Kromatografske in masno spektrometrične metode za karakterizacijo fenolnih spojin v ekstraktih japonskega dresnika (Fallopia japonica Houtt.) in drugih rastlinskih ekstraktih

Jug, Urška (avtor), Vovk, Irena (mentor)

Ukvarjali smo se z razvojem analiznih metod za določanje fenolnih spojin v propolisu in vzorcih rastlinskega izvora, pri čemer smo se osredotočili na korenike invazivne tujerodne rastlinske vrste japonski dresnik (Fallopia japonica Houtt.) in iskanje pozitivnih vidikov ter možnosti za njihovo izkoriščanje, saj predstavljajo obilno biomaso. Razvili smo HPTLC metodo za analizo fenolnih kislin, optimizirali HPTLC metodo za analizo flavonoidnih aglikonov in izbrali ustrezne pogoje za denzitometrične in HPTLC–MSn analize fenolnih kislin in flavonoidov. Te nove metode smo uporabili za analize ekstraktov propolisa, pražene kave, šipka, hibiskusa, rožmarina in žajblja. Razvili smo večstopenjski TLC postopek za izolacijo fenolnih spojin iz 70 % acetonskega(aq) ekstrakta lubja korenik japonskega dresnika, ki zajema frakcionacijo na PLC silikagelnih in HPTLC silikagelnih ali celuloznih ploščah v kombinaciji z različnimi topili za razvijanje. Kot prvi smo v korenikah japonskega dresnika zaznali procianidina B1 in B2, resveratrol-malonil-heksozid, resveratrol-acetil-heksozid, metilne derivate emodin biantrona in emodin biantron-heksoze ter derivate taksifolina in iz korenik kot prvi izolirali procianidina B1 in B2 ter emodin-8-O-malonil-glukozid. Izolirali smo tudi (+)-katehin, (–)-epikatehin, (–)-epikatehin galat, galat dimernega proantocianidina tipa B, procianidin B3, emodin in emodin-8-O-glukozid. Identiteto izoliranih spojin smo potrdili s HPTLC, HPTLC–MSn, večinoma pa tudi z 1H NMR. V izolate pa smo ujeli alifatske nečistote, ki izvirajo iz stacionarne faze HPTLC plošč in iz laboratorijskih potrošnih materialov, iz katerih se izlužuje drsilo oleamid. Izvora nečistot smo določili s pomočjo 1H NMR in HPTLC metode za ločbo razredov lipidnih spojin. Razvili smo UHPLC–ESI-MSn metodo za analize izlužkov in ekstraktov laboratorijskih materialov, za katere smo ugotovili, da predstavljajo interference analiznih in bioloških testiranj. Preverili smo provnetno oz. protivnetno delovanje 70 % etanolnih(aq) in 70 % acetonskih(aq) ekstraktov korenik japonskega dresnika in njihovega lubja prek vpliva na TLR4 signalne poti ter za vse dokazali provnetno delovanje v koncentraciji 100 μg mL–1. S testom DPPH smo določili antioksidativno delovanje ekstraktov lubja korenik japonskega dresnika, pripravljenih z osmimi različnimi topili, in za vse dobili nizke vrednosti IC50 (2,6–3,5 µg mL–1), primerljive z vrednostjo IC50 askorbinske kisline takoj po pripravi (3,1 µg mL–1). Razvili smo SEC-HPLC–UV metodo in izvedli frakcionacijo 70 % etanolnega(aq) ekstrakta lubja korenik japonskega dresnika, vodeno z on-line pokolonsko derivatizacijo z reagentom DPPH. Razvili smo RP-HPLC–UV–MSn metodo, z njo analizirali SEC-frakcije in v antioksidativni frakciji identificirali (–)-epikatehin, ki se je izkazal kot močnejši antioksidant (IC50 = 1,8 µg mL–1) kot ekstrakti in askorbinska kislina. Medtem ko je antioksidativnost askorbinske kisline upadala, sta (–)-epikatehin in izbrani ekstrakt ohranila stabilno antioksidativnost vsaj 14 dni. Antioksidativni 70 % etanolni(aq) ekstrakt smo vgradili v hitozanske folije, potrdili prehajanje antioksidantov iz folije v stično tekočino (simulant hrane) in s tem postavili temelje za razvoj biorazgradljivih vsebnikov za hrano/pijačo in zdravila, ki bi vsebino ščitili pred oksidacijo.

