Protective Effects of Arbutus unedo L. Honey in the Alleviation of Irinotecan-Induced Cytogenetic Damage in Human Lymphocytes-An In Vitro Study.

Strawberry tree (Arbutus unedo L.) honey (STH) has been used since ancient times as a folk medicine remedy, especially in certain Mediterranean countries. This honey, rich in phenolic content, is well recognized for its antioxidant, anti-inflammatory, and antimicrobial activities, and is used for the treatment of skin lesions as well as gastrointestinal and respiratory disorders. This study investigated whether STH alleviates genome damage in human peripheral blood lymphocytes produced by the cytotoxic drug irinotecan. The phenolic profile of STH was previously estimated by ultra-high-performance liquid chromatography coupled to a linear ion trap-Orbitrap hybrid mass spectrometer. The effects of STH were evaluated at three concentrations (1×, 5×, and 10×), based on the daily consumption of the honey by an adult person. After 2 h of in vitro exposure, standard lymphocyte cultures for the analysis of chromosome aberrations and the cytokinesis-block micronucleus cytome assay were established. Our results demonstrate that STH offered remarkable geno- and cytoprotection when administered with irinotecan. These findings are relevant for drawing preliminary conclusions regarding the in vitro safety of the tested honey. However, further studies are needed with the application of more complex experimental models.

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.

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.

Antifungal Activity of Oleuropein against Candida albicans-The In Vitro Study.

In the present study we investigated activity of oleuropein, a complex phenol present in large quantities in olive tree products, against opportunistic fungal pathogen Candida albicans. Oleuropein was found to have in vitro antifungal activity with a minimal inhibitory concentration (MIC) value of 12.5 mg·mL-1. Morphological changes in the nuclei after staining with fluorescent DNA-binding dyes revealed that apoptosis was a primary mode of cell death in the analyzed samples treated with subinhibitory concentrations of oleuropein. Our results suggest that this antifungal agent targets virulence factors essential for establishment of the fungal infection. We noticed that oleuropein modulates morphogenetic conversion and inhibits filamentation of C. albicans. The hydrophobicity assay showed that oleuropein in sub-MIC values has significantly decreased, in both aerobic and anaerobic conditions, the cellular surface hydrophobicity (CSH) of C. albicans, a factor associated with adhesion to epithelial cells. It was also demonstrated that the tested compound inhibits the activity of SAPs, cellular enzymes secreted by C. albicans, which are reported to be related to the pathogenicity of the fungi. Additionally, we detected that oleuropein causes a reduction in total sterol content in the membrane of C. albicans cells, which might be involved in the mechanism of its antifungal activity.

Olive leaf extract activity against Candida albicans and C. dubliniensis - the in vitro viability study.

Olive leaf extract is characterized by a high content of polyphenols (oleuropein, hydroxytyrosol and their derivatives), which is associated with its therapeutic properties. The objective of the present research was to evaluate the antifungal activity of olive leaf extract against Candida albicans ATCC 10231 and C. dubliniensis CBS 7987 strains. Minimum inhibitory concentrations (MIC) of the extract were determined by several in vitro assays. The extract showed a concentration depended effect on the viability of C. albicans with MIC value of 46.875 mg mL-1 and C. dubliniensis with MIC value 62.5 mg mL-1. Most sensitive methods for testing the antifungal effect of the extracts were the trypan blue exclusion method and fluorescent dye exclusion method while MIC could not be determined by the method according to the EUCAST recommendation suggesting that herbal preparations contain compounds that may interfere with this susceptibility testing. The fluorescent dye exclusion method was also used for the assessment of morphological changes in the nuclei of treated cells. According to the obtained results, olive leaf extract is less effective against the tested strains than hydroxytyrosol, an olive plant constituent tested in our previous study.

Hydroxytyrosol expresses antifungal activity in vitro.

Hydroxytyrosol (HT) is a potent antioxidant found in olive oil and leaves. Using several in vitro approaches, we tested antifungal activity of HT. HT showed broad spectrum of antifungal activity against medically important yeasts and dermatophyte strains with MIC values ranging between 97.6 µgml⁻¹ and 6.25 mgml⁻¹. The antimicrobial activity of HT was also tested using the time-kill methodology. Below the MIC value, HT showed potent damage of cell wall of Candida albicans ATCC 10231 using fluorescent dye-exclusion method. At the subinhibitory concentration, HT also influenced dimorphic transition of Candida indicating that HT is inhibitor of germ-tube formation as one of the most important virulence factor of C. albicans. Furthermore, HT showed disturbances in cell surface hydrophobicity (CSH) of C. albicans. The in vitro results indicate that HT caused a significant cell wall damage and changes in CSH as well as inhibition of germ-tube formation as virulence factor of C. albicans. The study indicates that HT has a considerable in vitro antifungal activity against medically important yeasts.

Antimicrobial activity of Willowherb (Epilobium angustifolium L.) leaves and flowers.

Since the aetiology of benign prostatic hyperplasia (BHP) is still unknown, the use of medicinal herb extracts and products prepared thereof are recommended due to their antimicrobial activity, especially during early stages of BHP. A comparison was performed of the in vitro antimicrobial activity (using broth microdilution assay) of flowers and leaves of willowherb (Epilobium angustifolium L., Onagraceae) from Mt. Velebit (Croatia). The strains (standard ATCC and clinical isolates) of Staphylococcus aureus (including MRSA), Bacillus subtilis, Escherichia coli (including p-fimbriae positive strain), Pseudomonas aeruginosa, Proteus mirabilis, Candida albicans, C. tropicalis, C. dubliniensis and Saccharomyces cerevisiae were susceptible with MIC values between 4.6±0.2 and 18.2±0.8 mg/mL. The results of in vitro studies showed that no differences were found in the antimicrobial activity between the ethanol extracts of leaves and flowers of E. angustifolium. Using the quantitative fluorescent assay with ethidium bromide and acridine orange, the viability of C. albicans ATCC 10231 was assessed after in vitro exposure to E. angustifolium leaf and flower ethanol extracts. Apoptosis of C. albicans blastospores dominated over necrosis in all treated samples after short-term exposure with 6 to 12 mg/mL of extracts. In addition to the valuable biological activity of E. angustifolium extracts, the data obtained from the in vitro diffusion, the dilution assay and antifungal viability fluorescent assay suggest that leaf and flower ethanol extracts of E. angustifolium L. are a promising complementary herbal therapy of conditions such as BHP.

