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Olive leaves as a main byproduct of olive oil and fruit industry are a valuable source of phytochemicals such as polyphenols, with multiple biomedical effects. Apart from leaves, olive branches and stems make up a significant amount of olive waste. It is well known that the drying process and long-term storage affect the stability and concentration of polyphenols present in raw materials. For that matter, two different means of storing olive waste, at room temperature and +4 °C, were compared by determining the content of the polyphenol oleuropein (OLE) in olive leaf, branch, and stem extracts (LE, BE, and SE) by HPLC-DAD method. Total phenols (TPC), o-diphenols (o-DPC), and total flavonoids (TFC) content in extracts were assessed by UV-Vis measurements. LE prepared from leaves stored at +4 °C had the highest OLE content, 30.7 mg g−1 of dry extract (DE). SE from stems stored at +4 °C was the richest in TPC and TFC (193 mg GAE/g DE and 82.9 mg CE/g DE, respectively), due to the higher purity of the extract. The biological activity of extracts was determined on cervical cancer (HeLa), melanoma (A375), metastatic melanoma (A375M) tumor cell lines, and on spontaneously immortalized cell line of keratinocytes (HaCaT), using the MTT assay. The data show that all extracts had a similar dose-dependent effect on cell viability in HeLa cells, while the effect of LE on melanoma A375 and A375M, and HaCaT cells was cell-line dependent.

Cancer presents one of the leading causes of death in the world. Current treatment includes the administration of one or more anticancer drugs, commonly known as chemotherapy. The biggest issue concerning the chemotherapeutics is their toxicity on normal cells and persisting side effects. One approach to the issue is chemoprevention and the other one is the discovery of more effective drugs or drug combinations, including combinations with polyphenols. Olive oil polyphenols (OOPs), especially hydroxytyrosol (HTyr), tyrosol (Tyr) and their derivatives oleuropein (Ole), oleacein and oleocanthal (Oc) express anticancer activity on different cancer models. Recent studies report that phenolic extract of virgin olive oil may be more effective than the individual phenolic compounds. Also, there is a growing body of evidence about the combined treatment of OOPs with various anticancer drugs, such as cisplatin, tamoxifen, doxorubicin and others. These novel approaches may present an advanced strategy in the prevention and treatment of cancer.

This review article describes studies published over the past five years on the combination of polyphenols, which are the most studied in the field of anticancer effects (curcumin, quercetin, resveratrol, epigallocatechin gallate, and apigenin) and chemotherapeutics such as cisplatin, 5-fluorouracil, oxaliplatin, paclitaxel, etc. According to WHO data, research has been limited to five cancers with the highest morbidity rate (lung, colorectal, liver, gastric, and breast cancer). A systematic review of articles published in the past five years (from January 2018 to January 2023) was carried out with the help of all Web of Science databases and the available base of clinical studies. Based on the preclinical studies presented in this review, polyphenols can enhance drug efficacy and reduce chemoresistance through different molecular mechanisms. Considering the large number of studies, curcumin could be a molecule in future chemotherapy cocktails. One of the main problems in clinical research is related to the limited bioavailability of most polyphenols. The design of a new co-delivery system for drugs and polyphenols is essential for future clinical research. Some polyphenols work in synergy with chemotherapeutic drugs, but some polyphenols can act antagonistically, so caution is always required.

The most common type of renal cell carcinoma (RCC) is clear cell renal cell carcinoma (ccRCC), which has a high metastatic potential. Even though the International Metastatic RCC Database Consortium (IMDC) risk model is conventionally utilized for selection and stratification of patients with metastatic RCC (mRCC), there remains an unmet demand for novel prognostic and predictive markers. The goal of this study was to analyze the expression of Vascular endothelial growth factor (VEGF), Cluster of Differentiation 31 (CD31) to determine microvessel density, and Angiopoietin-1 (Ang-1) in primary kidney tumors, as well as their predictive and prognostic value in patients with metastatic ccRCC (mccRCC) who were treated with first-line sunitinib. The study included 35 mccRCC patients who were treated with first-line sunitinib in period between 2009 and 2019. Immunofluorescence was used to examine biomarker expression in tissue specimens of the primary tumor and surrounding normal kidney tissue. Median disease-free survival (DFS) was longer in patients with negative and low tumor VEGF score than in patients with medium tumor VEGF score (p=0.02). Those with low tumor CD31 expression had a longer median DFS than patients with high tumor CD31 expression (p=0.019). There was no correlation between Ang-1 expression and DFS. The expression of biomarkers in normal kidney tissue was significantly lower than in tumor tissue (p<0.001). In conclusion, higher VEGF scores and greater CD31 expression were associated with longer DFS, but neither of these biomarkers correlated with progression-free survival or overall survival.

