kamešnica Sample) on the HeLa, HCT116, and U2OS cell lines.EO of V. anagalloides showed the highest antiproliferative activity among all tested EOs (on the MDA-MB-231 cancer cell line, it was 180.1 µg/mL and 243.4 µg/mL after 48 and 72 h, respectively, and on the T24 cancer cell line, it was 389.9 µg/mL and 158.1 µg/mL after 48 and 72 h, respectively).Nazlić et al. [19] reported found in oregano and thyme EO.Carvacrol induced apoptosis in the MDA-MB-231 breast cancer cell line via mitochondrial membrane permeabilization resulted in the release of cytochrome C, and the induction of caspases was indicated by DNA cleavage and fragmentation [24].According to Feng et al. [44], flavonoids extracted from Veronica sibirica (Vtfs) induced dose-dependent apoptosis in MCF-7 breast cancer cells with IC 50 of 42 µg/mL.In our present study, apoptotic activity was tested on the previously mentioned Veronica species on MDA-MB-231 and the T24 cancer cell line.Mastelić et al. [45] reported paclitaxel in a concentration of 40 nM (36.24 µg/mL) for apoptotic activity on the MDA-MB-231 cancer cell line.According to Mastelić et al. [45], apoptotic activity of paclitaxel was 7.80% and for control, 1.58%, in apoptosis for the MDA-MB-231 cancer cell line.According to Bilušić et al. [46], cisplatin was used as a positive control against the T24 and A549 cancer cell lines for apoptotic activity in a concentration of 50 µg/mL.Cisplatin showed 1.36 ± 0.82% and 0.86 ± 0.14% in early/late apoptosis, respectively, on the T24 cancer cell line.In the present study, the best apoptotic activity of all tested EOs showed V. agrestis EO on the MDA-MB-231 cancer cell line (10.47 ± 0.53% of early apoptotic and 9.06 ± 0.74% of late apoptotic cells, comparing to control 3.61 ± 0.62% and 0.80 ± 0.17% of early/late apoptosis, respectively), and among the HYs, V. cymbalaria showed 9.95 ± 1.05% of early apoptotic and 3.06 ± 0.28% of late apoptotic cells, and V. anagalloides 8.29 ± 1.09% of early apoptotic and 1.95 ± 0.36% of late apoptotic cells, comparing to control (for EO was 7.45 ± 1.01% and 0.54 ± 0.25%, and for HYs was 4.91 ± 1.97% and 0.70 ± 0.09% of early/late apoptosis, respectively) on the T24 cancer cell line.To our knowledge, no other study involved testing apoptotic activity of the Veronica species' EOs or HYs.
The results obtained in this research showed comparison of EO and HY composition from the above-mentioned Veronica species, and significant antiproliferative and apoptotic activities of tested extracts and their possible chemotherapeutic properties, which is why they deserve further investigation.Also, one of the most extensive and interesting fields of application of nanobiotechnology is medicine using plant extracts.Ahmadov et al. [47] reported the process of green synthesis by Scutellaria baicalensis extract, and the components present in plant extract were the most important in the formation and stabilization of silver nanoparticles.These bioactive components can be connected to the surface of the silver nanoparticles and function as nanodrugs.Therefore, future research should be focused on FVCs present in the Veronica species on antiproliferative and apoptotic activities, their possible encapsulation for in vivo studies, and as potential nanodrugs in medicine.1).The voucher specimens were deposited in the herbarium of the Laboratory of Botany (HPMF-HR) of the Faculty of Science, University of Split, Croatia.All specimens were air dried in a single layer for 10 days and protected from direct sunlight.

Extraction of Volatile Compounds
Plant material (30-50 g) of each Veronica species studied (Table 1) was hydrodistilled by microwave-assisted extraction (MAE) using an ETHOS X device (Milestone, Italy).MAE was performed at atmospheric pressure for 30 min (extraction process started after 10 min) at 800 W (98 • C).All extracts consisted of two layers: a lipophilic layer (essential oil) and a water layer (hydrosol).The lipophilic layer was collected in a side tube with a pentane/diethyl ether trap (VWR, Radnor, PA, USA), dried over anhydrous sodium sulfate, and stored at −20 • C until analysis.The pentane/diethyl ether trap was used because of the ease of evaporation (in order to exclude their toxicity), determining the yield of essential oil, and the certainty that there will be no loss of thermolabile components.The water extracts were also collected, and 2 g of HY from each sample was placed in a glass bottle and sealed with a stopper.The sample thus prepared was placed in a water bath, and a solid phase micro-extraction (SPME) needle was injected through the septum of the bottle cap.The first part of the process took place at 40 • C for 20 min to allow the compounds to evaporate from the water.The SPME fiber is directly above the liquid sample, which is stirred during the next 20 min of the process.The volatile compounds settled on the resin SPME fiber.The prepared sample was injected into the gas chromatography (GC) inlet and left there for 20 min to ensure that all volatile compounds were reabsorbed by the SPME fiber into the injection liner.

