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    Ultrasonic extraction of polyphenolic compounds from species Helichrysum italicum (Roth) G. Don. using (2-hydroxypropyl)-B-cyclodextrins

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    Smilje (Helichrysum italicum, (Roth), G. Don) je izvor visokovrijednih polifenola, prirodnih antioksidansa. Raznolikost tih kemijskih spojeva, koji pojedinačno ili u sinergiji pridonose farmakološkoj aktivnosti, nudi brojne mogućnosti praktične primjene ekstrakata biljke. U cilju učinkovitog izdvajanja navedenih spojeva iz biljnog materijala provedena je optimizacija ekstrakcijskih uvjeta. U radu je korištena metoda inkapsulacije (2-hidroksipropil)-β-ciklodekstrinom, te ultrazvukom potpomognuta ekstrakcija, u svrhu boljeg iskorištenja i skraćenog vremena ekstrakcije. U usporedbi sa konvencionalnom metodom ekstrakcije etanolom ili drugim organskim otapalima, ova metoda je ekološki prihvatljiva. Provedena je spektrofotometrijska analiza sadržaja ukupnih polifenola i podskupina polifenola, fenolnih kiselina i flavonoida u ekstraktima, te kvantifikacija sadržaja klorogenske kiseline korištenjem tekućinske kromatografije visoke djelotvornosti (engl. HPLC). Najboljim uvjetima ekstrakcije pokazali su se neosporno: snaga ultrazvuka 720 W, temperatura 70 °C, masa droge 0,6 g te voda kao otapalo. Parametri s najvećim utjecajem na ekstrakcijski kapacitet su temperatura i masa droge. Snaga ultrazvuka indirektno utječe na prinos dok glicerol gotovo nema utjecaja. Udio mliječne kiseline pozitivno utječe na ekstrakciju fenolnih kiselina pri nižim temperaturama, dok na koncentraciju flavonoida ima negativan utjecaj.Immortelle (Helichrysum italicum, (Roth), G. Don) is a source of high-value polyphenols, natural antioxidants. The diversity of these chemical compounds, which individually or synergistically contribute to pharmacological activity, offers many possibilities in practical use of plant extracts. In order to efficiently extract mentioned compounds from plant material, extraction conditions were optimised. In this paper, methods that were used were (2-hydroxypropyl)-β-cyclodextrin encapsulation and ultrasound-assisted extraction for the purpose of better extraction efficiency and shortened extraction time. In comparison to conventional extraction method with ethanol or other organic solvents, this method is environmentally friendly. Spectrophotometric analysis of total polyphenols, phenolic subclasses, phenolic acids and flavonoids in the extracts was conducted, as well as quantification of chlorogenic acid content using high performance liquid chromatography (HPLC). The best extraction conditions indisputably proved to be: ultrasound power 720 W, temperature 70 °C, drug weight 0,6 g and water as a solvent. Parameters with the greatest impact on the extraction capacity are temperature and drug mass. Ultrasound power affects the yield indirectly, while glycerol had almost no effect. The proportion of lactic acid has a positive effect on phenolic acids’ extraction at lower temperatures, while it has a negative effect on the flavonoid concentration

