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Enzymatic syntesis of flavonoid esters and controlled release from cosmetic formulation

dc.contributor.advisorBezbradica, Dejan
dc.contributor.otherPjanović, Rada
dc.contributor.otherKnežević-Jugović, Zorica
dc.contributor.otherĆorović, Marija
dc.contributor.otherAntov, Mirjana
dc.creatorMilivojević, Ana
dc.date.accessioned2020-11-02T11:21:52Z
dc.date.available2020-11-02T11:21:52Z
dc.date.issued2019-08-30
dc.identifier.urihttp://eteze.bg.ac.rs/application/showtheses?thesesId=7678
dc.identifier.urihttps://fedorabg.bg.ac.rs/fedora/get/o:22863/bdef:Content/download
dc.identifier.urihttp://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=24141321
dc.identifier.urihttps://nardus.mpn.gov.rs/handle/123456789/17584
dc.description.abstractU okviru ove doktorske disertacije ispitana je mogućnost efikasne i ekonomiĉne enzimske sinteze estara tri flavonoida (floridzina, naringina i eskulina) korišćenjem razliĉitih acil-donora i imobilisane lipaze tipa B iz C. antarctica kao biokatalizatora. Pored toga, ispitan je transdermalni prenos i odreĊene su difuzione karakteristike sintetisanih estara iz razliĉitih kozmetiĉkih formulacija. Sinteza estara tri pomenuta flavonoida korišćenjem oleinske kiseline kao acil-donora i Novozym® 435 kao biokatalizatora je optimizovana i pokazano je da su koncentracija flavonoida i molski odnos supstrata dva najbitnija faktora koja utiĉu na tok sinteze. Maksimalni stepen konverzije sva tri flavonoida postiže se pri najmanjoj poĉetnoj koncentraciji flavonoida, 10 mM. Sa druge strane, uoĉeno je da za postizanje maksimalnog prinosa proizvoda optimalni uslovi zavise od primenjenog acil-akceptora s obzirom da 30 mM naringina i 70 mM eskulina predstavljaju optimalne poĉetne koncentracije ova dva flavonoida za dobijanje visokog prinosa proizvoda. Kada je reĉ o molskom odnosu supstrata, pokazano je da reakciji sinteze floridzin-oleata pogoduje veliki višak oleinske kiseline (1:18 za stepen konverzije i 1:13 za specifiĉni prinos), dok je za esterifikaciju druga dva flavonoida optimalna pet puta veća koncentracija acil-donora u reakcionoj smeši. Što se tiĉe uticaja dužine lanca (C2-C18) i stepena nezasićenosti masne kiseline na sintezu floridzin-oleata, primenom svih kiselina ostvareni su visoki stepeni konverzije, preko 70 %. U cilju razvoja isplativijeg procesa esterifikacije flavonoida, u drugoj fazi ove disertacije, ispitani su i alternativni, jeftiniji acil-donori, kao što su triacetin i anhidrid sirćetne kiseline za sintezu flavonoid-acetata, i razliĉita biljna ulja (kokosovo, laneno i suncokretovo ulje) za sintezu estara viših masnih kiselina. Triacetin se pokazao kao odgovarajući supstrat za sintezu acetil-derivata flavonoida, pri ĉemu je postupak acetilovanja floridzina optimizovan u ekološki prihvatljivom sistemu bez rastvaraĉa. Kao proizvod reakcije acetilovanja floridzina dobijena je smeša estara floridzin-6"-O-acetata i floridzin-3",6"-O-diacetata. Kinetiĉka studija je pokazala da se proces acetilovanja flavonoida može opisati dvostepenom monosupstratnom reakcijom, a da su oba stupnja reakcije adekvatno opisana reverzibilnom Mihaelis-Mentenovom kinetikom u celom ispitanom reakcionom toku. Kada je reĉ o upotrebi prirodnih biljnih ulja kao acil-donora, sva ispitana ulja su se pokazala kao dobri supstrati u reakciji esterifikacije sva tri flavonoida (ostvareni stepeni konverzije preko 65 %) pri ĉemu kao proizvodi reakcije nastaju smeše estara flavonoida i kiselina prisutnih u ulju. Primenom statistiĉkih metoda planiranja i metode odzivnih površina reakcija esterifikacije eskulina primenom lanenog ulja je optimizovana i visok stepen konverzije (78,5 %), prinos (19,13 mg/ml), kao i specifiĉni prinos proizvoda (2,83 mmol/genzima) su postignuti pod optimalnim uslovima. OdreĊeni optimalni uslovi su uspešno primenjeni i u reakcijama transesterifikacije eskulina korišćenjem druga dva ulja. Kao drugi naĉin da se doprinese