In vitro propagacija i biološka aktivnost etarskog ulja i metanolnih ekstrakata povratiča (Tanacetum vulgare L.)

Abstract

Rodu Tanacetum pripadaju mnoge aromatične, lekovite i ukrasne vrste bogate biološki aktivnim sekundarnim metabolitima. Tanacetum vulgare L. (syn. Chrysanthemum vulgare L., povratič, vratiželja, konopljika) je višegodišnja, zeljasta biljka, poreklom iz Evrope i centralne Azije koja se obično sreće duž puteva, pruga, pašnjaka i polja ali i priobalnih područja. U Srbiji je ova vrsta deo sinurbane (ruderalne) flore. Predmet istraživanja ove disertacije bila ja fitohemijska karakterizacija sastava i bioloških aktivnosti etarskog ulja i metanolnih ekstrakata ove vrste. In vitro kultura T. vulgare je uspešno uspostavljena iz semena biljaka sakupljenih u prirodi. Kultura izdanaka je uspešno održavana i multiplikovana na MS hranljivoj podlozi uz dodatak 6-benziaminopurina (BAP) a prilikom ožiljavanja BAP je zamenjen indolsirćetnom kiselinom (IBA). In vitro kultura korenova održavana je u tečnoj MS hranljivoj podlozi sa dodatkom IBA koja je uticala na povećanje biomase gajenih korenova tokom subkultura. Histoločkom analizom sekretornih struktura in vitro gajenih izdanaka utvrđeno je da su na listovima i stablu prisutne biserijatne glandularne trihome sa sekrecionim sadržajem u kojem su detektovani lipidi, terpeni i alkaloidi. Hemijska analiza metanolnih ekstrakata herbe i korena T. vulgare ukazala je na to da su ekstrakti biljaka gajenih in vitro kvalitativno siromašniji u odnosu na metanolne ekstrakte biljaka iz prirode ali su se ekstrakti biljaka in vitro odlikovali višestruko većom zastupljenošću pojedinih jedinjenja, pre svih 3,5-O-dikafeoilhininskom kiselinom. Najzastupljenija jedinjenja su fenolne kiseline iz grupe derivata cimetne kiseline i to: neohlorogena, 3,5-O-dikafeoilhininska i dikafeoilhininska kiselina. Pored fenolnih kiselina detektovano je i 17 flavonoida. Relativni udeo fenolnih kiselina i flavonoida se razlikovao u zavisnosti od dela biljke korišćenog za pripremanje matanolnog ekstrakta. Uporedna analiza fitohemijskog sastava isparljivih komponenti etarskog ulja T. vugare sakupljenog sa prirodnog staništa i izdanaka gajenih in vitro pokazala je takođe velike razlike u zastupljenosti pojedinačnih ali i čitavih grupa jedinjenja. GC/MS analizom je pokazano da su kod biljaka iz prirode dominantne komponente u etarskom ulju pripadale grupi oksidovanih monoterpena, sa najzastupljenijim jedinjenjima trans-hrisantenil acetatom , trans-hrizantenolom , trans-tujonom i cis-tujonom. Sa druge strane, etarsko ulje in vitro gajenih biljaka T. vulgare karakterisalo se ujednačenim prisustvom monoterpena i seskviterpena. Svi monoterpeni identifikovani kod in vitro biljaka bili su prisutni i u etarskom ulju biljaka iz prirode dok su seskviterpenska jedinjenja bila daleko zastupljenija i raznovrsnija kod in vitro biljaka. Za analizu bioloških aktivnosti jedinjenja sekundarnog metabolizma T. vulgare korišćeni su etarsko ulje i metanolni ekstrakti dobijeni iz biljaka sakupljenih iz prirode. Rezultati su pokazali da svi analizirani metanolni ekstrakti ispoljavaju značajnu antioksidativnu aktivnost koja je korelirala sa sadržajem ukupnih fenola u ekstraktima. Metanolni ekstrakt korena posedovao je najveći antioksidativni kapacitet a ujedno je sadržavao i najviše ukupnih fenola, sa najvećom relativnom zastupljenošću neohlorogene, 3,5-Odikafeoilhininske i dikafeoilhininske