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Natural products have a key role in drug discovery in pharmacology and medicine. Prunus spinosa L. (blackthorn) grown in Çanakkale province in western Turkey, is known as a medicinal plant, a rich source of biologically active compounds such as phenolics, flavonoids and anthocyanidins. The flower and fruit extracts of the plant are subjects of many studies, but they usually lack in details of its potential for bio-inhibition studies. Thus, this study aimed to investigate the antioxidant, enzyme inhibition and antiproliferative activity studies of the methanol, ethyl acetate, dichloromethane, and n-hexane extracts of the plant. The ethyl acetate and methanol extracts demonstrated more better antioxidant activity with DPPH, FRAP, CUPRAC, and TEAC assays. Enzyme inhibition studies of the extracts were performed using β-lactamase and various proteases. The methanol (FL) and ethyl acetate (FL and L) extracts at the concentration of 10 mg/mL, showed good inhibition against α-chymotrypsin, trypsin, and papain with values of 22.6%, 34.7% and 92.1%, respectively. Furthermore, the methanol and ethyl acetate extracts have displayed higher cytotoxic effect against cancer cells such as Hep3B and HT29 when compared to healthy cells (HUVEC) using MTT assay. The findings suggest that P. spinosa L. extracts and their components may be potential for further investigations of novel drug candidates., Doğal ürünler, farmakoloji ve tıpta ilaç keşfinde anahtar bir role sahiptir. Türkiye'nin batısındaki Çanakkale ilinde yetişen Prunus spinosa L. (karaçalı), tıbbi bir bitki olarak bilinir ve fenolikler, flavonoidler ve antosiyanidinler gibi biyolojik olarak aktif bileşiklerin zengin bir kaynağıdır. Bitkinin çiçek ve meyve özleri birçok çalışmanın konusudur, ancak genellikle biyoinhibisyon çalışmaları için potansiyelinin ayrıntılarından yoksundurlar. Bu nedenle, bu çalışmada bitkinin metanol, etil asetat, diklorometan ve n-hekzan özütlerinin antioksidan, enzim inhibisyonu ve antiproliferatif etkinlik çalışmalarının incelenmesi amaçlanmıştır. Etil asetat ve metanol özütleri, DPPH, FRAP, CUPRAC ve TEAC analizlerinde daha iyi antioksidan etkinlik sergiledi. Özütlerin enzim inhibisyon çalışmaları, β-laktamaz ve çeşitli proteazlar kullanılarak yapıldı. 10 mg/mL derişimdeki metanol (FL) ve etil asetat (FL ve L) özütleri, sırasıyla % 22.6, % 34.7 ve % 92.1 değerleriyle a-kimotripsin, tripsin ve papine karşı iyi inhibisyon gösterdi. Ayrıca metanol ve etil asetat özütleri, MTT testi kullanılarak sağlıklı hücrelere (HUVEC) kıyasla Hep3B ve HT29 gibi kanser hücrelerine karşı daha yüksek sitotoksik etki göstermiştir. Bulgular, P. spinosa L. özütleri ve bileşenlerinin yeni ilaç adaylarının ileri incelemeleri için potansiyel olabileceğini gösterdi.

The purpose of this study was to analyze the phenolic compounds and the antioxidant activity of the aqueous extract obtained from the fruit of Prunus spinosa L.. The aqueous extract of 10% concentration was obtained from the pulp of dried fruits, harvested from Tulcea county, Romania. The tannin content was 3.38g % and the total polyphenols 6.95g %. Based on the HPLC analysis, the identified polyphenolic acids were chlorogenic acid, caffeic acid and gallic acid and the concentrations in mg per 100g of fruit pulp powder were 15.174, 10.93 and 81.468, respectively. The 100 mg/mL aqueous extract had a high DPPH radical scavenger ability (87.30%) which correlated with the polyphenol content and supports the possibility of using rich bioactive aqueous extracts for oxidative stress related conditions.

Anthocyanin compounds are pigments that have a red, blue, and/or violet coloration widely found in nature, mainly in fruits [1]. Given the current need to replace synthetic colorants with healthier natural alternatives, this study investigated the potential of haskap (Prunus spinosa L.) and blackthorn (Lonicera careulea L.) fruits as sources of anthocyanins. For this purpose, the characterization of these fruits (the hydroethanolic extract of blachthorn epicarp and the haskap juice) was carried out by high performance liquid chromatography coupled to a diode array detector and a mass spectrometer (HPLC-DAD/ESI-MS). The identification was carried out using standards, when available, comparing their retention times, UV-Vis spectra, and mass spectra. In the absence of standards, the identification was carried out by the fragmentation profile and by comparison with the information available in the literature. The quantification was performed from the peak areas recorded at a wavelength of 520 nm, compared to the standard calibration curves. The blackthorn epicarp showed two anthocyanin compounds, being the molecules present in the highest concentrations cyanidin-3-O-rutinoside ([H]+ at m/z 595) and peonidin-3-O-rutinoside ([H]+ at m/z 609). In haskap berries, six anthocyanins were identified, namely cyanidin-O-hexoside-O-hexoside ([H]+ at m/z 611), cyanidin-O-rhamnoside-O-hexoside ([H]+ at m/z 595), pelargonidin-3-O-glucoside ([H]+ at m/z 433), peonidin-3-O-glucoside ([H]+ at m/z 463), peonidin-O-rhamnoside-O-hexoside ([H]+ at m/z 609), and cyanidin-3-O-glucoside ([H]+ at m/z 449), with the latest as the most abundant one. These results demonstrate that the blackthorn fruit epicarp and haskap fruits can be considered great sources of pigments in the red-purple colour range, having applicability both in the food and pharmaceutical industries. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for C. Pereira and L. Barros contracts and A.K. Molina and M.G. Leichtweis PhD grants (2020.06231.BD and 2020.06706.BD, respectively). To FEDER-Interreg España-Portugal programme for financial support through TRANSCoLAB 0612_TRANS_CO_LAB_2_P project; to the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural® info:eu-repo/semantics/publishedVersion

Hiperglikemija je metabolički poremećaj karakteriziran kroničnom povišenom glukozom u krvi kao rezultat nedovoljnog ili nepravilnog lučenja inzulina, te nizom sekundarnih komplikacija. Mnogi biljni pripravci bogati polifenolima imaju dokazan hipoglikemijski i antioksidacijski potencijal putem različitih mehanizama djelovanja. U ovome radu su istraženi in vivo učinci polifenola iz ekstrakta cvijeta Prunus spinosa L. (ECT) na biodostupnost, biljege oksidacijskog stresa, antioksidacijske enzime te utjecaj na glikemijske parametre, u normalnog i hiperglikemijskog C57BL/6 miša, tijekom 10 dana eksperimenta. Miševi su bili podijeljeni u 4 skupine, 1. Kontrola (K), 2. Normoglikemija (ECT), 3. Hiperglikemija (AL, izazvana aloksanom), 4. Hiperglikemija (AL+ECT). Dobiveni rezultati sugeriraju da polifenoli kao i njihovi metaboliti iz ECT u tkivima jetre i bubrega povećavaju aktivnost antioksidacijskih enzima (SOD, GSH, CAT) te snižavaju biljege oksidacijskog stresa (MDA, AOPP) u hiperglikemijskoj skupini koja je tretirana sa ECT u odnosu na netretiranu hiperglikemijsku skupinu (AL). Rezultati analize glikemijskih parametara ukazuju na inhibitorno djelovanje na aktivnost α- amilaze, poboljšanu toleranciju glukoze (OGTT), sniženu glukozu u krvi na tašte te poboljšano lučenje inzulina u skupini AL+ECT u odnosu na AL skupinu, što se pripisuje djelovanju polifenola i njihovih metabolita iz ECT. Dobiveni rezultati mogu doprinijeti razumijevanju utjecaja polifenola iz ECT na oksido-redukcijski i glikemijski status kod oboljelih od hiperglikemije te kao potencijalna suportivna terapija u cilju postizanja bolje glikemijske kontrole i prevencije sekundarnih komplikacija kronične hiperglikemije. Hyperglycemia is a metabolic disorder characterized by chronically elevated blood glucose which is a result of insufficient or improper insulin secretion as well as a multitude of secondary complications. Many plant sources rich in polyphenols have a proven hypoglycemic and antioxidant effect through various mechanism. In this thesis the following were examined during a 10 day in vivo experimentation of the effect of polyphenols from Prunus spinosa L. flower extract (ECT) on C57BL/6 mice: bioavailability, influence on biomarkers of oxidative stress, antioxidant enzymes and glycemic parameters. Mice were divided into 4 groups: 1. Control (K), 2. Normoglycemic group (ECT), 3. Hyperglycemic group (AL, alloxan-induced), 4. Hyperglycemic group (AL+ECT). The given results suggest that polyphenols as well as their metabolites from ECT in liver and kidney tissue increase the activity of antioxidant enzymes (SOD, GSH, CAT) and lower the biomarkers of oxidative stress (MDA, AOPP) in the hyperglycemic group treated with ECT relative to the untreated hyperglycemic group (AL). Analysis results of glycemic parameters indicate an inhibitory effect on α-amylase activity, improved glucose tolerance (OGTT), decreased fasting blood glucose and increased insulin secretion in the AL+ECT group compared to the AL group, which is attributed to the efficiency of polyphenols and their metabolites from ECT. The obtained results can contribute to understanding the influence of polyphenols from ECT on the oxidative and glycemic status in patients with hyperglycemia and as a potential supportive therapy in order to achieve better glycemic control and prevention of secondary complication from chronic hyperglycemia.

