Naslov
Phytochemical analysis and biological activities of Jasione montana L. (Campanulaceae)

Autori
Juszczak, Aleksandra Maria

Vrsta, podvrsta i kategorija rada
Ocjenski radovi, doktorska disertacija

Mjesto
Białystok

Datum
15.09

Godina
2022

Stranica
193

Mentor
Tomczyk, Michał ; Zovko Končić, Marijana

Ključne riječi
Phytochemical analysis ; biological activity ; Jasione montana

Sažetak
Sheep’s bit scabious (Jasione montana L.) (bellflower, Campanulaceae), growing in sandy habitats in woodlands and forest-steppe areas, is a biennial stalk plant commonly found in Europe. Taking into account the scarce information on the phytochemical profile and pharmacological potential of J. montana, as well as low soil quality requirements, experimental work was undertaken to determine the chemical composition and biological activity of the J. montana herb. At the first stage of the study, a detailed qualitative analysis of the obtained extracts (JM1–JM3) and their fractions (JM4–JM6) was carried out using the LC–PDA–ESI–MS/TOF method, which resulted in the determination of the presence of 25 polyphenolic compounds – mainly flavonoids. Subsequently, an attempt was made to isolate compounds with potential pharmacological effects. As a result, three homogeneous, chromatographically pure compounds were obtained, and their full structural characterization was established based on the following spectral analyses: UV, 1H and 13C NMR, and MS. The isolated compounds were identified as luteolin (compound 22), luteolin 7-O-glucoside (compound 12), as well as luteolin 7-O-sambubioside (compound 9), which was isolated and described for the first time in the J. montana species. Based on a validated new method for quantification of the isolated compounds, compounds 22 and 12 were identified as the predominant compounds in JM4, while 22 and other flavonoid derivatives were found to be predominant in JM4. In subsequent steps, the biological activity of the extracts, fractions (JM1–JM6) and their main compounds (9, 12, 22) from J. montana were examined. Based on the results obtained in an in vitro model of C32 cutaneous melanoma cells, the JM4 fraction and its predominant compound 22 were assigned strong cytotoxic activity, represented by IC50 values of 119.7±3.2 µg/mL and 95.1±7.2 µg/mL, respectively. Moreover, the pro-apoptotic potential of the aforementioned fraction and its major component was established by cytometric determination of the apoptosis phase, mitochondrial membrane potential (MMP) and expression of caspase-3, -8, -9 and -10. The JM4 fraction was able to significantly reduce MMP in C32 cells, which was consistent with a significant percentage of cells in the early and later apoptosis phase documented by annexin V staining. Furthermore, the determination of caspase-9 activity confirmed activation of the intrinsic (mitochondrial) apoptosis pathway. Based on the observed increased expression of caspase-8 and caspase-10 under the influence of JM4 and its major component, 22, their effect on the extrinsic apoptotic pathway was assessed. Final confirmation of the pro-apoptotic activity was obtained through the determination of caspase-3, directly leading to DNA degradation and cancer cell death. In the next stage, the effect of J. montana extracts, fractions and isolated compounds on the mechanisms involved in the skin repair process was evaluated. Among all tested samples, fractions JM4 and JM5, which had the highest content of polyphenolic compounds, showed significant antioxidant potential in the DPPH and FRAP assays, as well as the highest elastase inhibition capacity. All tested extracts, fractions and isolated compounds were able to stimulate human skin fibroblast cell migration and proliferation in an in vitro "scratch" model. However, the effect on collagen synthesis was limited, and so a lack of collagen type I degradation was observed as concentration increased. Furthermore, anti-inflammatory potential was documented through the observed ability of compound 22 and fractions in which it was present, especially JM4, to modulate the expression of pro- and anti-inflammatory cytokines (IL-1β, IL-6 and IL-8), confirming the relationship between the content of flavonoid compounds and the activity of individual extracts/fractions. In conclusion, the research carried out for the purposes of the present dissertation suggests a potential application of the aboveground parts of Jasione montana in the development of new topical therapeutic preparations with potential anticancer and/or wound healing stimulating properties. However, further research on the molecular mechanism, selectivity, metabolism or toxicological properties of the plant material and compounds of natural origin in an in vivo model, as well as for the purposes of developing a suitable formulation, are necessary.

Izvorni jezik
Engleski

Znanstvena područja
Farmacija

POVEZANOST RADA