Novel procedures for olive leaves extracts processing: Selective isolation of oleuropein and elenolic acid.

Several processes have been developed in the past to selectively extract oleuropein and its aglycones from olive derived materials. In the present manuscript, we outline a novel approach for processing olive leaves aqueous extracts. This allowed first to select microwave irradiation as the methodology able to provide a large enrichment in oleuropein. Subsequently, the use of lamellar solids led to the selective and high yield concentration of the same. Adsorption on solids also largely contributed to the long term chemical stability of oleuropein. Finally, an eco-friendly, readily available, and reusable catalyst like H2SO4 supported on silica was applied for the hydrolysis of oleuropein into hydroxytyrosol and elenolic acid. This latter was in turn selectively isolated by an acid-base work-up providing its monoaldehydic dihydropyran form (7.8 % extractive yield), that was unequivocally characterized by GC-MS. The isolation of elenolic acid in pure form is described herein for the first time.

Prolidase activity assays. A survey of the reported literature methodologies.

Prolidase (EC.3.4.13.9) is a dipeptidase known nowadays to play a pivotal role in several physiological and pathological processes. More in particular, this enzyme is involved in the cleavage of proline- and hydroxyproline-containing dipeptides (imidodipeptides), thus finely regulating the homeostasis of free proline and hydroxyproline. Abnormally high or low levels of prolidase have been found in numerous acute and chronic syndromes affecting humans (chronic liver fibrosis, viral and acute hepatitis, cancer, neurological disorders, inflammation, skin diseases, intellectual disability, respiratory infection, and others) for which the content of proline is well recognized as a clinical marker. As a consequence, the accurate analytical determination of prolidase activity is of greatly significant importance in clinical diagnosis and therapy. Apart from the Chinard's assay, some other more sensitive and well validated methodologies have been published. These include colorimetric and spectrophotometric determinations of free proline produced by enzymatic reactions, capillary electrophoresis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, electrochemoluminescence, thin layer chromatography, and HPLC. The aim of this comprehensive review is to make a detailed survey of the in so far reported analytical techniques, highlighting their general features, as well as their advantages and possible drawbacks, providing in the meantime suggestions to stimulate further research in this intriguing field.

Separation and quantification of Tartrazine (E102) and Brilliant Blue FCF (E133) in green colored foods and beverages.

In the present study we investigated the capacities of a panel of 25 solid sorbents represented by layered structures, inorganic oxides and hydroxides, and phyllosilicates, to effectively remove in high yield Tartrazine (E102) and Brilliant Blue FCF (E133) from aqueous solutions, and more notable, green colored food matrices. Quantification of the title compounds have been achieved by HPLC-DAD analyses. Contents of E102 and E133 in real samples were in the range 1.3-36.5 µg/mL and 1.0-20.1 µg/mL, respectively. After a treatment of 1 min., in most cases a complete bleaching of solutions and deep coloring of the solid phase was recorded. The most effective solids to this aim were seen to be aluminium based ayered double hydroxides. In the case of magnesium oxide for E102, and magnesium aluminium D. benzensulfonate SDS 01 H8L and Florisil for E133, a selective adsorption (>99.9 %) of only one dye was observed. The adsorption recorded was strictly dependent on the loading of the sorbent. Related values were 300 mg for the separation of E102 by magnesium oxide from all the five food matrices under investigation, and in the range 200 mg-300 mg for magnesium aluminium D. benzensulfonate SDS 01 H8L and Florisil in the case of E133. The application of Langmuir and Freundlich models suggested that the adsorption may take place in the inner layers of the solids with a favourable thermodynamique outcome. Findings described herein offer the concrete possibility of quantifications of individual dyes in matrices containing more than one food colorant.

Novel insights into the biomolecular mechanism of action of 4'-geranyloxyferulic acid, a colon cancer chemopreventive agent.

