Development and Box-Behnken design optimization of a green extraction method natural deep eutectic solvent-based for phenolic compounds from barley malt rootlets.

In recent years, attention has been turned finding new sources of phenolic compounds, antioxidant molecules, main by-products from the agri-food chain like barley malt rootlets (BMRs). Traditionally, phenolic compounds are extracted from food matrices using different procedures, for example, solid-liquid, liquid-liquid, or solid-phase extraction techniques employing organic solvents. With the advent of green chemistry, attention has been paid to the search for green, nontoxic, inexpensive, and nonflammable solvents and the natural deep eutectic solvents (NADESs) respect these characteristics. The aim of this project was to develop and optimize an environmentally friendly, inexpensive, and rapid extraction method for phenolic compounds from BMRs using natural DESs as extractive solvents. Several natural DESs were tested as extractive solvents and, among them, the best results in terms of total phenolic content were obtained using a choline chloride-malic acid (1:2 molar ratio)-based mixture. Box-Behnken experimental design guaranteed the extraction of 9.51 ± 0.83 gallic acid equivalent/g of BMRs, under the following optimal extraction conditions: 1:21 solid-to-liquid ratio, 80°C as extraction temperature, 43 min as the time of extraction, and 29% as a percentage of added water in the NADESs. Phenolic acids and flavonoids were detected in the BMRs extract through HPLC-PDA/MS analysis.

Comparison between In Vitro Chemical and Ex Vivo Biological Assays to Evaluate Antioxidant Capacity of Botanical Extracts.

The anti-oxidative activity of plant-derived extracts is well-known and confers health-promoting effects on functional foods and food supplements. Aim of this work is to evaluate the capability of two different assays to predict the real biological antioxidant efficiency. At this purpose, extracts from five different plant-derived matrices and commercial purified phytochemicals were analyzed for their anti-oxidative properties by using well-standardized in vitro chemical method (TEAC) and an ex vivo biological assay. The biological assay, a cellular membrane system obtained from erythrocytes of healthy volunteers, is based on the capability of phytochemicals treatment to prevent membrane lipid peroxidation under oxidative stress by UV-B radiation. Plant extracts naturally rich in phenols with different structure and purified phytochemicals showed different in vitro and ex vivo antioxidant capacities. A high correlation between phenolic contents of the plant-derived extracts and their ability to prevent oxidative injuries in a biological system was found, thus underlying the relevance of this class of metabolites in preventing oxidative stress. On the other hand, a low correlation between the antioxidant capacities was shown between in vitro and ex vivo antioxidant assay. Moreover, data presented in this work show how food complex matrices are more effective in preventing oxidative damages at biological level than pure phytochemicals, even if for these latter, the antioxidant activity was generally higher than that observed for food complex matrices.

Blood orange (Citrus sinensis) as a rich source of nutraceuticals: investigation of bioactive compounds in different parts of the fruit by HPLC-PDA/MS.

This study is part of a wider investigation aimed to sustain the economical value of the by-products generated by the Citrus industry. In particular, the protected geographic indication (PGI) red orange of sicily (known as blood orange) has been analysed by HPLC and by the enzymatic AOAC method. All the by-products contain significant amounts of biologically active compounds (limonoids and flavonoids). The decanted pulps were the most abundant, with the highest amount of flavonoids (130 g/kg) and high amount of limonoids (5.5 g/kg). Seeds were the best source of limonoids with about 10 g/kg. Low amount of anthocyanins were found only in coarse pulps and waste water. The total, the insoluble and the soluble dietary fibre (TDF, IDF and SDF respectively) were also determined. The pulps resulted to be the best source of dietary fibre, based on the amount and on the best insoluble/soluble ratio.

Distribution of bioactives in entire mill chain from the drupe to the oil and wastes.

Industrial olive oil production generates large amounts of by-products, mainly pomaces and wastewaters. The latter, in particular, represents serious environmental problems requiring special treatments prior to disposal. While olive pomace finds use as alternative energy source, wastewaters still remain a task since it is not reusable, representing an additional cost on olive oil for its treatments. This study is a "comprehensive" overview on the distribution of bioactives in entire mill chain from the drupe to the oil and wastes. Identification was achieved through liquid chromatography coupled with mass spectrometry, spectrofotometric and fluorimetric detection. Phenols resulted the most abundant class of substances, with the highest hydroxytyrosol amounts in wastewater (214 mg/kg). Pomace contained a total of 304 mg/kg in terms of bioactives, thus representing a potential food supplement ingredient for functional foods with high nutritional values.

Choline-chloride and betaine-based deep eutectic solvents for green extraction of nutraceutical compounds from spent coffee ground.