    Amide-type harmicines as potential antimalarial agents

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    U prvom dijelu istraživanja ovog doktorskog rada sintetizirano je četrdeset harmicina amidnoga tipa (7a-h, 13a-h, 19a-h, 22a-h, 25a-f, i-p), hibridnih spojeva harmina/srodnih β-karbolinskih alkaloida i cimetne kiseline/odabranih derivata cimetne kiseline (DCK-a) međusobno povezanih amidnom vezom. U tu svrhu pripravljeni su amini 6, 12, 18, 21 i 24, čime je primarna amino skupina uvedena u pet položaja β-karbolina: 1, 3, 6, 7 i 9. U idućem koraku sintetizirani su harmicini amidnoga tipa reakcijama povezivanja dobivenih amina s cimetnom kiselinom/DCK-ima korištenjem standardnih reakcijskih uvjeta (HATU/DIEA). Harmicini su karakterizirani spektroskopskim (1H, 13C NMR, IR) i spektrometrijskim (MS) tehnikama te im je određeno talište. Svim spojevima ispitano je antiplazmodijsko djelovanje in vitro na eritrocitnu fazu životnog ciklusa dva soja Plasmodium falciparum (Pf3D7 i PfDd2), hepatocitnu fazu životnog ciklusa P. berghei i citotoksičnost na humanu staničnu liniju hepatocelularnog karcinoma (HepG2). U drugom dijelu istraživanja provedena je analiza kvantitativnog odnosa strukture i djelovanja (engl. quantitative structure-activity relationship, QSAR). Antiplazmodijsko djelovanje harmicina na eritrocitnu fazu Pf3D7 povezano je s pripadajućim izračunatim molekulskim deskriptorima te je primjenom metode višestruke linearne regresije dobiven prediktivni model ovisnosti log IC50 o dvije varijable: izoelektričnoj točki (pI) i Abrahamovom deskriptoru E (Abr E). Model je validiran unutarnjom 10-strukom unakrsnom validacijom. U trećem dijelu istraživanja, za provedbu vanjske validacije modela predložene su strukture 356 novih harmicina te su im, prema dobivenom modelu, izračunate predviđene IC50 vrijednosti. Potom je odabrano, sintetizirano i karakterizirano ukupno 19 harmicina: šest harmicina amidnoga tipa (25k-p), 11 harmicina karbamatnog tipa (28a-k) i dva harmicina ureidnog tipa (32b,c) u položaju N-9 β-karbolina, te je ispitano njihovo antiplazmodijsko djelovanje in vitro na eritrocitnu fazu životnog ciklusa plazmodija (Pf3D7 i PfDd2). Na temelju dobivenih rezultata izračunate su razlike između eksperimentalnih i predviđenih vrijednosti i srednja apsolutna pogreška predviđanja. Najjače djelovanje, gotovo dva reda veličine jače od harmina, pokazali su harmicini amidnoga tipa u položaju N-9 β-karbolina (25b-e,i,j). Rezultati ovog doktorskog rada predstavljaju temelj za daljnji razvoj novih harmicina amidnoga tipa s pojačanim antiplazmodijskim djelovanjem.The first part of this thesis includes the synthesis of forty amide-type harmicines (7a-h, 13a-h, 19a-h, 22a-h, 25a-f, i-p), hybrid compounds in which harmine/β-carboline alkaloids and cinnamic acid or cinnamic acid derivatives (CADs) are linked via an amide bond. First, the primary amino group was introduced in five positions of the β-carboline ring: 1, 3, 6, 7 and 9, which resulted in the synthesis of amines 6, 12, 18, 21 and 24. Subsequently, the coupling reaction of amines and cinnamic acid/CADs were preformed using standard conditions (HATU/DIEA). The synthesized compounds were characterized by spectroscopic (1H, 13C NMR, IR) and spectrometric (MS) techniques, as well as determination of their melting points. Finally, we evaluated biological activities of the prepared harmicines, i.e. in vitro antiplasmodial activity against the erythrocytic stage of the Plasmodium falciparum life cycle (Pf3D7 and PfDd2) and hepatic stage of the P. berghei life cycle, as well as cytotoxicity against human hepatocellular carcinoma cell line (HepG2). In the second part of this thesis quantitative structure-activity relationship (QSAR) analysis was performed using a multiple linear regression technique. A set of molecular descriptors underwent the forward stepwise regression procedure and the predictive model was built by selecting two molecular descriptors: isoelectric point (pI) and Abraham descriptor E (Abr E). The constructed model was internally validated using 10-fold cross-validation. For the purpose of external validation of the constructed model, in the third part of this thesis 356 novel harmicines were designed and their predicted logIC50 values calculated, followed by the synthesis and characterization of six amide-type harmicines (25k-p), eleven carbamate type-harmicines (28a-k) and two ureido-type harmicines (32b,c) in the position N-9 of the β-carboline. Next, we evaluated in vitro antiplasmodial activity of novel harmicines against the erythrocytic stage of the P. falciparum life cycle (Pf3D7 and PfDd2). Based on the obtained results, the absolute differences between the experimental and predicted values as well as mean absolute error were calculated. The most active compounds, amide-type harmicines in the position N-9 of the β-carboline (25b-e,i,j), displayed at least two orders of magnitude stronger activities than the parent compound, harmine. The results of this doctoral thesis represent the basis for the further development of novel amide-type harmicines with enhanced antiplasmodial activity

