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Caratterizzazione chimica e biologica di oli essenziali dell’Amazzonia orientale e definizione di un loro profilo applicativo in un contesto salutistico
No full textL’attività di ricerca svolta in questa tesi di dottorato è stata incentrata sulla valorizzazione della biodiversità della foresta amazzonica tramite lo studio di piante aromatiche dell’Ecuador. Tali piante sono particolarmente ricche di oli essenziali, metaboliti secondari costituiti principalmente da molecole volatili odorose, note per essere spesso biologicamente attive. Proprio per via della loro fragranza, gli oli essenziali sono sovente contemplati nelle tradizioni etnomediche delle popolazioni indigene. Questo studio ha come punto di partenza le informazioni della medicina tradizionale con lo scopo di investigare piante poco conosciute potenzialmente interessanti per la salute dell’uomo (in campo farmaceutico, cosmetico ed alimentare). Dopo un’accurata ricerca in letteratura, sono state selezionate per essere studiate le seguenti piante: Chenopodium ambrosioides, Dacryodes peruviana, Piper carpunya, Schinus molle, Tagetes minuta, Endlicheria klugii, Ocotea cernua, Cymbopogon citratus, Ocimum micranthum e Ocotea quixos. Queste specie vegetali fanno parte di differenti famiglie proprio per abbracciare una maggior biodiversità possibile.
Grazie ad una collaborazione con l’Universidad Politecnica Salesiana (Quito, Ecuador) e con l’Universidad Estatal Amazonica (Puyo, Ecuador), sono state distillate le droghe fresche delle piante ottenendone gli oli essenziali per arrivare poi ad un “fingerprinting” chimico e biologico. Gli oli essenziali sono stati caratterizzati chimicamente tramite gascromatografia accoppiata alla massa (GC-MS) e risonanza magnetica nucleare (NMR) per una determinazione qualitativa e quantitativa dei composti in essi presenti e successivamente sono stati testati per determinare diverse attività biologiche. Si è partiti dallo studio dell’attività antiossidante mediante il saggio del DPPH, dove l’olio essenziale di O. micranthum ha confermato eccellenti risultati. Sono stati eseguiti test di attività antimicrobica con batteri e funghi sia patogeni per l’uomo che fitopatogeni ed è stata approfondita la loro attività di sinergismo con farmaci di sintesi. Particolarmente interessante si è rilevata l’attività su funghi dermatofiti dove molti dei campioni sono risultati efficaci e il sinergisno con il fluconazolo degli oli essenziali di P. carpunya e S. molle nei confronti di vari ceppi di Candida spp. Sono stati effettuati anche esperimenti di attività antinfiammatoria, nei quali i risultati migliori si sono ottenuti con l’olio essenziale di C. ambrosioides. Per verificare invece la sicurezza per l’uomo sono stati eseguiti test di attività mutagena, dove tutti gli oli essenziali presi in considerazione sono risultati negativi. Infine, è stata realizzata una formulazione cosmentica contenente l’olio essenziale di O. micranthum, ossia quello che si è mostrato più attivo. Lo scopo era quello di trovare un’applicazione per i campioni più promettenti al fine di valorizzare la biodiversità della foresta Amazzonica.The research activities performed in this PhD thesis focuses to promotion of Amazonian rainforest biodiversity by studying aromatic plants of Ecuador. Aromatics plant are reach of essential oils, secondary metabolites consisting mainly in fragrant volatile molecules known to be usually biologically active. Just for their fragrance essential oils are often known by ethnomedicine of indigenous populations. This study starts from information of traditional medicine to investigate poorly known plants potentially interesting for human health (in pharmaceutical, cosmetic and food environment). After careful literature research were choosen these plants Chenopodium ambrosioides, Dacryodes peruviana, Piper carpunya, Schinus molle, Tagetes minuta, Endlicheria klugii, Ocotea cernua, Cymbopogon citratus, Ocimum micranthum and Ocotea quixos. All of different families to consider greater biodiversity possible.
