Discovery of a new class of potent pyrrolo[3,4-c]quinoline-1,3-diones based inhibitors of human dihydroorotate dehydrogenase: Synthesis, pharmacological and toxicological evaluation.

Twenty N-substituted pyrrolo[3,4-c]quinoline-1,3-diones 3a-t were synthesized by a cyclization reaction of Pfitzinger's quinoline ester precursor with the selected aromatic, heteroaromatic and aliphatic amines. The structures of all derivatives were confirmed by IR, 1H NMR, 13C NMR and HRMS spectra, while their purity was determined using HPLC techniques. Almost all compounds were identified as a new class ofpotent inhibitors against hDHODH among which 3a and 3t were the most active ones with the same IC50 values of 0.11 µM, about seven times better than reference drug leflunomide. These two derivatives also exhibited very low cytotoxic effects toward healthy HaCaT cells and the optimal lipophilic properties with logP value of 1.12 and 2.07 respectively, obtained experimentally at physiological pH. We further evaluated the comparative differences in toxicological impact of the three most active compounds 3a, 3n and 3t and reference drug leflunomide. The rats were divided into five groups and were treated intraperitoneally, control group (group I) with a single dose of leflunomide (20 mg/kg) group II and the other three groups, III, IV and V were treated with 3a, 3n and 3t (20 mg/kg bw) separately. The investigation was performed in liver, kidney and blood by examining serum biochemical parameters and parameters of oxidative stress.

Optimization and validation of an HS-SPME/GC-MS method for determining volatile organic compounds in dry-cured ham.

The formation of volatile organic compounds (VOCs) in dry-cured ham is a result of different biochemical and enzymatic processes. Moreover, accurately quantifying these VOCs is challenging since ham is a complex matrix, which contains compounds from various chemical families and a wide range of volatilities of different molecular masses. In this study, we systematically optimized and validated an analytical method for quantifying VOCs in dry-cured ham using headspace solid phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Optimal SPME conditions were determined through both an experimental procedure (one-factor-at-a-time) and response surface methodology (RSM), revealing that a 60-min equilibration at 70°C, a 60-min extraction at the same temperature, and a 4-min desorption time at 250°C provided the most favorable results. To enhance quantitation, twelve multiple internal standards (ISTDs) were employed to address and improve the quantitation of the 12 VOCs. Method validation covered aspects of linearity, limits of detection (LOD: 0.03-1.13 mg kg-1), limits of quantitation (LOQ: 0.09-3.41 mg kg-1), and working ranges (0.01-19.1 mg kg-1). The practical application of this optimized method was demonstrated by analyzing dry-cured ham samples (n = 4), sourced from the Slovenian market. The initial statistical evaluation indicates that different types of dry-cured hams can be differentiated (with an 83.1% of accuracy) according to their aromatic profile. However, a larger sample size would be required to provide a more comprehensive assessment.

Putative application of Najas marina L. extracts as a source of bioactive compounds and their antioxidant, antimicrobial, antibiofilm, and genotoxic properties.

In this research paper, the total phenols (TP), flavonoids (TF), and tannins (TT) content in the acetone and ethyl acetate extracts of Najas marina L. and the identification and quantification of phenolic acids and flavonoids from the ethyl acetate extract were performed. Antioxidant, antimicrobial, and antibiofilm properties of the mentioned extracts were investigated in vitro. The genotoxic potential was analyzed in cultured human peripheral blood lymphocytes (PBL). The TP and TF content was higher in the ethyl acetate extract, dominated by quercetin (172.4 µg mg-1) and ferulic acid (22.74 µg mg-1), while the TT content was slightly higher in the acetone extract. Both extracts tested showed limited antioxidant effects compared to ascorbic acid. The strongest antibacterial activity was observed with Gram-positive bacteria, particularly Staphylococcus aureus (MIC and MMC at 0.31 mg ml-1) and S. aureus ATCC 25923 (MIC at <0.02 mg ml-1), while antifungal activity was limited. Both extracts tested showed better activity on preformed biofilms. Acetone extract had no genotoxic activity but showed significant genoprotective activity against mitomycin C-induced DNA damage in cultured PBLs. Results of our research demonstrate the potential for the development of plant-based antibacterial and biofilm agents.

Green One-Pot Synthesis of Coumarin-Hydroxybenzohydrazide Hybrids and Their Antioxidant Potency.