    Pharmacological activity and application of isatin and isatin derivatives

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    Izatin je prirodni alkaloid narančaste boje izoliran iz biljaka roda Isatis prisutnih po cijelom svijetu. O.L. Erdman i A. Laurent prvi su puta 1841. godine sinetizirali izatin oksidacijom indiga smjesom dušične i kromne kiseline, a do danas je otkriven velik broj izatinskih derivata od kojih su neki pokazali značajna antitumorska, antibakterijska, antikonvulzivna, protuupalna i brojna druga djelovanja. Iako izatinski derivati imaju širok spektar djelovanja, do sada se antitumorsko djelovanje pokazalo najznačanijim. Sunitinib je derivat izatina koji je 2006. godine registrirala EMA u liječenju GIST-a, a kasnije i za liječenje neoperabilnih ili metastatski dobro diferenciranih tumora gušterače (pNET) i metastatskog karcinoma bubrežnih stanica (MRCC). Osim što je registriran kao lijek u navedenim indikacijama, sunitinib prolazi kroz klinička ispitivanja kao dvojna terapija s docetakselom, kapecitabinom i paklitakselom. Inhibitor je više tirozin kinaza te tako sprječava rast tumora, proliferaciju stanica i angiogenezu, a značajno inhibitorno djelovanje pokazao je prema VEGFR i PDGFR. Indirubin je poznat izatinski derivat s protuupalnim, antiproliferativnim, hepatoprotektivnim i protuleukemijskim djelovanjem. Iako njegova sigurnost i učinkovitost nije dokazana, u tradicionalnoj se kineskoj medicini koristi u terapiji psorijaze, prevenciji mastitisa uzrokovanog LPS-om E. Coli i S. aureus te kao dodatna terapija CML-a. Hibridi izatinskih derivata s fluorokinolonima ispituju se kao antibakterijska terapija u liječenju infekcija uzrokovanih H. influenzae, K. pneumonia, P.aeruginosa. U najnovijim istraživanjima pronađeno je da djelovanjem bakterijskih enzima u crijevima dolazi do sinteze izatina iz triptofana što uzrokuje povećane koncentracije izatina u tjelesnim tekućinama i mozgu. Do navedene promjene dolazi tijekom izloženosti akutnom stresu, tijekom Parkinsonove bolesti zbog povećanog broja bakterija roda Enterococcaceae te se često javlja kod pacijenata oboljelih od bulimije. Ovim diplomskim radom prikazana su najistaknutija farmakološka djelovanja izatina i izatinskih derivata i učinak bakterijske mikroflore na rad i funkciju mozga.Isatin is natural alkaloid, isolated as orange solid from plants of Isatis genus. First synthesis of isatin dates back to 1841. when O.L. Erdman and A. Laurent obtained it by oxidation of indigo with mixture of nitric and chromic acid. Up to now, large number of isatin derivatives with various pharmacological activities, some of which are antibacterial, antitumor, anticonvulsant, anti-inflammatory etc., have been reported. Isatin derivatives as antitumor agents have been mostly studied and Sunitinib is the one of isatin derivatives which EMA registrated in 2006. for treatment of GIST, pNet's and MRCC. At the moment, Sunitinib is in the clinical trial in combination with capecitabine, paclitaxel and docetaxel in treatment for the different types of tumours. It is multi-tyrosine receptor kinase inhibitor and therefore it prevents growth and proliferation of tumors cells and angiogenesis. Significant inhibitory activity has been reported with VEGFR and PDGFR. Indirubin is isatin derivative with anti-inflammatory, antiproliferative, hepatoprotective and antileukemic activity. Although it's safety and efficiency are not proven, in Chinese traditional medicine it is used in treatment of psoriasis, mastitis caused by E. Coli and S. aureus prevention and as additional therapy of the CML. Isatin hybrids of fluoroquinolones show antibacterial activity to H. influenze, K. pneumonie, P. aerugionosa and many others. Recent studies reported that isatin can be synthesized from triptofan by gastrointestinal bacterial enzymes which leads to high isatin concentarion in body fluids and brain. This is often caused by acute stress, large number of Enterococcaceae in patient with Parkinson disease and bulimia. This review shows the most prominent pharmacological effect of isatin and isatin derivatives and the effect of bacterial microflora on activity and function of brain