Objectives: The aim of the study was to evaluate the attitude and perspectives of dental students of four Croatian universities towards online learning during the COVID-19 pandemic., Material and Methods: An anonymous internet-based survey was administered to undergraduate dental medicine students at the Universities of Zagreb, Rijeka, Split and Osijek. The 29-item questionnaire collected data on students' demographics, online learning organization and management, and perception of online classes., Results: Five hundred and four participants (85.1% female) took part in the survey. The majority of the participants (63.5%) were from the University of Zagreb. 39.6% of students reported agreement regarding online learning satisfaction. Individual university satisfaction ratings on overall online learning were: Osijek 3.69, Zagreb 3.22, Split 3.05 and Rijeka 2.64. Most students considered that lectures (82.9%) and seminars (78.9%) could be successfully delivered in an online learning format. The online learning format cannot successfully deliver laboratory, preclinical, clinical practicals or clinical clerkship, as agreed by more than 80% of the total student sample. 60% of students consider online learning a valuable alternative to face-to-face instruction., Conclusion: Online learning was highly praised for educational formats such as lectures and seminars, and was considered a useful substitute for conventional learning. Conventional practical courses cannot be substituted with online learning. Overall perspective about online learning was mixed among the students of four universities. The findings of the present study can serve to help individual universities address the shortcomings and reinforce the strengths of their OL programs., Competing Interests: Conflict of Interest The authors have no conflicts of interest to declare.

Nowadays, many individuals, whether healthy or diagnosed with disease, tend to expose themselves to various easily accessible natural products in hopes of benefiting their health and well-being. Mediterranean populations have traditionally used olive oil not only in nutrition but also in cosmetics, including skincare. In this study, the phenolic profile-composed of twelve compounds altogether, including the secoiridoids oleocanthal (OCAL) and oleacein (OCEIN)-of extra virgin olive oil (EVOO) from autochthonous cultivars from Croatia was determined using 1 H qNMR spectroscopy and HPLC-DAD analysis, and its biological activity was investigated in melanoma cell lines. The EVOO with the highest OCEIN content had the strongest anti-cancer activity in A375 melanoma cells and the least toxic effect on the non-cancerous keratocyte cell line (HaCaT). On the other hand, pure OCAL was shown to be more effective and safer than pure OCEIN. Post-treatment with any of the EVOO phenolic extracts (EVOO-PEs) enhanced the anti-cancer effect of the anti-cancerous drug dacarbazine (DTIC) applied in pre-treatment, while they did not compromise the viability of non-cancerous cells. The metastatic melanoma A375M cell line was almost unresponsive to the EVOO-PEs themselves, as well as to pure OCEIN and OCAL. Our results demonstrate that olive oils and/or their compounds may have a potentially beneficial effect on melanoma treatment. However, their usage can be detrimental or futile, especially in healthy cells, due to inadequately applied concentrations/combinations or the presence of resistant cells.

Yeast Saccharomyces cerevisiae is an ideal model organism for studying molecular mechanisms of the stress response provoked by metals. In this work, yeast cells response to iron (Fe 3+ ) or lead (Pb 2+ ) exposure was tested and compared. Survival test was used to determine testing doses of metal ions-for Fe 3+ it was 4 mM and for Pb 2+ 8 mM. These (high, over-loaded) doses provoked comparable values of growth inhibition, but different values in vitality measurement. The percentage of metabolically active cells, determined by fluorescent FUN-1 dye, was lower in Pb 2+ than in Fe 3+ treated cells. Besides, endogenous antioxidant defence systems in the cells treated with Pb 2+ were less efficient compared to Fe 3+ . At the mitochondrial level, the effects of metal ions were in correlation with the results of cell metabolic activity. The mitochondrial proteome of Pb 2+ treated cells showed the domination of protein downregulation. Yeast cells treated either with Fe 3+ or Pb 2+ shared 19 common significantly changed proteins. The affected proteins were involved in different cellular process and amongst them only five proteins belong to energy and carbohydrate metabolism, and protein biosynthesis. Based on all obtained results, it is possible to conclude that the effects of Fe 3+ and Pb 2+ on yeast cells show rather specific patterns of toxicity and stress response.