The aim of this study was to investigate the crude extracts isolated from branches of Prunus spinosa L. (Rosaceae), in order to quantify polyphenols and tannins using instrumental methods (UV-Vis spectrometry and HPTLC densitometry). The qualitative analysis on biomass revealed the presence of valuable compounds such as polyphenols, flavonoids and tannins. HPTLC fingerprinting of crude methanol extract showed several peaks, with different Rf values, corresponding to phytocompounds such as chlorogenic acid (Rf 0.52), neochlorogenic acid (Rf 0.58), caffeic acid (Rf 0.95) and protocatechuic acid (Rf 0.96). The quantitative analysis revealed that the branches of P. spinosa contain polyphenols (2.97�0.059%g gallic acid equivalent/g dried vegetal material) and tannins (0.90�0.033%g tannic acid equivalent/g dried vegetal material). The HPTLC densitometry analysis offers information about the amount of neochlorogenic acid in the vegetal material 0.12% g/g. The content in polyphenols and tannins suggests the possibilities to use this species to separate compounds with antioxidant potential. These results open perspectives for complex valorization of Prunus spinosa species, a bioresource known only for phytotherapeutical and nutritional potential of their fruits.

Prunus spinosa L. (blackthorn) flower was most recently pointed out as a valuable source of bioactive compounds that have potent anticancer properties. To test for antineoplastic activity, hepatocarcinoma cells (Hepa 1–6) and corresponding non-neoplastic hepatocytes (AML 12) were subjected to Prunus spinosa L. blossom ethanolic extract (5–200 μg mL−1). Antiproliferative activity was determined by Trypan Blue (TB) and Kenacid Blue (KB) methods after 24 and 72 h. Inhibitory effects on Hepa 1–6 proliferation were observed at all concentrations with significant inhibition at ≥ 100 μg mL−1 (p < .025–p < .001) determined with both methods. Cytotoxicity was also detected in non-neoplastic hepatocytes with significant doses above 10 μg mL−1 (p < .05–p < .001) after 72 h. Cellular viability loss after 24 h was more pronounced in non-neoplastic vs. hepatocarcinoma cells as confirmed with IC50TB values (half maximal inhibitory concentration): 59.05 μg mL−1 in AML 12 vs. 166.87 μg mL−1 in Hepa 1–6 cells and IC50KB: 72.69 vs. 172.93 μg mL−1, respectively. Prolonged incubation (72 h) resulted in almost equal toxicity in both cell lines. Prooxidative activity of blackthorn flower extract was observed in both cell lines with higher ROS formation in neoplastic cells. Cell death was primarily through necrosis. The proportion of apoptotic cells in both cultures was low: fraction of early apoptosis – 0.04–1.65% of the total cell population and fraction of late apoptosis: only 0.05–3.25%.

The present study was undertaken to determine some pomological, physical and chemical properties of five apricot cultivars from University of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia—‘Novosadska kasnocvetna’, ‘Novosadska rodna’, NS-6, NS-4, and ‘Buda’—and two selections (SK-1 and SK-3) grafted on myrobalan as a rootstock, with blackthorn (Prunus spinosa L.) used as interstock. The experiment was conducted during the 2013–2017 period at the experimental field for fruit growing of the University of Novi Sad, Faculty of Agriculture. The orchard was established in 2012 with 4 × 2 m tree spacing. Phenological characteristics, yield, trunk circumference, fruit and stone size, as well as chemical properties of Novi Sad’s apricot cultivars and selections were determined. The earliest flowering onset was observed in the cultivar ‘Buda’ and the latest in the selection SK-3. In all examined cultivars and selections, fruit weight was above 60 g and ‘Buda’ was particularly distinguished by its fruit weight and stone to flesh ratio. ‘Novosadska kasnocvetna’ had the lowest yield efficiency (0.4 kg cm−2), yield per tree (4.4 kg) and per ha (5.5 t). Cultivar NS-6 and selection SK-1 were the most productive, with the cumulative yield per tree of 27.1 and 26.2 kg, respectively. The highest trunk cross-sectional area in all five experimental years was measured for the cultivar ‘Buda’ (58.1 cm2). The highest soluble solids content was detected in the cultivar ‘Buda’ (16.1°Brix), while ‘Novosadska kasnocvetna’ had the highest total acid content (1.4%). Novi Sad’s apricot cultivars and selections yield good fruit quality, which can thus serve as a very powerful source of phenolic compounds.

Natural matrices that are rich in anthocyanin compounds are increasingly explored by the food industry due to their colouring properties. As examples, Prunus spinosa L. and Lonicera careulea L. fruits are excellent sources of anthocyanins and are, therefore, increasingly explored for their colouring properties to be applied as food colorants, in addition to providing beneficial properties to the consumer [1,2]. Therefore, the aim of this study was to evaluate the anthocyanin profile (HPLC-DAD/ESI-MS) and the antioxidant (TBARS and OxHLIA) and antimicrobial properties of the hydroethanolic extract of P. spinosa fruit epicarp and L. careulea fruit juice. A high content of cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside was found in the juice of L. caerulea berries and in the hydroethanolic extract of the epicarp of P. spinosa, respectively. As for the antioxidant activity, in the TBARS assay, the L. caerulea berries showed a higher capacity (IC50 of 29.9±0.3 μg/mL) than the positive control, trolox (IC50 of 139±5 μg/mL). Similarly, the epicarp of P. spinosa showed the ability to inhibit lipid peroxidation, revealing an EC50 value of 204±2 μg/mL. Regarding the ability to retard oxidative haemolysis, both extracts showed activity not only at 60 min, but also at 120 min, allowing EC50 values of 145±5 μg/mL and 938±49 μg/mL, respectively, for L. caerulea, and 296±4 and 509±3 μg/mL, respectively, for P. spinosa. On the other hand, both colouring extracts revealed great antimicrobial properties. Through this work, it was possible to conclude that L. caerulea and P. spinosa berries have a high colouring capacity and bioactive potential, being suitable for the development of new products for food industry. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for C. Pereira and L. Barros contracts and A.K. Molina and M.G. Leichtweis PhD grants (2020.06231.BD and 2020.06706.BD, respectively). To FEDER-Interreg España-Portugal programme for financial support through TRANSCoLAB 0612_TRANS_CO_LAB_2_P project; to the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®. info:eu-repo/semantics/publishedVersion

The study examined the antioxidative physiological effects of phenolics from an ethanol-water extract of blackthorn flowers orally administrated to C57/BL6 mice for 28 days in daily doses of 25 mg of total phenolics/kg body weight. Contents of phenolics in the intestine, liver, and kidneys collected after 1, 7, 14, 21, and 28 days of extract administration were analyzed by UPLC-MS/MS method. In the same tissues, the antioxidative properties were determined as ferric reducing antioxidant power (FRAP), ABTS•+ scavenging activity, content of reduced glutathione (GSH), and activity of superoxide dismutase (SOD) and catalase (CAT). The lipid peroxidation in tissues was also evaluated by thiobarbituric acid reactive substances (TBARS) assay. The exposed mice (compared to the control ones) had a lower content of TBARS in all tissues mostly on the third/fourth week of daily consumption. SOD activity and GSH content increased on the 28th day in tissues. CAT activity was higher only in the liver after one week of consumption but remained unchanged in other organs throughout the experiment. Phenolic profiles were different in individual tissues. The most prominent increases compared to the control were determined for contents of 3- O -feruloylquinic acid, 4- O-p -coumaroylqiunic acid, kaempferol pentoside, and quercetin rhamnoside in the intestine; for ferulic acid and quercetin 3- O -rutinoside in the liver; and for quercetin 3- O -rutinoside, ferulic acid, and 4- O-p -coumaroylquinic acid in the kidneys. The screened phenolics with different distribution in tissues could be responsible for slight differences in the recorded antioxidative effects.

This paper presents data on the optimization and validation of an RP-HPLC-PDA method for quantification of 30 phenolic constituents of the blackthorn (Prunus spinosa L.) flower. The method development data cover detailed descriptions of the optimization process in terms of elution solvents, gradient profile, temperature, and flow rate. The validation data cover accuracy and precision (intra- and inter-day variability) for retention times and peak areas. Moreover, the quantification data for the commercial samples of blackthorn flower (different manufactures and years of collection), as well as for the extracts (of different polarity) prepared thereof, are included. The data presented here were related to the article: “Simultaneous quantification of thirty polyphenols in blackthorn flowers and dry extracts prepared thereof: HPLC-PDA method development and validation for quality control” [1]. Keywords: Prunus spinosa, Polyphenols, Quality control, High performance liquid chromatography, Optimization, Tertiary gradient, Validation, Quantification

The comparison of the antioxidant activity of the studied forest fruits emphasized a hierarchy of the antioxidant capacity in rosehip, blackthorn, lingonberry and cornelian cherry. The purpose of the study was to investigate the antioxidant capacity and nutritional value of rosehip, blackthorn, lingonberry and cornelian cherry. In the current study, the FT-IR spectroscopy technique was applied to detect molecular components in forest fruits samples. Antioxidant capacity was evaluated with photochemical assay as well as humidity, protein, fibre, lipid and carbohydrate content. The FT-IR results revealed the presence of different bio-active compounds in berries such as flavonoids, tannins, sugars, acids, proanthocyanidins, carotenoids, citric metabolites and others. The highest antioxidant capacity was observed in rosehip 105.67±1.38 and blackthorn 49.89±1.92 (μg/mg equivalent ascorbic acid). Regarding nutritional parameters, rosehip showed the most increased content of protein displaying average values of 1.60, carbohydrates 38.20 and fibre 24.10 (g/100 g/sample). These results can provide useful information providing a research interest for the identification of new molecular compounds from Romanian flora samples., Vaccinium vitis-idaea L. Cornus mas L. Prunus spinosa Rosa canina

Prunus spinosa L. is a shrub that produces berries—sloes—with a high content of phenolic compounds and anthocyanins. Numerous consumer products are made from sloes, such as “Pacharán” liquors or jams. For these reasons, it is crucial to design accurate analytical methods for the extraction of these bioactive compounds in order to promote their consumption and to improve our health. In this study, an ultrasound-assisted extraction method was developed to extract phenolic compounds and anthocyanins from sloes. Several process variables (methanol composition, pH, temperature, cycle, amplitude, and sample–solvent ratio) were optimized based on a Box–Behnken design. The most influential parameters for the extraction of total phenolic compounds were the cycle, the percentage of methanol, and pH of the extraction solvent, with 0.2 s, 67%, and pH 7 as their optimal values, respectively. For total anthocyanins, all parameters were influential, with 44% methanol at pH 2, 48 °C, 0.2 s cycles, 30% amplitude, and 1.5 g/20 mL ratio as the optimal values for their corresponding variables. Both methods showed high recoveries, short time use, and high precision (RSD < 5%). In addition, the sloe liqueur “Pacharán” and sloe jams were analyzed to demonstrate the applicability of these methods for the extraction of the real matrix.