In this manuscript the biomolecular mechanism of action of the natural colon cancer chemopreventive agent 4'-geranyloxyferulic acid in cultured Caco-2 cells has been investigated. It was first demonstrated how the application of this phytochemical led to a time- and dose-dependent decrease of cell viability and in parallel to a massive generation of reactive oxygen species and induction of caspases 3 and 9, finally providing apoptosis. This event is accompanied by deep modifications in key pro-apoptotic targets like CD95, DR4 and 5, cytochrome c, Apaf-1, Bcl-2, and Bax. Such effects can explain the large apoptosis recorded in Caco-2 cells treated with 4'-geranyloxyferulic acid.

Pro­differentiating compounds for human intervertebral disc cells are present in Violina pumpkin leaf extracts.

Intervertebral disc (IVD) degeneration (IDD) is closely associated with inflammation, oxidative stress and loss of the discogenic phenotype, which current therapies are unable to reverse. In the present study, the effects of acetone extract from Violina pumpkin (Cucurbita moschata) leaves on degenerated IVD cells were investigated. IVD cells were isolated from the degenerated disc tissue of patients undergoing spinal surgery and were exposed to acetone extract and three major thin layer chromatography subfractions. The results revealed that, in particular, the cells benefited from exposure to subfraction Fr7, which consisted almost entirely of p­Coumaric acid. Western blot and immunocytochemical analysis showed that Fr7 induced a significant increase in discogenic transcription factors (SOX9 and tricho­rhino­phalangeal syndrome type I protein, zinc finger protein), extracellular matrix components (aggrecan, collagen type II), cellular homeostasis and stress response regulators, such as FOXO3a, nuclear factor erythroid 2­related factor 2, superoxide dismutase 2 and sirtuin 1. Two important markers related to the presence and activity of stem cells, migratory capacity and OCT4 expression, were assessed by scratch assay and western blotting, respectively, and were significantly increased in Fr7­treated cells. Moreover, Fr7 counteracted H2O2­triggered cell damage, preventing increases in the pro­inflammatory and anti­chondrogenic microRNA (miR), miR­221. These findings strengthen the hypothesis that adequate stimuli can support resident cells to repopulate the degenerated IVD and restart the anabolic machinery. Taken together, these data contribute to the discovery of molecules potentially effective in slowing the progression of IDD, a disease for which there is currently no effective treatment. Moreover, the use of part of a plant, the pumpkin leaves, which is usually considered a waste product in the Western world, indicated that it contains substances with potential beneficial effects on human health.

Protection of Mitochondrial Potential and Activity by Oxyprenylated Phenylpropanoids.

A series of five naturally occurring oxyprenylated phenylpropanoids, namely, the coumarins auraptene (7-geranyloxycoumarin) 1 and 7-isopentenyloxycoumarin 2, and the coumaric acid and ferulic acid derivatives, 4'-isopentenyloxycoumaric acid 3, boropinic acid 4, and 4'-geranyloxyferulic acid 5 were tested for their effects on mitochondrial functionality using the organophosphate pesticides glyphosate and chlorpyrifos, and resveratrol, as the reference. While not showing an appreciable in vitro antioxidant activity, and virtually no or a little effect on the viability of non-cancer cell lines BEAS-2B and SHSY-5Y, all phytochemicals exhibited a marked protective effect on mitochondrial potential and activity, with values that were comparable to resveratrol. Auraptene 1 and 7-isopentenyloxycoumarin 2 were seen to be the most effective secondary metabolite to this concern, in particular in being able to completely abolish the decrease of mitochondrial potential induced by increasing concentration of both glyphosate and chlorpyrifos. All the compounds tested also exhibited a protective effect on mitochondrial activity. The potency displayed will shed more light on the molecular basis of the beneficial effects of auraptene, 7-isopentenyloxycoumarin, and structurally related oxyprenylated phenylpropanoids reported to date in the literature.

Solid-phase adsorption methodologies of naturally occurring anthraquinones: A review.