    Use of herbal preparations in pregnancy

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    S obzirom na moguće štetne učinke primjene pojedinih biljnih vrsta u trudnoći, upoznatost s učestalošću i navikama korištenja biljnih lijekova i pripravaka tijekom trudnoće važna je za sve zdravstvene djelatnike koji skrbe o trudnicama. Cilj ovog ispitivanja bio je utvrditi sklonost trudnica korištenju biljnih pripravaka za vanjsku i unutarnju primjenu i prikupiti njihove stavove i uvjerenja o sigurnosti, učinkovitosti i dostupnosti biljnih pripravaka u usporedbi s konvencionalnim lijekovima, te utvrditi načine informiranja o biljnim pripravcima. Kod trudnica koje su tijekom trudnoće koristile biljne pripravke, cilj je bio prikupiti podatke o tome u kojem su ih tromjesečju i koliko često u prosjeku koristile, prema čijoj preporuci, jesu li s time upoznale svog ginekologa te gdje su bili kupljeni i za koje smetnje i/ili namjene su bili korišteni koji biljni pripravci. U tu svrhu korišten je anketni upitnik koji su prije trudničkog pregleda na Odjelu za ginekologiju i porodništvo Opće bolnice Zabok i bolnice hrvatskih veterana u Zaboku ispunile ukupno 102 trudnice. Dobiveni rezultati ukazali su na to da je 56 % ispitanica, od kojih je većina bila s područja Krapinsko-zagorske županije, koristilo biljne pripravke tijekom trudnoće. Mnoge od njih koristile su ih svakodnevno (27 %) ili više od dva puta tjedno (20 %), najčešće bez preporuke (55 %), a često i bez znanja svog ginekologa (48 %). Iako se većina ispitanica nije smatrala dovoljno informiranima o biljnim pripravcima koji se smiju koristiti u trudnoći, 60 % svih trudnica vjerovalo je da su biljni pripravci sigurni za korištenje u trudnoći te sigurniji od konvencionalnih lijekova. Ispitanice su biljne pripravke najčešće koristile kod suhog i nadražajnog kašlja, problema sa suhom kožom, infekcija mokraćnog sustava, grlobolje te mučnine i povraćanja. Najčešće korištena biljna vrsta bio je badem, a navelo ga je 50 % trudnica koje su koristile biljne pripravke i to isključivo za vanjsku primjenu, dok su manje zastupljeni đumbir (27 %), brusnica (25 %), kamilica (23 %) i bijeli sljez (12,5 %) bili u pravilu korišteni za unutarnju primjenu. Kao najpouzdaniji izvor informacija o biljnim pripravcima trudnice su u najvećoj mjeri navodile liječnika (55 %) i ljekarnika (47 %), a najveći udio ispitanica naveo je i da biljne pripravke kupuju upravo u ljekarnama (52 %). Zato je na liječnicima i ljekarnicima odgovornost pripadnicama ove posebno osjetljive populacije pružati pravovremene i potpune informacije o biljnim lijekovima i biljnim pripravcima, a kako bi se izbjegli mogući rizici te osigurala njihova sigurna i učinkovita primjena.In view of potential adverse effects of the use of certain plant species during pregnancy, knowledge about the frequency and habits of using herbal medicines and preparations during pregnancy is important for all healthcare professionals who provide care to pregnant women. The aim of this study was to determine the tendencies of pregnant women to use herbal preparations for external and internal use and to collect their views and beliefs on the safety, efficacy and availability of herbal preparations in comparison to conventional drugs, as well as to determine their ways of informing about herbal preparations. The aim was to collect data on the trimester in which pregnant women had been using herbal preparations and how often on average, according to whose recommendation, whether their gynecologist had been informed about it or not, where the herbal preparations were bought and for which disturbances and/or for what purpose was each of them used. For this purpose, a questionnaire was used, which was filled out before the medical examination at the Department of Gynecology and Obstetrics of the General Hospital Zabok and the Hospital of Croatian Veterans in Zabok by a total of 102 pregnant women. The obtained results indicated that 56% of the respondents, most of whom were from Krapina-Zagorje County, had been using herbal preparations during pregnancy. Many of them used them on a daily basis (27%) or more than twice per week (20%), usually without recommendation (55%), and often without the knowledge of their gynecologist (48%). Although most respondents did not think of themselves as being sufficiently informed about herbal preparations that may be used during pregnancy, 60% of all pregnant women believed that herbal preparations were safe to use during pregnancy and safer than conventional drugs. The respondents most often used herbal preparations for dry and irritating cough, dry skin problems, urinary tract infections, sore throat as well as nausea and vomiting. The most commonly used plant species was almond, which was mentioned by 50% of pregnant women that had been using herbal preparations, exclusively for external use, while less represented ginger (27%), cranberry (25%), chamomile (23%) and marsh mallow (12,5%) were mainly used internally. Physicians (55%) and pharmacists (47%) were cited by pregnant women as the most reliable providers of information on herbal preparations, and the largest share of respondents also stated that they were buying herbal preparations in pharmacies (52%). It is therefore the responsibility of physicians and pharmacists to provide members of this particularly sensitive population with timely and complete information on herbal medicines and herbal preparations in order to avoid possible risks and to ensure their safe and effective use

