In vitro propagacija i biološka aktivnost etarskog ulja i metanolnih ekstrakata povratiča (Tanacetum vulgare L.)
In vitro propagation and biological activity of essential oil and methanol extracts of tansy (Tanacetum vulgare L.)
2017
Authors:
Devrnja, NinaContributors
Ćalić, DušicaCvetić Antić, Tijana
Savić, Jelena
Janošević, Dušica
Tešević, Vele
Document Type:
Doctoral thesis (Published version)
Metadata
Show full item recordAbstract:
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.
Keywords:
Tanacetum vulgare L.; In vitro culture; Essential oil; Methanol extracts; Antimicrobial activity; Cytotoxic activity; Insecticidal activity; Induction of defense mechanismsSource:
University of Belgrade, Faculty of Biology, 2017, 1-233URI
http://uvidok.rcub.bg.ac.rs/handle/123456789/2159https://radar.ibiss.bg.ac.rs/handle/123456789/2852