Collaborating with the Universidad Politecnica Salesiana (Quito, Ecuador) and with Universidad Estatal Amazonica (Puyo, Ecuador), fresh crude drugs have been distilled to obtain essential oils (EOs) for a phytochemical and biological fingerprinting. The EOs have been chemically characterized through GC-MS and NMR for qualitative and quantitative determination of the compounds and tested for various biological activities. Was performed antioxidant activity with DPPH assay, where O. micranthum essential oil confirmed excellent results. Was performed antimicrobial assays for human or plant pathogens bacteria and fungi, and synergistic studies between essential oil and synthetic drugs where essential oils have been particular efficient against dermatophyte and P. carpunya and S. molle essential oils shown good synergism with fluconazole in Candida spp. Strains. Were conducted antinflamatory tests in which C. ambrosioides essential oil gave best results and mutagenic experiments to verify human safety that excluded potential mutagenic activities. Finally was made a cosmetics formulation with O. micranthum, the more active essential oil. The aims was precisely to find an application for best samples to increase in value the Amazonian biodiversity
Bacillus stearothermophilus acetylacetoin synthase and diacetyl(acetoin)reductase in the synthesis of chiral a,b-dihydroxyketones
No full textC-C Bond forming reaction is one of the main goals in synthetic organic chemistry and
represents the critical step of many syntheses.1,2 In this field very attractive are the
enzymatic C-C bond forming reactions that are highly chemo-, regio- and
enantioselective. On the other hand the enzymatic reduction of prochiral carbonyl group
to chiral alcohols is widely used in organic synthesis.3
In the present communication Bacillus stearothermophilus acetylacetoin synthase
(AAS) has been used to synthesize α-hydroxy-1,3-diketones 2 and 3 (Figure 1) starting
from the corresponding 1,2-diketones 1.
Biotransformation of hyodeoxycholic acid with Rhodococcus spp.
No full textDuring the last decades the biotransformations of bile acids have been successfully used
to reduce the number of steps in the synthesis of ursodeoxycholic acid from cholic acid1
and to enhance the selectivity of the oxido-reduction reactions.2,3
The biotrasformation of hyodeoxycholic acid (3α,6α-dihydroxy-5β-cholan-24-oic acid)
with various Rhodococcus strains (collection of IEP GmbH) is reported and the
reactions are described in the Figure 1
CARATTERIZAZIONE CHIMICA E ATTIVITA' BIOLOGICA DELL'OLIO ESSENZIALE DA CORTECCE DI CRYPTOCARYA MASSOIA (Oken) Kostern (LAURACEAE)
No full textL'olio essenziale (o.e.) da cortecce di C. massoia (Lauraceae), specie endemica dell'isola di Nuova Guinea, utilizzata dall'industria dei profumi e scarsamente investigata sotto il profilo fitochimico e funzionale, è stato caratterizzato mediante GC-FID e GC-MS e valutato per le capacità antiossidanti ed antimicrobiche. Dall'indagine chimica è emersa la prevalente ed insolita presenza di diversi lattoni (82%), alcuni dei quali inediti per la specie, tra cui: massoia lattone C8=3.39±0.08%, C10=56.06±0.42%, C12=16.51±0.11%, C14=0.56±0.01%). È stata inoltre verificata la presenza minoritaria di composti benzilici (15%) con prevalenza di benzil benzoato 12.73±0.10% e di benzoil salicilato 1.78±0.01%. Per quanto riguarda i saggi di bioattività, l'o.e. è stato dapprima sottoposto a saggi di valutazione dell'attività antiossidante mediante test spettrofotometrici del DPPH e ABTS che hanno evidenziato una modesta efficacia se confrontata con i dati espressi dall'o.e. di timo commerciale preso a riferimento. La valutazione dell'attività biologica dell'o.e. è stata estesa alla capacità antimicrobica (disk diffusion assay) impiegando 12 ceppi di microrganismi, tra batteri Gram+, Gram- e lieviti. I valori di MIC più interessanti si sono rivelati quelli in riferimento a Klebsiella oxytoca (9.8±1.1μg/μl); con Bacillus brevi e Pseudomonas aeruginosa, l’olio di massoia ha evidenziato MIC (18.6±1.4μg/μl) confrontabili con quelle espresse dal controllo positivo (o.e. di timo). Di particolare interesse è risultata l'efficacia verso Candida albicans (MIC=4.9±0.8μg/μl) con valori circa 4 volte inferiori rispetto al riferimento positivo (o.e. timo commerciale). Le valutazioni di bioattività sono state estese al metodo bioautografico con lastre (HP)TLC per