The effect of freezing (M), freeze drying (L) and convection drying (S) of blackthorn fruits on the content of selected bioactive compounds and antioxidant properties compared to the control sample – fresh fruit (PK), was tested. Changes in the content of flavonoids, anthocyanins, pectins, general acidity and the ability to reduce the DPPPH radical, were determined. The obtained results showed that the freeze-dried fruits had the highest content of anthocyanins and flavonoids, respectively 0.07 and 0.17%. Pectin contents in freeze-dried and convectively dried fruits were similar at the level of 1.66 and 1.64% as well as for acidity respectively 0.076 and 0.071 g·100 g–1 infusion. Frozen fruits did not differ significantly compared to the control in terms of the content of anthocyanins, flavonoids, pectin and acidity. The highest scavenging capacity of free radical DPPH was characterized by the infusion obtained from fresh fruits.

This paper aims to analyse the variability of morphological characteristics of Prunus spinosa L. shrubs, identified and studied in the spontaneous flora in southern Oltenia, Romania (localities Calopar and Gura Văii). The observations made on trunk diameter, stem height, spinosity degree, ability to form basal shoots, have outlined the high variability, which is useful in breeding programs. Variability coefficient ranged between 26.02% and 30.87% for the stem diameter, between 23.48% and 32.32% for the plant height, between 30.93% and 37.81% for canopy diameter. Based on the obtained results, the C31, GV26, GV28, GV29, GV30, GV31, GV33, GV34, GV37 and GV8, GV9, GV27, GV35, C5, C24, C25, C26 and C29 genotypes are recommended for fixing the eroded lands, owing to their very strong, strong and medium ability to form basal shoots; while C10, C12, C23, C27, C28, C30, GV6, GV10, GV24, GV25, GV32 and GV36 genotypes are recommended to be studied as rootstocks in fruit tree species, having in view the low or missing vigor and ability to form basal shoots.

This study aimed to evaluate the effectiveness of natural casing treatment with ethanol and aqueous extract of the blackthorn fruits (Prunus spinosa L.) on the quality of vacuum packed Kranjska sausages. Three experimental groups of sample sausages were produced. First sample was a conventional sausage filled in a natural casing, second sample was sausage filled in a natural casing that was previously submerged in ethanol extract of the blackthorn and third sample was sausage filled in a natural casing that was previously submerged in aqueous extract of the blackthorn. The sausages were produced in a industrial conditions, stuffed into the pretreated natural casings, vacuum packaged and stored at 4°C for 60 days. There were no significant differences (p>0.05) in chemical composition and in sensory quality between the different sample sausages. This study has shown that extract of the blackthorn fruits (Prunus spinosa L.) incorporated into natural casing before the filling operation affects the reduction in the number of lactic acid bacteria on the outside surface of sausage samples, in vacuum packed Kranjska sausages, stored 60 days at low temperatures. The sausages with treated casings did not have much better oxidative stability during storage, because the herbal extracts probably did not diffuse into the filling, its amount was too small to significantly affect the decrease of the acid and peroxide number, and TBARS value.

The aim of this study was to determine the influence of sloe berry (SB) addition at different ratios on the physical, chemical, antioxidant, colour and sensory properties and mineral contents of ice creams. The increment of SB led to the an increase of titratable acidity, overrun, viscosity and ash values, first dripping and complete melting times; in contrast, it decreased the total solid, pH and fat values. Mn and Zn values were affected significantly (P L* and b* values decreased significantly in all samples as compared with control sample, while a*, H°, C* and ∆E* increased significantly with addition of SB. Antioxidant activity was the highest in the sample containing 15 % SB (w/w) among ice cream samples. The ice cream samples containing 15 % SB had higher score than the control sample in terms of colour and appearance, gumming structure and general acceptability.

The aim of the study was to determine the influence of selected methods of preservation and storage time on the content of antioxidants in blackthorn fruit (Prunus spinosa L.). Preserved fruits were stored for 13 and 26 weeks without light at room temperature (freeze-dried and convection dried) and at –20°C (frozen samples). It was shown that both the method of preservation and the storage time had significant influence on the content of polyphenols (anthocyanins, flavonoids, total polyphenols) and antioxidant properties (Fe3+ reduction strength) of blackthorn fruit. As the storage time progressed, the anthocyanins content decreased in all preservation variants. In the case of frozen fruit after 26 weeks of storage, the content of flavonoids (by 12.5%), total polyphenols (by 48%) and the ability to reduce the iron ions were increased (by 55%). In turn, in the dried fruits after this period, the anthocyanins content, the polyphenol content and reduction strength were reduced. The highest values of the parameters evaluated after the assumed storage time has been demonstrated in lyophilizates.

Polyphenol (PPH) organ distribution may explicite their physiological efficacy. Water extract of blackthorn (Prunus spinosa L.) flower (PSE) was used to model the tissue distribution of PPH in C57BL/6 mice following oral acute and repeated (subchronic 28 days) administration of 25 mg/kg bw of total PPH. 32 individual PPH's were measured by UPLC-MS (15 min, 30 min, 1h, 3h, 6h, 24h in acute and 1st, 7th, 14th, 21th, 28th day in subchronic treatment).Highest present PPH in the PSE were not necessarily the highest bioavialable in the tissues. Repeated treatment increased the number of significantly higher PPHv Cmax/AUCalast, innn PSE treated animals vs. control, by +6% in the intestine, +45.5% in the liver. +16.0% in kidney and +32% in the brain. Intestine had preference for bioaccumulation of ferulloylquinic acid and kaempherol-pentoside, liver for kaempherol- pentosid and quercetin-3-O-rutinosid, kidney for ferullic acid and quercetin-rutinoside and brain bioacumullated quercetin-rhamnoside and quercetin- rutinoside.

A recuperação de biorresíduos da indústria alimentar tem vindo a ganhar relevância pela crescente procura da sua valorização enquanto fontes de compostos de interesse para posterior aplicação em diversos ramos industriais, como é o caso da indústria alimentar, têxtil, e farmacêutica, entre outras [1,2]. Assim, o presente estudo visa a recuperação de antocianinas a partir de biorresíduos de frutos: a casca de figo, rica em cianidina 3-rutinósido (C3R), e o epicarpo de abrunho, rico em C3R e peonidina 3-rutinósido (P3R). Foram testadas duas técnicas de extração, maceração (EM) e assistida por ultrassons (EAU), às quais se aplicou uma metodologia de superfície de resposta usando o desenho composto central circunscrito com cinco níveis em cada uma das variáveis independentes estudadas (tempo, proporção de água-etanol como solvente e temperatura (EM) ou potência (EAU)). O perfil de antocianinas dos diferentes extratos foi obtido por HPLC-DAD-ESI/MS e utilizou-se a concentração total de antocianinas (C) no resíduo de extração (R; mg C/g R) e na amostra desidratada (A; mg C/g A) e o rendimento de resíduo obtido (g R/g A) como respostas para o modelo. Foi ainda avaliada a bioatividade (citotoxicidade e atividade antioxidante e antimicrobiana) dos extratos obtidos em condições ótimas, bem como o poder corante dos mesmos avaliados pela incorporação do extrato ótimo em produtos de pastelaria. Para ambas as amostras, a EAU revelou ser a técnica mais eficiente, com condições ótimas de recuperação de C3R aos 21,3±0,6 min, 310±26 W e 100±1% de etanol no caso da casca de figo, com um rendimento de 0,5±0,2 g R/g A e um total de antocianinas de 9,0±0,8 mg C/g R e 4,3±0,1 mg C/g A. Por sua vez, a partir do epicarpo de abrunho foi possível obter um total de C3R e P3R de 18±2 mg C/g R e 11,8±0,8 mg C/g A, e um rendimento de 0,69±0,02 g R/g A, nas condições ideais de extração de 5,0±0,2 min, 400±32 W e 48±3% de etanol. Relativamente às propriedades bioativas, o extrato de casca de figo revelou um valor de EC50 de 2447±2 μg/mL no método TBARS e valores de EC80 de 515±31 e 1624±56 μg/mL, para 60 e 120 min, respetivamente, no ensaio OxHLIA. O extrato de epicarpo de abrunho apresentou valores de EC50 (TBARS; 204,22±0,02 μg/mL) e EC80 (OxHLIA; 303±3 e 540±3 μg/mL, para 60 e 120 min, respetivamente) inferiores, o que indica uma maior atividade antioxidante em ambos os ensaios. Ambos os extratos revelaram propriedades antibacterianas, inibindo o crescimento de 9 estirpes bacterianas (5 Gram-negativo e 4 Grampositivo), em concentrações entre 2,5 e 20 mg/mL. Para além disso, não apresentaram toxicidade em células não tumorais de fígado de porco, o que torna viável a sua aplicação em produtos alimentares sem problemas de toxicidade. Assim, o poder corante dos extratos antociânicos foi testado em produtos de pastelaria: o extrato de casca de figo foi incorporado em cobertura de “donut” e o de epicarpo de abrunho em “beijinhos”, um doce típico do Brasil. Esta aplicação não alterou significativamente o valor nutricional das matrizes em estudo e, para além de conferir ao alimento a cor desejada, proporciona também propriedades antioxidantes e antimicrobianas. FCT e FEDER sob o Programa PT2020 pelo apoio financeiro ao CIMO (UID/AGR/00690/2019), LA LSRE-LCM (POCI-01-0145-FEDER-006984), contrato de Pereira C no âmbito da celebração do contrato-programa previsto nos números 4, 5 e 6 do art. 23.º do D.L. n.º 57/2016, de 29 de agosto, alterado pela Lei n.º 57/2017, de 19 de julho e contrato de Barros L no âmbito da celebração do contrato-programa de emprego científico institucional; FEDER-Interreg España-Portugal pelo financiamento ao projeto 0377_Iberphenol_6_E; ao FEDER através do Programa Operacional Regional Norte 2020, no âmbito do Projeto NORTE-01- 0145-FEDER-023289 (DeCodE) e projeto Mobilizador Norte-01-0247-FEDER-024479: ValorNatural®. Xunta de Galicia pelo apoio financeiro a Prieto MA. info:eu-repo/semantics/publishedVersion

