Elemental and Radiological Characterisation of Arbutus unedo L. Leaves and Tea: Impact of Preparation Method on Nutritional Risk/Benefit.

The aim of this study was to characterise the elemental and radiological composition of strawberry tree (Arbutus unedo L.) leaves and tea preparations and compare it with commercial Uvin H herbal mixture, widely used in treatment of urinary tract infections. The concentration of 17 elements and the activity concentration of selected radionuclides were measured in strawberry tree leaves/Uvin H herbal mixture, as well as in herbal tea prepared by infusion or decoction of leaves for 5 or 10 min. In both leaves and tea preparations, Ca, K, Mg, and Na were the most abundant elements, while the lowest levels were measured for As, Co, Mo, and Se. Only 137Cs and 40K were detected in analysed leaves/herbal mixture, while the activity of radionuclides in tea preparations was below the detection limit. The maximum possible health benefits can be obtained by a 10-min decoction of leaves, which resulted in the highest total phenolic content and antioxidant activity and levels of K, Ca, Mg, Na, Fe, Mn, and Se in comparison to the other preparation methods evaluated in this study. The calculated intake of potentially toxic elements and radionuclides does not represent a health risk to consumers.

Development of a Pressurized Green Liquid Extraction Procedure to Recover Antioxidant Bioactive Compounds from Strawberry Tree Fruit (Arbutus unedo L.).

Strawberry tree fruits (Arbutus unedo L.) are a natural source of valuable biologically active compounds. Therefore, the aim of this work was to develop a green extraction approach using pressurized liquid extraction (PLE) to provide the highest yield of bioactive compounds. Different extraction solvents (distilled water, 50% and 96% ethanol) and different PLE parameters were investigated: temperature (40, 80, and 120 °C), static extraction time (5 and 10 min), and number of cycles (1 and 2). Total phenolic contents (TPC), hydroxycinnamic acids (HCA), flavonols (FL), total flavonoids (TF), condensed tannins (CT), and antioxidant capacity (DPPH and FRAP) were determined in PLE extracts. Solvent type, temperature, static extraction time, and number of cycles had a statistically significant effect on all bioactive compounds and antioxidant capacity (p ≤ 0.05). All bioactive compounds were positively correlated with temperature, except for TPC and DPPH. For all polyphenols studied, the optimal PLE parameters were a temperature of 120 °C, a static extraction time of 10 min and 2 cycles. The best solvent for most bioactive compounds was 96% ethanol, except for TPC, for which 50% ethanol was better. This study suggests that PLE is a promising tool to intensify the extraction of bioactive compounds from strawberry tree fruits.

Unauthorized Food Manipulation as a Criminal Offense: Food Authenticity, Legal Frameworks, Analytical Tools and Cases.

Food fraud is a criminal intent motivated by economic gain to adulterate or misrepresent food ingredients and packaging. The development of a reliable food supply system is at great risk under globalization, but Food Business Operators (FBOs) have a legal obligation to implement and maintain food traceability and quality at all stages of food production, processing, and distribution. Incidents of food fraud have a strong negative impact on consumer confidence in the food industry. Therefore, local and international regulatory mechanisms are established to prevent or mitigate food fraud. This review brings new perspectives linking EU and US legislation, as well as new definitions and descriptions of the criminal aspect of food fraud incidents. It also describes certain new insights into the application of state-of-the-art methods and techniques that provide valuable tools for geographic, botanical, or other chemical markers of food authenticity. The review also provides an overview of the most common cases of food fraud worldwide from 2010 to 2020. Further research is needed to support the development of predictive models for innovative approaches to adulteration, especially when some valuable nutrients are replaced by toxic ingredients. A possible solution to minimize food fraud incidents is to increase the level of risk-based inspections, establish more productive monitoring and implementation of food protection systems in the supply chain, and implement better ingredient control and certification. National and international (e.g., regional) police offices for food fraud should be introduced, possessing knowledge and skills in food, food safety, food processing, and food products, as initial positive results have emerged in some countries.

Comparative Study on the Phenolic Fingerprint and Antioxidant Activity of Strawberry Tree (Arbutus unedo L.) Leaves and Fruits.