kiseline u odnosu na ostale ekstrakte. Etarsko ulje i metanolni ekstrakti cveta, lista, stabljike i korena T. vulgare ispoljili su i značajnu antimikrobnu aktivnost testiranu na 8 bakterijskih i 6 vrsta mikromiceta. Etarsko ulje T. vulgare ispoljilo je jako antimikrobno dejstvo prema većini ispitivanih bakterijskih (5 od analiziranih 8) i svim vrstama mikromiceta. S druge strane, metanolni ekstrakti korena, lista, cveta i stabljike T. vulgare ispoljili su jaku aktivnost na sve testirane vrste bakterija i većinu mikromiceta. U većini slučajeva efekti testiranih ekstrakata su bili na nivou efekata referentnih antibiotika i antimikotika korišćenih kao pozitivne kontrole, te se etarsko ulje i metanolni ekstrakti T. vulgare mogu preporučiti u prevenciji i lečenju infekcija izazvanih vrstama prema kojima je pokazana visoka aktivnost ali i kao agensi u konzerviranju i očuvanju kvaliteta namirnica u prehrambenoj industriji. U daljem radu ispitivan je citotoksični potencijal etarskog ulja i metanolnih ekstrakata na maligne ćelijske linije humanog adenokarcinoma cerviksa (HeLa). Radi utvrđivanja nivoa selektivnog efekta ispitivana je i aktivnost na zdrave humane ćelijske linije fetalnih fibroblasta pluća (MRC-5). Rezultati in vitro testa su pokazali da je etarsko ulje ispoljilo najslabiju citotoksičnost dok su metanolni ekstrakti stabla i korena pokazali slabije citotoksično delovanje na ciljne ćelije u odnosu na metanolne ekstrakte lista i cveta. Nakon tretmana metanolnim ekstraktima lista i cveta kod većine HeLa ćelija došlo je do gubitka adhezije, skupljanja membrane i zaokrugljivanja ćelija što je ukazivalo na ćelijsku smrt. Praćenje efekta etarskog ulja T. vulgare na larve gubara (Lymantria dispar L.) pokazalo je da etarsko ulje T. vulgare nije ispoljilo akutnu toksičnost na gusenice drugog stadijuma razvića ali je izazvalo odlaganje presvlačenja i značajno smanjenje procenta presvučenih gusenica. Ingestija etarskog ulja uticala je i na druge parametre rastenja i razvića posmatrane kod gusenica četvrtog stupnja razvića. Etarsko ulje je smanjilo dnevni prirast mase gusenica četvrtog stupnja kao i brzinu konzumacije hrane. Iako je etarsko ulje uticalo i na smanjenje konverzije svarene hrane u biomasu gusenica, to smanjenje se nije pokazalo kao statistički značajno pa se može zaključiti da je time smanjen negativni efekat ingestije etarskog ulja i delom kompenzovan smanjeni unos hrane izazvan prisustvom etarskog ulja. U cilju ispitivanja potencijala etarskog ulja T. vulgare da indukuje mehanizme odbrane in vitro gajenih biljaka krompira koje su rasle u atmosferi ispunjenoj isparljivim komponentama ovog ulja, analizirana je ekspresija 4 gena uključena u mehanizme odbrane protiv patogena i herbivornih insekata. Prisustvo etarskog ulja izazvalo je intenzivnu indukciju dva analizirana PR (eng. pathogenesis-related) gena, PR-2 i PR-5. Prve promene u ekspresiji uočene su nakon 8 sati od izlaganja, a maksimum ekspresije od oko 50 puta zabeležen je za PR-2 gen nakon 12 h. Na osnovu preliminarne PTR-MS analize i merenja zastupljenosti pojedinačnih jedinjenja etarskog ulja u atmosferi teglica u kojima je bio krompir dominantna jedinjenja su bila monoterpenski ugljovodonici α-pinen i p-cimen. Pored ovih jedinjenja i artemizia-keton, cis-tujon, trans-tujon i kamfor su bili veoma zastupljeni, nagoveštavajući da bi možda upravo ove grupe jedinjenja mogli biti odgovorni za indukciju odbrane krompira.