Compounds from the plant world that possess antioxidant abilities are of special importance for the food and pharmaceutical industry. Coumarins are a large, widely distributed group of natural compounds, usually found in plants, often with good antioxidant capacity. The coumarin-hydroxybenzohydrazide derivatives were synthesized using a green, one-pot protocol. This procedure includes the use of an environmentally benign mixture (vinegar and ethanol) as a catalyst and solvent, as well as very easy isolation of the desired products. The obtained compounds were structurally characterized by IR and NMR spectroscopy. The purity of all compounds was determined by HPLC and by elemental microanalysis. In addition, these compounds were evaluated for their in vitro antioxidant activity. Mechanisms of antioxidative activity were theoretically investigated by the density functional theory approach and the calculated values of various thermodynamic parameters, such as bond dissociation enthalpy, proton affinity, frontier molecular orbitals, and ionization potential. In silico calculations indicated that hydrogen atom transfer and sequential proton loss-electron transfer reaction mechanisms are probable, in non-polar and polar solvents respectively. Additionally, it was found that the single-electron transfer followed by proton transfer was not an operative mechanism in either solvent. The conducted tests indicate the excellent antioxidant activity, as well as the low potential toxicity, of the investigated compounds, which makes them good candidates for potential use in food chemistry.

Genotoxic and cytotoxic properties of two medical plants (Teucrium arduini L.and Teucrium flavum L.) in relation to their polyphenolic contents.

A large number of species belonging to the genus Teucrium are used in pharmacy and traditional medicine for the treatment of different diseases. This study aimed to evaluate the polyphenolic composition as well as genotoxic and cytotoxic effects of methanolic extracts from T. arduini and T. flavum, two native species found in Montenegro. We determined the total phenolic and flavonoid contents of these plants using spectrophotometric methods; the qualitative content of polyphenolic compounds was investigated by high-performance liquid chromatography (HPLC). Genotoxicity in cultured human lymphocytes was measured in the cytokinesis-block micronucleus assay (CBMN) and comet assay in the range between 125 and 1000 µg/mL. Cytotoxicity was assessed by the MTT viability assay in normal human MRC-5 fibroblasts and MDA-MB-231 breast carcinoma cells. The content of total phenolics and flavonoids in T. arduini extract was higher than in T. flavum (200.35 mg GA/g vs. 171.08 mg GA/g; 96.32 mg RU/g vs. 78.14 mg RU/g). The polyphenolic composition of both extracts was qualitatively similar and eight phenol compounds were identified. The most commonly present phenol was caffeic acid and among four flavonoids, the most common was quercetin. Both plant extracts were genotoxic in both the CBMN and comet assays at concentrations of 250, 500 and 1000 µg/mL. After 72 h of exposure, the extracts of T. arduini and T. flavum were found to induce cytotoxicity in MRC-5 fibroblasts but not in MDA-MB-231 breast cancer cells. The results suggest that the constituents of both plant species are genotoxic and cytotoxic, therefore these extracts warrant additional evaluation to be safely applied in humans.

Response surface methodology and artificial neural network approach for the optimization of ultrasound-assisted extraction of polyphenols from garlic.

This paper aimed to establish the optimal conditions for ultrasound-assisted extraction of polyphenols from domestic garlic (Allium sativum L.) using response surface methodology (RSM) and artificial neural network (ANN) approach. A 4-factor-3-level central composite design was used to optimize ultrasound-assisted extraction (UAE) to obtain a maximum yield of target responses. Maximum values of the two output parameters: 19.498 mg GAE/g fresh weight of sample total phenolic content and 1.422 mg RUT/g fresh weight of sample total flavonoid content were obtained under optimum extraction conditions: 13.50 min X1, 59.00 °C X2, 71.00% X3 and 20.00 mL/g X4. Root mean square error for training, validation, and testing were 0.0209, 3.6819 and 1.8341, respectively. The correlation coefficient between experimentally obtained total phenolic content and total flavonoid content and values predicted by ANN were 0.9998 for training, 0.9733 for validation, and 0.9821 for testing, indicating the good predictive ability of the model. The ANN model had a higher prediction efficiency than the RSM model. Hence, RSM can demonstrate the interaction effects of basic inherent UAE parameters on target responses, whereas ANN can reliably model the UAE process with better predictive and estimation capabilities.