Identification of Volatile Compounds
Chromatographic analyses were performed using a GC (model 3900; Varian Inc., Lake Forest, CA, USA) equipped with a flame ionization detector and a mass spectrometer (model 2100 T; Varian Inc., Lake Forest, CA, USA), a nonpolar capillary column VF-5 ms (30 m × 0.25 mm i.d., coating thickness 0.25 µm, Palo Alto, CA, USA), and a polar CP Wax 52 (30 m × 0.25 mm i.d., coating thickness 0.25 µm, Palo Alto, CA, USA).The chromatographic methods and conditions for hydrosol fraction analysis were the same as described in the article by Dunkić et al. [2]: the condition for the VF-5-ms column was a temperature of 60 • C (isothermal) for 3 min, which was then increased to 246 • C at a rate of 3 • C min −1 and maintained for 25 min (isothermal).The condition for the CP Wax 52 column was a temperature of 70 • C (isothermal) for 5 min, which was then increased to 240 • C at a rate of 3 • C min −1 and maintained for 25 min (isothermal).The injection volume was 2 µL, and the split ratio was 1:20.The MS conditions were as follows: ion source temperature, 200 • C; ionization voltage, 70 eV; mass scan range, 40-350 mass units.The individual peaks of all samples were identified by comparing their retention indices of n-alkanes with those of authentic samples and the studies [27,48] by comparison with our libraries from previous work and by comparison with other previously published material for the Veronica species [49][50][51].Results are given as the mean of three analyses with standard deviation (n = 3 ± SD).

Cell Viability and Proliferation Were Determined by Measuring Cellular Metabolism Using MTT Assay
Cell viability and proliferation after treatment with essential oils (EOs) and hydrosols (HYs) of six different Veronica species (V.agrestis, V. anagalloides, V. austriaca ssp.jacquinii, V. beccabunga, V. cymbalaria, and V. officinalis) were performed against two human cancer cell lines, breast cancer cell line MDA-MB-231 and bladder cancer cell line T24, to determine which sample has the best antiproliferative activity.Stock solutions of EOs were prepared in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/mL.Cell lines MDA-MB-231 and T24 were grown in Dulbecco's modified Eagle's medium (DMEM, Euroclone, Milano, Italy) in a humidified incubator at 37 • C with 5% CO 2 .The DMEM medium contained 10% fetal bovine serum (FBS, Euroclone, Milano, Italy) and 1% antibiotics (penicillin and streptomycin, Euroclone, Milano, Italy) and was used as a negative control.An equal number of cells (1 × 10 4 ) were transferred into 96 wells and left overnight.The cells were then treated with EOs of the above-mentioned Veronica species at concentrations of 50, 100, 250, 500, and 1000 µg/mL, while HYs were tested at different dilutions (10%, 20%, 30%, 40%, and 50%) after MAE for 4, 24, 48, and 72 h.Cell viability and proliferation were determined by measuring cellular metabolism using an MTT assay.Yellow tetrazoline MTT (3-(4,5dimethylthiazolid-2)-2,5-diphenyltetrazoline bromide) is reduced in metabolically active cells to the purple formazan.After 2 h, the medium with MTT was removed, and DMSO was added.The plates were incubated for 10 min at 37 • C with shaking.The absorbance was measured at 570 nm by a HiPo MPP-96 microplate photometer (Biosan, Riga, Latvia).All samples were run on three different plates in triplicate per plate.Solvent control was measured: the highest concentration of DMSO was adjusted to 1% (v/v) and did not show antiproliferative activity.According to Mastelić et al. [45], the antiproliferative activity of paclitaxel as positive control on MDA-MB-231 after 4, 24, 48, and 72 h in percentage of metabolically active cells was 82.04%, 83.85%, 49.21%, and 33.11%.According to Bilušić et al. [46], cisplatin was used as a positive control against T24 and A549 cancer cell lines for antiproliferative activity in a concentration of 0.05 mg/mL.Cisplatin showed antiproliferative activity after 4, 24, 48, and 72 h (percentages of metabolically active cells were 91.56%, 86.33%, 56.18%, and 52.32 for the T24 cancer cell line).The experiments and procedures are in accordance with ethical and safety guidelines.For statistical analyses, a t-test with unequal variances was performed using GraphPad Prism 9.0 statistical software (San Diego, CA, USA) with the significance set at * p < 0.05.The calculation of IC 50 values was performed with GraphPad Prism software version 9.0 (San Diego, CA, USA), normalizing the data by three independent measurements of untreated controls.