INTRODUCTION: Solid-phase extraction applied to plant matrices is nowadays a well-validated technique allowing to concentrate and purify different secondary metabolites. Several classes of phytochemicals have been selectively extracted by this methodology. During the last decade attention has been focused on biologically active anthraquinones from numerous sources like edible, healthy, and medicinal plants. OBJECTIVES: The aim of this review is to provide a detailed literature survey of the solid-phase adsorption methodologies for the extraction of natural anthraquinones reported so far and to discuss and propose future directions in this field of research. MATERIALS AND METHODS: Substructure search was performed in the SciFinder Scholar, PubMed, Medline, and Scopus databases. RESULTS: The first report about application of solid-phase adsorption for the purification of anthraquinones appeared in the literature in 2002. From this date, and in particular during recent years, the most notable examples included the use of chitin- and chitosan-based polymers, of molecularly imprinted polymers, of coated magnetic nanoparticles, of miniaturized matrix solid-phase dispersion, of functionalized resins, of differently structured lamellar solids, and finally of vortex-synchronized matrix solid-phase dispersion. CONCLUSIONS: The herein detailed solid-phase adsorption methodologies are powerful tools to selectively extract natural anthraquinones and/or provide anthraquinone-enriched phytopreparations. Nevertheless, many other important methods have been applied to synthetic anthraquinones (e.g., azo dyes). These could be conveniently employed also for natural anthranoids. Studies in this field are discussed in this review article.

The Evanescent Bouquet of Individual Bear Fingerprint.

The evanescent and invisible communication carried by chemical signals, pheromones, or signature mixtures or, as we prefer, the pheromonal individual fingerprint, between members of the same species is poorly studied in mammals, mainly because of the lack of identification of the molecules. The difference between pheromones and the pheromonal individual fingerprint is that the former generate stereotyped innate responses while the latter requires learning, i.e., different receivers can learn different signature mixtures from the same individual. Furthermore, pheromones are usually produced by a particular gland, while the pheromonal individual fingerprint is the entire bouquet produced by the entire secreting gland of the body. In the present study, we aim to investigate the pheromonal individual fingerprint of brown bears in northern Italy. We collected the entire putative pheromone bouquet from all production sites in free-ranging bears and analyzed the entire crude extract to profile the individual fingerprint according to species-, sex- and subjective-specific characteristics. We were able to putatively characterize the brown bears' pheromonal individual fingerprints and compare them with the partial pheromone identifications published by other studies. This work is a step forward in the study of the complexity of chemical communication, particularly in a solitary endangered species.

Cognitive and biological effects of citrus phytochemicals in subjective cognitive decline: a 36-week, randomized, placebo-controlled trial.

BACKGROUND: Auraptene (AUR) and naringenin (NAR) are citrus-derived phytochemicals that influence several biological mechanisms associated with cognitive decline, including neuronal damage, oxidative stress and inflammation. Clinical evidence of the efficacy of a nutraceutical with the potential to enhance cognitive function in cohorts at risk of cognitive decline would be of great value from a preventive perspective. The primary aim of this study is to determine the cognitive effects of a 36-week treatment with citrus peel extract standardized in levels of AUR and NAR in older adults experiencing subjective cognitive decline (SCD). The secondary aim is to determine the effects of these phytochemicals on blood-based biomarkers indicative of neuronal damage, oxidative stress, and inflammation. METHODS: Eighty older persons with SCD will be recruited and randomly assigned to receive the active treatment (400 mg of citrus peel extract containing 0.1 mg of AUR and 3 mg of NAR) or the placebo at a 1:1 ratio for 36 weeks. The primary endpoint is a change in the Repeatable Battery for the Assessment of Neuropsychological Status score from baseline to weeks 18 and 36. Other cognitive outcomes will include changes in verbal and nonverbal memory, attention, executive and visuospatial functions. Blood samples will be collected from a consecutive subsample of 60 participants. The secondary endpoint is a change in interleukin-8 levels over the 36-week period. Other biological outcomes include changes in markers of neuronal damage, oxidative stress, and pro- and anti-inflammatory cytokines. CONCLUSION: This study will evaluate whether an intervention with citrus peel extract standardized in levels of AUR and NAR has cognitive and biological effects in older adults with SCD, facilitating the establishment of nutrition intervention in people at risk of cognitive decline. TRIAL REGISTRATION: The trial is registered with the United States National Library of Medicine at the National Institutes of Health Registry of Clinical Trials under the code NCT04744922 on February 9th, 2021 ( https://www. CLINICALTRIALS: gov/ct2/show/NCT04744922 ).