    Immortelle (Helichrysum italicum (Roth) G.Don) in cosmetics and phytopharmacy

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    Cvijet primorskog smilja (Helichrysum italicum) i ostali nadzemni dijelovi biljke predstavljaju bogat izvor bioaktivnih sekundarnih metabolita koji su posljedica prilagodbe biljke na izazovne uvjete u kojima raste. Iako je danas zbog karakterističnog mirisa popularno u parfemskoj industriji, a zahvaljujući antioksidativnim i regenerativnim svojstvima dobro poznato kao sastojak anti-age kozmetike te proizvoda za njegu i revitalizaciju kože, primorsko smilje se u tradicionalnoj medicini koristi u tretiranju rana, ožiljaka, hematoma i upalnih stanja kože te terapiji bolesti dišnog i probavnog sustava. U posljednje vrijeme sve je češće predmet znanstvenih istraživanja kojima je dokazano protuupalno, antimikrobno i antioksidativno djelovanju ove mediteranske biljke i time opravdana tradicionalna uporaba. Navedena istraživanja otvaraju mogućnosti primjene smilja i u indikacijama koje nisu dio narodne medicine.The flowers of immortelle (Helichrysum italicum), as well as the other aerial parts of the plant, are a rich source of bioactive secondary metabolites that are synthesized as a result of the plant's adaptation to the challenging growing environment. Although nowadays it is popular in perfume industry due to its characteristic scent and owing to its antioxidant and regenerative properties it is a well-known ingredient in anti-age cosmetics, skin care and revitalization products, in traditional medicine immortelle is used in the treatment of wounds, scars, hematomas, and inflammatory skin conditions as well as in therapy of respiratory and gastrointestinal disorders. Recently, it has been the subject of scientific research which has proved the anti-inflammatory, antimicrobial and antioxidant action of this Mediterranean plant and thus justified its traditional use. New findings open the possibility of using immortelle for the new indications that are not part of folk medicine

    Centrifugal partition chromatography in isolation of natural products

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    Centrifugalna particijska kromatografija (CPC) je novija preparativna tehnika koja se sve više uspješno koristi u izolaciji, pročišćavanju i ukoncentriravanju prirodnih produkata. U okviru ovog diplomskog rada prikazana su dosadašnja teorijska saznanja o tom tipu tekućinske kromatografiji bez čvrstog nosača te mogućnostima njezine primjene u području prirodnih produkata. Obzirom da su ekstrakti dobiveni iz biljnih sirovina kompleksne smjese, odjeljivanje njihovih bioaktivnih sastavnica složen je i dugotrajan proces. CPC metoda kombinira načela ekstrakcije i kromatografije i može se koristiti za izolaciju tvari sa širokim rasponom polarnosti. Značajka ovog sustava su tekuće stacionarne i mobilne faze pa se analiza i odjeljivanje sastavnica prirodnih produkata zasniva na raspodjeli otopljenih tvari između te dvije nemješljive tekuće faze. Najčešće korišten dvofazni sustav otapala koji se koristi u području prirodnih produkata je heksan/etil acetat/ metanol/ voda. Tehnika se pokazala kao učinkovita alternativa konvencionalnim preparativnim kromatografskim tehnikama, a karakterizira ju kraće vrijeme pročišćavanja i mogućnost injektiranja veće količine uzorka, kao i dobivanje veće količine spojeva visoke čistoće (> 90 %). Znanstvena istraživanja dokazuju uspješnost primjene u izolaciji i pročišćavanje različitih skupina prirodnih spojeva kao što su kanabinoidi, lignani, kumarini, antocijani, alkaloidi, flavonoidi, stilbeni, ksantoni, monoterpeni, seskviterpeni, saponini i različiti pigmenti. Obzirom na učinkovitost preparativnog pročišćavanja, ova bi vrsta kromatografije mogla zadovoljiti potražnju za velikim količinama čistih prirodnih spojeva potrebnim za daljnja biološka, farmakološka i klinička istraživanja odnosno za proizvodnju standarda za potrebe kemijskih analiza ili pak za dobivanje frakcioniranih i ukoncentriranih prirodnih produkata od interesa za farmaceutsku, prehrambenu i kozmetičku industriju.Centrifugal partition chromatography (CPC) is a recent preparative technique that is increasingly used in isolation, purification and concentration of natural products. In this thesis, the theoretical finding about this type of liquid chromatography without a solid support and the possibilities of its application in the field of natural products are presented. Since extracts obtained from plant raw material are quite complex mixtures, the separation of their bioactive components is a complex process. The CPC method combines extraction and chromatography principles and it can be used to isolate substances with a wide range of polarity. The characteristics of this system are liquid stationary and mobile phases, so analysis and separation of the components of natural products are based on the distribution of solutes between two immiscible liquid phases. The most commonly used two phase solvent system for this purpose is hexane / ethyl acetate / methanol / water. The technique has proven to be an effective alternative to conventional preparative chromatographic techniques and it is characterized by a shorter purification time and possibility of injecting a larger quantity of samples, as well as by obtaining a higher quantity of high purity compounds (> 90 %). Scientific researches prove the success of application in the isolation and purification of various group of natural compounds such as cannabinoids, lignans, coumarins, anthocyanins, alkaloids, flavonoids, stilbenes, xanthones, monoterpens, sesquiterpens, saponins and various pigments. Given the efficiency of preparative purification, this type of chromatography could satisfy the demand for large quantities of pure natural compounds necessary for further biological, pharmacological and clinical research, of for the production of standards for the purposes of chemical analyses or for the production of fractionated and concentrated natural products of interest for pharmaceutical, food and cosmetic industry