razvoju isplativijeg procesa esterifikacije estara, u narednoj fazi optimizovana je imobilizacija CAL B na tri odabrana LifeTechTM nosaĉa sa epoksi (ECR 8285F), oktadecil (ECR 8806M) i amino (ECR 8409F) funkcionalnim grupama. Pripremljeni imobilisani preparati primenjeni su u reakciji sinteze estara tri pomenuta flavonoida, pri ĉemu lipaze imobilisane na hidrofobni i nosaĉ sa epoksi grupama pokazuju veoma dobru esterifikacionu aktivnost i odliĉnu operativnu stabilnost i tokom 15 ciklusa reakcije esterifikacije. U cilju proširenja primene estara flavonoida, ispitana je mogućnost njihovog inkorporiranja u razliĉite kozmetiĉke formulacije i praćena je kinetika otpuštanja sintetisanih estara kroz Strat-MTM membranu koja služi kao model sistem za transdermalni prenos. U svim eksperimentima primećeno je da do prenosa komponente dolazi sa zakašnjenjem (lag faza) i da koeficijent difuzije estara opada sa povećanjem broja C atoma i smanjenjem broja dvostrukih veza u lancu masne kiseline. Formulacije u kojima je voda kontinualna faza su se pokazale kao najpogodniji sistemi za inkorporiranje estara, a najveći efektivni koeficijenti difuzije ostvareni su prilikom otpuštanja estara iz gel-emulzija. I na kraju, u cilju ispitivanja mogućnosti komercijalne primene estara flavonoida dobijenih u reakciji sa prirodnim uljima procenjena je stabilnost pripremljenih gel-emulzija, odreĊen efekat hidratacije i stepen iritabilnosti nakon topikalne primene. Pripremljene gel-emulzije pokazuju dobru stabilnost i nakon 2 meseca skladištenja. Štaviše, sve ispitane formulacije pokazuju dobar efekat hidratacije kože, uz minimalnu iritabilnost pojedinih formulacija i odliĉnu dermatološku kompatibilnost.sr
dc.description.abstractWithin this doctoral dissertation, the possibility of efficient and economical enzymatic synthesis of esters of three flavonoids (floridzin, naringin and esculin) using different acyl donors and immobilized lipases B from C. antarctica as a biocatalyst was investigated. Additionally, transdermal diffusion characteristics of synthesized esters from various cosmetic formulations were determined. Synthesis of esters of the three aforementioned flavonoids using oleic acid as an acyl donor and Novozym® 435 as a biocatalyst was optimized and it was shown that the concentration of flavonoid and substrate molar ratio were two the most important factors influencing the synthesis process. The maximum conversion degree of all three flavonoids was achieved at a minimum initial flavonoid concentration, 10 mM. On the other hand, the optimum conditions for product yield as output depend on the acyl acceptor used, given that 30 mM and 70 mM were optimal concentrations of naringin and esculin for achieving the highest product yields. When it comes to the substrate molar ratio, it was shown that the large excess of oleic acid (1:18 for the conversion degree and 1:13 for the specific yield) was preferable for synthesis of floridzin-oleate, while for the esterification of other two flavonoids, five fold excess of oleic acid was optimal for high product yields. When it comes to the effect of fatty acids chain length (C2-C18) and unsaturation degree on floridzin-oleate synthesis, it was shown that high conversion degrees, over 70 % were obtained in reactions with all examined fatty acids. In order to develop the more economical process of flavonoid esterification, in the second phase od this thesis, alternative, cheaper acyl donors, such as triacetin and acetic anhydride for the synthesis of flavonoid acetate, and various vegetable oils (coconut, linseed and sunflower oil) for the synthesis of long chain fatty acid esters were examined. Triacetin proved to be the appropriate substrate for the synthesis of the acetyl derivative of flavonoids and eco-friendly, solvent-free enzymatic acetylation of floridzin was optimized. The mixture of floridzin-6 "-O-acetate and floridzin-3", 6 "-O-diacetate was obtained as a product