• Jezik: Slovenski jezik
• Ključne besede: Polygonum cuspidatum, Fallopia japonica, propolis, kava, šipek, hibiskus, rožmarin, žajbelj, rdeči dren, fenolne kisline, flavonoidi, flavan-3-oli, proantocianidini, antrakinoni, HPTLC, HPTLC–MS, denzitometrija, (U)HPLC–MS, SEC-HPLC–UV, test DPPH, antioksidativno delovanje, izolacija fenolnih spojin
• Vrsta gradiva: Doktorsko delo/naloga
• Tipologija: 2.08 - Doktorska disertacija
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2021
• PID: 20.500.12556/RUL-125876
• COBISS.SI-ID: 67564547
• Datum objave v RUL: 08.04.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Chromatographic and mass spectrometric methods for characterization of phenolic compounds in extracts of Japanese knotweed (Fallopia japonica Houtt.) and other plant extracts

Izvleček

Analytical methods for the determination of phenolic compounds in propolis and plant materials were developed. Most of our research was focused on rhizomes of invasive alien plant species Japanese knotweed (Fallopia japonica Houtt.) to exploit their abundant biomass and explore other positive aspects. We developed the HPTLC method for the analysis of phenolic acids, optimized the HPTLC method for the analysis of flavonoid aglycones and optimized conditions for densitometric and HPTLC–MSn analyses of phenolic acids and flavonoids. Using these new methods we analysed propolis, roasted coffee, rose hip, hibiscus, rosemary and sage extracts. A multi-step TLC fractionation protocol on PLC silica gel and HPTLC silica gel or cellulose plates in combination with various developing solvents was developed to isolate phenolic compounds from 70% acetone(aq) extract of Japanese knotweed rhizome bark. We detected procyanidins B1 and B2, resveratrol-malonyl-hexoside, resveratrol-acetyl-hexoside, methyl derivatives of emodin bianthrone and emodin bianthrone-hexose, and taxifoline derivatives and isolated procyanidins B1 and B2 and emodin-O-malonyl glucoside for the first time in Japanese knotweed rhizomes. (+)-Catechin, (–)-epicatechin, (–)-epicatechin gallate, dimeric proanthocyanidin type B gallate, procyanidin B3, emodin and emodin-8-O-glucoside were also isolated. Isolated compounds were identified by HPTLC, HPTLC–MSn and 1H NMR (most compounds). Aliphatic contaminants originating from the HPTLC stationary phase and from laboratory consumables (leaching of the lubricant oleamide), were collected in isolates and their origins were confirmed by 1H NMR and the HPTLC method for the separation of classes of lipid compounds. The UHPLC–ESI-MS method was developed for analyses of leachates and extracts of laboratory consumables, which were found to interfere with analytical and bioassay results. We tested the pro-inflammatory and anti-inflammatory activity of 70% ethanol(aq) and 70% acetone(aq) extracts from Japanese knotweed rhizomes and rhizome bark via the TLR4 signalling pathways and for all detected pro-inflammatory activity at 100 μg mL–1. A DPPH test was used to determine the antioxidant activity of Japanese knotweed rhizome bark extracts prepared with eight different solvents. All extracts gave low IC50 values (2.6–3.5 µg mL–1) comparable to the IC50 value of ascorbic acid immediately after preparation (3.1 μg mL–1). We developed a SEC-HPLC–UV method and performed on-line DPPH guided fractionation of 70% ethanol(aq) extract using post-column derivatization with the DPPH reagent. We also developed a RP-HPLC–UV–MSn method, analyzed SEC fractions and identified (–)-epicatechin in the fraction with the antioxidant activity. (–)-Epicatechin showed stronger antioxidant activity (IC50 = 1.8 μg mL–1) than the extracts and ascorbic acid. The antioxidant activity of (–)-epicatechin and the 70% ethanol(aq) extract remained stable for at least 14 days, while the antioxidant activity of ascorbic acid decreased. The 70% ethanol(aq) extract was incorporated in the chitosan foils. The transition of antioxidants from the foil to the contact fluid (food simulant) was confirmed, thereby laying the foundation for development of biodegradable containers for food/drink and pharmaceutic forms, which would protect the contents against oxidation.

• Ključne besede: Polygonum cuspidatum, Fallopia japonica, propolis, coffee, rose hip, hibiscus, rosemary, sage, red dogwood, phenolic acids, flavonoids, flavan-3-ols, proanthocyanidins, anthraquinones, HPTLC, HPTLC–MS, densitometry, (U)HPLC–MS, SEC-HPLC–UV, DPPH assay, antioxidant activity, isolation of phenolic compounds