Ispitivanje genotoksičnih i citotoksičnih efekata vrsta Artemisia vulgaris i Artemisia Alba na humane limfocite in vitro i SW-480 ćelijsku liniju karcinoma kolona

Abstract

Artemisia vrste se tradicionalno koriste u tretmanu različitih bolesti. Cilj ove doktorske disertacije bio je da se ispita fitohemijski sastav i genotoksični i citotoksični efekat ekstrakata (metanolski, acetonski i vodeni) Artemisia vulgaris L. i Artemisia alba Turra pojedinačno i u kombinovanom tretmanu sa mitomicinom C (MMC). Fitohemijski sastav je utvrđen spektrofotometrijski i primenom HPLC metode. Citotoksičnost je procenjena MTT testom na ćelijskoj liniji kancera kolona (SW-480) i mezenhimalnim matičnim ćelijama periodoncijuma (PDLS), dok je tip ćelijske smrti utvrđen primenom protočne citometrije. Citokinezis-blok mikronukleus (MN) test u kultivisanim humanim limfocitima je korišćen za procenu genotoksičnog i antimutagenog efekta. Ekstrakti su bili bogati ukupnim fenolima i flavonoidima, dok su se kvantitativnom analizom izdvojili metanolski ekstrakti obe biljke kao najbogatiji. Najjači citostatski efekat posredovan ranom apoptozom postignut je nakon tretmana malignih ćelija acetonskim ekstraktom A. alba. Obe biljke ostvarile su sinergističko dejstvo sa MMC indukujući snažan citotoksični i proapoptotski efekat na maligne ćelije. Vodeni ekstrakt A. alba nije delovao genotoksično na humane limfocite, dok su u kombinovanom tretmanu sa MMC ekstrakti obe biljke ispoljili antimutageni efekat. Analizom efekta najzastupljenije fenolne kiseline (3,5-dihidroksibenzoeve kiseline) i flavonoida (kvercetin-3-Oglukopiranozida) uočeno je da je fenolna kiselina bila genotoksična samo u najvišoj, dok je flavonoid povećavao MN frekvencu u svim testiranim koncentracijama. MMCindukovana MN frekvenca se redukovala nakon tretmana fenolnom kiselinom, dok je flavonoid ispoljio komutageni efekat. Dobijeni rezultati ukazuju da je korišćenje vodenog ekstrakta A. alba u lekovite svrhe bezbedno.

Аrtemisia species are used in traditional medicine to treat various diseases. The aim of this dissertation was to investigate the phytochemical composition and genotoxic and cytotoxic effect of different extracts (methanolic, acetone, and aqueous) of Artemisia vulgaris L. and Artemisia alba Turra, separately and in combination with mitomycin C (MMC). The phytochemical composition was determined spectrophotometrically and using the HPLC method. Cytotoxicity was assessed by the MTT assay on a colon cancer cell line (SW-480) and stem cells derived from the human periodontal ligament (PDLSCs), while the type of cell death was analyzed by flow cytometry. The cytokinesis-block micronucleus (MN) assay in peripheral blood lymphocytes was used to evaluate the genotoxic and antimutagen effect. The extracts were rich in total phenolic and flavonoid, while quantitative analysis revealed that the methanolic extracts were the most abundant. The great cytostatic effect mediated by early apoptosis was achieved after treatment with acetone extract A. alba. Both plants achieved a synergistic effect with MMC, inducing strong cytotoxic and proapoptotic effect on malignant cells. Aqueous extract of A. alba was not genotoxic on human lymphocytes, whereaes in the combined treatment with MMC, both plants showed antimutagenic effect. Analysis of the effect of the most abundant phenolic acid (3,5- dihydroxybenzoic acid) and flavonoid (quercetin-3-O-glucopyranosid) showed that phenolic acid showed a genotoxic effect in the highest, whereas flavonoid increased MN frequency at all tested concentrations. MMC-induced MN frequency was reduced after treatment with phenolic acid, whereas flavonoid exhibited significant co-mutagenic effect. The results showed that the use of aqueous extract of A. alba for medicinal purposes is safe.