Physicochemical Characterization and Biological Activities of Black and White Garlic: In Vivo and In Vitro Assays.

White and three types of black garlic (13, 32, and 45 days of aging, named 0C1, 1C2, and 2C1, respectively) were selected to study possible differences in their nutraceutic potential. For this purpose, garlic were physicochemically characterized (Brix, pH, aW, L, polyphenol, and antioxidant capacity), and both in vivo and in vitro assays were carried out. Black garlic samples showed higher polyphenol content and antioxidant capacity than the white ones. The biological assays showed that none of the samples (neither raw nor black garlic) produced toxic effects in the Drosophila melanogaster animal genetic model, nor exerted protective effects against H2O2, with the exception of the 0C1 black garlic. Moreover, only white garlic was genotoxic at the highest concentration. On the other hand, 0C1 black garlic was the most antigenotoxic substance. The in vivo longevity assays showed significant extension of lifespan at some concentrations of white and 0C1and 1C2 black garlic. The in vitro experiments showed that all of the garlic samples induced a decrease in leukemia cell growth. However, no type of garlic was able to induce proapoptotic internucleosomal DNA fragmentation. Taking into account the physicochemical and biological data, black garlic could be considered a potential functional food and used in the preventive treatment of age-related diseases. In addition, our findings could be relevant for black-garlic-processing agrifood companies, as the economical and timing costs can significantly be shortened from 45 to 13 days of aging.

Nutraceutic Potential of Two Allium Species and Their Distinctive Organosulfur Compounds: A Multi-Assay Evaluation.

This study aimed to evaluate the biological activities of two Allium species (garlic and onion) as well as diallyl disulphide (DADS) and dipropyl disulphide (DPDS) as their representative bioactive compounds in a multi-assay experimental design. The genotoxic, antigenotoxic, and lifespan effects of garlic, onion, DADS, and DPDS were checked in Drosophila melanogaster and their cytotoxic, pro-apoptotic, and DNA-clastogenic activities were analyzed using HL60 tumoral cells. All compounds were non-genotoxic and antigenotoxic against H2O2-induced DNA damage with a positive dose-response effect and different inhibition percentages (the highest value: 95% for DADS) at all tested concentrations. Daily intake of Allium vegetables, DADS, or DPDS had no positive effects on flies' lifespan and health span. Garlic and DADS exerted the highest cytotoxic effects in a positive dose-dependent manner. Garlic and DADS exerted a DNA-internucleosomal fragmentation as an index of induced proapoptotic activity on HL60 cells. Allium vegetables and DADS were able to induce clastogenic strand breaks in the DNA of HL60 cells. This study showed the genomic safety of the assayed substances and their protective genetic effects against the hydrogen peroxide genotoxine. Long-term treatments during the whole life of the Drosophila genetic model were beneficial only at low-median concentrations. The chemo-preventive activity of garlic could be associated with its distinctive organosulfur DADS. We suggest that supplementary studies are needed to clarify the cell death pathway against garlic and DADS.

Red and White Wine Lees Show Inhibitory Effects on Liver Carcinogenesis.

SCOPE: Wine has shown anticarcinogenic benefits in hepatocarcinoma and polyphenols seem to be responsible for these effects. Wine lees are the sediments produced during fermentation and they endow wine with organoleptic and physicochemical properties. However, the anticarcinogenic role of these compounds is still unknown. Thus, the purpose of this work is to determine the phytochemical profiles of wine lees and then to analyze their anticarcinogenic effect and DNA methylation on a model of hepatocarcinogenesis. METHODS AND RESULTS: The phytochemical composition of lees is determined by the Folin-Ciocalteu method and high-performance liquid chromatography. An in vivo study using a diethyl nitrosamine-hepatocarcinogenesis-induced model is performed to investigate the hepatoprotective properties of different doses of wine lees. For the DNA methylation analysis, a bisulfite-based method is used. Both types of lees mostly contain pyrogallol, gallic, and syringic acid with a high content of catechins in red lees. The carcinogen hypermethylates the Alu-M2 repetitive sequence and white lees decreases the hypermethylation at all tested concentrations. Low concentration of red and white lees and high concentration of white lees significantly improve the hepatocellular architecture and decrease the mitotic index in the murine model. CONCLUSION: These findings suggest that wine lees are promising agents for chemoprevention of hepatocarcinoma.

Anti/genotoxic, longevity inductive, cytotoxic, and clastogenic-related bioactivities of tomato and lycopene.