Assessment of genotoxic potency of sulfate-rich surface waters on medicinal leech and human leukocytes using different versions of the Comet assay.

The aim of the present study was to investigate how exposure to sulfate-rich surface waters affects the level of primary DNA damage in hemocytes of leech Hirudo medicinalis. Samples of surface water were collected at two sites near a gypsum factory (Knin, Croatia) and two reference sites. In the laboratory, samples were subjected to detailed chemical analysis and used in toxicity testing. For that purpose, previously acclimatized individuals of H. medicinalis were sub-chronically exposed (for 28 days) to tested water samples. Levels of primary DNA damage were evaluated using the alkaline Comet assay in hemocytes collected on days 7, 14, 21 and 28 of exposure and compared with their baseline values. Genotoxic potency of the water sample with the highest sulfate concentration was further evaluated using the alkaline, neutral and hOGG1-modified Comet assay on human peripheral blood leukocytes exposed ex vivo for 30 min. The purpose was to explore which mechanisms are responsible for DNA damage. Chemical analysis revealed that sulfate concentrations in two water samples collected in Mali Kukar Lake (1630 mg/L SO4) and Kosovcica River (823.3 mg/L SO4) exceeded the WHO and US EPA defined limits for sulfate in drinking water. Increased levels of metals were found only in the water sample collected in Mali Kukar Lake. However, of the 65 elements analyzed, only nickel and titanium exceed the value legally accepted in Croatia for drinking water. The levels of DNA damage, estimated by the alkaline Comet assay in hemocytes of medicinal leech, increased with the duration of exposure to two sulfate-rich water samples. Since hemocytes responded sensitively to treatment, they could be used for biomonitoring purposes. As observed on treated human peripheral blood leukocytes, all versions of the Comet assay were effective in detecting DNA damage, which was measured in samples with sulfate concentrations equal to or higher than the legally accepted levels for drinking water. Based on the obtained results, it can be assumed that genotoxicity was a consequence both of direct (single- and double-strand DNA breaks) and indirect effects (oxidative damage) caused by the combined effects of all contaminants present in the tested water samples. Our results indicate the need for in situ monitoring and purification of gypsum mine water prior to its release in the natural environment.

Application of the comet assay and detection of DNA damage in haemocytes of medicinal leech affected by aluminium pollution: a case study.

This report describes an investigation of genotoxic effects in medicinal leech (Hirudo verbana) exposed to water and sediment of Lake Njivice (Krk Island, Croatia) contaminated by aluminium compounds. The levels of primary DNA damage in leech haemocytes and loss of DNA integrity caused by acute and chronic exposure to contaminated water and sediment were investigated using the alkaline comet assay. Genotoxic effects induced by acute exposure to contaminants were evaluated on leech haemocytes and blood cells of fish and mouse treated ex vivo. The effects of chronic exposure were assessed on haemocytes sampled from an animal kept under laboratory conditions on contaminated water and sediment for 180 days. The results indicate the DNA damaging potential of aluminium compounds present in an excess amount in tested samples.

Evaluation of radioprotective effects of propolis and its flavonoid constituents: in vitro study on human white blood cells.

This in vitro study aimed to evaluate the possible radioprotective effects of the natural substances WSDP, caffeic acid, chrysin and naringin on gamma-irradiated human white blood cells. The effectiveness of tested compounds was evaluated using the alkaline comet assay, the analysis of structural chromosome aberration and the cytokinesis-block micronucleus assay. The results obtained by the alkaline comet study indicate favourable toxicity profiles of propolis and its polyphenolic components, and confirmed the radioprotective abilities comparable to the chemical radioprotector AET. WSDP and its polyphenolic components were able to reduce the number of necrotic cells. None of tested compounds induced significant genotoxicity, but all of them offered a quite measurable protection against DNA damage. WSDP was found to be the most effective in diminishing the levels of primary and more complex cytogenetic DNA damage in white blood cells. Considering its complex composition, to undoubtedly explain the underlying mechanisms of cyto/radioprotective effects, further studies are needed.

Assessment by survival analysis of the radioprotective properties of propolis and its polyphenolic compounds.

The radioprotective effects of propolis and polyphenolic compounds from propolis on the radiation-induced mortality of mice exposed to 9 Gy of gamma-irradiation were studied. Intraperitoneal (i.p.) treatment of mice at doses of 100 mg kg(-1) body weight of propolis (water or ethanolic extract; WSDP or EEP) or its polyphenolic compounds (quercetin, naringin caffeic acid, chrysin) consecutively for 3 d before irradiation, delayed the onset of mortality and reduced the symptoms of radiation sickness. All test compounds provided protection against hematopoietic death (death within 30 d after irradiation). The greatest protection was achieved with quercetin; the number of survivors at the termination of the experiment was 63%. According to statistical analyses by the Kaplan-Meier method and the log-rank test, a significant difference between test components and control was found (p<0.001). Treatment with test components after lethal irradiation was ineffective. These results suggest that propolis and its polyphenolic compounds given to mice before irradiation protect mice from the lethal effects of whole-body irradiation.