Analysis of Wild Raspberries (Rubus idaeus L.): Optimization of the Ultrasonic-Assisted Extraction of Phenolics and a New Insight in Phenolics Bioaccessibility.

A simple and efficient ultrasonic-assisted extraction (UAE) technique was developed in order to find optimal conditions for the extraction of total phenolic compounds, flavonoids and anthocyanins in wild raspberry (Rubus idaeus L.) fruits. Several extraction variables, including methanol composition (v/v, %), solid-solvent ratio (g/mL), time (min) and extraction temperature (°C) were optimized using response surface methodology (RSM). Under optimal conditions for extraction, the total phenolics were found in the concentration of 383 mg GAE/100 g of fresh fruit weight, while HPLC-PDA analysis of the optimized extract showed the presence of cyanidin-3-glucoside, cyanidin-3-sophoroside, catechin, gallic and ellagic acid. The experimental values of DPPH and ABTS radical scavenging activities were 29.0 and 39.5 µmol Trolox/g of fresh fruit weight, respectively. In vitro simulated gastrointestinal digestion showed great raspberry phenolics stability. Our study assessed the bioaccessible phenolics in wild raspberry fruits and showed optimal conditions for the effective extraction of bioactive compounds for their analysis.

Statistical optimization of an RP-HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities.

An isocratic RP-HPLC method for the separation and identification of selected flavonoids (quercetin, rutin, luteolin-7-O-glucoside, kaempferol and kaempferol-3-O-glucoside) in commercial berry juices (blackcurrant, blueberry, red raspberry and cherry) was developed with the aid of central composite design and response surface methodology. The optimal separation conditions were a mobile phase of 85:15 (% v/v) water-acetonitrile, pH 2.8 (adjusted with formic acid), flow rate 0.5 mL min-1 and column temperature 35°C. The obtained levels of bioflavonoids (mg per 100 mL of juice) were as follows: for quercetin, ca. 0.21-5.12; for kaempferol, ca. 0.05-1.2; for rutin, ca. 0.4-6.5; for luteolin-7-O-glucoside, ca. 5.6-10.2; and for kaempferol-3-O-glucoside, ca. 0.02-0.12. These are considerably lower than the values in fresh fruits. Total phenolic, flavonoid and anthocyanin contents were determined spectrophotometrically. Total flavonoid content varied as follows: blackcurrant > blueberry > red raspberry > cherry. The antioxidant activity of juice extracts (DPPH and ABTS methods) expressed as IC50 values varied from 8.56 to 14.05 mg L-1 . These values are ~2.5-3 times lower than quercetin, ascorbic acid and Trolox®, but compared with rutin and butylhydroxytoluene, berries show similar or better antioxidant activity by both the DPPH and ABTS methods.

Polyphenolic contents of Teucrium polium L. and Teucrium scordium L. associated with their protective effects against MMC-induced chromosomal damage in cultured human peripheral blood lymphocytes.

Teucrium species have been used in traditional medicine for treatment of different diseases. The aim of this study was to investigate polyphenolic contents by high-performance liquid chromatography (HPLC), and the genotoxic effect of methanolic extracts of Teucrium polium and Teucrium scordium using the cytokinesis-block micronucleus (CBMN) assay on human peripheral blood lymphocytes (PBLs) from healthy donors. The HPLC  analysis  showed that extracts consist of phenolic acid (gallic, vanillic, caefic, chlorogenic, p-coumaric, sinapic) and flavonoids (catechin, rutin, myricetin, luteolin, quercetin and apigenin). Cultures were treated with extracts of both plants separately and in combinations with mitomycin C (MMC). In separate treatments, both herbal extracts significantly induced micronucleus (MN) frequency only at the highest concentrations. All concentrations of T. scordium , except the lowest, and all concentrations of T. polium extracts in combined treatment with MMC significantly reduced the frequency of MN. The extract of T. polium did not significantly aefct the nuclear division index (NDI), whereas T. scordium in higher concentrations, separately and in combined treatment with MMC, significantly decreased the NDI value. Our results suggest that both herbal extracts in combination with MMC have antimutagenic (T. polium) and proapoptotic effects (T. scordium), which indicates their protective effects in PBLs.

Impact of the toxicity of Cylindrospermopsis raciborskii (Woloszynska) Seenayya & Subba Raju on laboratory rats in vivo.