Virgin olive oil (VOO) is a functional food specific to the Mediterranean diet and related to human health, especially as a protector of cardiovascular health, in the prevention of several types of cancers, and in modification of immune and inflammatory response. Phenolic compounds have central importance for these extraordinary health benefits. In the production of high-quality olive oils, it is very important to process freshly picked olives and avoid any storage of fruits. However, in Croatia there is a very traditional and environmentally friendly method of olive oil production, where olive fruits are stored in seawater for some time prior to processing. This practice is also notable nowadays since there are people who prefer the characteristic flavor of the "seawater olive oil", although some people argue against its quality and biomedical relevance. In this study, the phenolic contents of VOO prepared from the immediately processed fresh olives and olives processed after storage in seawater were compared with the use of high-performance liquid chromatography-mass spectrometry (HPLC-MS) and spectrophotometric analysis. The results suggest that "seawater olive oil" should be considered as a safe food of biomedical relevance, as it still contains a significant proportion of important phenolics like hydroxytyrosol, tyrosol and oleacein (e.g., 63.2% of total phenols in comparison to VOO).

The roles of phenolics from olive oils as effective anticancer agents have been documented in various in vitro studies of different cancer cells lines, but the relationship between the phenolic profile of olive oil and its biological activity needs more elucidation. In this study, we analysed phenolic profiles of extra virgin olive oils (EVOOs) from different autochthonous cultivars from Croatia (Oblica, Bjelica, Buža, Žižolera) and investigated the biological effect of EVOO phenolic extracts (EVOO-PEs) on human cervical (HeLa) and human colon (SW48) cancer cell lines alone and in combination with cisplatin (cDDP), carboplatin (CBP), 5-fluorouracil (5-FU) and irinotecan. The quantitative evaluation of olive oil polyphenols was performed by HPLC-DAD and spectrophotometric analysis. The biological effect of EVOO-PEs alone and in combination with anticancer drugs was measured by MTT assay. Analysed EVOO-PEs differ in phenolic profile and inhibited HeLa and SW48 cells in a dose-dependent manner. Further, it is shown that EVOO-PEs (Oblica-Sea, Buža and Žižolera), in combination with anticancer drugs, increase the metabolic activity of HeLa and SW48 cells and have a protective role. These data imply careful consummation of olive oil during chemotherapy of cancer patients.

The Mediterranean diet and olive oil as its quintessential part are almost synonymous with a healthy way of eating and living nowadays. This kind of diet has been highly appreciated and is widely recognized for being associated with many favorable effects, such as reduced incidence of different chronic diseases and prolonged longevity. Although olive oil polyphenols present a minor fraction in the composition of olive oil, they seem to be of great importance when it comes to the health benefits, and interest in their biological and potential therapeutic effects is huge. There is a growing body of in vitro and in vivo studies, as well as intervention-based clinical trials, revealing new aspects of already known and many new, previously unknown activities and health effects of these compounds. This review summarizes recent findings regarding biological activities, metabolism and bioavailability of the major olive oil phenolic compounds-hydroxytyrosol, tyrosol, oleuropein, oleocanthal and oleacein-the most important being their antiatherogenic, cardioprotective, anticancer, neuroprotective and endocrine effects. The evidence presented in the review concludes that these phenolic compounds have great pharmacological potential, however, further studies are still required., Competing Interests: The authors declare no conflict of interest.

