Chemical, Antioxidant, and Antimicrobial Properties of the Peel and Male Flower By-Products of Four Varieties of Punica granatum L. Cultivated in the Marche Region for Their Use in Cosmetic Products.

We are now seeing an increase in the production of agri-food waste, which is an essential resource for the recovery of bioactive compounds that may be employed as innovative natural ingredients in cosmetics. To date, the approach to cosmetics preservation has seen a significant shift in the search for biological components that give healthier alternatives for customers and help businesses operate in an environmentally friendly manner. To achieve this goal, we studied pomegranate extracts using the peel and, for the first time, extracts from the male flowers of a wide pomegranate variety cultivated in the Marche region, specifically, the Wonderful, Mollar de Elche, Parfianka, and less-studied G1 varieties. We studied the phenol compounds profile, antioxidant capacity, antimicrobial activity, and cell viability of the obtained pomegranate extracts. The identification and quantification of phenol compounds belonging to different classes, such as hydrolysable tannins, hydroxybenzoic acid, hydroxycinnamic acid, dihydroflavonol, gallocatechin, and anthocyanins, were performed using UPLC-ESI-MS/MS. Punicalagin isomers and punicalin resulted in the most abundant polyphenols found in the peel and male flower extracts. Mollar de Elche 2020 peel extract revealed a high concentration of punicalagin A and B (7206.4 mg/kg and 5812.9), while the content of gallic acid revealed high results in the G1 and Parfianka varieties. All extracts were spectrophotometrically analysed to determine their total phenol content (TPC) using the Folin-Ciocalteu method and their antioxidant capacity (AC). In terms of the total phenol obtained by the Folin-Ciocalteu colorimetric method, Mollar de Elche 2020 extracts reported the highest TPC content of 12.341 µmol GAE/g. Results revealed that the Mollar de Elche and Wonderful 2020 peel extracts demonstrated the highest TPC and AC. Furthermore, AC results indicated that the peel extracts displayed higher AC than the male flower extract due to the high punicalagin content detected by UPLC analysis. The antimicrobial activity testing revealed that the Wonderful and G1 2020 peel extracts resulted active against Escherichia coli, while all extracts exhibited promising anticandidal activity. Additionally, the cytocompatibility was evaluated in keratinocytes HaCaT cells by testing concentrations of pomegranate extracts ranging from 0.15 to 5.00 mg/mL. Extracts were non-toxic for the cells in the tested concentration range. The acquired results may help exploit pomegranate agri-food waste products provided by the Marche region's short supply chain for their use as an antimicrobial and antioxidant booster in the formulation of cosmetic products.

Circulating Extracellular Vesicles Contain Liver-Derived RNA Species as Indicators of Severe Cholestasis-Induced Early Liver Fibrosis in Mice.

Aims: Biliary diseases represent around 10% of all chronic liver diseases and affect both adults and children. Currently available biochemical tests detect cholestasis but not early liver fibrosis. Circulating extracellular vesicles (EVs) provide a noninvasive, real-time molecular snapshot of the injured organ. We thus aimed at searching for a panel of EV-based biomarkers for cholestasis-induced early liver fibrosis using mouse models. Results: Progressive and detectable histological evidence of collagen deposition and liver fibrosis was observed from day 8 after bile duct ligation (BDL) in mice. Whole transcriptome and small RNA sequencing analyses of circulating EVs revealed differentially enriched RNA species after BDL versus sham controls. Unsupervised hierarchical clustering identified a signature that allowed for discrimination between BDL and controls. In particular, 151 microRNAs (miRNAs) enriched in BDL-derived EVs were identified, of which 66 were conserved in humans. The liver was an important source of circulating EVs in BDL animals as evidenced by the enrichment of several hepatic mRNAs, such as Albumin and Haptoglobin. Interestingly, among experimentally validated miRNAs, miR192-5p, miR194-5p, miR22-3p, and miR29a-3p showed similar enrichment patterns also in EVs derived from 3,5-diethoxycarboncyl-1,4-dihydrocollidine-treated (drug-induced severe cholestasis) but not in mice with mild phenotype or non-cholestatic liver fibrosis. Innovation: A panel of mRNAs and miRNAs contained in circulating EVs, when combined, indicates hepatic damage and fibrosis in mice and represents promising biomarkers for human severe cholestasis-induced liver fibrosis. Conclusion: Analysis of EV-based miRNAs, in combination with hepatic injury RNA markers, can detect early cholestatic liver injury and fibrosis in mice. Antioxid. Redox Signal. 36, 480-504.

