Plants have been used by man since ancient times. In a first moment, people used vegetables for their nutritive purposes but after the discovery of therapeutic properties, different human communities used plants for illness cure and health progress. Milk thistle (Silybum marianum L.) is an officinal herb, famous for silymarin, a pharmaceutical compound present in its fruits (achenes). Terpenes, other bioactive compounds, are also present in this plant. Despite the important roles of terpenes in the Asteraceae family, the biosynthetic pathway responsible for terpenes biosynthesis in milk thistle is unknown and few articles are present in the literature concerning the content of terpenes in the tissues of this plant. For this reason, in this research, ecophysiological and metabolomic studies were carried out to investigate the metabolomic profile of different silymarin constituents (silybin, silychristin and silydianin) in various milk thistle tissues. Metabolomic analyses were also integrated with bioinformatic and genetic methodologies to examine the terpenes metabolomic profile in different milk thistle tissues and to characterize the genes involved in the biosynthesis of these bioactive compounds. Seed germination physiology was investigated and a correlation between afterripening and percentage of germination was detected. Metabolomic profile of silymarin constituents and terpenes were carried out in flowers from different development stages, leaves, stems and roots of milk thistle. Silymarin constituents were detected by liquid chromatography-mass spectrometry (LC-MS) in flowers at the third development stage. Terpenes were detected by gas chromatography-quadrupole mass spectrometry (GC-qMS) of the headspace sampled by solid-phase microextraction (SPME). A different distribution of sesquiterpenes, monoterpenes and terpenes derivates was found in the different milk thistle tissues. The D-Limonene was the most detected terpenes in milk thistle tissues. β-elemene was found mainly in the roots. The biosynthetic pathway responsible for sesquiterpenes biosynthesis in milk thistle (Silybum marianum L. Gaernt.) is unknown, for this reason in this study, bioinformatic, metabolomic, biochemical and molecular methodologies were integrated to investigate the candidate genes involved in the biosynthesis of these volatile compounds. Five SmTPS genes were isolated and two were found to be functional: SmTps1 was a multi-product enzyme, catalyzing the formation of amorpha 4,11-diene (small peak), β-Curcumene, β-Sesquiphellandrene and Zingiberene, while SmTPS2 was a single-product enzyme catalyzing the formation of Germacrene A. This study provides a molecular basis for the production of volatile terpenes in milk thistle tissues. In our study, for the first time the presence of terpenes was detected not only in the seed extracts, but also in other milk thistle tissues and we also report for the first time the isolation and expression of key genes involved in sesquiterpenes biosynthesis in this species. This study is very important both for the scientific community and for possible pharmacological applications. In fact, apart from their importance in plant physiology and ecology, volatile terpenoids are also utilized as natural flavor and aroma compounds and have beneficial effect on humans as health promoting compounds. Therefore, these results could lead to further uses of milk thistle not only for therapeutic purposes, but also as insecticide. Further studies are needed to understand better: the expression of silymarin in the different chemotypes; the functions of terpenes in the different milk thistle tissues and the expression of the genes involved in the biosynthesis of terpenes in this species.
L’uomo ha utilizzato le piante fin dall’antichità. In un primo momento per scopi nutritivi e successivamente, dopo la scoperta delle loro proprietà terapeutiche, anche per la cura di malattie. Il cardo mariano (Silybum marianum L.) è una pianta officinale, nota principalmente per la presenza nei suoi frutti (acheni) della silimarina, una mistura di composti con proprietà farmaceutiche. Altri composti bioattivi presenti in questa pianta sono i terpene. Nonostante l’importante ruolo dei terpeni nelle Asteraceae, pochi articoli riguardanti il contenuto terpenico nei tessuti del cardo mariano sono presenti in letteratura e ad oggi, nessun dato è noto sul pathway di biosintesi di tali composti nella pianta. Per tale motivo, in questa ricerca, studi ecofisiologici e metabolomici sono stati effettuati per analizzare il profilo metabolico dei diversi costituenti della silimarina (silibina, silicristina e silidianina) nei differenti tessuti di cardo mariano. Per esaminare il profilo metabolico dei terpeni nei tessuti di tale pianta ed i geni coinvolti nella loro biosintesi, analisi metaboliche sono state integrate con metodologie sia di bioinformatica che di genetica. Per quanto concerne lo studio fisiologico sulla germinazione dei semi, è stata individuata una correlazione tra la dormienza dei semi e la percentuale di germinazione. È stato identificato il profilo metabolico della silimarina e dei terpeni in tre differenti stadi di sviluppo delle infiorescenze, foglie, steli e radici di cardo mariano. I costituenti della silimarina sono stati individuati, mediante l’utilizzo della cromatografia liquida accoppiata alla spettrometria di massa (LC-MS), principalmente nelle infiorescenze al terzo stadio di sviluppo. Il profilo metabolico dei terpeni è stato analizzato mediante un tipo di gas-cromatografia accoppiata alla spettrometria di massa specifica per i composti volatili (gas chromatography quadrupole mass spectometry - GCqMS - of the headspace sampled by solid-phase micro extraction - SPME). Una differente distribuzione dei sesquiterpeni, monoterpeni e terpeni derivati è stata osservata nei differenti tessuti di cardo mariano presi in esame. Il D-Limonene è stato il composto principalmente identificato nei