U ovome radu je istražena in vivo farmakokinetika-apsorpcija, metabolizam i raspodjela polifenolnih spojeva iz vodenog ekstrakta cvijeta trnine (Prunus spinosa L.), te vodenog ekstrakta cvijeta trnine u kombinaciji s prehranom obogaćenom proteinima, mastima i ugljikohidratima u organima (tanko crijevo, jetra, mozak, bubreg) miša C57BL/6. Također je istražen bioakumulacijski potencijal nakupljanja molekula polifenola u navedenim organima, te biološki raspoložive maksimalne koncentracije pojedinih skupina molekula polifenola. Osim navedenog, istraženi su glavni biomarkeri oksidacijskog stresa (MDA, PC), aktivacija antioksidacijskih enzima (SOD, GSH, CAT) i antioksidacijski kapaciteti navedenih tkiva. Dobiveni rezultati pokazuju da se od ukupno 32 vrste polifenolnih spojeva koje nalazimo u ekstraktu cvijeta trnine, apsorpcijom i bioraspodjelom po organima u različitim vremenima bioakumuliraju različite vrste polifenolnih spojeva što potvrđuje postavljenu hipotezu o selektivnoj preferenciji za bioakumulacijom i zadržavanjem pojedinog tipa polifenolnih spojeva unutar različitih organa. Rezultati analize antioksidacijskog sustava su također pokazali koje pojedine skupine polifenola i njihovih metabolita aktiviraju enzime uključene u antioksidacijski sustav obrane (SOD, GSH, CAT), a izmjereni antioksidacijski kapaciteti tkiva ukazuju da pojedini organi u pokusnim skupinama pokazuju relativno visoki antioksidacijski kapacitet u odnosu na kontrolnu skupinu životinja. Dobiveni rezultati ovog istraživanja pridonose boljem razumijevanju afiniteta pojedinih skupina polifenola prema pojedinim tkivima i organima s temeljnim ciljem postizanja boljeg antioksidacijskog statusa organizma. This thesis deals with the in vivo pharmacokinetics-absorption, metabolism and distribution of polyphenolic compounds from the sloe (Prunus spinosa L.) flower water extract and the water extract of sloe in combination with a diet enriched by proteins, fat and carbohydrates in the organs (small intestine, liver, brain, kidney) of the mouse C57BL/6. Furthermore, the bioaccumulative potential of polyphenolic compounds in these organs was also investigated, as well as the bioavailable maximum concentration of polyphenolic molecules. In addition, major oxidative stress biomarkers (MDA, PC), antioxidant enzyme activation (SOD, GSH, CAT), and antioxidative capacity of selected tissues were investigated. The obtained results show that out of a total of 32 types of polyphenolic compounds found in the sloe flower extract, various types of polyphenolic compounds bioaccumulate by absorption and biodistribution within organs at different times which confirms the set hypothesis of selective preference for bioaccumulation and retention of a certain type of polyphenolic compounds within different organs. The results of the antioxidation system analysis have also shown which groups of polyphenols and their metabolites activate specific enzymes involved in the antioxidative defence system (SOD, GSH, CAT) and the measured antioxidant tissue capacity indicates that certain organs in the test groups exhibit a relatively high antioxidative capacity in relation to the animal control group. The results of this study contribute to a better understanding of the affinity of certain groups of polyphenols to certain tissues and organs with the fundamental aim of achieving a better antioxidative status of the organism.

Blackthorn (Prunus spinosa L.) germination is often low, so new methods need to be developed with a view to improving nursery yields and to inform decision-making on natural regeneration. To this end, the effects of seed moisture content (MC) levels in combination with warm and chilling treatments on blackthorn seed dormancy release were investigated. In another experiment, the effect on seed germination of warm and chilling treatments in combination with exogenous hormones was investigated. Following treatment, the seeds were allowed to germinate at a constant 15°C with 8 h lighting per day or 20 (dark)/30°C (light). Seed lot effects were evident, but were consistent across treatments. Seeds adjusted to the lower target MC level (TMC) maintained high germination potential over a longer period of treatment than in those held in the fully imbibed (FI) state. The highest germination was achieved in the TMC seeds that were given six weeks warm treatment followed by 32 weeks chilling. Hormone treatmen...

The aim of this work was to investigate and compare the phenolic profile of 15 wild growing blackthorn (Prunus spinosa L.) genotypes from the slopes of Fruska Gora mountain in north Serbia. Their effect in inhibiting i) α-amylase and α-glucosidase activities and ii) colorectal cancer cell line (HT29) growth was also studied. Blackthorn fruit extracts exhibited high phenolic content being enrich in anthocyanins. Principal component analysis was used to correlate the bioactive response with phenolic composition. It was found that derivatives quercetin and anthocyanin peonidin are the major contributors of the inhibition of carbohydrates hydrolyzing enzymes as well as with the antiproliferative effect of blackthorn. Among all samples, the genotype from Beska locality showed the higher capacity in inhibiting alpha-amylase, alpha-glucosidase and HT29 cell growth. Because of high anthocyanin content and higher bioactive response, these genotypes could be recommended for the further cultivation and investigation.

Šumske drvenaste vrste (potencijalno) jestivih i/ili korisnih plodova, označene kao samoniklo voće, predstavljaju važan deo specijskog diverztiteta u šumskim ekosistemima Srbije. Vrste Rosa canina L. (Rosaceae), Crataegus monogyna Jacq. (Rosaceae), Prunus spinosa L. (Rosaceae), Cornus mas L. (Cornaceae) i Cornus sanguinea L. (Cornaceae), iako relativno česte u flori Srbije, još uvek nisu dovoljno proučene sa fitohemijskog, kao ni sa ekološko-hemijskog aspekta. Kvalitativna i kvantitativna LC/MS-MS analiza sadržaja određenih hemijskih jedinjenja u populacijama sa međusobno udaljenih staništa, kao i u pojedinačnim jedinkama na mikrostaništu, kao osnovni cilj ovog rada, pružila je informacije o sintezi specifičnih sekundarnih metabolita kao odgovoru na kompleks ekoloških faktora na staništu. Hemijska karakterizacija odabranih vrsta urađena je identifikacijom i kvatifikacijom fenolne kiseline (neohlorogena kiselina [5-Okafeoilhinska kiselina]) i pet flavonoida (kvercituron [kvercetin-3-O-glukuronid], hiperozid [kvercetin-3-O-galaktozid], rutin [kvercetin-3-O-rutinozid], izokvercetin [kvercetin-3-Oglukozid] i kvercitrin [kvercetin-3-O- ramnozid]) u metanolnim ekstraktima svežih plodova uzorkovanih iz tri prirodne populacije svake vrste. Geografska udaljenost istraživanih populacija (Avala, Zlatarsko jezero i Ozren) omogućila je analizu povezanosti sadržaja ispitivanih fenolnih jedinjenja sa različitim sredinskim uslovima. U metanolnim ekstraktima svežih plodova vrsta R.canina, C. mas i C. sanguinea detektovano je prisustvo svih šest analiziranih fenolnih jedinjenja: neohlorogene kiseline, kvercetin-glukuronida, -galaktozida, -rutinozida, -glukozida i -ramnozida, dok kod vrsta C. monogyna i P. spinosa prisustvo kvercetin-glukuronida nije utvrđeno. U poređenju sa literaturnim podacima ispitivanja plodova kultivisanih i divljih vrsta koje se smatraju bogatim izvorima analiziranih fenolnih jedinjenja (prirodnih antioksidanata), predstavljeni rezultati pokazuju da se prirodne populacije pet šumskih vrsta sa teritorije Srbije odlikuju veoma visokim sadržajem ovih jedinjenja. Ispitivane vrste se značajno razlikuju kako po sadržaju analiziranih fenolnih jedinjenja u ukupnom uzorku, tako i po njihovom procentualnom učešću i varijabilnosti sadržaja. Kod svih ispitivanih vrsta je utvrđen manji ili veći stepen interpopulacione varijabilnosti, a hemometrijskom analizom je utvrđeno koja od ispitivanih jedinjenja najviše doprinose razlikama između populacija...