Ageratum conyzoides L. inhibits 5-alpha-reductase gene expression in human prostate cells and reduces symptoms of benign prostatic hypertrophy in otherwise healthy men in a double blind randomized placebo controlled clinical study.

A double-blind, randomized, placebo-controlled clinical trial assessed the efficacy and safety of Ageratum conyzoides in treating benign prostatic hypertrophy (BPH). In this study, 109 men with medically diagnosed BPH, aged 41-76 years, were administered the investigational product, A. conyzoides extract at a dose of 250 mg/d or placebo, q.d. for 12 weeks. The primary outcome measures were the International Prostate Symptom Score (IPSS), daily urinary frequency and safety evaluations. The secondary outcome measures were testosterone, dihydrotestosterone, oestradiol, sex hormone binding globulin (SHBG), Dehydroepiandrosterone sulfate (DHEA-S) and cortisol levels, and prostate specific antigen (PSA), lipids, blood glucose, the Aging Male's Symptom (AMS) Score and sexual function assessed by Derogatis Interview for Sexual Functioning-Self Report (DISF-SR). The effect of A. conyzoides L extract on gene expression of 5-alpha-reductase in human prostate cells was also investigated to elucidate a potential mechanism of action. The clinical study, showed a significant reduction in total IPSS score (p < 0.01) and day- and night-time urinary frequency (P < 0.01) over time after treatment with A. conyzoides. Steroid hormones, SHBG, PSA levels, lipids, and blood glucose remained within healthy reference range in both groups. There were no changes in AMS or DISF-SR in either group. Gene arrays demonstrated that A. conyzoides extract was effective in reducing the expression of mRNA coding for 5-alpha-reductase types 2 and 1 in human prostate epithelial cells. The overall results indicate that A. conyzoides may be an effective treatment for reducing symptoms of BPH in healthy men, in part, through inhibition of 5-alpha-reductase enzyme activity. © 2017 BioFactors, 43(6):789-800, 2017.

Comparison of the cytotoxic potential of cigarette smoke and electronic cigarette vapour extract on cultured myocardial cells.

