The main focus of my PhD project is to valorize the biodiversity of both countries, Italy and Ecuador, studying two species belonging to Lamiaceae family: Hyssopus officinalis subsp. aristatus (Godr.) Nyman an endemic plant of Italy and Ocimum campechianum Willd., a native species from Ecuador. Botanical identification and characterization of the aerial parts of these species through macroscopic and microscopic approaches was done. Screening and structural determination of main phenolics components through TLC and HPLC-DAD was performed, as well as the GC-MS characterization of essential oils (EOs) obtained by hydrodistillation. The main compounds were separated through flash chromatography and structurally identified through NMR. The potential use of the EOs and the alcoholic extracts (AEs) (ethanol 70%, methanol) in health applications was evaluated through in vitro antioxidant, antimicrobial, cytotoxic and mutagenic tests. H. officinalis subsp. aristatus has been harvested in natural habitats in Abruzzo, Civitaretenga (AQ) in 2016, Navelli and on Majella in 2018. The EOs obtained form hydrodistillation were characterized by different main compounds: Civitaretenga EO was characterized by limonen-10-yl-acetate, Navelli EO by cis- (43.2%) and trans-pinocamphone (11.0%) and Majella EO by methyleugenol (41.5%) and 1,8-cineole (39.7%). These evidences confirm the fundamental importance of the area in which plant grow in guiding the synthesis of secondary metabolites and directing the research towards further investigations into the area of Civitaretenga and other parts of Abruzzo. All AEs showed, in order of relative abundance of the main phenols, chlorogenic, rosmarinic and caftaric acid. The amount of chlorogenic acid for the specimen collected in Civitaretenga was about twice as much as in the other extracts. The AEs highlighted an interesting antioxidant capacity (DPPH test). Regarding the cytotoxicity, no significant results were observed for EOs on A549 cell line, while for the ethanolic extract an encouraging reduction in both the viability and the migration was observed. The cytotoxicity of the main molecules characterizing the extracts did not provide any appreciable results at the tested concentrations. The extracts did not show any cytotoxicity against HaCat cell line, giving indication of safe use, confirmed also by Ames test. The aerial parts of O. campechianum were collected from a wild population in the Amazonian region (Pastaza, Ecuador). The EO obtained by hydrodistillation was characterized by eugenol (46.55%), β-caryophyllene (11.94 %), β-elemene (9.06%) and 1,8-cineole (5.35%). The methanolic and ethanolic extracts, chemically characterized for the first time in this research, showed a similar fingerprinting, with the main presence of rosmarinic acid, followed by caftaric, chlorogenic acids as minor components. The ethanolic extract exhibited the greatest abundance of every compound. In particular, the quantities of caftaric and chlorogenic acid were about twice as much as in the other extract, while rosmarinic acid was nearly four times more abundant. A preliminary screening of the extracts against A549 cell line were performed but with negative outcomes. On the other hand, extracts did not show any cytotoxicity against HaCat cell line, giving indication of their safe of use. Regarding the antibacterial activity of essential oil resulted the most active preparation with moderate antimicrobial properties. All extracts exhibited a noteworthy antioxidant capacity (DPPH): in particular, the IC50 values of the EO (7.7±0.1 µg/ml) and ethanolic extract (11.1±0.01µg/ml) were close to the one of the positive control (Trolox). Rosmarinic acid and eugenol, respectively tested as main components of AEs and EO showed values close to Trolox. AEs and EO did not reveal mutagenic activity. The results confirmed very promising properties for the formulation of cosmetic and food supplements products.