valutare qualitativamente quali classi di metaboliti dell'o.e. di massoia fossero maggiormente responsabili dell'attività del fitocomplesso. I massoia lattoni corrispondenti alle bande risultate più attive sono stati successivamente isolati e sottoposti a caratterizzazione, tutt'ora in corso. Relativamente ad espressioni di sicurezza, sono state effettuate indagini di genotossicità (Ames test) che hanno escluso proprietà mutagene dell'olio essenziale. Da questa indagine preliminare, emerge il singolare profilo fitochimico di questo olio essenziale caratterizzato prevalentemente da composti lattonici, e da una bioattività interessante, degna di ulteriori approfondimenti rispetto ad un potenziale impiego salutistico
PRELIMINARY PHYTOCHEMICAL AND BIOLOGICAL PROPERTIES OF A SARDINIAN ENDEMISM: RIBES SANDALIOTICUM ARRIGONI
No full textRibes sandalioticum (basionim Ribes multiflorum Kit. ex Roem. & Shult. subsp. sandalioticum Arrigoni, Grossulariaceae) represents one of the almost 300 plant endemisms in Sardinia, with an areal comprised among Monte Limbara, Punta Balestrieri, Cima Giugantinu, Marghine and Gennargentu chains (Ballero & Appendino, 2000, and references therein). Since R. sandalioticum (RS) has been never investigated before under a phytochemical and functional point of view, the present research reports preliminary data regarding polyphenols, procyanidins and flavonoids content, antioxidant and antimicrobial properties of hydro-alcoholic extracts (Mother Tinctures, MTs) prepared following European Pharmacopoeia (7th ed.) indications. Fresh leaves from spontaneous plants collected at Monte Limbara (Nuoro) were freeze dried and extracted; the same procedures were performed for fresh leaves from R. nigrum Sardinian spontaneous plants (RNS) and for commercial Croatian crude drug (leaves) (RNC), in order to have phytochemical, biological and healthy reference with the most used herbal drug belonging to the genus Ribes. HPTLC qualitative analyses revealed the presence of kaempferol-3-glucoside, hyperoside, isoquercitrin and chlorogenic acid. Further investigations are in progress to identify two compounds evidenced at Rf 0.35 and 0.80; most probably the latter corresponds to quercetin-pentose. HPTLC performed for RNS and RNC samples revealed the same chromatographic profile except for the detection of chlorogenic acid and the absence of the two bands under investigation, which therefore emerged as chemo-typical of RS. Total polyphenols, procyanidins and flavonoids of Ribes MTs were then checked employing spectrophotometric methods (Porter et al., 1986). RS revealed a total polyphenol content of 11.99±1.07 μg gallic acid/μl MT). The value was slightly lower (9.37%) than that expressed by Sardinian RNS (13.23±0.95 μg gallic acid/μl MT) and 20.60% higher than that of RNC (9.52±0.78 μg gallic acid/μl MT). For total flavonoids, considered as μg hyperoside/μl MT, RS revealed a 10.46% (4.45±0.30) lower content than that of RNS (4.97±0.39), but 54.83% higher than that of commercial RNC (2.01±0.11). Procyanidins were most abundant in RS (2.18±0.18 μg cyanidin chloride/μl MT), 6.88% higher than in RNS (2.03±0.27) and 83.03% higher than RNC (0.37±0.04). Antioxidant capacity (IC50 μg/ml) was performed through spectrophotometric assays (DPPH, ABTS) and HPTLC-bioautographic assay (Rossi et al., 2011) revealing a bioactivity of RS always intermediate between RNS and RNC samples. HPTLC-bioautography evidenced the flavonoids detected as the most responsible and, interestingly, with an increasing antioxidant capacity in the following 24hs. Antimicrobial activity (bacteria and yeasts) of RS extracts, performed through disk diffusion and HPTLC-bioautographic assays, evidenced very low efficacies (for e.g. B. subtilis, MIC: 30.8μg/μl corresponding to MT as is; C. albicans, MIC: 15.4μg/μl, corresponding to MT 50% diluted). The involvement of the detected flavonoids and of the chemicals corresponding to Rf 0.35 and 0.80 has been assessed through bioautographic assays (Rossi et al., 2011). In conclusion, this is the first report about the Sardinian endemism Ribes sandalioticum and further investigations are in progress in order to better clarify its chemodiversity and possible functional perspectives in herbal healthy market
UN PROFILO CHIMICO E DI BIOATTIVITÀ DELL'OLIO ESSENZIALE DI CROTON LECHLERI MÜLL. ARG. (EUPHORBIACEAE): DALLA FORESTA AMAZZONICA ALLA FARMACIA?