In vivo laboratory studies of toxicity were performed on Wistar rats using a methanol extract produced by the natural population of Cylindrospermopsis raciborskii (abundance of 2.13 × 105 trichomes mL-1) collected at Aleksandrovac Lake (Serbia). HPLC analysis showed that the extract contains 6.65 µg cylindrospermopsin (CYN) mg-1. The rats were killed 24 or 72 h after a single intraperitoneal injection of C. raciborskii extract in concentrations of 1500, 3000, 6000 and 12,000 µg kg-1 body weight (bw) and an equivalent amount of CYN as present in the highest dose of the extract (79.80 µg CYN kg-1 bw). The genotoxic effect on the livers treated with C. raciborskii was evaluated using comet assay and potential induction of oxidative stress as the toxicity mechanism associated with the presence of CYN in extract. The results from the analyses of DNA damage in the comet tail length, tail moment and percentage of DNA in the tail in the liver indicated that administration of extract and CYN present statistically significant difference when compared with the negative control group. Although an increase in the frequency of selected parameters induced by the CYN was observed in the liver, this damage was less than the damage resulting from the administration of the highest dose of extract. The changes in the biochemical parameters of the hepatic damage showed that the application of single doses of the extract and CYN did not cause serious liver damage in rats. The extract and CYN significantly increased oxidative stress in rats' liver after a single exposure.

Evaluation of matrix effect in determination of some bioflavonoids in food samples by LC-MS/MS method.

In the present work the LC-MS/MS method with solid phase extraction for simultaneous determination of bioflavonoids rutin, quercetin, hesperidin, hesperetin and kaempferol in some food samples (red onion, orange peel and honey) was developed and the matrix effect accompanying this determination was quantified. The matrix effect evaluated using a postextraction addition method was found to be negative in the range -44 to -0.5%, indicating ionization suppression and strongly depended on bioflavonoid concentration. The observed matrix effect was explained taking into account the co-elution of phenolic acids, in terms of their acid-base and hydrophilic properties. The efficacy of extraction expressed as the absolute recoveries of flavonoids were 88-96%, indicating very good efficiency of extraction. The extracts of food samples obtained either by Soxhlet or ultrasonic extraction were analyzed for bioflavonoid content by the LC-MS/MS method in selected reaction monitoring mode using a triple quadrupole detector and standard addition method, which was found to be the most suitable calibration approach for these samples. The optimized separation was achieved on a Phenomenex Gemini C18 column with gradient elution and mobile phase composition A: 2% acetic acid in water and B: acetonitrile. R(s) values were in the range from 1.3 to 3.1, indicating good selectivity of the method. The obtained results (mg/100g fresh weight) for different bioflavonids were for rutin 0.16, for quercetin in the range 0.65-56, for hesperidin 0.016-24, for hesperetin 0.0068-36.4 and for kaempferol 0.14-1.63 and generally show good agreement with published data. Low detection limits (0.014-0.063 µg/mL) were obtained with acceptable recoveries (86-114%). Total time of analysis was less than 40 min, therefore the proposed method represents significant improvement over existing methods.

Optimization of separation and determination of moxifloxacin and its related substances by RP-HPLC.

A RP-HPLC method for the separation and determination of impurities of moxifloxacin, in its pharmaceutical forms as well as moxifloxacin degradation products, was developed with the aid of DryLab software and chemometric (response surface) approach. The separation of four synthesis-related impurities was achieved on a Waters C(18) XTerra column using a mobile phase of (water+triethylamine (2%, v/v)): acetonitrile=90:10 (v/v%); the pH of water phase being adjusted with phosphoric acid to 6.0. Flow rate of the mobile phase was 1.5 ml/min and UV detection at 290 nm was employed. The column was thermostated at 45 degrees C. The resolution between the two least resolved impurity peaks was in average, R(s,min) > 1.5. Method validation parameters indicate linear dynamic range 0.2-2.0 microg/ml with LOQ ca. 0.20 microg/ml and LOD ca. 0.05 microg/ml for all analytes. The method was applied for the impurities determination in drug tablets and infusion (Avelox), Bayer AG) and for degradation products determination in a stability study of moxifloxacin. The impurity content in the tablets and infusion was quantified as 0.1% of total drug. Two degradation products were noted under hydrolytic conditions. The method can also be used for rapid and accurate quantification of moxifloxacin hydrochloride in its tablets during stability testing.