The health effects of green tea are associated with catechins: (-)-epigallocatechin-3-O-gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin-3-O-gallate, and (-)-epicatechin. An understanding of compound absorption, distribution, metabolism, excretion, and toxicity characteristics is essential for explaining its biological activities. Herein, absorption, distribution, metabolism, excretion, and toxicity properties of in vivo detected metabolites of green tea catechins (GTCs) have been analyzed in silico. The influence of metabolic transformations on absorption, distribution, metabolism, and excretion profiles of GTCs corresponds to the effects of size, charge, and lipophilicity, as already observed for other small molecules. Mutagenic, carcinogenic, or liver toxic effects were predicted only for a few metabolites. Similar to galloylated GTCs EGCG and (--)-epicatechin-3-O-gallate, the sulfo-conjugates were predicted to bind at the warfarin binding site. The low free plasma concentration of these derivatives may be consequential to their serum albumin binding. The activity cliff detected for methylated conjugates of EGCG indicates that GTCs' pro-oxidative activity in bound state comes primarily from free hydroxyl groups of the pyrogallol ring B., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Reactive oxygen species, along with reactive nitrogen species, may play an important role in the pathogenesis and progress of many diseases, including cancer, diabetes and sickle cell disease. It has been postulated that hydroxyurea, one of the main treatments in sickle cell disease, achieves its activity partly also through its antioxidant properties. A series of hydroxyurea derivatives of L- and D-amino acid amides and cycloalkyl-N-aryl-hydroxamic acids was synthesized and investigated for their radical scavenging activity, chelating properties and antioxidant activity. All the compounds showed exceptional antiradical activities. For example, free radical scavenging activities of investigated hydroxyureas were higher than the activity of standard antioxidant, butylated hydroxyanisole (BHA). Moreover, most of the investigated hydroxamic acids were stronger Fe²⁺ ion chelators than quercetin. In addition, the investigated compounds, especially hydroxamic acids, were proven to be excellent antioxidants. They were as effective as BHA in inhibiting β-carotene-linoleic acid coupled oxidation. It is reasonable to assume that the antioxidant activity of the investigated compounds could contribute to their previously proven biological properties as cytostatic and antiviral agents.

Ethnopharmacological Relevance: Propolis is a resinous hive product collected by honeybees from various plant sources. It is widely used in traditional medicine and is reported to have a broad spectrum of pharmacological effects (antibacterial, antihepatoxic, antioxidative, anti-inflammatory, etc.). Thus the aim of this study was to assess cytotoxic effect of various ethanol propolis extractions on the cervical tumor cell line (HeLa) and compare it with their phenolic acids and flavonoids composition., Materials and Methods: Twenty samples of raw propolis were collected from 17 localities of Croatia (I-XVII), 2 of Bosnia and Hercegovina (XVIII, XIX) and 1 of Macedonia (XX). Reverse phase HPLC was used to determine the chemical composition of polyphenols. Biological experiments were carried out in vitro on cervix adenocarcinoma cell line (HeLa)., Results: Phenolic acids (ferulic acid, p-coumaric acid, caffeic acid) and flavonoids (tectochrysin, galangin, pinocembrin, pinocembrin-7-methylether, chrysin, apigenin, kaempferol, quercetin) have been determined using HPLC analysis in 20 ethanolic propolis extracts. All samples contain tectochrysin in ranges of 0.1988 mg/g (XVIII) to 1.2004 mg/g (III), while caffeic acid and quercetin have not been found. Flavonoid that is most abundant is galangin in ranges from 0.3706 mg/g (XVII) to 47.4879 mg/g (IX). The samples of propolis numbers I, VI and X applied in the investigated concentration range manifested significant reduction of cell growth. GI(50) value as indicator of cytotoxicity among propolis samples showed that propolis number VII is the most effective (GI(50) =76 μg/ml) followed by propolis nos. XV, XVIII and I., Conclusion: Antiproliferative and cytotoxic effect of propolis on the HeLa cells is not correlating with the concentration of particular components but on establishing the possible synergistic antiproliferative activity of individual phenolic acid and flavonoids. Differences in the chemical composition lead to diversity in biological activity of propolis samples., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Two types of polymer-drug conjugates were synthesized starting from styrene-maleic acid anhydride copolymer (SMA). Fenoprofen and gemfibrozil were chosen as model drugs because of their short plasma half lives. Both drugs were first converted to their 2-aminoethylamides, which possess free amino groups capable of reacting with SMA anhydride rings. By modifying the degree and type of substitution, lipophilic and hydrophilic conjugates were obtained. Drug loading in the conjugates was between 17 and 47%.

In vitro evaluation of the novel cycloalkyl-N-(4-chlorophenyl)-hydroxamic acids (2a-g) demonstrated that 2b,d,e exhibited rather marked inhibitory activity (IC50 = 7-10 microM) against pancreatic carcinoma, 2b-d against colon carcinoma, 2d against laryngeal carcinoma, and 2b,d against breast carcinoma. 2e showed the most pronounced anti-cytomegalovirus activity (EC50 = 1.5 and 0.8 microg mL(-1)) only at > or = 5-fold lower than the cytotoxic concentration. 2d and 2f showed modest, albeit selective, activity against cytomegalovirus (2d, EC50 = 7.3-8.9 microg mL(-1), selectivity index 7-10; 2f, EC50 = 7-13 microg mL(-1), selectivity index 10).