The strawberry tree (Arbutus unedo L., Ericaceae family) is an evergreen Mediterranean shrub whose leaves and fruits are used in traditional medicine due to their antioxidant, antimicrobial, antidiabetic, diuretic, and antiproliferative properties. The health benefits are mainly attributed to the presence of phenolic compounds. The aim of this study was to compare the phenolic profiles, total phenolic content (TPC), and radical scavenging activity (RSA) of A. unedo leaves and fruits collected at two locations in Croatia. Phenolic profiles were identified using an ultra-high-performance liquid chromatograph (UHPLC) coupled with a hybrid mass spectrometer (LTQ Orbitrap MS). TPC was determined by Folin-Ciocalteu's assay, while RSA was investigated using DPPH reagent. A total of 64 phenolics (60 and 42 compounds in leaves and fruits, respectively) were identified. Hyperoside and flavan-3-ols were predominant compounds in leaves, while gallocatechin and catechin were the major compounds found in fruits. To the authors' knowledge, 16 and 5 phenolics in leaves and fruits, respectively, were reported for the first time. Principal component analysis (PCA) showed that UHPLC-LTQ Orbitrap MS could be used to identify which phenolics were able to discriminate samples regarding plant tissue and geographical origin. TPC in leaves and fruits were in the ranges of 67.07-104.74 and 16.78-25.86 mg gallic acid equivalents (GAE)/g dried weight (dw), respectively. RSA for leaves and fruits were in the ranges of 408.92-430.98 and 74.30-104.04 µmol Trolox equivalents (TE)/g dw, respectively. The number of identified phenolics was lower in fruits compared to leaves. Such a large number of bioactive phenolics identified and the strong antioxidant activity pointed to A. unedo as a promising health-promoting plant and natural food preservative.

The effects of strawberry tree (Arbutus unedo L.) water leaf extract and arbutin upon kidney function and primary DNA damage in renal cells of rats.

Although strawberry tree (Arbutus unedo L.) leaves have long been used as a herbal remedy, insufficient information is available on their nephrotoxicity. We assessed the safety of strawberry tree water leaf extract and its key component arbutin, administered per os to Lewis rats of both genders at 200 mg/kg b.w./day for 14 and 28 days. The effects of the tested compounds on DNA integrity in renal cells was evaluated using alkaline comet assay, while kidney function was studied using serum creatinine and urea levels. Strawberry tree water leaf extract showed high biocompatibility with kidney tissue. It did not impair DNA integrity of renal cells and kidney function, either in male or female rats. However, exposure to single arbutin affected the levels of primary DNA damage in renal cells which could be related to metabolic conversion of arbutin into hydroquinone, whose nephrotoxicity has previously been proven.

Liver function and DNA integrity in hepatocytes of rats evaluated after treatments with strawberry tree (Arbutus unedo L.) water leaf extract and arbutin.

Due to their beneficial health effects, strawberry tree (Arbutus unedo L.) leaves have for decades been used as herbal remedy in countries of the Mediterranean region. This pilot study is the first to investigate the liver function and DNA integrity in rat hepatocytes evaluated after 14 and 28 day treatments with strawberry tree water leaf extract and arbutin, administered per os to Lewis rats of both genders at a daily dose 200 mg/kg b.w. We focused on two types of biomarkers: enzyme serum markers of liver function (AST, ALT, and LDH), and primary DNA damage in the liver cells, which was estimated using the alkaline comet assay. At the tested dose, strawberry tree water leaf extract showed acceptable biocompatibility with liver tissue both in male and female rats, especially after shorter exposure. Our results also suggest that oral administration of single arbutin to rats was not associated with significant impairments either in the liver function or DNA integrity in hepatocytes. Considering that prolonged exposure to the tested compounds revealed minor changes in the studied biomarkers, future in vivo studies have to further clarify the biological and physiological relevance of these findings.

Food defence systems as an answer to food terrorism.

Terrorist attacks on critical infrastructures can cause problems to a national stability and functioning. Food and water supply chains are some of the most important infrastructures, and it is the country's (government's) obligation to provide sufficient quantities of food and water to its population. Intentional food contamination can, among other motives, originate from an act of terrorism (with political or ideological motives) with the aim of causing fear (terror) among people. Food defence systems can help assess vulnerabilities, determine mitigation strategies for terrorist attack, estimate risks, and prevent a terrorist attack. Risk assessment and prevention also include control over the production and distribution of potential chemical, biological, radiological or nuclear (CBRN) agents or their related materials. When a terrorist attack occurs, rapid and organised response is essential in terms of determining the type of agent used, managing the diseased, ensuring the functioning of the food and water supply, and the recovery of the infrastructure system under attack. Food defence planning as part of a food counterterrorism strategy should include considerations regarding the global food market and the fact that ingredients are supplied from all over the world (vendor certificates). Preventing terrorist attacks on sources of food and water is a far better option than crisis management once an attack had already been committed, but governments should have a response to any scenario.