No full textCortecce di C. lechleri, raccolte da piante adulte cresciute in tre differenti siti nella provincia Morona-Santiago (Ecuador) ai margini della foresta amazzonica sono state distillate in corrente di vapore [olio essenziale (o.e.)=0.61 ml/kg]. L'o.e. presentava una composizione (GC, GC-MS, NMR) di 74 costituenti, con una prevalenza di sesquiterpeni (sesquicineolo, 17,29%; α-calacorene, 11,29%; 1,10-di-epi-cubenolo, 4,75%; β-calacorene, 4,34%; epi-cedrolo, 4,09%). Il profilo di bioattività è stato determinato valutando attività antiossidante, antimicrobica, citotossicità, mutagenicità e mutageno protezione. DPPH assay e β-carotene bleaching test evidenziavano IC50 coerenti con una efficacia antiossidante significativamente superiore ai riferimenti. Il saggio TLC-DPPH-bioautographic assay ha poi evidenziato che 3 frazioni dell’olio caratterizzate da α- (24%), β- (8%) e δ-cadalene (15%) erano le principali responsabili dell'attività. L’attività antibatterica su TLC ha espresso MIC variabili tra 10 μg/spot (E. coli) e 100 μg/spot (P. aeruginosa) e un principale coinvolgimento dei calacoreni. Il saggio di Ames su ceppi di Salmonella typhimurium TA98 e TA100 con e senza attivazione metabolica (S9) ha dimostrato la non citotossicità e la non mutagenicità dell’o.e. (10-2 e 100 mg/piastra) e, per contro, una riduzione delle colonie revertenti sul ceppo TA98 del 30% (+S9) e del 33% (-S9) in presenza di 2-aminoanthracene e nitro fluorene (2 μg/piastra). Il test di mutageno-protezione è stato esteso a mutageni di interesse dietistico (ammine eterocicliche, HCAs) con quantità di o.e. comprese tra 10-2 e 1.0 mg/piastra, tenendo conto della massima dose inattiva (HUD: Highest Uneffective Dose). In presenza di S9, l'o.e. ha evidenziato riduzione dei revertenti prodotti dalla esposizione alle HCAs a 5•10-2 mg/piastra; alla stessa concentrazione, ma in assenza di S9, ha manifestato la stessa capacità verso 2-amino-3-methylimidazo[4,5-f’]quinoline (IQ) e 2-amino-3,4-dimethylimidazo[4,5-f’]quinoline (MeIQ) (10-7 mol/piastra); nessuna efficacia con 2-amino- 3,8-dimethylimidazo[4,5-f’]quinoxaline (MeIQx) e 2-amino-6-methyldipyrido[l,2-a:3',2'-d]imidazole (Glu-P-1). L'o.e. è stato poi saggiato su linee cellulari tumorali LoVo ed HepG2, evidenziando IC50 di 74.95±0.05 μg/ml ed 82.28±0.03 μg/ml rispettivamente, delineandone un possibile utilizzo come ingrediente in prodotti salutistici con efficacia preventiva rispetto all'azione cancerogenetica
CHEMICAL CHARACTERIZATION AND BIOACTIVITY OF AYURVEDIC CRUDE DRUGS AND RELATED PREPARATIONS: BOERHAAVIA DIFFUSA L., CONVOLVULUS PLURICAULIS CHOISY AND CURCULIGO ORCHIOIDES
No full textAyurveda, the traditional Indian medicine, is recognized by the European Union as non-conventional medicine and it includes more than 7000 plants used for therapeutic purposes in complex formulations poorly investigated under a chemical and biological point of view (Guerrini and Sacchetti, 2012). On these premises, preparations (decoction, DEC, and hydro-alcoholic extract, HE) of Boerhaavia diffusa L. (roots), Convolvulus pluricaulis Choisy (whole plant) and Curculigo orchioides Gaertn. (roots) were studied for the first time through a chemical and bioactivity approach. GC-FID, GC-MS, HPLC and HPTLC, and NMR strategies were employed to identify and quantify the main chemical compounds and to verify the repeatability of their fingerprinting. The study of biological activities was driven both by the ethnomedical traditional uses of the species in the Ayurvedic culture (Agrawal et al., 2011; Bhowmik et al., 2012; Chauhan et al., 2010) and by those aspects related to modern western phytotherapic