Valorizzare la biodiversità dei paesi, Italia e Ecuador, studiando due specie appartenenti alla famiglia delle Lamiaceae: Hyssopus officinalis subsp. aristatus (Godr.) Nyman una specie endemica italiana e Ocimum campechianum Willd. autoctona dell'Ecuador. È stata effettuata l'identificazione botanica e la caratterizzazione delle parti aeree di queste specie attraverso osservazioni al microscopio, lo screening e la determinazione strutturale dei principali componenti fenolici attraverso TLC e HPLC-DAD, nonché la caratterizzazione GC-MS di oli essenziali (EO) ottenuti per idrodistillazione. I principali composti sono stati separati mediante flash cromatografia e identificati strutturalmente tramite NMR. Il potenziale utilizzo degli OE e degli estratti alcolici (EA) (etanolo 70%, metanolo) per applicazioni nell’ambito salutistico è stato valutato mediante test antiossidanti, antimicrobici, citotossici e mutageni in vitro. Da H. officinalis subsp. aristatus, raccolto in habitat naturali in Abruzzo (Civitaretenga (AQ) nel 2016, Navelli e in Majella nel 2018), sono stati ottenuti OE caratterizzati da diversi composti principali: Civitaretenga OE da limonen-10-il-acetato, Navelli OE da cis- (43,2%) e trans-pinocanfone (11,0%) e Majella OE da methileugenolo (41,5%) e 1,8-cineolo (39,7%). Queste risultati sottolineano l'importanza fondamentale dell'area in cui le piante crescono nel guidare la sintesi dei metaboliti secondari e nel dirigere la ricerca verso ulteriori indagini nell'area di Civitaretenga e in altre parti dell'Abruzzo. Tutti gli EA hanno mostrato, come principali fenoli, acido clorogenico, rosmarinico e caftarico . La quantità di acido clorogenico per il campione raccolto in Civitaretenga era circa il doppio rispetto agli altri estratti. Gli EA hanno evidenziato un'interessante capacità antiossidante (test DPPH). Per quanto riguarda la citotossicità, non sono stati osservati risultati significativi per gli OE sulla linea cellulare A549, mentre per l'estratto etanolico è stata osservata una incoraggiante riduzione sia della vitalità che della migrazione. Lo studio della citotossicità delle principali molecole che caratterizzano gli estratti non ha fornito risultati apprezzabili alle concentrazioni testate. Gli estratti non hanno mostrato alcuna citotossicità nei confronti della linea cellulare HaCat, fornendo indicazioni per un uso sicuro, confermato anche dal test di Ames. Le parti aeree di O. campechianum sono state raccolte allo stato spontaneo nella regione amazzonica di Pastaza (Ecuador). L'OE ottenuto per idrodistillazione era caratterizzato da eugenolo (46,55%), β-cariofillene (11,94%), β-elemene (9,06%) e 1,8-cineolo (5,35%). Gli estratti metanolici ed etanolici, caratterizzati chimicamente per la prima volta in questa ricerca, hanno mostrato come componente principale acido rosmarinico, seguito da acido caftarico e clorogenico. L'estratto etanolico possedeva quantità di acido caftarico e clorogenico circa il doppio rispetto all'altro estratto, mentre l'acido rosmarinico era quasi quattro volte più abbondante. Uno screening preliminare degli estratti contro la linea cellulare A549 ha con esiti negativi Gli estratti non hanno mostrato alcuna citotossicità nei confronti della linea cellulare HaCat, fornendo indicazioni sulla loro sicurezza d'uso. L’OE è risultato la preparazione più attiva per le proprietà antimicrobiche, comunque moderate. Tutti gli estratti hanno mostrato una notevole capacità antiossidante (DPPH): in particolare, i valori IC50 di OE (7,7 ± 0,1 µg/ml) ed estratto etanolico (11,1 ± 0,01µg/ml) erano vicini a quello del controllo positivo (Trolox), come pure quelli dell’acido rosmarinico e dell'eugenolo, rispettivamente componenti principali degli EA e dell'OE. Tutte le preparazioni non hanno rivelato attività mutagena. I risultati hanno confermato proprietà molto promettenti per la formulazione di prodotti cosmetici e integratori alimentari