The effects of strawberry tree water leaf extract, arbutin and hydroquinone on haematological parameters and levels of primary DNA damage in white blood cells of rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Strawberry tree (Arbutus unedo L., Ericaceae) leaves represent a potent source of biologically active compounds and have been used for a long to relieve symptoms of various health impairments and diseases. Two major compounds related to their beneficial activities in animals and humans are arbutin and hydroquinone. AIM OF THE STUDY: To establish potential benefit/risk ratio associated with daily oral administration of strawberry tree water leaf extract, arbutin and hydroquinone in doses expected to be non-toxic. MATERIALS AND METHODS: We performed a 14-day and a 28-day study on male and female Lewis rats and evaluated main haematological parameters and the effects of treatments on the levels of primary DNA damage in white blood cells (WBC) using the alkaline comet assay. RESULTS: Our findings suggest no significant changes in the haematological parameters following prolonged exposure to strawberry tree water leaf extract, arbutin, and hydroquinone. However, hydroquinone causes increased, and extract as well as arbutin decreased WBC count in male rats compared to control after 14 days of treatment. DNA damage measured in WBC of rats treated with all compounds was below 10% of the DNA in the comet tail, which indicates low genotoxicity. The genotoxic potential of strawberry water leaf extract was within acceptable limits and reflected effects of a complex chemical composition upon DNA. We also observed slight gender- and exposure time- related differences in primary DNA damage in the leucocytes of control and treated rats. CONCLUSIONS: Future studies should investigate which doses of strawberry tree water leaf extract would be most promising for the potential use as a substitute for bearberry leaves for treatment of urinary infection.

Multi-elemental composition and antioxidant properties of strawberry tree (Arbutus unedo L.) honey from the coastal region of Croatia: Risk-benefit analysis.

The concentration of 23 major and trace elements, total phenolic content (TPC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity were determined in nine samples of strawberry tree honey and compared to other types of unifloral honeys. The most abundant elements in strawberry tree honey were potassium, calcium, magnesium and sodium, ranging between 1276 and 2367, 95.2-154, 14.4-74.4 and 13.4-64.3mg/kg, respectively. Strawberry tree honey had generally higher TPC (range: 0.314-0.522g GA/kg) and DPPH (1.94-4.45mM TE/kg) compared to other analysed unifloral honeys. A strong positive relationship was found between TPC and DPPH, TPC and concentration of homogentisic acid (HGA), chemical marker of strawberry tree honey, and between DPPH and HGA. Regarding daily intake of essential elements, strawberry tree honey can be considered nutritionally richer than the majority of unifloral honeys available in Croatia, while contribution to tolerable intake set for potentially toxic elements was very low, corresponding to pristine areas.

Facts about phthalate toxicity in humans and their occurrence in alcoholic beverages.

Phthalates are esters of phthalic acid and aliphatic alcohol added to plastic to improve its softness, flexibility, and extensibility. They easily migrate from plastic products into the environment because of their physical and chemical properties. This review summarises their characteristics, distribution in the environment, monitoring, use, toxic effects on human health, regulatory limits in different matrices and products, and tolerable daily intake. The studies we have reviewed suggest that phthalates have a potential to affect reproduction and development in humans. Due to the inconsistent data, further studies are needed and, in the meantime, precautionary policies must be implemented. Here we draw attention to the methods of determining phthalate levels in alcoholic beverages and reported levels in plum spirits produced in Croatia. Legally produced and moderately consumed plum spirits do not seem to increase the risk of phthalate toxicity for human health. We conclude with recommendations for the effective monitoring of phthalate exposure in humans and for the implementation of alternative materials in alcohol production.

Development and Validation of a GC-MS Method for the Analysis of Homogentisic Acid in Strawberry Tree (Arbutus unedo L.) Honey.

To confirm the botanical origin of strawberry tree (Arbutus unedo L.) honey, a liquid-liquid extraction followed by GC-MS method was developed for the quantitative determination of homogentisic acid (HGA), the main phenolic compound in this honey. Different parameters affecting extraction, such as the type and volume of extraction solvents, pH of the solution, and amount of salt, were optimized. The method showed good linearity (r2 = 0.9990) over the tested concentration range (50-500 mg/kg) and a low LOD (0.3 mg/kg). Precision expressed as RSD was <7%. The average accuracy was 95%. The optimized method was applied for determining the HGA content in strawberry tree honey samples from Croatia. The HGA content in analyzed samples (n = 7) ranged from 245.1 to 485.9 mg/kg. The proposed method provided reliable performance and can be easily implemented for the routine monitoring of HGA in strawberry tree honey in order to assure honey QC.