tessuti del cardo mariano, mentre la β-elemene è stata trovata principalmente nelle radici. Come detto sopra, il pathway di biosintesi dei sesquiterpeni nel cardo mariano non è ancora noto, per questo motivo, metodologie bioinformatiche, metabolomiche e molecolari sono state sfruttate per analizzare i geni coinvolti nella biosintesi di questi composti volatili. Cinque geni SmTPS sono stati isolati, due dei quali erano funzionali: SmTPS1, un enzima capace di produrre più di un composto, ha prodotto amorpha 4,11-diene, β-Curcumene, β-Sesquiphellandrene e Zingiberene; mentre SmTPS2 è stato capace di produrre un solo composto, la Germacrene A. Questo studio fornisce delle basi molecolari sulla produzione dei terpeni nei tessuti di cardo mariano. In questa ricerca, per la prima volta, è stata individuata la presenza dei terpeni non solo in estratti ottenuti dai semi, ma anche in altri tessuti di cardo mariano e sono stati anche identificati ed espressi, per la prima volta, alcuni dei geni coinvolti nella biosintesi dei sesquiterpeni di questa importante specie. Tale studio assume una grande rilevanza oltre che per la comunità scientifica anche per le applicazioni farmacologiche. Infatti, i terpeni hanno rilevanti ruoli non solo per la fisiologia ed ecologia della pianta, ma sono anche utilizzati come composti aromatici ed hanno effetti benefici sulla salute dell’uomo. Per cui, i risultati ottenuti potrebbero aprire nuovi orizzonti nella ricerca scientifica per eventuali ed ulteriori utilizzi del cardo mariano non solo a scopo terapeutico, ma anche come insetticida. Ulteriori studi sono necessari per comprendere meglio: l’espressione della silimarina nei diversi chemiotipi; le funzioni dei terpeni nei differenti tessuti del cardo mariano e l’espressione dei geni coinvolti nella biosintesi dei terpeni in questa specie.
Studio di composti bioattivi in Silybum marianum L. e caratterizzazione dei geni coinvolti nella biosintesi dei terpeni / D'Amante, Giusi. - (2020 Jan 30).
Studio di composti bioattivi in Silybum marianum L. e caratterizzazione dei geni coinvolti nella biosintesi dei terpeni
D'AMANTE, GIUSI
2020-01-30
Abstract
Plants have been used by man since ancient times. In a first moment, people used vegetables for their nutritive purposes but after the discovery of therapeutic properties, different human communities used plants for illness cure and health progress. Milk thistle (Silybum marianum L.) is an officinal herb, famous for silymarin, a pharmaceutical compound present in its fruits (achenes). Terpenes, other bioactive compounds, are also present in this plant. Despite the important roles of terpenes in the Asteraceae family, the biosynthetic pathway responsible for terpenes biosynthesis in milk thistle is unknown and few articles are present in the literature concerning the content of terpenes in the tissues of this plant. For this reason, in this research, ecophysiological and metabolomic studies were carried out to investigate the metabolomic profile of different silymarin constituents (silybin, silychristin and silydianin) in various milk thistle tissues. Metabolomic analyses were also integrated with bioinformatic and genetic methodologies to examine the terpenes metabolomic profile in different milk thistle tissues and to characterize the genes involved in the biosynthesis of these bioactive compounds. Seed germination physiology was investigated and a correlation between afterripening and percentage of germination was detected. Metabolomic profile of silymarin constituents and terpenes were carried out in flowers from different development stages, leaves, stems and roots of milk thistle. Silymarin constituents were detected by liquid chromatography-mass spectrometry (LC-MS) in flowers at the third development stage. Terpenes were detected by gas chromatography-quadrupole mass spectrometry (GC-qMS) of the headspace sampled by solid-phase microextraction (SPME). A different distribution of sesquiterpenes, monoterpenes and terpenes derivates was found in the different milk thistle tissues. The D-Limonene was the most detected terpenes in milk thistle tissues. β-elemene was found mainly in the roots. The biosynthetic pathway responsible for sesquiterpenes biosynthesis in milk thistle (Silybum marianum L. Gaernt.) is unknown, for this reason in this study, bioinformatic, metabolomic, biochemical and molecular methodologies were integrated to investigate the candidate genes involved in the biosynthesis of these volatile compounds. Five SmTPS genes were isolated and two were found to be functional: SmTps1 was a multi-product enzyme, catalyzing the formation of amorpha 4,11-diene (small peak), β-Curcumene, β-Sesquiphellandrene and Zingiberene, while SmTPS2 was a single-product enzyme catalyzing the formation of Germacrene A. This study provides a molecular basis for the production of volatile terpenes in milk thistle tissues. In our study, for the first time the presence of terpenes was detected not only in the seed extracts, but also in other milk thistle tissues and we also report for the first time the isolation and expression of key genes involved in sesquiterpenes biosynthesis in this species. This study is very important both for the scientific community and for possible pharmacological applications. In fact, apart from their importance in plant physiology and ecology, volatile terpenoids are also utilized as natural flavor and aroma compounds and have beneficial effect on humans as health promoting compounds. Therefore, these results could lead to further uses of milk thistle not only for therapeutic purposes, but also as insecticide. Further studies are needed to understand better: the expression of silymarin in the different chemotypes; the functions of terpenes in the different milk thistle tissues and the expression of the genes involved in the biosynthesis of terpenes in this species.File | Dimensione | Formato | |
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