The aim of this study was to evaluate some biological activities of tomato as well as lycopene and to consider a new nutraceutic value for this fruit regarding to the protection against genetic damage and as a chemopreventive agent. Genotoxicity, DNA-protection against hydrogen peroxide, and lifespan properties of tomato and lycopene were assessed through wing spot test and longevity assay using the Drosophila in vivo model. Additionally, chemopreventive activity was investigated through cytotoxicity, DNA-fragmentation comet and annexin V FITC/PI assays using HL60 in vitro model. Results showed that: (i) tomato and lycopene are not genotoxic and protect against H2 O2 -induced damage; (ii) with respect to the lifespan, tomato and lycopene are harmless at the lowest concentration; (iii) tomato is cytotoxic in a dose-dependent manner, but not lycopene; (iv) tomato and lycopene do not induce internucleosomal DNA-fragmentation although they induce significant clastogenic activity at low level in the leukemia cells. To sum up, tomato is a good candidate to be considered as a nutraceutical substance. Furthermore, synergistic action among other components within tomato matrix could be the cause of the health effects observed in this vegetable, which are not fully explained by lycopene. Environ. Mol. Mutagen. 59:427-437, 2018. © 2018 Wiley Periodicals, Inc.

Role of Zucchini and Its Distinctive Components in the Modulation of Degenerative Processes: Genotoxicity, Anti-Genotoxicity, Cytotoxicity and Apoptotic Effects.

Zucchini (Cucurbita pepo subsp. pepo) is a seasonal vegetable with high nutritional and medical values. Many useful properties of this fruit are attributed to bioactive compounds. Zucchini fruits ("Yellow" and "Light Green" varieties) and four distinctive components (lutein, ß-carotene, zeaxanthin and dehydroascorbic acid) were selected. Firstly, the lutein, ß-carotene, zeaxanthin and dehydroascorbic acid contents were determined in these fruits. Then, in order to evaluate the safety and suitability of their use, different assays were carried out: (i) genotoxicity and anti-genotoxicity tests to determine the safety and DNA-protection against hydrogen peroxide; (ii) cytotoxicity; and (iii) DNA fragmentation and Annexin V/PI (Propidium Iodide) assays to evaluate the pro-apoptotic effect. Results showed that: (i) all the substances were non-genotoxic; (ii) all the substances were anti-genotoxic except the highest concentration of lutein; (iii) "Yellow" zucchini epicarp and mesocarp exhibited the highest cytotoxic activity (IC50 > 0.1 mg/mL and 0.2 mg/mL, respectively); and (iv) "Light Green" zucchini skin induced internucleosomal DNA fragmentation, ß-carotene being the possible molecule responsible for its pro-apoptotic activity. To sum up, zucchini fruit could play a positive role in human health and nutrition due to this fruit and its components were safe, able to inhibit significantly the H2O2-induced damage and exhibit anti-proliferative and pro-apoptotic activities toward HL60 (human promyelocytic leukemia cells) tumor cells. The information generated from this research should be considered when selecting potential accessions for breeding program purposes.

Demethylating and anti-hepatocarcinogenic potential of hesperidin, a natural polyphenol of Citrus juices.

Hepatocellular carcinoma (HCC) is a neoplasia representing the fifth most common malignancy worldwide and the third cause of death from cancer. Diets with high content in fruits and vegetables are widely recommended for their health-promoting properties, among them, the protection against diabetes, cancer, and cardiovascular diseases. Hesperidin is the most important phenol in the orange fruit with well-known health benefits. Diet components have been used as possible modulator agents of DNA methylation in cancer cells and epigenetic therapy against their harmful effects could be a potential tool in chemotherapy. The purpose of the present study was to evaluate the methylation patterns induced by hesperidin in HL60 cell line as an in vitro model in order to analyze its chemopreventive effects in epigenetic cancer therapies. A parallel in vivo pilot experience using a rat diethyl nitrosamine hepatocarcinogenesis-induced model was carried out to validate the therapeutic efficacy of this orange flavonol. Results showed that: (i) Hesperidin is cytotoxic in a dose-dependent manner and the IC50 was 12.5 mM; (ii) Hesperidin exerts a significant hypomethylating effect on the LINE-1 sequence (up to 47% hypomethylation at 12.5 mM) and on the ALU-M2 repetitive sequences (up to 32% at 6 mM) in HL60 tumor cells. (iii) Hesperidin does not affect the rat body and liver weight and it is able to reduce the diethyl nitrosamine-induced nodules at 1,000, 500, and 250 ppm. In conclusion, hesperidin could be proposed as a candidate molecule in chemoprevention in epigenetic therapy purposes.

