Use of Secondary Metabolites Profiling and Antioxidant Activity to Unravel the Differences between Two Species of Nettle.

In recent years, the interest in natural remedies has increased, so it is important to analyze the plants widely distributed in nature but whose composition is little known. The main objective of the present work is to obtain information based on the profiles of secondary metabolites and antioxidant activity in Lamium album, a very widespread but little studied plant, with the aim of revealing the differences compared to Urtica dioica. First, the optimization of enzymatic extraction assisted by ultrasound was carried out by the Box-Behnken method. The optimized parameters were: concentration of the enzyme-3.3% cellulase, temperature-55 °C, and the extraction time-40.00 min. The efficiency was estimated based on the content of iridoids, the main class of secondary metabolites from Lamium album. Second, the secondary metabolites profiles of the nettle extracts were obtained by thin-layer chromatography using both normal and reverse phases and by RP-UHPLC. The antioxidant activity was evaluated using DPPH and ABTS+ radicals. The obtained results revealed significant differences between the two nettle species, both in terms of the phytochemical compounds, as well as the antioxidant activity, confirming the fact that Lamium album has a high potential to be used in phytomedicine.

Sweeteners' Influence on In Vitro α-Glucosidase Inhibitory Activity, Cytotoxicity, Stability and In Vivo Bioavailability of the Anthocyanins from Lingonberry Jams.

Several lines of evidence demonstrate the multiple health-promoting properties of anthocyanins, but little is known regarding the bioavailability of these phytochemicals. Therefore, the stability during storage and bioavailability of anthocyanins from lingonberries jams were determined by HPLC, together with the impact of used sweeteners on their adsorption. Further, the in vitro α-glucosidase inhibition using spectrophotometric methods and cytotoxicity determined on normal and colon cancer cells were communicated. The content of anthocyanins was significantly decreased during storage in coconut sugar-based jam, but was best preserved in jam with fructose and stevia. Fructose and stevia-based jams showed the highest inhibition activity upon α-glucosidase. Lingonberry jams showed no cytotoxic effects on normal cells, but at low concentration reduced the tumor cells viability. Anthocyanins were still detectable in rats' blood streams after 24 h, showing a prolonged bioavailability in rats. This study brings important results that will enable the development of functional food products.

Estimation of degradation kinetics of bioactive compounds in several lingonberry jams as affected by different sweeteners and storage conditions.

Lingonberry fruits are considered to play an important role in nutrition, as they comprise a variety of health-promoting components. Because of lingonberries seasonal availability and also due to their rapid degradation, their stability during processing is a continuous challenge for the food industry. Lingonberries are ideal fruits in making jam due to their natural deep reddish color, but recently, increased demand for low-calorie jams with alternative sweeteners has gained special attention. In this line, the objective of this study was to monitor the changes in anthocyanins, vitamin C, total phenolics, total reducing sugars and antioxidant capacity of several lingonberry jams formulated with different sweeteners (sucrose, fructose, erythritol, brown sugar, coconut sugar, stevia, saccharin). Due to the fact that storage conditions are important factors for jam quality, the jams were stored for 180 days at 4 °C and 25 °C (both under light and dark conditions). The rate constants (k) and the half time values (t1/2) of the degradation processes were determined and degradation kinetics was studied. For all analyzed conditions, first-order reaction kinetics was established for the degradation process of anthocyanins, whereas a second-order kinetic model described the degradation of the other compounds. Kinetic parameters showed that the stability of the studied compounds was highly influenced by the type of sweetener used in jam formulation. Total phenolics and antioxidants were best preserved in the presence of stevia, coconut sugar and fructose, whereas a destabilizing effect of erythritol on vitamin C and anthocyanins content during storage was observed. Among all the studied compounds, anthocyanins presented the fastest degradation, regardless storage conditions. The stability of studied compounds was higher at lower storage temperature (4 °C), while increasing the temperature at 25 °C and exposure to light determined higher rate of the degradation processes. The results provide useful information for understanding some bioactive compounds degradation in real foods, contributing to the development of new food products and providing information of commercial importance.

Effect of in vitro simulated gastrointestinal digestion on some nutritional characteristics of several dried fruits.

