Valerija Dunkić

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.

(1) Background: The purpose of this study was to compare the free volatile compounds of 18 Veronica species (Plantaginaceae), as previously analyzed by gas chromatography coupled with mass spectrometry, with their DNA sequences for internal transcribed spacers ITS2 and ITS1-5.8S-ITS2 of the nuclear ribosomal DNA. (2) Methods: Two sets of DNA sequence data were generated and used for phylogenetic analysis: ITS2 sequences (~360 bp) obtained by next-generation sequencing and ITS1-5.8S-ITS2 sequences (~580 bp) sequenced by the Sanger sequencing method. Clustering from previously analyzed free volatile compounds was performed by Ward’s method. (3) Results: Both sets of DNA sequence data showed that the 18 analyzed Veronica species were grouped into eight main groups corresponding to the following subgenera: Pentasepalae, Pocilla, Chamaedrys, Veronica, Beccabunga, Cochlidiosperma, Stenocarpon and Pseudolysimachium. Results of the clustering analysis of free volatile compounds showed better clustering when using microwave-extracted volatiles. Three clusters were detected with the following main compounds: hexahydrofarnesyl acetone, hexadecanoic acid, phytol, caryophyllene oxide and (E)-caryophyllene. (4) Conclusion: The phylogenetic analysis of ITS2 data obtained by NGS technology and ITS1-5.8S-ITS2 data obtained by Sanger sequencing resulted in the grouping of 18 Veronica species into eight subgenera, which is in accordance with the existing classification. Statistical testing showed that there was no correlation between such clustering of Veronica species and clustering that was based on free volatile compounds. The achieved results can be viewed in the light of parallel evolution among some of the species of the Veronica genus as well as the fact that volatile compound composition can be influenced by environmental factors or epigenetic modifications.