culture: antioxidant activity; genotoxic and anti-genotoxic potential; and cytotoxicity against cancer and normal human cell lines. Ferulic acid and vanillin were identified and quantified in DEC and in HE of B. diffusa. However, HE showed the presence of other two characterising molecules: boeravinone B and eupalitin. β-sitosterol was identified and quantified in both preparations, but resulted more abundant in DEC. Ferulic acid showed antioxidant activity by ABTS assay higher than positive control. Preparations and pure compounds did not show anti-genotoxic activity towards two compounds with mutagenic properties. The study of cytotoxicity using DEC, HE and pure molecules against human cancer cell lines (A549, CaCo-2, MCF7, LoVo and HepG2), showed once more data far from those stated by the American National Cancer Institute (Suffness and Pezzuto, 1991). DEC of Convolvulus pluricaulis was characterised by the presence of phenolic acids (caftaric acid, caffeic acid, p-coumaric acid, iso-ferulic acid and tr-ferulic acid). Stigmasterol, β-sitosterol, lupeol and vanillin isomers and derivatives (2H4MB, 3H4MB and acetovanillone) were also detected in the same preparation. HE showed the presence of p-coumaric acid, vanillin, 2H4MB, acetovanillone, and vanillic acid, but the absence of phytosterols. The antioxidant activity evaluation indicated DEC as the preparation that exhibited always the best efficacy. HE showed instead an higher activity than DEC in the ABTS assay, no activity against DPPH radical and a lower capacity of blocking the radical propagation than DEC in the β-carotene bleaching test. Cytotoxic activity of the considered preparations against A549, CaCo-2, MCF7, LoVo and HepG2 cancer cell lines are not significant. The tests carried out with DEC and HE extracts against two leukaemia cell lines, drug-sensitive (CCRF-CEM) and multi-drug-resistant (CEM/ADR5000), showed, instead, interesting results: DEC CHCl3 extract and the HE soxhlet extract exhibit experimental data in line with the one stated by the American National Cancer Institute against the CCRF-CEM cell line, but respectively about 2 and 3 fold higher in the tests against CEM/ADR5000, showing cross-resistance phenomena. Curculigoside A and orcinol-β-D-glucoside were identified and quantified through HPLC methods in C. orchioides, and chosen as characterising compounds of preparations. β-sitosterol was also identified and quantified using GC-FID. The HE samples showed the highest antioxidant activity in every performed test. Regarding the anti-genotoxic activity, DEC resulted slightly more active than HE, but the highest activity was showed by orcinol-β-D-glucoside, even if it did not reach the 50% inhibition at the maximum concentration tested. The evaluation of the cytotoxic activity against human cancer cell lines exhibited an opposite trend, pointing out DEC as more active than HE for A549, CaCo-2, and HepG2. HE was instead more active against CCRF-CEM and CEM/ADR5000, but with IC50 data far from significant indication given by the American National Cancer Institute. Pure molecules did not exhibit any activity. However, further chemical and biological investigations will be carried out to shed an even more clear light on efficacy and safety of Ayurvedic preparations
Flavonoids in Ecuadorian Oreocallis grandiflora (Lam.) R. Br.: Perspectives of use of this species as a food supplement