To provide new information on potential health benefits of dried fruits, the effects of in vitro digestion on minerals, total phenolics, total sugars and the antioxidant capacities were evaluated. For the first time, the bioaccessibility of these compounds during in vitro digestion was assessed by multivariate statistical analysis. Although the amount of all minerals decreased after digestion, moderate bioaccessibility was found, excepting Zn. The highest bioaccessibility of phenolics was obtained in prunes and the lowest in dates and cranberries. Total sugars content increased after in vitro digestion of dates, raisins and coconut, but decreased for cranberries, prunes and banana. The in vitro digestion led to an increase in the antioxidant capacity for the majority of dried fruits. The similarities/dis-similarities in pattern of analyzed parameters during digestion was revealed by heat map. Two-way joining cluster and principal component analysis were used to highlight the most relevant parameters in each digestion phase.

Sweeteners from Different Lingonberry Jams Influence on Bioaccessibility of Vitamin C, Anthocyanins and Antioxidant Capacity under In Vitro Gastrointestinal Digestion.

Lingonberries are considered anot fully exploited major source of antioxidants. Their health benefits are closely linked to their bioavailability. Due to growing health concerns, consumers are looking for jams prepared with sweeteners other than white sugar, which could be a good alternative to meet their needs. The aim of this research was to evaluate the influence of sucrose, fructose, erythritol, brown sugar, coconut sugar, stevia and saccharine on the bioaccessibility of vitamin C, anthocyanins andthe antioxidant capacity of lingonberry jams under in vitro gastrointestinal digestion. The vitamin C, total anthocyanin content and antioxidant capacity measured by ABTS and FRAP assays were determined spectrophotometrically. Individual anthocyanins were determined by high performance liquid chromatography. All analyzed compounds were highly altered during gastrointestinal digestion and this effect was more visible in the case of the anthocyanins. Antioxidant capacity decreased after gastric digestion, but after the gastrointestinal step, radical scavenging capacity increased, while reducing power decreased. Vitamin C bioaccessibility was negatively affected by coconut sugar addition, while stevia addition showed a protective effect. Fructose and sucrose increased the total anthocyanin stability during the intestinal phase. Stevia, fructose and coconut sugar exhibited high protection of the antioxidant capacity of lingonberry jams during digestion.

The Influence of in Vitro Gastrointestinal Digestion of Brassica oleracea Florets on the Antioxidant Activity and Chlorophyll, Carotenoid and Phenolic Content.

Brassica oleracea L. var. Italica is known to contain a wide variety of antioxidants and due to the protection against various diseases its consumption has been increasing over the years. Thus, knowledge of the changes that occur during the digestion process is of great interest. The aim of this study was to investigate the influence of in vitro gastrointestinal digestion of broccoli on antioxidant activity and on the chlorophyll, carotenoid and phenolic content. First, the ultrasound-assisted extraction of bioactive compounds was optimized and the kinetic model was evaluated. Then, the broccoli was subjected to a static simulated digestion. The antioxidant activity was monitored by ABTS [2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate)] assay and the contents of target compounds were investigated by UV-Vis spectrophotometry and thin-layer chromatography. The optimum conditions were: solvent-ethanol; time-20 min and temperature-30 °C, and a second order kinetic model was found to describe the mechanism of extraction. The antioxidant activity and carotenoid, chlorophyll and total phenolic content was significantly decreased after simulated gastric and intestinal digestion. The gastric digestion considerably decreased carotenoid and chlorophyll content, meanwhile the intestinal digestion significantly decreased the total phenolic content (TPC). The antioxidant activity was equally affected by both gastric and intestinal digestion.

Chemo-mapping and biochemical-modulatory and antioxidant/prooxidant effect of Galium verum extract during acute restraint and dark stress in female rats.