BACKGROUND: Electronic cigarettes (ECs) have been marketed as an alternative-to-smoking habit. Besides chemical studies of the content of EC liquids or vapour, little research has been conducted on their in vitro effects. Smoking is an important risk factor for cardiovascular disease and cigarette smoke (CS) has well-established cytotoxic effects on myocardial cells. The purpose of this study was to evaluate the cytotoxic potential of the vapour of 20 EC liquid samples and a "base" liquid sample (50% glycerol and 50% propylene glycol, with no nicotine or flavourings) on cultured myocardial cells. Included were 4 samples produced by using cured tobacco leaves in order to extract the tobacco flavour. METHODS: Cytotoxicity was tested according to the ISO 10993-5 standard. By activating an EC device at 3.7 volts (6.2 watts-all samples, including the "base" liquid) and at 4.5 volts (9.2 watts-four randomly selected samples), 200 mg of liquid evaporated and was extracted in 20 mL of culture medium. Cigarette smoke (CS) extract from three tobacco cigarettes was produced according to ISO 3308 method (2 s puffs of 35 mL volume, one puff every 60 s). The extracts, undiluted (100%) and in four dilutions (50%, 25%, 12.5%, and 6.25%), were applied to myocardial cells (H9c2); percent-viability was measured after 24 h incubation. According to ISO 10993-5, viability of <70% was considered cytotoxic. RESULTS: CS extract was cytotoxic at extract concentrations >6.25% (viability: 76.9 ± 2.0% at 6.25%, 38.2 ± 0.5% at 12.5%, 3.1 ± 0.2% at 25%, 5.2 ± 0.8% at 50%, and 3.9 ± 0.2% at 100% extract concentration). Three EC extracts (produced by tobacco leaves) were cytotoxic at 100% and 50% extract concentrations (viability range: 2.2%-39.1% and 7.4%-66.9% respectively) and one ("Cinnamon-Cookies" flavour) was cytotoxic at 100% concentration only (viability: 64.8 ± 2.5%). Inhibitory concentration 50 was >3 times lower in CS extract compared to the worst-performing EC vapour extract. For EC extracts produced by high-voltage and energy, viability was reduced but no sample was cytotoxic according to ISO 10993-5 definition. Vapour produced by the "base" liquid was not cytotoxic at any extract concentration. Cell survival was not associated with nicotine concentration of EC liquids. CONCLUSIONS: This study indicates that some EC samples have cytotoxic properties on cultured cardiomyoblasts, associated with the production process and materials used in flavourings. However, all EC vapour extracts were significantly less cytotoxic compared to CS extract.

Cytotoxicity evaluation of electronic cigarette vapor extract on cultured mammalian fibroblasts (ClearStream-LIFE): comparison with tobacco cigarette smoke extract.

CONTEXT: Electronic cigarettes (ECs) are used as alternatives to smoking; however, data on their cytotoxic potential are scarce. OBJECTIVE: To evaluate the cytotoxic potential of 21 EC liquids compared to the effects of cigarette smoke (CS). METHODS: Cytotoxicity was evaluated according to UNI EN ISO 10993-5 standard. By activating an EC device, 200 mg of liquid was evaporated and was extracted in 20 ml of culture medium. CS extract from one cigarette was also produced. The extracts, undiluted (100%) and in five dilutions (50%, 25%, 12.5%, 6.25% and 3.125%), were applied to cultured murine fibroblasts (3T3), and viability was measured after 24-hour incubation by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Viability of less than 70% was considered cytotoxic. RESULTS: CS extract showed cytotoxic effects at extract concentrations above 12.5% (viability: 89.1 ± 3.5% at 3.125%, 77.8 ± 1.8% at 6.25%, 72.8 ± 9.7% at 12.5%, 5.9 ± 0.9% at 25%, 9.4 ± 5.3% at 50% and 5.7 ± 0.7% at 100% extract concentration). Range of fibroblast viability for EC vapor extracts was 88.5-117.8% at 3.125%, 86.4-115.3% at 6.25%, 85.8-111.7% at 12.5%, 78.1-106.2% at 25%, 79.0-103.7% at 50% and 51.0-102.2% at 100% extract concentration. One vapor extract was cytotoxic at 100% extract concentration only (viability: 51.0 ± 2.6%). However, even for that liquid, viability was 795% higher relative to CS extract. CONCLUSIONS: This study indicates that EC vapor is significantly less cytotoxic compared tobacco CS. These results should be validated by clinical studies.

Decontamination of tetramethylammonium hydroxide (TMAH) splashes: promising results with Diphoterine in vitro.

Tetramethylammonium hydroxide (TMAH), used in microelectronic industries and research and development, has both corrosive properties and systemic toxicity. Two fatal TMAH occupational exposure cases have been published. Studies comparing initial TMAH decontamination with Diphoterine versus tap water were performed: an in vitro pH titration study and an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in vitro cytotoxicity cell viability assay. For pH normalization, 17 times more tap water than Diphoterine was required. In the cytotoxicity test, two-thirds of the cells remained viable after Diphoterine washing, compared with only one-third after tap water washing (p < .001). Diphoterine washing is a promising TMAH decontamination method.