    Occurrence of moulds on crops during vegetation period and after harvest

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    Utjecaj klimatskih promjena na svakodnevni život jasno je uočljiv. Značajan porast temperatura, sve češće varijacije između ekstremnih količina padalina i sušnih perioda te sve veće koncentracije ugljikovog dioksida u atmosferi ostvaruju efekte na kvantitativni i kvalitativni sastav mikotoksikotvornih plijesni te na njihovu proizvodnju mikotoksina. Prikupljeni su uzorci kukuruza, ječma i pšenice iz tri predjela Hrvatske u tri vremenska perioda – sjeverna, istočna i središnja Hrvatska u srpnju 2020. i 2021. godine, tijekom vegetacijskog perioda, i u prosincu 2020. godine, u periodu žetve. Analizom koncentracije plijesni utvrđen je statistički značajan porast srednje vrijednosti koncentracija na uzorcima ječma iz istočne Hrvatske iz srpnja 2020. (103400 CFU/g na podlozi DG-18 te 286600 CFU/g na podlozi DRBC), uzorcima pšenice iz središnje Hrvatske iz srpnja 2021. (520000 CFU/g na podlozi DRBC) i uzorcima kukuruza iz središnje Hrvatske iz srpnja 2021. (394600 CFU/g na podlozi DRBC).Plijesni koje su dominantno prisutne u uzorcima iz srpnja 2020. godine su kvasci i Fusarium, uzorci iz prosinca 2020. godine primarno sadrže plijesni iz rodova Fusarium, Cladosporium, kvasce, Aspergillus i Penicillium, a uzorci iz srpnja 2021. u velikoj mjeri sadrže kvasce te plijesni iz rodova Cladosporium, i Penicillium.U budućnosti je od presudne važnosti uzeti u obzir utjecaj promjena klimatskih uvjeta na mikotoksikotvorne plijesni koje rastu na žitaricama kako bi efekt istih na zdravlje i ekonomiju bio minimiziran.Effects of climate change can be observed in everyday life. Changes in climate such as temperatures rising, occuring variations in precipitations and rising levels of carbon dioxide in Earth's atmosphere can affect qualitativeand quantitative composition of mycotoxin producing molds. Samples of corn, wheat and barley were collected from warehouses in three parts of Croatia - northern, eastern and central in July 2020. and 2021., during the vegetation period, and in December 2020., during harvest. The analysis of mold concentration has shown statistically significant increase in the average concentration on barley from the area of eastern Croatia in July 2020. (103400 CFU/g on DG-18 agar and 286600 CFU/g on DRBC agar), on wheat from the area of central Croatia from July 2021. (520000 CFU/g oni DRBC agar) and on corn from the area of central Croati from July 2021. (394600 CFU/g on DRBC agar).Samples from July of 2020. mostly contain genera Fusarium and yeasts, samples from December of 2020. mostly contain genera Fusarium, Cladosporium, yeasts, Aspergillus and Penicillium and samples from July of 2021. mostly contain genera Cladosporium, Penicillium. And yeasts.It is of great importance to take the effects of climate change on the production of mycotxins into consideration in order to avoid health and economic problems