Coffee beans contain several compounds which have shown nutraceutial properties; among them chlorogenic acids (CGAs) are the most studied, have shown nutraceutical properties. These molecules are also present in the waste by-product known as spent coffee grounds (SCG). SCG are generated during the production of coffee drink, whereby roasted, ground coffee beans are heat or steam treated. A HPLC-PDA/ESI-MS method has been developed for the analysis of the most representative CGAs in SCG samples using a green extraction approach based on deep eutectic solvents (DESs). We examined fifteen different DESs based on choline chloride and betaine as hydrogen bond acceptor in combination with different hydrogen bond donors being seven alcohols, two organic acids, one sugar and urea. Fifteen CGAs were detected, identified and quantified for the evaluation of extraction efficiency. A betaine-based DES with triethylene glycol (molar ratio 1:2) was the most effective one at extracting CGAs from SCG also compared to a conventional organic solvent. Thirty percent of water added to DES and sample to solvent ratio 1:15 (g/mL) were selected as the best extraction conditions. The chromatographic method was validated studying LOD, LOQ, retention time and peak area precision, and linearity range. Retention time and peak area repeatability were determined obtaining RSDs values below 0.63 % and 8.51 %, respectively. Inter-day precision was determined obtaining RSDs value below 0.58 % and 8.80 % for retention time and peak area, respectively.

Application of deep eutectic solvents for the extraction of phenolic compounds from extra-virgin olive oil.

A HPLC-DAD/ESI-MS method has been developed and validated for the analysis of the most representative phenolic compounds in extra-virgin olive oil (EVOO) samples using a green extraction approach based on deep eutectic solvents (DESs) at room temperature. We examined ten DESs based on choline chloride and betaine in combination with different hydrogen bond donors comprising six alcohols, two organic acids, and one urea. Five phenolic compounds, belonging to the classes of secoiridoids and phenolic alcohols, were selected for the evaluation of extraction efficiency. A betaine-based DES with glycerol (molar ratio 1:2) was found to be the most effective for extracting phenolic compounds as compared to a conventional solvent. The optimization of the extraction method involved the study of the quantity of water to be added to the DES and evaluation of the sample-to-solvent ratio optimal condition. Thirty percent of water added to DES and sample to solvent ratio 1:1 (w/v) were selected as the best conditions. The chromatographic method was validated by studying LOD, LOQ, intraday and interday retention time precision, and linearity range. Recovery values obtained spiking seed oil sample aliquots with standard compounds at 5 and 100 µg/g concentration were in the range between 75.2% and 98.7%.

Characterization of the polyphenolic fraction of pomegranate samples by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry detection.

Punica granatum L., commonly known as pomegranate, is an ancient fruit widely consumed all over the world as fresh fruit or juice. In addition, it is extensively used in therapeutic formulas, cosmetics and food seasonings. The fruit is native to Afghanistan, Iran, China and the Indian sub-continent. The pomegranate market has steadily grown, presumably due to the increasing demand of health-conscious consumers for products with potential beneficial effects on human health, due to the synergistic presence of a unique and complex phytochemical composition that enclose anthocyanins, phenolic acids and hydrolysable tannins. Conventionally, for their analysis liquid chromatography is employed even though it can present some drawbacks in terms of resolving power. In this contribution, as a valuable alternative, comprehensive two-dimensional liquid chromatography with "shifted gradients" in the second dimension, was applied for the characterization of three pomegranate samples, leading to the identification of 37 different polyphenolic compounds.

Effects of ionizing radiation on bio-active plant extracts useful for preventing oxidative damages.

Humans are exposed to ionizing radiations in medical radiodiagnosis and radiotherapy that cause oxidative damages and degenerative diseases. Airplane pilots, and even more astronauts, are exposed to a variety of potentially harmful factors, including cosmic radiations. Among the phytochemicals, phenols are particularly efficient in countering the oxidative stress. In the present study, different extracts obtained from plant food, plant by-products and dietary supplements, have been compared for their antioxidant properties before and after irradiation of 140 cGy, a dose absorbed during a hypothetical stay of three years in the space. All the dry extracts, characterized in terms of vitamin C and phenolic content, remained chemically unaltered and maintained their antioxidant capability after irradiation. Our results suggest the potential use of these extracts as nutraceuticals to protect humans from oxidative damages, even when these extracts must be stored in an environment exposed to cosmic radiations as in a space station.

Extraction, Analysis, and Antioxidant Activity Evaluation of Phenolic Compounds in Different Italian Extra-Virgin Olive Oils.