The present work describes the evaluation of fig peels and blackthorn fruit extracts as natural purple colorants in doughnuts (icing) and in a typical Brazilian pastry called “beijinho”. The extracts were screened for their antioxidant activity as well as their antibacterial capacity. Nutritionally, the employed extracts did not induce significant changes, contrarily to the observed for the rheological features, mainly the darker purple tone observed when blackthorn extract was used in the icing solution. After 24 h, both prepared formulations showed a decrease in color intensity, with no significant differences between fig and blackthorn extracts. In turn, the firmness and consistency of the doughnuts benefited from using natural colorants in the icing solution, while “beijinhos” became softer and chewier, which are valued attributes. A significant increase in the antioxidant and antimicrobial activities was also observed for both natural extracts. Accordingly, the evaluated extracts are promising candidates as natural food colorants. The authors would like to thank the Foundation for Science and Technology (FCT, Portugal) and FEDER, under Program PT2020 for financing CIMO (Centro de Investigação de Montanha) (UID/AGR/ 00690/2019). This work was further funded by the European Structural and Investment Funds (FEEI) through the Regional Operational Program North 2020 within the scope of the Mobilizador ValorNatural® project, to whom the author M. Carocho thanks for his contract. Further acknowledgments are due to the national funding by FCT, P.I., through the institutional scientific employment program-contract for L. Barros’ contracts, and though the celebration of program-contract foreseen in No. 4, 5 and 6 of article 23° of Decree-Law No. 57/2016, of 29th August, amended by Law No. 57/2017, of 19th July for C. Pereira’s contract. info:eu-repo/semantics/publishedVersion

U ovome radu je istražena in vivo farmakokinetika- apsorpcija, metabolizam i raspodjela polifenolnih spojeva iz vodenog ekstrakta cvijeta trnine (Prunus spinosa L.) u organima (tanko crijevo, jetra, mozak, bubreg) miša C57BL/6. Također je istražen bioakumulacijski potencijal nakupljanja molekula polifenola u navedenim organima te biološki raspoložive maksimalne koncentracije pojedinih skupina molekula polifenola. Dobiveni rezultati pokazuju da se od ukupno 32 vrste polifenolnih spojeva koje nalazimo u ekstraktu cvijeta trnine, apsorpcijom i bioraspodjelom po organima u različitim vremenima bioakumuliraju različite vrste polifenolnih spojeva što potvrđuje postavljenu hipotezu o selektivnoj preferenciji za bioakumulacijom i zadržavanjem pojedinog tipa polifenolnih spojeva unutar različitih organa. Dobiveni rezultati ovog istraživanja pridonose boljem razumijevanju afiniteta pojedinih skupina polifenola prema pojedinim tkivima i organima s temeljnim ciljem postizanja boljeg antioksidacijskog statusa organizma.

Prunus spinosa L. is a wild shrub with bitter and astringent fruits with poor commercial value. Nevertheless, they are rich sources of anthocyanins, more specifically cyanidin 3-rutinoside and peonidin 3-rutinoside, which are concentrated in the epicarp. These compounds possess recognized colouring properties and have been increasingly explored for substitution of artificial colorants, for being safer alternatives. In this context, the present work aimed to develop an anthocyanin-based food colorant obtained from P. spinosa fruit epicarp. The conditions that maximize the extraction were optimized, comparing heat (HAE) and ultrasound (UAE) assisted extraction techniques. For that purpose, a response surface methodology was applied, using a circumscribed central composite design with three variables and five levels, being the relevant variables time, temperature (HAE) or power (UAE), and ethanol concentration. The anthocyanin compounds were quantified by HPLC-DAD-ESI/MS. Furthermore, the bioactivity of the optimal extract was assessed through antioxidant, antimicrobial, and cytotoxic activity assays. UAE was the most efficient method, under optimal conditions of 5.0 ± 0.2 min, 400 ± 32 W, and 48 ± 3% ethanol, where the extraction yield was 68.60 ± 2.1%, with a total anthocyanin content of 18 ± 2 mg/g (dried extracted residue-basis) and 11,8 ± 0,8 mg/g (dried epicarp-basis). Additionally, these response values were slightly improved when the solid-to-liquid ratio effect at the optimal conditions in a dose-response format was tested, showing a steady decreasing pattern in the range of 5 to 250 g/L. Regarding bioactive properties, the obtained extract presented antioxidant activity, with EC50 values of 204.22 ± 0.02 μg/mL for the TBARS assay and 296 ± 4 and 509 ± 3 μg/mL for OxHLIA method at 60 and 120 min of reaction, respectively; and antimicrobial properties, allowing the growth inhibition of 9 bacteria strains of public health interest in concentrations from 2.5 to 20 mg/mL. Besides, the obtained extract did not show hepatotoxic effects on a porcine liver primary cell culture (PLP2), being thus safe for incorporation into foodstuff. Lastly, the anthocyanin-based extract was introduced in a traditional Brazilian confectionary product, “beijinho”, where its colouring capacity was confirmed. FCT and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2013), LA LSRE-LCM (POCI-01-0145-FEDER-006984), Carla Pereira (SFRH/BPD/122650/2016) grant, and L. Barros contract. Interreg España-Portugal for financial support to project 0377_Iberphenol_6_E. FEEI through the Regional Operational Program North 2020, within the scope of Project NORTE-01-0145-FEDER-023289: DeCodE and Norte-01-0247-FEDER-024479: ValorNatural®. Xunta de Galicia for financial support to M.A. Prieto grant. info:eu-repo/semantics/publishedVersion

Os corantes alimentares são amplamente utilizados em todo o mundo para melhorar as características dos alimentos, nomeadamente conferir uma nova coloração ou intensificar a cor natural do alimento. No entanto, a maioria destes compostos é de origem artificial e muitos deles têm revelado problemas de toxicidade para a organismo humano. Entre eles, destacam-se reações alérgicas, hiperaüvidade e défice de atenção em crianças, ou até mesmo propriedades cancerígenas [l]. Por outro lado, existem moléculas de origem natural com elevado poder corante que não revelam toxicidade, podendo assim ser aplicadas na indústria alimentar em detrimento das artificiais. O presente estudo surge no seguimento desta problemática e visa recuperar antocianinas, uma classe de compostos fenólicos com reconhecidas propriedades corantes, a partir de fontes naturais muito pouco exploradas. Assim, o epicarpo dos frutos de Prunus spinosa L. (abrunho), rico em cianidina 3-rutinósido e peonidina 3-rutinósido, e a casca dos frutos de Ficus carica L. (figo), rica em cianidina 3-rutinósido, foram utilizados para extração destes compostos. Para assegurar uma recuperação máxima, foram testadas duas técnicas de extração, assistida por maceração (EAM) e por ultrassons (EAU), às quais se aplicou uma metodologia de superfície de resposta usando o desenho composto central circunscrito com cinco níveis em cada uma das variáveis independentes estudadas (tempo, proporção de água-etanol como solvente e temperatura (EAM) ou potência (EAU)). O perfil antociânico dos diferentes extratos foi obtido por HPLC-DAD-ESI/MS e como respostas para o modelo foram utüizadas a concentração total de antocianinas (C) no resíduo de extração (R; mg C/g R) e na amostra desidratada (A; mg C/g A), bem como o rendimento de resíduo obtido (g R/g A). Em ambas as matrizes, a técnica mais eficiente foi a extração assistida por ultrassons, obtendo-se condições ótimas de recuperação de compostos aos 5 min, 400 W e 48% de etanol no caso do epicarpo de abrunho, com um rendimento de extração de 0,69 g R/g A e um total de antocianinas de 18, 17 mg C/g Re 11,76 mg C/g A. Por outro lado, a partir da casca de figo foi possível obter 17,0 mg C/g R e 3, 11 mg C/g A, e um rendimento de 0,19 g R/g A, nas condições ideais de extração de 21 min, 310 W e 100% de etanol. Assim, a extração de antocianinas a partir de biorresíduos dos frutos de abrunho e figo, com recurso a técnicas de extração não convencionais, revelaram ser uma boa alternativa para recuperação destes compostos com vista à sua utilização na indústria alimentar. AGRADECIMENTOS: FCT e FEDER sob o Programa PT2020 pelo apoio financeiro ao CIMO (UID/ AGR/00690/2013), LA LSRE-LCM^ (POCI-01-0145-FEDER-006984), bolsa de C. Pereira (SFRH/ BPD/122650/2016) e contrato de L. Barras; FEDER-Interreg Espana-Portugal pelo financiamento ao projeto 0377_Iberphenpl_6_E; ao FEDER através do Programa Operacional Regional Norte 2020, no âmbito do Projeto NORTE-01-0145-FEDER-023289 (DeCodE) e projeto Mobilizador Norte-01-0247-FEDER-024479: ValorNatural'. Xunta de Galicia pelo apoio financeiro a M.A. Prieto. info:eu-repo/semantics/publishedVersion