In vitro assessment of the cytotoxic, DNA damaging, and cytogenetic effects of hydroquinone in human peripheral blood lymphocytes.

This study investigated the mechanisms of hydroquinone toxicity and assessed the relationships between its cytotoxic, genotoxic, and cytogenetic effects tested at 8, 140, and 280 µg mL-1 in human peripheral blood lymphocytes exposed for 24 h. The outcomes of the treatments were evaluated using the apoptosis/necrosis assay, the alkaline comet assay, and the cytokinesis-block micronucleus (CBMN) cytome assay. The tested hydroquinone concentrations produced relatively weak cytotoxicity in resting lymphocytes, which mostly died via apoptosis. Hydroquinone's marked genotoxic effects were detected using the alkaline comet assay. Significantly decreased values of all comet parameters compared to controls indicated specific mechanisms of hydroquinone-DNA interactions. Our results suggest that the two higher hydroquinone concentrations possibly led to cross-linking and adduct formation. Increased levels of DNA breakage measured following exposure to the lowest concentration suggested mechanisms related to oxidative stress and inhibition of topoisomerase II. At 8 µg mL-1, hydroquinone did not significantly affect MN formation. At 140 and 280 µg mL-1, it completely blocked lymphocyte division. The two latter concentrations also led to erythrocyte stabilization and prevented their lysis. At least two facts contribute to this study's relevance: (I) this is the first study that quantifies the degree of reduction in total comet area measured in lymphocyte DNA after hydroquinone treatment, (II) it is also the first one on a lymphocyte model that adopted the "cytome" protocol in an MN assay and found that lymphocytes exposure even to low hydroquinone concentration resulted in a significant increase of nuclear bud frequency. Considering the limitations of the lymphocyte model, which does not possess intrinsic metabolic activation, in order to unequivocally prove the obtained results further studies using other appropriate cell lines are advised.

Quantitative analysis of arbutin and hydroquinone in strawberry tree (Arbutus unedo L., Ericaceae) leaves by gas chromatography-mass spectrometry.

The phenolic glycoside arbutin and its metabolite with uroantiseptic activity hydroquinone occur naturally in the leaves of various medicinal plants and spices. In this study, an extraction procedure coupled with gas chromatography-mass spectrometry (GC-MS) was developed to determine arbutin and hydroquinone content in strawberry tree (Arbutus unedo L., Ericaceae) leaves. The method showed good linearity (R2>0.9987) in the tested concentration range (0.5-200 µg mL(-1)), as well as good precision (RSD<5%), analytical recovery (96.2-98.0%), and sensitivity (limit of detection=0.009 and 0.004 µg mL(-1) for arbutin and hydroquinone, respectively). The results obtained by the validated GC-MS method corresponded well to those obtained by high performance liquid chromatography (HPLC) method. The proposed method was then applied for determining arbutin and hydroquinone content in methanolic leaf extracts. The amount of arbutin in the leaves collected on the island of Kolocep (6.82 mg g(-1) dry weight) was found to be higher (tpaired=43.57, tc=2.92) in comparison to the amount of arbutin in the leaves collected on the island of Mali Losinj (2.75 mg g(-1) dry weight). Hydroquinone was not detected in any of the samples. The analytical features of the proposed GC-MS method demonstrated that arbutin and hydroquinone could be determined alternatively by gas chromatography. Due to its wide concentration range, the method could also be suitable for arbutin and hydroquinone analysis in leaves of other plant families (Rosaceae, Lamiaceae, etc.).

[Exposure to phtalates and their presence in alcoholic beverages]. / Izlozenost ftalatima i njihova pojavnost u alkoholnim picima.

Phthalates are phthalic acid and aliphatic alcohol esters used as additives to plastic in order to improve its softness, flexibility, and elongation. Phthalates are highly mobile and migrate easily from plastic products into the environment due to their physical and chemical properties. This study briefly describes the characteristics and distribution of phthalates in the environment, their toxic effects on human health, the legislation regarding the maximum allowed concentration of phthalates in drinking water and products intended for infants, as well as the tolerable daily intake. Special attention is given to the methods of determining phthalates and their levels in alcoholic beverages, with an overview of phthalate occurrences and concentrations in plum brandy made in Croatia. A segment on denatured alcohol and illegally marketed alcohol is also included, as well as guidelines for the effective monitoring of the routes of human exposure to phthalates.