The analysis of phenolic compounds in extra virgin olive oils was carried out by high-performance liquid chromatography utilizing photodiode array and mass spectrometry detectors. The chromatographic profile of thirty samples from four Italian Regions highlighted the presence of secoiridoids, phenolic alcohols, flavonoids, and phenolic acid classes. A similar qualitative profile was observed with some differences in peak area and fifteen compounds were tentatively identified. Quantitative analysis was performed by UV detection considering eight standard phenolic compounds. The chromatographic method, after optimization, was validated studying some parameters, e.g., intra-day and inter-day retention time precision, limit of detection, limit of quantification, and linearity. Recovery of the method was performed achieving good results (10 and 50 g·g-1 with recovery of 72.9⁻92.1% (w/w) and 79.1⁻102.8% (w/w), respectively). In all samples secoiridoids were the main compounds ranging from 85 to more than 99% (w/w) of the total concentration of detected phenolic compounds while phenolic acids accounted for the lowest percentage (0.1⁻0.6%, w/w). Finally, total concentration of phenolic compounds and antioxidant activity were determined with different chemical assays. A good and significant correlation among total phenolic compound concentration and antioxidant activity was observed. A significant different phenolic compound concentration and antioxidant activity was determined between samples from Puglia and Sicily. This was studied performing statistical analysis by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test.

Selection and use of autochthonous mixed starter for lactic acid fermentation of carrots, French beans or marrows.

Strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Weissella soli/Weissella koreensis, Enterococcus faecalis, Pediococcus pentosaceus and Lactobacillus fermentum were identified from raw carrots, French beans and marrows by partial 16S rRNA gene sequence. L. plantarum M1, Leuc. mesenteroides C1 and P. pentosaceus F4 were selected based on the rates of growth and acidification in vegetable juice media, and used as the autochthonous mixed starter for the fermentation of carrots, French beans or marrows. An allochthonous starter, consisting of the same species, was also used for fermentation. A two-step fermentation process (1 day at 25 degrees C and 7 days at 15 degrees C) in brine (1% w/v) followed by storage at room temperature in olive oil until 40 days was set up. Unstarted vegetables subjected to the same treatments were used as the controls. Cell numbers of lactic acid bacteria in the started vegetables were ca. 10,000 (autochthonous starter) and 1000 (allochthonous starter) times higher than unstarted samples throughout the process. When fermented with the autochthonous starter, carrots, French beans or marrows were characterized by the rapid decrease of pH (<4.5), marked consumption of fermentable carbohydrates, and inhibition of Enterobacteriaceae and yeasts. Fermentation with the allochthonous starter did not acidify and inhibit bacteria and yeasts so rapidly. After 40 days, carrots, French beans and marrows fermented with the autochthonous starter had significantly (P<0.05) higher total concentration of vitamin C (ascorbate+dehydroascorbate) with respect to those fermented with the allochthonous starter and, especially unstarted vegetables. The same was found for the indexes of color. Firmness of both started vegetables was higher than unstarted vegetables. Sensory analysis differentiated started vegetables. Carrots and French beans fermented with the autochthonous starter were, especially, appreciated for fragrance. Appearance was the sensory attribute that mainly distinguished marrows fermented with the autochthonous starter.

Ectopic expression of maize polyamine oxidase and pea copper amine oxidase in the cell wall of tobacco plants.

To test the feasibility of altering polyamine levels by influencing their catabolic pathway, we obtained transgenic tobacco (Nicotiana tabacum) plants constitutively expressing either maize (Zea mays) polyamine oxidase (MPAO) or pea (Pisum sativum) copper amine oxidase (PCuAO), two extracellular and H(2)O(2)-producing enzymes. Despite the high expression levels of the transgenes in the extracellular space, the amount of free polyamines in the homozygous transgenic plants was similar to that in the wild-type ones, suggesting either a tight regulation of polyamine levels or a different compartmentalization of the two recombinant proteins and the bulk amount of endogenous polyamines. Furthermore, no change in lignification levels and plant morphology was observed in the transgenic plants compared to untransformed plants, while a small but significant change in reactive oxygen species-scavenging capacity was verified. Both the MPAO and the PCuAO tobacco transgenic plants produced high amounts of H(2)O(2) only in the presence of exogenously added enzyme substrates. These observations provided evidence for the limiting amount of freely available polyamines in the extracellular space in tobacco plants under physiological conditions, which was further confirmed for untransformed maize and pea plants. The amount of H(2)O(2) produced by exogenously added polyamines in cell suspensions from the MPAO transgenic plants was sufficient to induce programmed cell death, which was sensitive to catalase treatment and required gene expression and caspase-like activity. The MPAO and PCuAO transgenic plants represent excellent tools to study polyamine secretion and conjugation in the extracellular space, as well as to determine when and how polyamine catabolism actually intervenes both in cell wall development and in response to stress.