Dupla diplomação coma UTFPR - Universidade Tecnológica Federal do Paraná Entre as propriedades organoléticas, a cor exerce um papel muito importante na perceção global de um alimento, uma vez que a visão é normalmente o primeiro contato que o consumidor tem com o produto. Neste sentido, os corantes têm sido amplamente utilizados como aditivos alimentares, para suprir ou intensificar os aspetos de coloração dos alimentos, tornando-os mais atrativos e agradáveis. No entanto, devido à preocupação que os consumidores têm com a saúde e o registo de efeitos adversos de alguns corantes artificiais, existe um grande interesse na sua substituição por alternativas naturais mais seguras. As antocianinas são um grupo de pigmentos de coloração vermelha, azul e violeta, presentes em muitas flores e frutos, podendo ser consideradas alternativas aos corantes artificiais. Prunus spinosa L. é um arbusto selvagem que dá origem a frutos de sabor amargo e adstringente pouco explorados comercialmente, mas que apresentam níveis elevados de antocianinas, mais especificamente cianidina 3-rutinósido e peonidina 3-rutinósido, concentradas no epicarpo. Assim, o presente trabalho teve como objetivo desenvolver um corante alimentar natural a partir da extração de antocianinas dos frutos de P. spinosa, aliando os crescentes interesses na substituição dos corantes artificiais por alternativas naturais mais seguras à valorização de produtos agrícolas de fontes subexploradas. Foram otimizadas as condições que maximizam a extração de antocianinas do epicarpo de frutos de P. spinosa, comparando-se duas técnicas de extração, sendo uma convencional assistida por calor (HAE) e outra alternativa assistida por ultrassons (UAE), e tendo como variáveis relevantes tempo, temperatura (HAE) ou potência (UAE) e concentração de solvente. Para isso, foi aplicada uma metodologia de superfície de resposta, utilizando um desenho composto central circunscrito de três variáveis com cinco níveis, e os compostos antociânicos foram identificados e quantificados por HPLC-DAD-ESI/MS. A extração assistida por ultrassons foi o método mais eficaz, nas condições ótimas de 5,00 ± 0,15 min, 400,00 ± 32,00 W e 47,98 ± 2,88% de etanol (v/v), onde o rendimento de extração foi de 68,60 ± 2,06%, com um teor total de antocianinas de 18,17 ± 1,82 mg/g de extrato e 11,76 ± 0,82 mg/g de epicarpo seco, originando um extrato de cor roxo-avermelhado. Além disso, estes valores de resposta foram ligeiramente melhorados quando se testou o efeito da relação sólido-líquido nas condições ótimas num formato dose-resposta, mostrando um padrão decrescente constante na faixa de 5 a 250 g/L. Para além de estudar o potencial corante deste ingrediente natural, também se pretendeu avaliar o seu potencial funcional, para isso realizou-se uma avalição das propriedades bioativas e efectuou-se um estudo de aplicação num produto alimentar do extrato obtido nas condições otimizadas. A avaliação das bioatividades foi realizada através da análise da capacidade antioxidante, anti-inflamatória, antimicrobiana e citotóxica. O extrato apresentou uma atividade antioxidade considerável, com valores de EC50 de 204 ± 2 μg/mL para o método de TBARS e EC50 de 296 ± 4 e 509 ± 3 μg/mL para o método OxHLIA aos 60 e 120 min de reação, respetivamente. A capacidade antimicrobiana foi testada em 9 estirpes de bactérias de interesse na área da saúde, nas quais o extrato não apresentou efeito bactericida, mas teve a capacidade de inibir o crescimento bacteriano em concetrações de 2,5 a 20 mg/mL. A avaliação da atividade anti-inflamatória foi realizada em células de macrófagos de rato RAW 264.7 e a citotoxicidade foi testada em 4 linhas celulares tumorais humanas e numa cultura primária de células hepáticas não tumorais, para as quais o extrato não apresentou atividade, nem toxicidade. O extrato corante foi incorporado em “beijinhos”, um produto de confeitaria tipico brasileiro que tem como ingredientes básicos leite condensado, natas e manteiga, para avaliação da sua aplicabilidade numa matriz alimentar como corante alimentar. Foram determinados os parâmetros de cor, pH e textura, a composição nutricional e o valor energético das amostras de “beijinhos” logo após a sua confeção e depois de um dia de armazenamento. Em geral, o extrato otimizado incorporado revelou uma boa capacidade corante sem alterar as caraterísticas nutricionais do produto alimentar. Com isto, foi possível evidenciar o potencial da exploração do epicarpo dos frutos de P. spinosa como fonte de antocianinas a serem utilizadas no setor alimentar, tendo como valor agregado ao poder corante, a sua capacidade bioativa. Among organoleptic properties, colour plays a very important role in the overall perception of a foodstuff, since sight is usually the first contact that consumers have with the product. In this context, colorants have been widely used as food additives, to supply or intensify food colouring aspects, making them more attractive and pleasant. However, due to consumers' concerns about the health and adverse effects of some artificial colorants, there is a strong interest in replacing them with safer natural alternatives. Anthocyanins are a group of pigments with red, blue and violet coloration, being present in many flowers and fruits and increasingly considered as potential alternatives to artificial colorants. Prunus spinosa L. is a wild shrub with bitter and astringent fruits that are poorly commercially exploited but have high levels of anthocyanins, more specifically cyanidin 3-rutinoside and peonidin 3-rutinoside, concentrated in the epicarp. The objective of this study was to develop a natural food colorant, from the extraction of anthocyanins from the fruit epicarps of P. spinosa, combining the growing interest of substituting artificial colorants for safer natural alternatives and the valorization of agricultural products from underexploited sources. The conditions that maximize the extraction of anthocyanins from the fruit epicarp of P. spinosa were optimized, comparing two extractive techniques, one conventional heat-assisted (HAE) and another alternative ultrasound assisted extraction (UAE), testing as relevant variables time, temperature (HAE) or power (UAE), and solvent concentration. For that purpose, a response surface methodology was applied, using a circumscribed central composite design with three variables and five levels, and the anthocyanin compounds were identified and quantified by HPLC-DAD-ESI/MS. Ultrasound assisted extraction was the most efficient method, under optimum conditions of 5.00 ± 0.15 min, 400.00 ± 32.00 W and 47.98 ± 2.88% ethanol, where the extraction yield was 68.60 ± 2.06% (v/v), with a total anthocyanin content of 18.17 ± 1.82 mg/g of dry extract and 11.76 ± 0.82 mg/g of dry epicarp, wich gives rise to a purple-reddish extract. Additionally, these response values were slightly improved when the solid-to-liquid ratio effect at the optimal conditions in a dose-response format was tested, showing a steady decreasing pattern in the range 5 to 250 g/L. In addition to studying the colorant potential of this natural ingredient, it was also intended to evaluate its functional potential, for which an evaluation of the bioactive properties was determined, and an application study was carried out using a food product incorporated with the extract obtained under optimized conditions The evaluation of the bioativities was carried out through analyzes of the antioxidant, anti-inflammatory, antimicrobial and cytotoxic capacity. The extract presented a considerable antioxidant activity, with EC50 values of 204 ± 2 μg/mL for the TBARS assay and EC50 of 296 ± 4 and 509 ± 3 μg/mL for OxHLIA method at 60 and 120 min of reaction, respectively. The antimicrobial capacity was tested in 9 strains of bacteria of interest in the health area, in which the extract did not present bactericidal effect, but all had their growth inhibited with an extract concentration between of 2.5 to 20 mg/mL. The anti-inflammatory activity was assessed on RAW 264.7 mouse macrophages and cytotoxicity was tested using 4 human tumor cell lines and on a primary culture of non-tumor liver cells, for which the extract showed no activity and no toxicity. The dye extract was incorporated into "beijinhos", a typical brazilian confectionery product, that has as basic ingredients condensed milk, cream and butter, to evaluate its applicability as a natural colorant ingredient for a food matrix. The color, pH, and texture parameters, nutritional composition, and energetic value of "beijinhos" samples were determined after cooking and after one day of storage. In general, the incorporated optimized extract revealed a good coloring ability without altering the nutritional characteristics of the food product. With this, it was possible to evidence the potential exploitation of P. spinosa fruit epicarp as a source of anthocyanins to be used in the food industry, having as added value to the dye power, its bioactive capacity. Este trabalho é financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa Operacional Regional Norte 2020, no âmbito do Projeto NORTE-01-0145-FEDER-023289 (DeCodE) e Norte-01-0247-FEDER-024479 (projeto Mobilizador ValorNatural), e pelo programa FEDER-Interreg España-Portugal, no âmbito do projeto 0377_Iberphenol_6_E.

U radu je ispitan učinak etanolnog ekstrakta cvijeta trnine (Prunus spinosa L.) na vijabilnost i proliferaciju mišjih kancerogenih Hepa 1-6 i nekancerogenih AML 12 stanica jetre, nakon 24 i 72 sata, Trypan Blue metodom i protočnom citometrijom. Za tretman Hepa 1-6 stanica korištene su koncentracije ekstrakta cvijeta trnine od 10 do 200 μg mL-1, a za AML 12 stanice od 5 do 100 μg mL-1. Utvrđeno je da ekstrakt cvijeta trnine ima statistički značajan inhibicijski učinak na proliferaciju stanica nakon 24-satne inkubacije, kod Hepa 1-6 stanica pri koncentracijama većim od 100 μg mL-1, a kod AML 12 stanica pri svim ispitanim koncentracijama izuzev 10 μg mL-1. Tijekom perioda od 72 sata kod obje stanične linije pri svim ispitanim koncentracijama utvrđen je inhibitorni učinak ekstrakta cvijeta trnine. Iz izračunatih i uspoređenih IC50 vrijednosti, ekstrakt cvijeta trnine pokazao je jači inhibitorni učinak na AML 12 staničnu liniju. Protočnom citometrijom utvrđeno je da ekstrakt cvijeta trnine uzrokuje nekrozu stanica kod obje stanične linije. In this study effects of the blackthorn flower (Prunus spinosa L.) extract on cell proliferation and viability were examined in mouse cancer Hepa 1-6 and normal AML 12 liver cell lines using Trypan Blue method and flow cytometry after 24 and 72 hours. Different concentrations of blackthorn flower extract were used in treatment of cells (for Hepa 1–6 10-200 μg mL-1, and for AML 12 5-100 μg mL-1). Statistically significant decrease of Hepa 1-6 cell proliferation was detected at concentrations greather than 100 μg mL-1 and AML 12 cell proliferation at all examined concentrations, excluding 10 μg mL-1, after 24 hours. Statistically significant decrease of Hepa 1–6 and AML 12 cell proliferation was detected after 72 hours at all concentrations. According to calculated and compared IC50 values blackthorn flower extract has shown stronger inhibitory effect on AML 12 cell line. Results obtained with flow cytometry have shown that blackthorn flower extract causes necrosis of both cell lines.