    The impact of climate change on mold contamination of cereal crops

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    Znanstvenici posljednjih desetljeća ukazuju na sve izraženije klimatske promjene. Povećane razine ugljikovog dioksida, povišenje temperature na globalnoj razini i velike količine oborina imaju utjecaj na pojavu novih vrsta plijesni koje proizvode mikotoksine, kao i na povećanu kontaminaciju usjeva. Razvoj preventivnih modela za sprječavanje kontaminacije plijesnima bitni su iz gospodarskog, ali i zdravstvenog aspekta. Prikupljeni su uzorci kukuruza, pšenice i ječma iz skladišta sjeverne, istočne i središnje Hrvatske na proljeće 2021. godine. Analizom koncentracije plijesni utvrđen je statistički značajan porast srednje vrijednosti koncentracija na uzorcima kukuruza i to s područja središnje Hrvatske (1,07 x 107 CFU/g na DG-18 podlozi i 9,1 x 106 CFU/g na DRBC podlozi). Plijesni s najučestalijom pojavom su one iz roda Penicillium, Aspergillus sekcija Aspergillus i rod Fusarium (koji prevladava na kukuruzu). Također, na nekoliko uzoraka se pojavljuju plijesni iz roda Aspergillus sekcije Flavi koje su poznate po produkciji izrazito toksičnih aflatoksina. Kako bi se spriječila kontaminacija u skladištima, potrebno je osigurati dobru proizvođačku praksu (GMP). Žitarice je potrebno sušiti na način da razina vlage bude ispod razine pogodne za razvoj plijesni (aw ≤ 0,65), potrebno je spriječiti prisustvo insekata koji bi oštetili zrno i važno je omogućiti adekvatno strujanje zraka. Važno je na globalnoj razini smanjiti emisiju stakleničkih plinova kako bi se ublažile sve izraženije klimatske promjene koje nepogodno mogu utjecati na razvoj plijesni.In the last decades scientists have been warning about negative effects of climate change. Increased carbon dioxide levels, rising global temperature and high rainfall have caused the appearance of the new types of molds which produce mycotoxin. Also, it has caused increased crop contamination. It is important to develop the models for the prevention of mold contamination in order to avoid negative impact on the economy and health. Samples of corn, wheat and barley were collected from warehouses in northern, eastern and central Croatia in the spring of 2021. The analysis of mold concentration have showed statistically significant increase in the average concentration on maize from the area of central Croatia (1,07 x 107 CFU/g on DG-18 agar and 9,1 x 106 CFU/g on DRBC agar). The most frequently detected genera included Penicillium, Aspergillus section Aspergillus and the genera Fusarium (dominant on the maize). Also, there were molds of the genera Aspergillus section Flavi on several samples which are known for the production of extremely toxic aflatoxins. Good manufacturing practice (GMP) plays an important role in prevention of growth of the molds in storages. It is important for the crop to be dried on low humidity level (aw ≤ 0,65). Also, it is important to prevent damage of the crops by insects and to ensure adequate airflow in warehouses. Finally, efforts must be strengthened to ensure effective control of greenhouse gas emissions, otherwise it could lead to increase of climate change which has bad influence on mold growth

    Optimization of hyaluronidase inhibition method and determination of inhibition by olive leaf extracts (Olea europaea, L.)

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    Hijaluronska kiselina jedna je od esencijalnih komponenata tkiva koja sudjeluje u održavanju hidratacije, međustaničnoj komunikaciji i djeluje kao fiziološki lubrikant. Hijaluronidaza, kao enzim koji razgrađuje hijaluronsku kiselinu, sve više je u fokusu znanstvenih istraživanja u svrhu prevencije preuranjenog starenja kože i usporavanja progresije pojedinih bolesti. Cilj ovog diplomskog rada bila je optimizacija spektrofotometrijske metode inhibicije hijaluronidaze i njezina primjena na ekstraktima lista masline. U eksperimentima je korištena hijaluronidaza iz goveđih testisa i natrijeva sol hijaluronske kiseline. Metoda je prilagođena laboratorijskim uvjetima i primijenjena na ekstraktima lista masline te je ispitan njihov inhibicijski potencijal za enzim hijaluronidazu. Pozitivna kontrola bila je taninska kiselina, no najniži IC50, odnosno ukupno najjaču inhibiciju ostvarilo je otapalo B (IC50=3,47 ± 1,45 μg/mL; sastav: 28,8% PPG, 63,8% destilirana voda, 7,4% mliječna kiselina). Najsnažniju inhibiciju među ekstraktima pokazao je uzorak B042M, s IC50 vrijednošću od 4,91 ± 0,56 μg/mL. IC50 vrijednosti čistih otapala (A i B) usporedive su s IC50 vrijednostima dobivenima za ekstrakte priređene s pripadajućim otapalima, stoga se može zaključiti kako sama otapala najviše utječu na izmjerenu inhibiciju. Za potvrdu i točnije definiranje inhibicijskog učinka sastavnica iz lista masline potrebno je provesti dodatna istraživanja.Hyaluronic acid is an essential component of tissues and some of its roles are retaining hydration, intercellular communication, and lubrication. Hyaluronidase, as an enzyme that degrades hyaluronic acid, attracts the attention of researchers who study premature skin aging and different diseases connected to disbalanced hyaluronan homeostasis. The goal of this diploma thesis is to optimize the spectrophotometric method for determining hyaluronidase inhibition. Bovine testicular hyaluronidase was the experimental enzyme and sodium hyaluronate was its substrate. The method has been adjusted to laboratory requirements and was used for measuring hyaluronidase - inhibiting potential of olive leaf extracts. Tannic acid was used as positive control, but the lowest IC50 and overall strongest inhibition was exhibited by solvent B (IC50=3,47 ± 1,45 μg/mL; 28,8% PPG, 63,8% purified water, 7,4% lactic acid). Sample B042M exhibited the strongest inhibition of all the extracts, with IC50 of 4.91 ± 0.56 μg/mL. IC50 values of pure solvents (A and B) is comparable to the values measured for the extracts. This result indicates that the solvents alone act as strong inhibitors of the hyaluronidase enzyme. For exact defining of inhibition by olive leaf extracts, more research needs to be done

    Ultrasound-assisted extraction of polyphenolics of Plantago major L.