Galium verum is a well-known medicinal plant which is used in various pathologies. G. verum extracts are characterized here using chromatography, where among the rich pool of phenolic acids of flavonoids two known anti-stress modulators, chlorogenic acid and rutin are identified in high quantities. Additionally, the extracts are characterized using a series of in vitro assays (EPR, DPPH, TPC and TEAC). Considering the chemical findings, the potential beneficial effects of the G. verum extract are explored here in a living organism exposed to stress induced oxidative damages. Thus, the biochemical-modulatory and antioxidant roles of two doses of G. verum extract are examined in animals exposed to acute restraint and dark stress (S). The animals were divided in groups [control, S, SG1 (exposed to 25 mg G. verum extract), SG2 (50 mg extract)]. Increased levels of lipid peroxidation (TBARS from 4.43 to 8.06 nmol/mL), corticosterone from 0.43 to 1.96 µg/dL and epinephrine from 44.43 to 126.7 µg/mL, as well as decreased antioxidant enzymes activities (SOD/CAT) were observed in the S group. The G. verum extract afforded a near-normal equilibrium within the biochemical parameters of animals exposed to RS, by reducing oxidative damage (TBARS at a 3.73 nmol/mL; CS at 0.90 µg/dL; EP at 63.72 µg/mL) and by restoring the antioxidant balance.

Study of the Antioxidant Property Variation of Cornelian Cherry Fruits during Storage Using HPTLC and Spectrophotometric Assays.

Cornus species fruits are well known as a rich source of antioxidant compounds responsible for their diverse health benefits. The present study aims to investigate the variation of the total antioxidant capacity of Cornelian cherry (Cornus mas L.) fruits during storage, using high-performance thin-layer chromatography (HPTLC) and two spectrophotometric assays based on different mechanisms: the 2,2-azinobis(3-ethylbenzothiazolyne-6-sulphonic acid) radical cation (ABTS+∙) assay and the ferric reducing antioxidant power (FRAP) assay. The fruit extract was stored at room temperature (22°C) for 19 days. No major differences in the total antioxidant capacity were observed during this period, indicating that storage does not have any deleterious effect on the antioxidant properties of the investigated fruits extract. The antioxidant capacity varied between 12.91 and 12.83 µmol Trolox/g fruit as determined by the HPTLC method and from 36.13 to 33.93 µmol Trolox/g fruit as determined by the ABTS assay.

The Influence of the Variety, Vineyard, and Vintage on the Romanian White Wines Quality.

The wine is one of the most consumed drinks over the world, being subjected to falsification or adulteration regarding the variety, vintage, and geographical region. In this study, the influence of different characteristics of wines (type, production year, and origin) on the total phenolic content, total flavonoids content, antioxidant activity, total sugars content, pH, and 18O/16O isotopic ratio was investigated. The differentiation of selected wines on the basis of tested parameters was investigated using chemometric techniques, such as analysis of variance, cluster analysis, and principal component analysis. The experimental results are in agreement with other outcomes and allow concluding that variety and vineyard have the major influence on the studied parameters, but, also, statistical interaction effect between year and vineyard and year and variety is observed in some cases. The obtained results have demonstrated that these parameters together with chemometric techniques show a significant potential to be used for discrimination of white wines.

Total Phenolics, Total Anthocyanins, Antioxidant and Pro-oxidant Activity of Some Red Fruits Teas.

Fruits represent one of the main dietary sources of bioactive compounds. Due to their remarkable health benefits, many functional foods of fruit origin, including fruit teas, are present on the market and there is an increased interest regarding the investigation of their nutritional parameters and quality. The aims of our study were: 1) to determine the total phenolic content (TPC), total anthocyanins content (TAC), antioxidant activity (AA), the scavenging capacity (IC50), the pro-oxidant activity (Pro-ox) and Pro-Antidex of 12 commercially available red fruit teas, 2) to classify the analysed teas and 3) to evaluate the similarities between samples. The TPC was between 12.5 and 29.3 mg gallic acid equivalents (GAE)/g tea, the TAC varied between 2.6 and 5.6 mg cyanidin-3-glucoside (Cy-3-glu)/g tea and AA was in the range of 10.9-19.1 mg ascorbic acid equivalents (AAE)/g tea. The Pro-ox activity varied between 3.9 and 10.0 mg/mL tea extract and Pro-Antidex was between 3.3 and 7.3.

Analysis of total phenolic, flavonoids, anthocyanins and tannins content in Romanian red wines: prediction of antioxidant activities and classification of wines using artificial neural networks.