of this reaction. Kinetic study revealed that acetylation process could be regarded as a two-step mono-substrate reaction and, within entire reaction time, both stages were adequately described by reversible Michaelis-Menten kinetics. When it comes to the usage of natural vegetable oils as an alternative acyl donors, it was shown that all examined oils were good substrates for esterification of all three flavonoids (conversion degree above 65%), whereby the mixtures of different fatty acid esters of flavonoids were obtained as products of reactions. By using experimental design and response surface methodology (RSM), the esterification of esculin using linseed oil as acyl donor was optimized and under optimal conditions high molar conversion (78.5%), product yield (19.13 mg/ml), as well as specific yield (2.83 mmol/genzyme) were achieved. Determined optimums were successfully applied in esculin transesterification with other oils. As an another way to contribute to the development of a more cost-effective esterification process, CAL B immobilization on the three selected LifeTechTM carriers with epoxy (ECR 8285F), octadecyl (ECR 8806M) and amino (ECR 8409F) functional groups was optimized. Prepared immobilized enzymes were applied as biocatalyst for esterification of the three aforementioned flavonoids, and the lipase immobilized on the carriers with hydrophobic and epoxy group have shown very good esterification activity and excellent operational stability during the 15 reaction cycles. In order to extend the use of flavonoid esters, the possibility of their incorporation into various cosmetic formulations was examined and transdermal diffusion of synthesized esters using Strat-MTM membrane was investigated. The presence of lag phase was observed in all experiments and it was shown that the effective diffusion coefficient of esters decreased with an increase in the number of C atoms and with decrease in the number of double bonds in the fatty acid chain. Emulsions in which the water was continuous phase proved to be the most suitable systems for the incorporation of flavonoid esters, and the highest effective diffusion coefficients were achieved by release of esters from gel emulsions. Finally, in order to investigate the possibility of commercial application of flavonoid esters obtained in reaction with natural oils, the stability of the prepared gel emulsions, their hydration effect and irritability after topical administration was assessed. The prepared gel emulsions have shown good stability even after 2 months of storage. Moreover, all the examined formulations have shown excellent hydration effect, nonirritant character and overall dermatological compatibility.en
dc.formatapplication/pdf
dc.languagesr
dc.publisherУниверзитет у Београду, Технолошко-металуршки факултетsr
dc.relationinfo:eu-repo/grantAgreement/MESTD/Technological Development (TD or TR)/31035/RS//
dc.relationinfo:eu-repo/grantAgreement/MESTD/Integrated and Interdisciplinary Research (IIR or III)/46010/RS//
dc.rightsopenAccessen
dc.sourceУниверзитет у Београдуsr
dc.subjectflavonoidisr
dc.subjectflavonoidsen
dc.subjectlipaseen
dc.subjectesterificationen
dc.subjectimmobilizationen
dc.subjectvagetable oilsen
dc.subjectdiffusionen
dc.subjecttransdermal diffusionen
dc.subjectcosmetic formulationen
dc.subjectlipazasr
dc.subjectesterifikacijasr
dc.subjectimobilizacijasr
dc.subjectbiljna uljasr
dc.subjecttransdermalna difuzijasr
dc.subjectkozmetiĉke formulacijesr
dc.titleEnzimska sinteza estara flavonoida i kontrolisano otpuštanje iz kozmetičkih formulacijasr
dc.title.alternativeEnzymatic syntesis of flavonoid esters and controlled release from cosmetic formulationen
dc.typedoctoralThesisen
dc.rights.licenseBY-NC-ND
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/66478/IzvestajKomisije23366.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/66477/Disertacija.pdf


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