The novel L- and D-amino acid derivatives of hydroxyurea 5a-o were prepared by aminolysis of N-(1-benzotriazolecarbonyl)amino acid amides 4a-o with hydroxylamine. The hydantoin derivatives 6a-e,m,p were synthesized by base-catalyzed cyclization of amides 4, common precursors for 5 and 6. X-ray crystal structure analysis shows that the C5 atom in 6e possesses the S configuration, which is consistent with the configuration of the starting reagent, l-leucine. Among L-amino acid derivatives of hydroxyurea, 5h and 5i inhibited specifically murine leukemia and human T-lymphocytes (IC(50) = 10-19 microM) and showed selectivity with respect to normal human fibroblasts (WI 38). d-Amino acid derivatives of hydroxyurea 5m and 5o inhibited the growth of all tumor cell lines (IC(50) = 4.8-83.9 microM), but not the growth of normal fibroblasts (WI 38; IC(50) > 100 microM). Results on antiviral evaluations showed that N-(1-benzotriazolecarbonyl)amino acid amide 4m and hydantoin 6m had marked activity against the Davis strain of CMV (4m, EC(50) = 3.2 microg/mL; 6m, EC(50) = 4.0 microg/mL). However, these compounds showed also rather expressed cytotoxicity (4m, CC(50) = 43.4 microg/mL; 6m, CC(50) = 12.5 microg/mL(-1)).

A new synthetic approach to the 1,2,5-oxadiazine ring system is described. 2-Substituted or 2,4-disubstituted 2H-1,2,5-oxadiazine-3,6(4H,5H)-dione derivatives 4 were prepared by cyclisation of hydroxamic acids 3 derived from N-(1-benzotriazolylcarbonyl)-amino acids 1. The structures of the synthesised compounds were fully characterised by IR, 1H and 13C NMR spectroscopy and elemental analysis. The aim of this study was to evaluate biological activity of the newly synthesised oxadiazine derivatives. Cytotoxic and cytostatic activities were tested on two cell lines (HeLa and GMK) and evaluated by MTT-test. Two human DNA viruses (adenovirus 7 and herpesvirus 1) and two human RNA viruses (coxsackievirus B5 and echovirus 7) were used in the antiviral test. Selected biological studies indicated that 2-phenyl- -2H-1,2,5-oxadiazine-3,6(4H,5H)-dione (4a) and 4-benzyl-2-phenyl-2H-1,2,5-oxadiazine-3,6(4H,5H)-dione (4c) statistically significantly inhibited cell growth. A minor antiviral effect was observed upon adenovirus, herpesvirus and enteroviruses.

The purpose of this study was to evaluate and compare the ability of the macromolecular conjugates and microspheres to modify the release rate of gemfibrozil (Gem). Gem was covalently linked to two similar polymers: poly[alpha,beta-(N-2-hydroxyethyl-DL-aspartamide)] (PHEA) and poly[alpha,beta-(N-3-hydroxypropyl-DL-aspartamide)] (PHPA) by an ester linkage. The polymer-drug conjugates obtained (PHEA-G(1-3) and PHPA-G) differ in weight-average molecular weight, length of spacer and Gem content. Microspheres, composed of chitosans of different molecular weight alone or as a mixture with (2-hydroxypropyl)methylcellulose (HPMC), PHEA or PHPA and with different theoretical polymer/drug ratio (2:1 and 3:1, w/w) were prepared by spray drying. The microparticulate systems were morphologically characterised by scanning electron microscopy, particle size analysis and Gem content was determined. In vitro dissolution tests were performed to evaluate the feasibility of conjugates and microspheres in modulating Gem release. The results obtained show that microspheres are always suitable to modulate Gem release and that the best conditions are achieved by microspheres composed of the low molecular weight chitosan (CL) combined with PHPA or HPMC with either 2:1 or 3:1 (w/w) polymer/drug ratio. The PHEA-G conjugates exhibited rapid Gem release within less than 2 h, while the PHPA-G conjugate showed sustained Gem release profiles over a 10-h period.