In vivo and in vitro evaluation for nutraceutical purposes of capsaicin, capsanthin, lutein and four pepper varieties.

The purpose of this study is to determine the nutraceutic potential of different Capsicum sp, capsaicin, capsanthin and lutein and provide data in order to clarify the conflicting results obtained for capsaicin by different authors. To achieve these objectives, in vivo (geno/antigenotoxicity and lifespan assays in the animal model Drosophila) and in vitro (cytotoxicity and DNA-fragmentation assays in HL60 promyelocytic cell line) assays were carried out. Results showed that i) none of the tested substances were genotoxic except green hot pepper and capsaicin at the highest tested concentration (5 mg/mL and 11.5 µM respectively), ii) all tested substances except green hot pepper are antimutagenic against H2O2-induced damage, iii) only red sweet pepper significantly extend the lifespan and healthspan of D. melanogaster at 1.25 and 2.5 mg/mL, iv) all pepper varieties induce dose-depended cytotoxic effect in HL60 cells with different IC50, and v) all pepper varieties and capsaicin exerted proapoptotic effect on HL60 cells. IN CONCLUSION: (i) sweet peppers could be suggested as nutraceutical food, (ii) hot peppers should be moderately consumed, and (iii) supplementary studies are necessary to clarify the synergic effect of the carotenoids and capsaicinoids in the hot pepper food matrix.

Protective effect of borage seed oil and gamma linolenic acid on DNA: in vivo and in vitro studies.

Borage (Borago officinalis L.) seed oil has been used as a treatment for various degenerative diseases. Many useful properties of this oil are attributed to its high gamma linolenic acid content (GLA, 18:3 ω-6). The purpose of this study was to demonstrate the safety and suitability of the use of borage seed oil, along with one of its active components, GLA, with respect to DNA integrity, and to establish possible in vivo toxic and in vitro cytotoxic effects. In order to measure these properties, five types of assays were carried out: toxicity, genotoxicity, antigenotoxicity, cytotoxicity (using the promyelocytic leukaemia HL60 cell line), and life span (in vivo analysis using the Drosophila model). Results showed that i) Borage seed oil is not toxic to D. melanogaster at physiological concentrations below 125 µl/ml and the studies on GLA indicated non-toxicity at the lowest concentration analyzed ii) Borage seed oil and GLA are DNA safe (non-genotoxic) and antimutagenic compared to hydrogen peroxide, thereby confirming its antioxidant capacity; iii) Borage seed oil and GLA exhibited cytotoxic activity in low doses (IC50 of 1 µl/ml and 0.087 mM, respectively) iv) Low doses of borage seed oil (0.19%) increased the health span of D. melanogaster; and v) GLA significantly decreased the life span of D. melanogaster.Based on the antimutagenic and cytotoxic effects along with the ability to increase the health span, we propose supplementation with borage seed oil rather than GLA, because it protects DNA by modulating oxidative genetic damage in D. melanogaster, increases the health span and exerts cytotoxic activity towards promyelocytic HL60 cells.

Role of citrus juices and distinctive components in the modulation of degenerative processes: genotoxicity, antigenotoxicity, cytotoxicity, and longevity in Drosophila.

It is well established that breakfast beverages contain high quantities of Citrus juices. The purpose of the present study was to assess the nutraceutical value of orange and lemon juices as well as two of their active compounds: hesperidin and limonene. Indicator assays were performed at three levels to evaluate different biological health promoter activities: (i) determination of the safety and DNA-damage protecting ability against free radicals by using the somatic mutation and recombination test (SMART) in Drosophila melanogaster, (ii) study of the modulating role for life span in Drosophila melanogaster, and (iii) measurement of the cytotoxic activity against the human tumor cell line HL60. The highest concentrations assayed for lemon juice and limonene (50% v/v and 0.73 mM, respectively) showed genotoxic activity as evidenced from SMART. Orange and lemon juices as well as hesperidin and limonene exhibit antigenotoxic activity against hydrogen peroxide used as an oxidative genotoxin. Life-span experiments revealed that the lower concentrations of orange juice, hesperidin, and limonene exerted a positive influence on the life span of Drosophila. Finally all substances showed cytotoxic activity, with hesperidin being least active. Taking into account the safety, antigenotoxicity, longevity, and cytotoxicity data obtained in the different assays, orange juice may be a candidate as a nutraceutical food as it (1) is not genotoxic, (2) is able to protect DNA against free radicals, and (3) inhibits growth of tumor cells.

A pilot study on the DNA-protective, cytotoxic, and apoptosis-inducing properties of olive-leaf extracts.