No full textOreocallis grandiflora (Lam.) R. Br. is an Ecuadorian species belonging to the Proteaceae family, commonly known as cucharillo (Loja and Zamora provinces), cucharilla (Sierra region), gañal (Bolívar province), and algil (Chimborazo province). Its leaves and flowers, collected during blooming, are traditionally used for oral administration to treat liver diseases, vaginal bleeding, and ovary/uterus inflammation and as digestive, diuretic, and hypoglycemic remedy. Related literature does not report any scientific evidences regarding the chemical composition of the used parts of this species (leaves and flowers), while few indications are reported about the healthy properties of their preparations. Based on these premises, the present research was performed with the objectives to fill the gaps of the chemical and biological knowledge about this species, enriching the knowledge related to the plant biodiversity of Amazonian Ecuador and to the ethnobotanical tradition of Andean communities. Chemical and biological investigation (in vitro antioxidant and anti-inflammatory activity) of flower and leaf hydroalcoholic extracts shed a light on the functional metabolites putatively involved in healthy properties of the O. grandiflora traditional preparations. The chemical fingerprinting achieved by HPTLC and 1HNMR analyses showed the presence of flavonoids, subsequently quantitatively estimated by AlCl3 complexation assay and HPLC-DAD. Silica gel chromatography allowed the isolation of the main compounds of the flower extract: quercetin 3-O-glucuronide and myricetin 3-O-glucuronide. RP-HPLC-DAD-MS analyses showed the presence of quercetin 3-O-rutinoside and isorhamnetin 3-O-rutinoside, in addition to the above-mentioned molecules, in the leaf extract. Regarding the antioxidant (DPPH test, a radical scavenging assay) and anti-inflammatory (WST-1 assay, an oxidative burst test) activities, leaf extract showed the most promising results when compared to the positive controls. The same extract, however, exhibited a higher cytotoxicity compared to the flower extract, indicating the latter preparation as the most interesting anti-inflammatory crude drug
Polyphenols From Vitis vinifera Lambrusco By-Products (Leaves From Pruning): Extraction Parameters Evaluation Through Design of Experiment
No full textVitis vinifera L. leaves from pruning are by-products of the wine industry and represent an important source of secondary raw material,
thanks to their polyphenols content. Optimization of the extraction processes is a key factor for their valorization, and
Design of Experiment (DOE) could be a tool to obtain the most performing extract in terms of polyphenols quality/quantity and
bioactivity. Vitis vinifera Lambrusco leaves were subjected to ultrasound-assisted extractions guided by a 23 factorial design. Three
independent parameters (% solvent, time of extraction, and solvent:solid ratio) were considered to evaluate the extraction process
by analyzing the extraction yield, the total phenolic content (Folin-Ciocalteu assay), and the antioxidant capacity (DPPH assay).
Moreover, the content of the main molecules was identified and quantified by reversed-phase high-performance liquid chromatography
coupled with diode array detection and mass spectrometry. The DOE highlighted the best extraction conditions that showed
slight changes considering the different evaluating parameters. The highest extraction yield was obtained by extraction with 100%
water, 60 minutes of extraction time, and 30:1 solvent:solid ratio, but it was neither the richest in polyphenols nor antioxidant capacity.
The latter 2 characteristics were associated with the extraction performed using 50% ethanol, 35 minutes of extraction time,
and a 20:1 solvent:solid ratio. That extract also exhibited the highest quantity of flavonols
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