Many aromatic, medicinal and ornamental species rich in biologically active secondary metabolites belong to the genus Tanacetum. Tanacetum vulgare L. (syn. Chrysanthemum vulgare L., tansy) is a perennial, herbaceous plant originating in Europe and Central Asia, commonly encountered along roads, stripes, pastures, fields, and coastal areas. In Serbia, this species is part of the ruderal flora. The research subject of this dissertation was the phytochemical characterization of the composition and biological activities of T. vulgare essential oil and methanol extracts. In vitro culture of T. vulgare was successfully established from seeds of plants harvested in nature. Shoot cultures have been successfully maintained and multiplied on the nutrient medium enriched with 6-benziaminopurine (BAP); for needs of rooting, BAP was replaced with indole buteric-acid (IBA). The root in vitro cultures were maintained in a liquid nutrient medium supplemented with IBA which influenced the increase in the biomass of cultivated roots during subcultures. Histological analysis of secretory structures of in vitro plantlets showed presence of biseriate glandular trichomes with detected lipids, terpenes and alkaloids as secretory content,, both on the leaves and the stalk. The chemical analysis of methanol extracts of T. vulgare herb and roots indicated that the extracts of in vitro cultivated plants were qualitatively poorer than methanol extracts of plants harvested in nature, but the extracts isolated from in vitro plants were characterized by several times higher presence of certain compounds, primarily 3,5-O-dicaffeoylquinic acid. The most common compounds were phenolic acids from the derivatives of cinnamic acid group, such as neohlorogenic, 3,5-O-dicaffeoylquinic,and dicaffeoylquinic acid. In addition, 17 flavonoids were detected. The relative proportion of phenolic acids and flavanoids differed depending on the part of the plant used to prepare the methanol extract. Comparative analysis of the chemical composition of the volatile components in essential oil of T. vulgare collected from the natural habitat and in vitro cultured plants has also shown great differences in the composition both of individual and entire groups of compounds. In the plants harvested in nature, GC/MS analysis showed that the dominant components belonged to the group of oxidized monoterpenes, with the most prevalent compounds of trans-chrysanthenyl acetate, trans-chryzanthenol, trans-thujone and cisthujone. On the other hand, the essential oil obtained from in vitro grown T. vulgare plants was characterized by a uniform presence of monoterpenes and sesquiterpens. All monoterpenes identified in in vitro plantlets were also present in essential oil of plants collected from nature, on the other hand, sesquiterpene compounds were more diverse and more represent in in vitro plants. Essential oil and methanol extracts isolated from plants harvested from natural habitat were used to analyze the biological activities of the T. vulgare secondary metabolism compounds. The results showed that all the analyzed methanol extracts exhibited a significant antioxidant activity that correlated with the total phenol content in the extracts. The root extract showed the highest antioxidant capacity, containing thehighest amount oftotal phenols, with the maximum relative content of neochlorogenic, dicaffeoylquinic, and dicaffeoylquinic acids compared to other extracts. The essential oil and methanol extracts of T. vulgare flowers, leaves, stalks and roots exhibited significant antimicrobial activity tested on 8 bacterial and 6 micromycetes species. T. vulgare essential oil exhibited a strong antimicrobial effect on most bacterial strains (5 of the 8 analyzed) and all strains of micromycetes. On the other hand, methanol extracts of T. vulgare roots, leaves, flowers and stalks exhibited strong activity on all tested bacterial species and most of the micromycetes. In most cases the effects of the tested extracts were at the level of the effects of the reference antibiotics and antimycotics used as positive controls, so both essential oil and methanol extracts of T. vulgare can be recommended in the prevention and treatment of infections caused by species on which high activity is shown and also as preserving agents in the food industry. In further research, the cytotoxic potential of essential oil and methanol extracts on human cervical adenocarcinoma (HeLa) cells was investigated. To determine the level of selective effect, the activity on healthy human cell lines of fetal lung fibroblasts (MRC-5) was also studied. In vitro results showed that the essential oil exhibited the weakest cytotoxicity while methanol extracts of the stalk and root showed poor cytotoxic action on the target cells relative to the methanol extracts of leaf and flower. The most of HeLa cells exhibited loss of adhesion after leaf and flower methanol extracts treatment, also shrinking of membrane and rounding of cells were observed, indicating cell death. Monitoring the effects of T. vulgare essential oil on gypsy moth (Lymantria dispar L.) larvae showed that this essential oil did not exhibit acute toxicity to the caterpillar of the second developmental stage , but caused a delay in the coating and a significant reduction in the percentage of coated caterpillars. Ingestion of essential oil influenced on the other parameters of growth and development observed in the fourth stage of caterpillar development. The essential oil reduced the daily increment of the mass of caterpillars of the fourth developmental stage as well as the rate of food consumption. Although the essential oil influenced the reduction in the conversion of digested food into the caterpillar biomass, this reduction did not appear to be statistically significant. Hence, it can be concluded that the negative effect of essential oil on ingestion was reduced and that reduced intake of food was partially compensated. In order to investigate the potential of T. vulgare essential oil to induce the mechanisms of defense of in vitro grown potato plants (grown in an atmosphere filled with volatile components of this oil), the expression of 4 genes involved in the mechanisms of defense against pathogens and herbivore insects was analyzed. The presence of essential oil induced intense induction of two analyzed PR (pathogenesisrelated) genes, PR-2 and PR-5. The first changes in expression were observed after 8 hours of exposure, and the maximum increase of expression of about 50 times was recorded for the PR-2 gene after 12 h. Based on the preliminary PTR-MS analysis and measurements of the representation of individual essential oil compounds in the atmosphere in jars where the potatoes were grown, the dominant compounds were the monoterpen hydrocarbons α-pinene and pcymene. Beside these compounds, artemisia-ketone, cis-thujone, trans-thujone and camphor were highly represented, suggesting that these groups of compounds might be responsible for induction of potato defenses.