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    Veliki ili širokolisni trputac (Plantago major L., Plantaginaceae) je široko rasprostranjena biljka, koja je poznata u narodnoj medicini već dugi niz godina. Sadrži brojne biološki aktivne spojeve, a to su: flavonoidi, alkaloidi, terpenoidi, iridoidi, vitamini, polisaharidi, masne i fenolne kiseline. Zahvaljujući svom kemijskom sastavu posjedu brojne biološke učinke. Primjerice, pomaže u zacjeljivanju rana, koristan je kod dijareje, pomaže u prevenciji virusnih i bakterijskih infekcija, te snižava razinu glukoze i kolesterola u krvi. Polifenoli djeluju protuupalno, antioksidacijski, te smanjuju rizik od nekih kroničnih bolesti, npr. kardiovaskularnih. U ovom radu je za ekstrakciju polifenola iz P.major korištena brza i učinkovita metoda ekstrakcije, ultrazvučna ekstrakcija. Spektrofotometrijski je određena količina ukupnih polifenola, flavonoida i fenolnih kiselina. Koncentracija verbaskozida određena je korištenjem HPLC-a. Usporedbom koncentracija spojeva i uvjeta pripreme ekstrakta ustanovilo se koji su parametri uvjetovali najefikasniju ekstrakciju pojedinih polifenola. Najveći sadržaj ukupnih polifenola imao je ekstrakt koji sadrži 70 % GL, 5 % mliječne kiseline, a ekstrahirao se 30 min pri temperaturi od 70°C i snazi ultrazvuka od 144 W. Flavonoidi su se najbolje ekstrahirali sa 70 % GL, bez mliječne kiseline, a ekstrakcija je trajala 30 min pri 70 °C i snazi ultrazvuka od 720 W. Fenolnim kiselinama je odgovarao medij s 10 % GL i 0,3 mmol HP-β- CD, a ekstrakcija je trajala 30 min pri 20 °C i snazi ultrazvuka od 144 W. Ekstrakcija verbaskozida je najučinkovitije provedena s 10 % GL, 5% mliječne kiseline i 0,3 mmol HP-β-CD, a ekstrakcijsko vrijeme je bilo 10 min na 20 °C i pri snazi ultrazvuka od 144 W. Potrebno je statistički obraditi rezultate kako bi se preciznije odredilo koliko pojedini parametri utječu na ekstrakciju svih navedenih polifenolnih sastavnica.Greater or broadleaf plantain (Plantago major L., Plantaginaceae) is a widespread plant, known in folk medicine for many years. It contains numerous biologically active compounds including flavonoids, alkaloids, terpenoids, iridoids, vitamins, polysaccharides, fatty and phenolic acids. Due to its chemical constituents, it has numerous biological effects. For example, it promotes wound healing, prevents viral and bacterial infections, it is useful in treatment of diarrhea, and lowers the level of glucose and cholesterol in the blood. Polyphenols act as antioxidants, anti-inflammatory and reduce the risk of some chronic diseases, eg cardiovascular. In this work, a fast and efficient extraction method, ultrasonic extraction, was used for the extraction of polyphenols from P. major. The amount of total polyphenols, flavonoids and phenolic acids was determined spectrophotometrically. Verbascoside concentration was determined using HPLC. By comparing the concentrations of the target compounds and the conditions in which extracts were prepared, the parameters necesary for the most efficient extraction of individual polyphenols were established. The extract with the highest content of total polyphenols was prepared using 70 % GL, 5 % lactic acid and was extracted for 30 min at a temperature of 70 °C with an ultrasound power of 144 W. Flavonoids were best extracted using 70 % GL, without lactic acid, and the extraction lasted 30 min at 70 °C with an ultrasound power of 720 W. The most suitable medium for phenolic acid was with 10 % GL, 0.3 mmol HP-β- CD, and the extraction lasted 30 min at 20 °C with an ultrasound power of 144 W. The extract with 10 % GL, 5% lactic acid, 0.3 mmol HP-β-CD showed the highest verbascoside extraction, and the extraction time was 10 min at 20 °C and with an ultrasound power of 144 W. It is still necessary to statistically process the results in order to precisely determine how certain parameters affect the extraction of all the mentioned polyphenol components

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