Leaves of olive trees are an abundant raw material in the Mediterranean basin. They contain large amounts of potentially useful phytochemicals and could play beneficial roles in health care. In the present study, the principal bioactive phenols in olive-leaf extracts (OLEs) have been identified and quantified, and their genotoxic/antigenotoxic, cytotoxic and apoptotic effects have been assessed. The Somatic Mutation and Recombination Test (SMART) in wing imaginal discs of Drosophila melanogaster has been performed to test the possible genotoxicity of overall OLE and the individual components oleuropein and luteolin at different concentrations. The same assay was able to detect antigenotoxic activity against hydrogen peroxide as oxidative genotoxicant. None of the extracts/phenols tested showed significant mutagenic activity. This fact, together with the antigenotoxic activity against H(2)O(2) detected for all these extracts/phenols, confirmed the safety of OLE, oleuropein and luteolin in terms of DNA protection. HL60 human promyelocytic leukemia cells were used to assess the cytotoxic effects of the extracts/phenols. OLE, oleuropein and luteolin showed a dose-dependent cytotoxic effect with different IC50 (10µl/ml, 170µM, and 40µM, respectively). DNA fragmentation patterns and cell staining with acridine orange and ethidium bromide indicated that the mechanism for the cytotoxic effect of OLE, oleuropein and luteolin was the apoptotic pathway, with DNA laddering and cytoplasmic and nuclear changes. These results could help explain the mechanism of action that underlies the beneficial effect of OLE, proposed as a nutraceutical in the prevention of human cancer.

Targets of red grapes: oxidative damage of DNA and leukaemia cells.

Vitis vinifera is a widespread crop all over the world. The biophenols present in grapes have a remarkable influence on wine quality and also confer potential health-protecting properties to this fruit. The aim of the present work was to assess the beneficial properties of skin, seeds and pulp of red table grapes (RTG) (Vitis vinifera, Palieri Cadiz variety). Two potential beneficial activities of red table grapes (RTG) were analyzed: (i) The maintenance of genomic stability studying their genotoxic/antigenotoxic effects, and (ii) the in vitro cytotoxic effect against tumor cells of RTG components. The genotoxic and/or antigenotoxic effect was measured applying the somatic mutation and recombination test on wing imaginal discs of Drosophila melanogaster. The cytotoxic effect was monitored using the HL60 human leukemia model to evaluate the antiproliferative potential of the different parts of RTG. The three major parts (skin, seeds and pulp) are not genotoxic. When antigenotoxicity assays were performed using hydrogen peroxide as the oxidative genotoxin, skin, seed and pulp exerted a desmutagenic effect, with seeds and skin showing the most potent effect. The cytotoxicity tests using HL60 cells indicated that only skin and pulp fractions are able to inhibit the tumor growth, with skin having the lowest IC50 (1.8 mg/mL versus 8 mg/mL of pulp). These results suggest that RTG are potent anti-mutagens that protect DNA from oxidative damage as well as being cytotoxic toward the HL60 tumor cell line.

Antigenotoxicity, cytotoxicity, and apoptosis induction by apigenin, bisabolol, and protocatechuic acid.

Medicinal plants represent an important resource in new drug research. Antioxidant properties of plants can help to scavenge reactive oxygen species. The objective of this work was to evaluate the genotoxic, antigenotoxic, tumoricidal, and apoptotic effect of some major phenols (apigenin, bisabolol, and protocatechuic acid) from two medicinal plants, Matricaria chamomilla and Uncaria tomentosa. The wing spot test of Drosophila melanogaster was used to evaluate the genotoxicity and antigenotoxicity of the three phenols. The human model of HL-60 leukemia cells was used for the assessment of the cytotoxic effect, growth, and cellular viability. The apoptotic effect was evaluated using a DNA fragmentation assay based on the formation of internucleosomal units. Protocatechuic acid (0.25 and 1 mM), apigenin (0.46 and 1.85 mM), and bisabolol (0.56 and 2.24 mM) did not exhibit any genotoxic effect. The three phenols showed an antigenotoxic effect against the hydrogen peroxide effect and also exhibited tumoricidal activity. Apigenin (2.24-35.96 mM) showed a lower 50% inhibitory concentration (0.75 and 3.87 mM for the trypan blue test and WST-8 colorimetric assay, respectively) than bisabolol and protocatechuic acid. These phenolics also induced apoptosis in HL-60 leukemia cells. This study suggests that the antioxidant activity of Chamomilla and Uncaria could be partially responsible of their beneficial activity.