Wine is one of the most consumed beverages over the world containing large quantities of polyphenolic compounds. These compounds are responsible for quality of red wines, influencing the antioxidant activity, astringency, bitterness and colour, their composition in wine being influenced by the varieties, the vintage and the wineries. The aim of the present work is to build software instruments intended to work as data-mining tools for predicting valuable properties of wine and for revealing different wine classes. The developed ANNs are able to reveal the relationships between the concentration of total phenolic, flavonoids, anthocyanins, and tannins content, associated to the antioxidant activity, and the wine distinctive classes determined by the wine variety, harvesting year or winery. The presented ANNs proved to be reliable software tools for assessment or validation of the wine essential characteristics and authenticity and may be further used to establish a database of analytical characteristics of wines.

Determination of the floral origin of some Romanian honeys on the basis of physical and biochemical properties.

The aim of this study was to determine the physical and biochemical properties of some Romanian honeys in order to discriminate between their floral origins. The evaluated properties were total phenolic content, total protein content, total free amino acids content, color intensity (ABS(450)), pH, ash content, antioxidant activity. Twenty-six commercial honeys from six types of flowers (acacia, sunflower, forest, polyfloral, lime and Sea Buckthorn) were investigated. All samples showed considerable variations with reference to their properties. The properties values were in the range of approved limits (according to EU legislation). The total phenolic, total protein and total free amino acids contents and color intensity varied considerably. Similarly, forest honey had the highest antioxidant activity while the lowest was found in acacia honey. Correlation between the floral origin of honeys and the physical and biochemical properties, respectively, was observed. Moreover, this study demonstrates remarkable variation in DPPH scavenging activity and content of total phenols in honey, depending on its botanic source.

Application of a newly developed and validated high-performance thin-layer chromatographic method to control honey adulteration.

Honey is a saturated solution of sugars, used for a long time as a natural source of sugars and is an important ingredient in traditional medicine due to its antimicrobial, anti-inflammatory and antioxidant effects. Therefore, methods for quality control of honey and detection of its adulteration are very important. For this reason, the aim of this study is to develop and validate a new, simple and economical analytical method for detecting the adulteration of some Romanian honeys based on high-performance thin-layer chromatography (HPTLC) combined with image analysis. The proposed method involved the chromatographic separations of glucose, fructose and sucrose on silica gel HPTLC plates, developed twice with ethyl acetate-pyridine-water-acetic acid, 6:3:1:0.5 (v/v/v/v), followed by dipping in an immersion solution. The documentation of plates was performed using TLC visualization device and the images of plates were processed using a digital processor. The developed HPTLC method was validated for selectivity, linearity and range, LOD and LOQ, precision, robustness and accuracy. The method was then applied for quantitative determination of glucose, fructose and sucrose from different types of Romanian honeys, commercially available.

Degradation kinetics of anthocyanins from European cranberrybush (Viburnum opulus L.) fruit extracts. Effects of temperature, pH and storage solvent.

European cranberrybush (Viburnum opulus L.) fruits are well known for their biological properties, of which some are due to the presence of anthocyanins in the berries. Current literature provides little information concerning these fruits. The stability of anthocyanins from Viburnum opulus fruits, in aqueous and ethanolic extracts, stored under darkness for 7 days at different temperatures (2 °C, 37 °C and 75 °C) and pH values (pH = 3 and 7), was studied here. The lowest stability was showed by the anthocyanins from the water extract stored at 75 °C and pH = 7, with half-life and constant rate values of 1.98 h and 0.3488 h⁻¹, respectively. The results showed a good correlation between the total anthocyanin content (determined using the pH differential method) and the time of storage, with determination coefficients varying from R² = 0.9298 to R² = 0.9971. Results indicate that the storage degradation of anthocyanins followed first-order reaction kinetics under all investigated conditions.

Thin-layer chromatography with stationary phase gradient as a method for separation of water-soluble vitamins.

The group of hydrophilic vitamins play an important role in human health, and their lack or excess produces specific diseases. Therefore, the analysis of these compounds is indispensable for monitoring their content in pharmaceuticals and food in order to prevent some human diseases. TLC was successfully applied in the analysis of hydrophilic vitamins, but the most difficult problem in the simultaneous analysis of all these compounds is to find an optimum stationary phase-mobile phase system due to different chemical characteristics of analytes. Unfortunately structural analogues are difficult to separate in one chromatographic run, and this is the case in hydrophilic vitamins investigations. TLC gives the possibility to perform two-dimensional separations by using stationary phase gradient achieving the highest resolution by combining two systems with different selectivity. The goal of this work was to develop a method of analysis enabling separation of hydrophilic vitamins using TLC with adsorbent gradient. The developed method was used for identifying the water-soluble vitamins in alcoholic extracts of Hippophae rhamnoides and of Ribes nigrum.