Bu tez kapsamında elektron transfer metoduna dayanan ve literatürde yaygın olarak kullanılan antioksidan kapasite tayin metotlarına alternatif olabilecek yeni bir antioksidan kapasite tayin metodu geliştirilmiştir. Geliştirilen Cr(VI) antioksidan kapasite tayin (CHROMAC) metodu Cr(VI)’nın asidik ortamda organik maddeleri yükseltgeme ve 1,5-difenilkarbazid ile kararlı diaquabis (1,5-difenilkarbazon) krom(III) kompleksi oluşturma özelliğine dayanır. Materyallerin ve standart maddelerin antioksidan kapasite değerlerinin CHROMAC metoduyla tayin edilebilmesi için ortamın pH/E0 değeri (pH= 2,8/E0 =0,95V), Cr(VI)’nın 1,5-difenilkarbazid ile girdiği reaksiyondaki mol oranı (1:1), reaksiyon süresi (50 dakika), fenolik maddeler için doğrusal çalışma aralığının (6,0×10-5 -4,5×10-4 mol/L) ve girişim etkilerinin belirlenmesi çalışmaları yapılmıştır. Aynı zamanda geliştirilen CHROMAC metodunun doğal örneklere uygulanabilirliği incelenmiştir. Standart maddeler ile yapılan çalışmalarla metodun TEAC değeri ABTS ve CUPRAC metotlarıyla karşılaştırılmıştır. Ayrıca Bursa’da doğal olarak yetişen Sorbus torminalis L., Sorbus domestica L., Prunus spinosa L., Prunus divaricata Ledeb.subsp. divaricata., Diospyros lotus L., türlerinin meyveleri için ultrasonik ve çözücü ekstraksiyonları (asidik, bazik, asidik-bazik ve enzimatik) uygulanarak örnek hazırlama teknikleri geliştirilmiş ve bu meyvelerde bulunan fenolik maddeler, kantitatif olarak HPLC-DAD ile belirlenmiştir. Yabani meyvelerin antioksidan kapasite değerleri ABTS, CUPRAC ve CHROMAC metotları ile belirlenerek elde edilen sonuçlar karşılaştırılmıştır. In this thesis, based on electron transfer and a novel antioxidant capacity method, Cr (VI) antioxidant capacity (CHROMAC) method, which may be an alternative to widely used antioxidant capacity determination methods available in the literature, has been developed. The developed Cr(VI) antioxidant capacity (CHROMAC) method is based on the property of Cr (VI) to oxidize the organic materials in the acidic medium and to form the stable diaquabis (1,5-diphenylcarbazone) chromium (III) complex with 1,5- diphenylcarbazide. To determination of antioxidant capacity values of samples and standards with CHROMAC method, the working conditions need to be in proper pH/E0 ratio. For this purpose of developing CHROMAC method experimental studies is performed with determine of the pH/E0 (pH=2,8/E0 =0,95V) value of the medium, the molar ratio of Cr (VI) to the reactant with 1,5-diphenylcarbazide (1:1), the reaction time (50 minutes), the linear working range for phenolic substances (6,0×10-5 -4,5×10-4 mol/L) and interference effect. At the same time the applicability of the CHROMAC method to natural samples has been examined. The mentioned developed method TEAC values was compared with at worked method of ABTS and CUPRAC via standards. In addition, sample preparation techniques were developed by applying ultrasonic and solvent extractions (acidic, basic, acidic-basic and enzymatic) for the fruits of naturally grown of Sorbus torminalis L., Sorbus domestica L., Prunus spinosa L., Prunus divaricata Ledeb.subsp. divaricata, Diospyros lotus L. wild fruit species in Bursa and the phenolic materials present in these fruits were quantitatively determined by HPLCDAD. Antioxidant capacity values of wild fruits were determined by ABTS, CUPRAC and CHROMAC methods and the results obtained were compared.

We investigated the wound healing efficacy of the methanol extracts of Prunus spinosa L. fruits using an excisional cutaneous wound model in mice. We used 32 C57/Black mice equally divided into four groups. Group 1 Control, Group 2 vehicle (glyce-rine), Group 3 Prunus spinosa L., Group 4 Madecassol. Prunus spinosa L.and Madecassol were applied for 10 days by creating a excisional wound model on the back of each mouse in each group. At the end of the 10th day, samples from wound areas were taken for histologic follow-up, scoring with hematoxylin-eosin and immunohistochemical (TGF-b, COL1A1) staining. Wound healing was evaluated and scored by newly formed collagen structure, increase in TGF b and epidermal and dermal regeneration, granulation tissue thickness, angiogenesis (new vessel formation). Prunus spinosa L. increased wound healing rates by promoting granulation tissue, epidermal regeneration and angiogenesis. In addition there was a significant increase in collagen and TGF [beta] ratios in immunohistochemical staining. At the end of the 4 and 10 days after treatment, a significant increase was observed in wound contraction and re-epithelialization in both Prunus spinosa and madecassol treated groups compared to the control and vehicle groups. Our findings support the beneficial effects of Prunus spinosa for augmenting wound healing. The anti-inflammatory, antioxidant and antibacterial activities of Prunus spinosa by way of increasing collagen synthesis and decreasing the number of inflammatory cells during wound healing have suggested that it may be useful for treating skin wounds. © 2017, Logos Medical Publishing. All rights reserved.

We investigated the wound healing efficacy of the methanol extracts of Prunus spinosa L. fruits using an excisional cutaneous wound model in mice. We used 32 C57/Black mice equally divided into four groups. Group 1 Control, Group 2 vehicle (glyce-rine), Group 3 Prunus spinosa L., Group 4 Madecassol. Prunus spinosa L.and Madecassol were applied for 10 days by creating a excisional wound model on the back of each mouse in each group. At the end of the 10th day, samples from wound areas were taken for histologic follow-up, scoring with hematoxylin-eosin and immunohistochemical (TGF-b, COL1A1) staining. Wound healing was evaluated and scored by newly formed collagen structure, increase in TGF b and epidermal and dermal regeneration, granulation tissue thickness, angiogenesis (new vessel formation). Prunus spinosa L. increased wound healing rates by promoting granulation tissue, epidermal regeneration and angiogenesis. In addition there was a significant increase in collagen and TGF [beta] ratios in immunohistochemical staining. At the end of the 4 and 10 days after treatment, a significant increase was observed in wound contraction and re-epithelialization in both Prunus spinosa and madecassol treated groups compared to the control and vehicle groups. Our findings support the beneficial effects of Prunus spinosa for augmenting wound healing. The anti-inflammatory, antioxidant and antibacterial activities of Prunus spinosa by way of increasing collagen synthesis and decreasing the number of inflammatory cells during wound healing have suggested that it may be useful for treating skin wounds

In vivo farmakodinamički pokus apsorpcije i distribucije polifenolnih spojeva iz ekstrakta cvijeta trnine (Prunus spinosa L.) proveden je na modelu C57BL/6 miša. Rezultati pokazuju da je od ukupno detektiranih 30 vrsta polifenolnih spojeva koje nalazimo u ekstraktu trnine, apsorpcijom i biodistribucijom po organima u različitim vremenima bioakumulirane su različite vrste polifenolnih spojeva. Navedeni rezultati potvrđuju postavljenu hipotezu o selektivnoj preferenciji za bioakumulacijom i zadržavanjem pojedinog tipa polifenolnih spojeva unutar različitih organa. Primjerice u serumu i tankom crijevu je detektirano 18, u jetri 12, u bubregu i mozgu 16 polifenolnih spojeva i metabolita. U serumu je najzastupljeniji spoj 3-O-CaffeoQunic Acid, u svim analiziranim vremenima, u koncentracijama 3-3, 27 µg/mL. U tankom crijevu, bubregu i mozgu je najzastupljeniji spoj protoancijanidin B1, u koncentraciji 30-33, 13 µg/g tkiva. U jetri najzastupljenija je galna kiselina u koncentracijama 30-32, 95 µg/g tkiva. Drugi najzastupljeniji spojevi po koncentracijama su: katehin u serumu, tankom crijevu, bubregu i mozgu u koncentracijama 0, 6-1, 59 µg/g tkiva. U jetri drugi po koncentraciji najzastupljeniji spoj je 3-O-CaffeoQunic Acid sa rasponom 0, 7-1, 45 µg/g tkiva. Također, rezultati su pokazali koji polifenolni spojevi uopće ne ulaze u organizam ili pojedine organe ili su prisutni u vrlo niskim koncentracijama i kratko se zadržavaju u pojedinim organima.

WOS: 000423507700016 Polyphenol oxidase (PPO) enzyme was extracted from Prunus spinosa L. subsp. dasyphylla plum, partially purified by acetone precipitation, and its biochemical properties were investigated. Different substrates (p-coumaric acid, L-tyrosine, p-hydroxyphenyl propionic acid (PHPPA), catechol, 4-methylcatechol (4-MTC), hydrocaffeic acid, gallic acid, quercetin, catechin, and epicatechin) were analysed to determine their affinities with Prunus spinosa PPO (PsPPO). the substrate specificity was in the following order: 4-MTC > catechol > hydrocaffeic acid > catechin > epicatechin. 4-MTC was the most suitable substrate (K-m = 0.97 mM and V-max = 4753 U/mg protein). the optimal pH values were 7.0 for 4-MTC and catechol, 5.0 for catechin and epicatechin, and 4.0-6.0 for hydrocaffeic acid. Optimal temperatures were 40 degrees C for 4-MTC, 30 degrees C for catechol, and 60 degrees C for catechin, epicatechin, and hydrocaffeic acid. in the inhibition tests, the most potent inhibitor was found to be sodium metabisulphite (IC50 = 0.01 mM), followed by ascorbic acid, thiourea, benzoic acid, L-cysteine, and sodium azide. Approximately 80 and 75% of the diphenolase activity was conserved at pH 5.0 and 7.0, respectively, at 4 degrees C after 7 d incubation. Moreover, native polyacrylamide gel electrophoresis (Native-PAGE) of the enriched extract revealed the presence of at least six bands with PPO activity, suggesting the existence of different PPO isoforms. However, the oxidation of diphenol related to browning significantly, so the data obtained in this research provided a basis for the prevention of enzymatic browning of Prunus spinosa during processing. High-performance liquid chromatography (HPLC) revealed the plum extract contained protocatechuic acid, p-OH benzoic acid, vanillic acid, syringic acid, epicatechin, p-coumaric acid, and luteolin.