Hyssopus officinalis L. subsp. aristatus (Godr.) Nyman (Italy) and Ocimum campechianum Willd. (Ecuador): botanical identification, chemical and biological characterization

-
2020

Abstract

The main focus of my PhD project is to valorize the biodiversity of both countries, Italy and Ecuador, studying two species belonging to Lamiaceae family: Hyssopus officinalis subsp. aristatus (Godr.) Nyman an endemic plant of Italy and Ocimum campechianum Willd., a native species from Ecuador. Botanical identification and characterization of the aerial parts of these species through macroscopic and microscopic approaches was done. Screening and structural determination of main phenolics components through TLC and HPLC-DAD was performed, as well as the GC-MS characterization of essential oils (EOs) obtained by hydrodistillation. The main compounds were separated through flash chromatography and structurally identified through NMR. The potential use of the EOs and the alcoholic extracts (AEs) (ethanol 70%, methanol) in health applications was evaluated through in vitro antioxidant, antimicrobial, cytotoxic and mutagenic tests. H. officinalis subsp. aristatus has been harvested in natural habitats in Abruzzo, Civitaretenga (AQ) in 2016, Navelli and on Majella in 2018. The EOs obtained form hydrodistillation were characterized by different main compounds: Civitaretenga EO was characterized by limonen-10-yl-acetate, Navelli EO by cis- (43.2%) and trans-pinocamphone (11.0%) and Majella EO by methyleugenol (41.5%) and 1,8-cineole (39.7%). These evidences confirm the fundamental importance of the area in which plant grow in guiding the synthesis of secondary metabolites and directing the research towards further investigations into the area of Civitaretenga and other parts of Abruzzo. All AEs showed, in order of relative abundance of the main phenols, chlorogenic, rosmarinic and caftaric acid. The amount of chlorogenic acid for the specimen collected in Civitaretenga was about twice as much as in the other extracts. The AEs highlighted an interesting antioxidant capacity (DPPH test). Regarding the cytotoxicity, no significant results were observed for EOs on A549 cell line, while for the ethanolic extract an encouraging reduction in both the viability and the migration was observed. The cytotoxicity of the main molecules characterizing the extracts did not provide any appreciable results at the tested concentrations. The extracts did not show any cytotoxicity against HaCat cell line, giving indication of safe use, confirmed also by Ames test. The aerial parts of O. campechianum were collected from a wild population in the Amazonian region (Pastaza, Ecuador). The EO obtained by hydrodistillation was characterized by eugenol (46.55%), β-caryophyllene (11.94 %), β-elemene (9.06%) and 1,8-cineole (5.35%). The methanolic and ethanolic extracts, chemically characterized for the first time in this research, showed a similar fingerprinting, with the main presence of rosmarinic acid, followed by caftaric, chlorogenic acids as minor components. The ethanolic extract exhibited the greatest abundance of every compound. In particular, the quantities of caftaric and chlorogenic acid were about twice as much as in the other extract, while rosmarinic acid was nearly four times more abundant. A preliminary screening of the extracts against A549 cell line were performed but with negative outcomes. On the other hand, extracts did not show any cytotoxicity against HaCat cell line, giving indication of their safe of use. Regarding the antibacterial activity of essential oil resulted the most active preparation with moderate antimicrobial properties. All extracts exhibited a noteworthy antioxidant capacity (DPPH): in particular, the IC50 values of the EO (7.7±0.1 µg/ml) and ethanolic extract (11.1±0.01µg/ml) were close to the one of the positive control (Trolox). Rosmarinic acid and eugenol, respectively tested as main components of AEs and EO showed values close to Trolox. AEs and EO did not reveal mutagenic activity. The results confirmed very promising properties for the formulation of cosmetic and food supplements products.
ECHEVERRIA GUEVARA, MONICA PAULINA
GUERRINI, Alessandra
PINTON, Paolo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/2479169
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