Antioxidant activity prediction and classification of some teas using artificial neural networks.

In order to characterise and to classify some teas a simple, rapid and economical method based on composition, antioxidant activity and artificial neural networks (ANNs) is proposed. For these purpose two types of ANN based applications have been developed: one for predicting the antioxidant activity and a second one for establishing the class of the teas. The complex relationship between the total antioxidant activity (AA) depending on the total flavonoids content (F), total catechins content (C) and total methyl-xanthines content (MX) of commercial teas was revealed by the first designed feed-forward ANN. Secondly, using a probabilistic ANN, successful tea classification in various classes (green tea, black tea and express black tea) was also performed.

Simultaneous determination of methylxanthines in different types of tea by a newly developed and validated TLC method.

The aim of this paper is to develop a new simple, fast and economical method for simultaneous quantitative determination of methylxanthine compounds based on TLC combined with image analysis. To obtain certain results, both extraction and chromatographic separation were optimized. The optimum extraction conditions were maceration in ethanol-water 8:2, v/v. The chromatographic separations were done on the silica gel F(254) TLC plates developed with chloroform-dichloromethane-isopropanol, 4:2:1 v/v/v. Detection was performed under UV lamp at 254 nm and the evaluation of the chromatographic plate was based on digital processing of chromatographic images. The developed TLC method was validated for parameters such as specificity, linearity and range, LOD and LOQ, precision, robustness and accuracy. This method was then applied for determination of caffeine, theobromine and theophylline in different types of tea, commercially available. Moreover, the content of methylxanthines detected and determined in commercial tea samples can be used as chemical marker in quality control.

Statistical assessment of solvent mixture models used for separation of biological active compounds.

Two mathematical models with seven and six parameters have been created for use as methods for identification of the optimum mobile phase in chromatographic separations. A series of chromatographic response functions were proposed and implemented in order to assess and validate the models. The assessment was performed on a set of androstane isomers. Pearson, Spearman, Kendall tau-a,b,c and Goodman-Kruskal correlation coefficients were used in order to identify and to quantify the link and its nature (quantitative, categorical, semi-quantitative, both quantitative and categorical) between experimental values and the values estimated by the mathematical models. The study revealed that the six parameter model is valid and reliable for five chromatographic response factors (retardation factor, retardation factor ordered ascending by the chromatographic peak, resolution of pairs of compound, resolution matrix of successive chromatographic peaks, and quality factor). Furthermore, the model could be used as an instrument in analysis of the quality of experimental data. The results obtained by applying the model with six parameters for deviations of rank sums suggest that the data of the experiment no. 8 are questionable.

Analysis of some steroids by thin-layer chromatography using optimum mobile phases.

The aim of this paper was the analysis of five androstane isomers by thin-layer chromatography (TLC). The choice of proper mobile phase and the optimization of the mobile phase composition are very important because the chromatographic separation is difficult to achieve. In the first step, the proper mobile phase system was chosen from seven elution systems presented in the literature using numerical taxonomy method. The proper solvent system was found to be the mixture of chloroform, acetone, and petroleum ether. In the second step the composition of mobile phase was optimized by "simplex" method and "prisma" method. The optimum TLC system can be applied for the separation of androstane isomers from real samples such as drug formulation, biological and natural sources.

Separation of N-alkyl phenothiazine sulfones by HPTLC using an optimum mobile phase.

In high performance thin layer chromatography some form of optimization is necessary if complete separation of all components is required. The selection of mobile phase composition is one of the most important components of an optimization strategies. The aim of this paper is the separation of the N-alkyl phenothiazine sulfones by high performance thin layer chromatography using an optimum mobile phase system. The optimum composition of mobile phase (toluene-ethyl ether-chloroform, 30:50:20, v/v) are given by the maximum of objective function (F(obj)=10.6110).