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Regulacija produkcije labdanskih diterpena u kulturi in vitro Cistus creticus L. i analiza njihove biološke aktivnosti

Regulation of labdane diterpenes production in Cistus creticus L. in vitro culture and analysis of their biological activity.

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2014
Disertacija2722.pdf (9.056Mb)
Author
Skorić, Marijana V.
Mentor
Todorović, Slađana
Committee members
Sabovljević, Aneta
Glamočlija, Jasmina
Simonović, Ana
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Abstract
Cistus creticus L. je višegodišnja, žbunasta biljka koja nastanjuje suva i topla područja Mediterana. Smolu ove vrste lokalno stanovništvo Krita sakuplja od davnina zbog izraženih aromatičnih i lekovitih svojstava. Dominantna biološki aktivna komponenta smole i izdanaka ove biljke su labdanski diterpeni, dok je koren biljke još uvek nepoznatog sastava. U cilju iznalaženja optimalnih uslova za produkciju labdanskih diterpena predmet istraživanja ove disertacije bio je ispitivanje regulacije produkcije labdanskih diterpena u biljkama C. creticus gajenim in vitro pod uticajem različitih abiotičkih faktora i hranljivih podloga sa varirajućim sadržajem različitih šećera. Uspešno je uspostavljena in vitro kultura biljaka C. creticus na MS medijumu bez fitohormona, pri čemu parametri rastenja i razvića eksplantata nisu pokazali značajne razlike između ispitivanih klonova. Optimalne koncentracije ugljenih hidrata za rastenje i razviće biljaka C. creticus u uslovima in vitro su od 0,...03 M do 0,1 M saharoze i glukoze. Histohemijskim analizama listova izdanaka gajenih in vitro je utvrđeno da kapitatne glandularne trihome tipa II C. creticus predstavljaju mesto akumulacije labdanskih diterpena i fenolnih jedinjenja. Genetička transformacija C. creticus je izvršena pomoću Agrobacterium rhizogenes A4M70GUS, pri čemu je uspostavljena kultura transformisanih korenova. Fitohemijska karakterizacija biljaka C. creticus gajenih in vitro, omogućila je identifikaciju i kvantifikaciju glavnih grupa sekundarnih metabolita (terpena i fenolnih jedinjenja) koji se u ovim uslovima produkuju. Labdanski diterpeni su dominantna jedinjenja u izdancima biljaka C. creticus gajenim in vitro, a među njima su najzastupljeniji labd-7,13-dien-15-ol i labd-7,13-dien-15-ol acetat. Od isparljivih sekundarnih metabolita najzastupljeniji su monoterpeni i seskviterpeni. U dihlormetanskim ekstraktima eksudata kapitatnih trihoma tipa II najzastupljenije jedinjenje je labd-7,13-dien-15-ol acetat. U korenovima C. creticus potvrđeno je prisustvo fenolnih jedinjenja i masnih kiselina, dok labdanski diterpeni nisu detektovani. Analiza sadržaja fenolnih jedinjenja ukazala je da su u etanolnim ekstraktima izadanaka i korenova prisutna fenolna jedinjenja kao što su hininska kiselina, galokatehin i katehin...

Cistus creticus L. is a perennial, bushy plant that inhabits mainly dry and warm areas of the Mediterranean. The resin of this species has been actively collected by the local inhabitants of Crete since ancient times due to its aromatic and medicinal properties. The predominant bioactive component of the resin and the shoots of this plant are labdane diterpenes, while the chemical composition of the roots is still unknown. In order to find the optimal conditions for the labdane diterpenes production, the subject of this thesis was to investigate the regulation of labdane-type diterpenes production in in vitro grown plants of C. creticus, as influenced by various abiotic factors and growth media with varying content of different carbohydrates. In vitro culture of C. creticus was successfully established on hormone-free MS medium, and the parameters of growth and development of explants showed no significant differences among analyzed clones. Optimal concentrations of carbohyd...rates for in vitro growth and development of C. creticus ranged from 0.03 M to 0.1 M sucrose or glucose. Genetic transformation of C. creticus was performed with Agrobacterium rhizogenes A4M70GUS and the culture of transformed roots was established. Regeneration of transformed plants was unsuccessful. Histochemical analysis of the leaves from in vitro cultured C. creticus shoots showed that labdane diterpenes and phenolic compounds accumulate in the type II capitate glandular trichomes. Phytochemical characterization of in vitro grown C. creticus has enabled the identification and quantification of two major groups of secondary metabolites (terpenes and phenols), which are produced under these conditions. Predominant compounds accumulating in shoots of C. creticus grown in vitro were labdane diterpenes, the most abundant among them being labd-7,13-diene-15-ol and labd-7,13-dien-15-ol acetate. Amongst the volatile secondary metabolites, the most abundant were monoterpenes and sesquiterpenes. Labd-7,13-dien-15-ol acetate was the most abundant compound in dichloromethane extracts of type II capitate trichomes exudates. In the roots of C. creticus, the presence of the phenolic compounds and fatty acids was determined, whereas labdane diterpenes were not detected...

Faculty:
University of Belgrade, Faculty of Biology
Date:
16-07-2014
Projects:
  • Physiological, chemical and molecular analysis of the diversity of selected rare and endangered plant species and application of biotechnology for ex situ conservation and production of biologically active compounds (RS-173024)
Keywords:
Cistus creticus L. / Cistus creticus L. / labdane diterpenes / in vitro culture / phenolic compounds / MVA and MEP biosynthetic pathway / antioxidant activity / antimicrobial activity / cytotoxic activity / labdanski diterpeni / in vitro kultura / fenolna jedinjenja / MVA i MEP biosintetski put / antioksidativna aktivnost / antimikrobna aktivnost / citotoksična aktivnost
[ Google Scholar ]
URI
http://nardus.mpn.gov.rs/handle/123456789/5446
http://eteze.bg.ac.rs/application/showtheses?thesesId=2922
https://fedorabg.bg.ac.rs/fedora/get/o:11142/bdef:Content/download
http://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=1024924338

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