Apoptotic Activity
An equal number of cells (1 × 10 4 ) were seeded in 6-well plates and treated with essential oils (EOs) and hydrosols (HYs) of V. agrestis, V. anagalloides, V. austriaca ssp.jacquinii, V. beccabunga, V. cymbalaria, and V. officinalis for 48 h and then analyzed for apoptosis to determine which sample has the best apoptotic activity.The antiproliferative activities of V. agrestis EO and HY on the MDA-MB-231 cancer cell line after 48 were IC 50 572.1 µg/mL and 28.43%.V. anagalloides EO showed antiproliferative activity after 48 on the MDA-MB-231 and T24 cancer cell lines (IC 50 180.1 µg/mL and 389.9 µg/mL, respectively).HY of V. anagalloides showed antiproliferative activity of 19.82% on MDA-MB-231 and of 26.96% after 48 h on T24 cancer cell lines.V. austriaca ssp.jacquinii HY showed better antiproliferative effect, especially on the T24 cancer cell line.After 48 h on MDA-MB-231, it showed antiproliferative effect of 42.05%, but it pointed to the T24 cell line antiproliferative activity of 26.72% after 48 h.V. austriaca ssp.jacquinii HY showed on MDA-MB-231 an antiproliferative effect of 42.05% and pointed to the T24 cell line an antiproliferative activity of 26.72% after 48 h.V. beccabunga HY showed an antiproliferative effect on the T24 cancer cell line of 29.64% after 48 h.EO of V. cymbalaria also showed antiproliferative activity on the MDA-MB-231 cancer cell line (IC 50 249.9µg/mL) after 48 h.HY of V. cymbalaria showed antiproliferative effect on the MDA-MB-231 and T24 cancer cell lines (17.41% and 18.64% after 48 h, respectively).V. officinalis HY showed on the MDA-MB-231 and T24 cancer cell lines significant antiproliferative effect after 48 h (34.28% and 13.41%, respectively).A combination of Annexin-V-FITC and propidium iodide staining allows the distinction between early (Annexin-V + /PI − ) and late (Annexin-V + /PI + ) apoptotic cells, necrotic cells, and live cells.After treatment with EOs and HYs, the cells were trypsinized, washed with PBS, and resuspended in 100 µL of the binding buffer containing 5 µL of Annexin-V-FITC and/or 10 µL of PI (FITC Annexin V Apoptosis Detection Kit with PI, BioLegend, San Diego, CA, USA).The cells were incubated for 15 min at room temperature in the dark and, thereafter, analyzed by flow cytometry (BD Accuri C6, BD Biosciences).Using the FlowLogic Software (Inivai, Mentone, VIC, Australia), the percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).For statistical analyses of the apoptosis rate, a t-test with unequal variances, one-way ANOVA followed by a post hoc Tukey test or Kruskal-Wallis test, followed by Dunn's post hoc test, were performed using GraphPad Prism 7.0 statistical software (San Diego, CA, USA), with the significance set at p < 0.05, p < 0.01 and p < 0.001.

Plants 2023 , 26 Figure 1 .
Figure 1.Metabolic activity of the cells (%) after treatment with V. agrestis EO (a,b) and V. agrestis HY (c) on MDA-MB-231 and T24 cancer cell lines.The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 1 . 26 Figure 2 .
Figure 1.Metabolic activity of the cells (%) after treatment with V. agrestis EO (a,b) and V. agrestis HY (c) on MDA-MB-231 and T24 cancer cell lines.The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 2 .
Figure 2. Metabolic activity of the cells (%) after treatment: with V. anagalloides EO (a,b) and V. anagalloides HY(c,d) on MDA-MB-231 and T24 cancer cell lines.The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 3 .
Figure 3. Metabolic activity of the cells (%) after treatment with V. austriaca ssp.jacquini EO (a,b) and V. jacquini HY (c,d) on MDA-MB-231 and T24 cancer cell lines.The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 3 .
Figure 3. Metabolic activity of the cells (%) after treatment with V. austriaca ssp.jacquini EO (a,b) and V. jacquini HY (c,d) on MDA-MB-231 and T24 cancer cell lines.The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 3 .
Figure 3. Metabolic activity of the cells (%) after treatment with V. austriaca ssp.jacquini EO (a,b) and V. jacquini HY (c,d) on MDA-MB-231 and T24 cancer cell lines.The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 4 .
Figure 4. Metabolic activity of the cells (%) after treatment with V. beccabunga HY on the T24 cancer cell line.The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 5 .
Figure 5. Metabolic activity of the cells (%) after treatment with V. cymbalaria EO (a,b) and V. cymbalaria HY (c,d) on MDA-MB-231 and T24 cancer cell lines.The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 5 .
Figure 5. Metabolic activity of the cells (%) after treatment with V. cymbalaria EO (a,b) and V. cymbalaria HY (c,d) on MDA-MB-231 and T24 cancer cell lines.The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 6 .
Figure 6.Metabolic activity of the cells (%) after V. officinalis HY on MDA-MB-231 (a) and T24 cancer cell lines (b).The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 6 .
Figure 6.Metabolic activity of the cells (%) after V. officinalis HY on MDA-MB-231 (a) and T24 cancer cell lines (b).The results are expressed as means of three independent experiments with SD values (presented as error bars).For statistical analyses, a t-test with unequal variances was performed with the significance set at * p < 0.05.