Farelerde oluşturduğumuz deneysel yara modellerinde, Prunus Spinosa L. metanollü meyve ekstresini kullanarak, bu ekstrenin yara iyileşmesindeki etkisini araştırdık. Otuz iki adet C57/Black fare ile 4 deney grubu oluşturuldu. Birinci grup kontrol grubu, 2. grup taşıyıcı (gliserin) grup, 3. grup Prunus spinosa, 4. grup ma- decassol grubu idi ve her bir grupta farelerin sırtında eksizyonel yara modeli oluşturularak Prunus Spinosa L. ve madecassol 10. gün boyunca uygulandı. Onuncu gün sonunda yara bölgeleri his- tolojik takip için alındı, yara dokuları hematoksilen-eosin ve im- munohistokimyasal (TGF-?, COL1A1) boyalarla boyanarak, yara iyileşmesi için epidermal ve dermal rejenerasyon, granülasyon doku oluşumu, anjiogenezis (yeni damar oluşumu), immunohis- tokimyasal boyalarla yeni oluşan kollajen yapılanması ve TGF ? artışı değerlendirilerek skorlandı. Prunus spinosa L’nin yara do- kusunda iyileşme oranlarını, granülasyon dokusunu, epidermal rejenerasyonu ve anjiogenezisi artırarak gösterdiği yine immü- nohistokimyasal boyamalarda kollajen ve TGF ? oranlarında ista- tistiksel olarak anlamlı bir artışın olduğu gözlendi. Yine 4. günün ve 10. günün sonunda yara kontraksiyonunda ve yara yerinin reepitelizasyonunda ilaç ve madecassol grubunda kontrol gru- buna kıyasla anlamlı bir artış gözlenmiştir. Bulgularımız, Prunus spinosa L’nin yara iyileşmesini desteklediğini göstermiştir. Prunus spinosanın antiinflamatuvar, antioksidan ve antibakteriyel etki- si ile, kollajen sentezini artırarak ve inflamatuvar hücre sayısını azaltarak yara iyileşmesinin tedavisinde kullanılabileceğini dü- şündürdü., We investigated the wound healing efficacy of the methanol extracts of Prunus spinosa L. fruits using an excisional cutane- ous wound model in mice. We used 32 C57/Black mice equally divided into four groups. Group 1 Control, Group 2 vehicle (glyce- rine), Group 3 Prunus spinosa L., Group 4 Madecassol. Prunus spinosa L.and Madecassol were applied for 10 days by creating a excisional wound model on the back of each mouse in each group. At the end of the 10th day, samples from wound areas were taken for histologic follow-up, scoring with hematoxylin- eosin and immunohistochemical (TGF-b, COL1A1) staining. Wo- und healing was evaluated and scored by newly formed collagen structure, increase in TGF b and epidermal and dermal regene- ration, granulation tissue thickness, angiogenesis (new vessel formation). Prunus spinosa L. increased wound healing rates by promoting granulation tissue, epidermal regeneration and angi- ogenesis. In addition there was a significant increase in collagen and TGF [beta] ratios in immunohistochemical staining. At the end of the 4 and 10 days after treatment, a significant increase was observed in wound contraction and re-epithelialization in both Prunus spinosa and madecassol treated groups compared to the control and vehicle groups. Our findings support the be- neficial effects of Prunus spinosa for augmenting wound healing. The anti-inflammatory, antioxidant and antibacterial activities of Prunus spinosa by way of increasing collagen synthesis and dec- reasing the number of inflammatory cells during wound healing have suggested that it may be useful for treating skin wounds.

The concentrations of Zn, Mn, Fe, Pb, Ni, Cu and Cd in soil, leaves and edible wild fruit (Crataegus laevigata L., Cornus mas L. and Prunus spinosa L.) from southeast Serbia were determined by atomic absorption spectroscopy. Metal translocations from soil to fruit were calculated as well as their oral intake and health risk indices. Positive correlations were found among metal concentrations in soil, leaves and fruit.

U ovom radu ispitan je učinak ekstrakta cvijeta trnine (Prunus spinosa L.) na vijabilnost mišjih kanceroznih Hepa 1-6 i nekanceroznih AML 12 stanica tijekom 72 sata metodom Kenacid Blue. Za tretman su korištene različite koncentracije ekstrakta cvijeta trnine (za Hepa 1-6 staničnu liniju od 10 do 200 µg mL-1, a za AML 12 staničnu liniju od 5 do 100 µg mL-1 ). Utvrđeno je da ekstrakt cvijeta trnine ima statistički značajan inhibicijski učinak na proliferaciju Hepa 1-6 stanica tijekom cijelog perioda inkubacije od 72 sata pri koncentracijama većim od 100 µg mL-1. Statistički značajan inhibicijski učinak i na proliferaciju AML 12 stanica, ekstrakt cvijeta Prunus spinosa L. pokazao je nakon 48 sati pri koncentracijama većim od 30 µg mL-1 , a nakon 72 sata pri koncentracijama većim od 10 µg mL-1. Iz izračunatih i uspoređenih IC50 vrijednosti, ekstrakt cvijeta trnine pokazao je jači inhibitorni učinak na AML 12 staničnu liniju. Istraživanje je pokazalo da ekstrakt cvijeta Prunus spinosa L., u ispitivanim koncentracijama inducira nastajanje ROS-a (eng. reactive oxygen species, kisikove reaktivne vrste) u Hepa 1-6 stanicama, što ukazuje na prooksidacijsku aktivnost. In this study effects of the blackthorn flower (Prunus spinosa L.) extract on cell proliferation and viability was examined in mouse cancer Hepa 1-6 and normal AML 12 cell lines using Kenacid Blue method during 72 hours. Different concentrations of blackthorn flower extract were used in treatment of cells (for Hepa 1-6 10 - 200 µg mL-1, and for AML 12 5-100 µg mL-1). Statistically significant decrease of Hepa 1-6 cell proliferation was detected at concentrations greater than 100 µg mL-1 during whole period of incubation. Blackthorn flower extract has shown statistically significant inhibitory effect on AML 12 cell proliferation, after 48 hours at concentrations greater than 30 µg mL-1, and after 72 hours at concentrations greater than 10 µg mL-1. According to calculated and compared IC50 values, blackthorn flower extract has shown stronger toxic effect on AML 12 cell line. This study has shown that Prunus spinosa L. extracts at applied concentration range induces ROS (reactive oxygen species) formation in Hepa 1-6 cells, indicating prooxidant activity.

U ovom radu ispitan je učinak ekstrakta cvijeta trnine, koncentracije ukupnih fenola izraženih kao ekvivalenti galne kiseline u rasponu 10-200 μg/mL, na vijabilnost i proliferaciju Hep G2 stanica metodama Kenacid Blue i Neutral Red tijekom 72 sata. Statistički značajna inhibicija stanične proliferacije utvrđena je metodom Kenacid Blue nakon 24, 48 i 72 sata kod koncentracije ekstrakta od 50-200 µg/mL. IC50 vrijednost određena metodom Kenacid Blue, nakon 24 h iznosi 148,11 µg/mL, nakon 48 h 94,49 µg/mL i nakon 72 sata 59,87 µg/mL. Metodom Neutral Red određeno je da ekstrakt cvijeta trnine u koncentracijama od 50-200 µg/mL statistički značajno inhibira staničnu proliferaciju već nakon 24 sata inkubacije. IC50 vrijednost određena metodom Neutral Red, nakon 24 h iznosi 92,50 µg/mL, nakon 48 h 84,02 µg/mL i nakon 72 sata 54,93 µg/mL. Ovim istraživanjem utvrđeno je da ekstrakt cvijeta trnine inducira nastajanje ROS-a (reaktivnih kisikovih vrsta) u Hep G2 stanicama što ukazuje na izostanak antioksidacijske aktivnosti te biljke pri ispitanim koncetracijama. In this study we examined the effect of blackthorn flower extract, the concentration of total phenolics expressed as gallic acid equivalents in the range 10-200 mg/mL, on the viability and proliferation of Hep G2 cells by Kenacid Blue and Neutral Red methods for 72 hours. Statistically significant inhibition of cell proliferation was determined according Kenacid Blue method after 24, 48 and 72 hours at a concentration of 50-200 µg/mL. The IC50 value determined by the method Kenacid Blue, after 24 h was 148.11 µg/mL after 48 h 94.49 µg/mL and after 72 hours 59.87 µg/mL. By Neutral Red method was determined that the blackthorn flower extract at concentrations of 50-200 µg/mL significantly inhibited cell proliferation after 24 hours of incubation. The IC50 value determined by the method Neutral Red, after 24 h was 92.50 µg/mL after 48 h 84.02 µg/mL and after 72 hours 54.93 µg/mL. This study has established that an blackthorn flower extract induces the formation of ROS (reactive oxygen species) in Hep G2 cells, indicating the absence of antioxidant activity of that plant in the concentrations which are tested.