Figure 8 .
Figure 8. Apoptotic activity of V. anagalloides EO and V. anagalloides HY on the MDA-MB-231 cancer cell line (a,b).The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD) with the significance set at * p < 0.05.

Figure 9 .Figure 8 .
Figure 9. Apoptotic activity of V. austriaca ssp.jacquinii HY on the MDA-MB-231 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD) with the significance set at * p < 0.05, ** p < 0.01.

Figure 8 .
Figure 8. Apoptotic activity of V. anagalloides EO and V. anagalloides HY on the MDA-MB-231 cancer cell line (a,b).The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD) with the significance set at * p < 0.05.

Figure 10 .
Figure 10.Apoptotic activity of V. cymbalaria EO and V. cymbalaria HY on the MDA-MB-231 cancer cell line (a,b).The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD) with the significance set at * p < 0.05.

Figure 10 .
Figure 10.Apoptotic activity of V. cymbalaria EO and V. cymbalaria HY on the MDA-MB-231 ca cell line (a,b).The percentages of apoptotic cells (Annexin-V-positive cells) were determined presented as mean ± standard deviation (SD) with the significance set at * p < 0.05.

Figure 11 .
Figure 11.Apoptotic activity of V. officinalis HY on the MDA-MB-231 cancer cell line.percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as me standard deviation (SD).

Figure 11 .
Figure 11.Apoptotic activity of V. officinalis HY on the MDA-MB-231 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).

2 Figure 12 .
Figure 12.Apoptotic activity of V. anagalloides EO and V. anagalloides HY on the T24 cancer cell (a,b) The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented a mean ± standard deviation (SD) with the significance set at * p < 0.05, ** p < 0.01.

Figure 12 .
Figure 12.Apoptotic activity of V. anagalloides EO and V. anagalloides HY on the T24 cancer cell (a,b).The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD) with the significance set at * p < 0.05, ** p < 0.01.

Figure 12 .
Figure 12.Apoptotic activity of V. anagalloides EO and V. anagalloides HY on the T24 cancer cell (a,b).The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD) with the significance set at * p < 0.05, ** p < 0.01.

Figure 13 .
Figure 13.Apoptotic activity of V. austriaca ssp.jacquinii HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).

Figure 14 .
Figure 14.Apoptotic activity of V. beccabunga HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).

Figure 13 .
Figure 13.Apoptotic activity of V. austriaca ssp.jacquinii HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).

Figure 12 .
Figure 12.Apoptotic activity of V. anagalloides EO and V. anagalloides HY on the T24 cancer cell (a,b).The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD) with the significance set at * p < 0.05, ** p < 0.01.

Figure 13 .
Figure 13.Apoptotic activity of V. austriaca ssp.jacquinii HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).

Figure 14 .
Figure 14.Apoptotic activity of V. beccabunga HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).

Figure 14 .
Figure 14.Apoptotic activity of V. beccabunga HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).

Figure 15 .
Figure 15.Apoptotic activity of V. cymbalaria HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD) with the significance set at * p < 0.05, ***p < 0.001.

Figure 15 .
Figure 15.Apoptotic activity of V. cymbalaria HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD) with the significance set at * p < 0.05, ***p < 0.001.

Figure 16 .
Figure 16.Apoptotic activity of V. officinalis HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).

Figure 16 .
Figure 16.Apoptotic activity of V. officinalis HY on the T24 cancer cell line.The percentages of apoptotic cells (Annexin-V-positive cells) were determined and presented as mean ± standard deviation (SD).

Table 1 .
Details on collection data and origin of investigated Veronica species.

Table 2 .
Constituents of the essential oils (EOs, %) obtained by microwave extraction of six Veronica species.

Table 3 .
Constituents of the hydrosols (HYs, %) obtained by microwave extraction of six Veronica species.
4.1.Preparation, Extraction, and Identification of Volatile Compounds from Six Veronica Species 4.1.1.Preparation of Plant Material from Six Veronica Species All six Veronica species were collected during the flowering period in May and June 2022 at various locations in Croatia (Table