Estrogen Receptor 1 (ESR1) and the Wnt/ß-Catenin Pathway Mediate the Effect of the Coumarin Derivative Umbelliferon on Bone Mineralization.

Bone physiology is regulated by osteoblast and osteoclast activities, both involved in the bone remodeling process, through deposition and resorption mechanisms, respectively. The imbalance between these two phenomena contributes to the onset of bone diseases. Among these, osteoporosis is the most common metabolic bone disorder. The therapies currently used for its treatment include antiresorptive and anabolic agents associated with side effects. Therefore, alternative therapeutic approaches, including natural molecules such as coumarin and their derivatives, have recently shown positive results. Thus, our proposal was to investigate the effect of the coumarin derivative umbelliferon (UF) using an interesting model of human osteoblasts (hOBs) isolated from osteoporotic patients. UF significantly improved the activity of osteoporotic-patient-derived hOBs via estrogen receptor 1 (ESR1) and the downstream activation of ß-catenin pathway. Additionally, hOBs were co-cultured in microgravity with human osteoclasts (hOCs) using a 3D system bioreactor, able to reproduce the bone remodeling unit in bone loss conditions in vitro. Notably, UF exerted its anabolic role by reducing the multinucleated cells. Overall, our study confirms the potential efficacy of UF in bone health, and identified, for the first time, a prospective alternative natural compound useful to prevent/treat bone loss diseases such as osteoporosis.

Leaves of Yellow Gentian (Gentiana lutea) as an Alternative Source of Bitter Secoiridoid Glycosides.

In a search for methods of manufacturing bitter principles from Gentiana lutea, mainly represented by gentiopicroside (1) and amarogentin (2), as an alternative to extraction from the roots of this plant, in this short communication it is shown that the leaves of this plant can be regarded as an additional source of such phytochemicals. Extraction of G. lutea leaves was coupled to solid-phase adsorption by differently structured solids as a separation technology step, providing a selective isolation of both these secondary metabolites in good to excellent yields. Thus, the extraction of bitter secoiridoids can be achieved in an equivalent or improved way rather than processing the roots of G. lutea while preserving the biodiversity of the species.

A novel and efficient concentration of pomegranate juice with enhanced antioxidant activity.

In the present study we investigated the performance of a panel of 13 solid sorbents comprising layered double hydroxides, zirconium phosphate-based materials, and phyllosilicates as heterogeneous supports for the concentration of pomegranate (Punica granatum L.) juice. Mg-containing clays exhibited an almost complete bleaching capacityof pomegranate juice and more interestingly provided blends with an increased antioxidant capacity (around 1.5-fold) respect to the parent juice when assayed for the Trolox equivalent antioxidant capacity (TEAC) coupled to ABTS decolorization test. Such an activity remained practically unaltered after 4 days during which the pomegranate concentrated preparations remained supported on clays. The approach investigated herein and used for the concentration of pomegranate juice and the discovery of the preservation for long periods of the antioxidant activities of pomegranate extracts when supported on solid sorbents have been reported herein for the first time in the literature to the best of our knowledge.

A revised version of the Iwaoka's assay: Application of hyphenated techniques.

Assessing the effects of glutathione peroxidase (GPx)-like catalytic activities of natural, semi-synthetic, and synthetic compounds is nowadays well recognized of importance for the preliminary screening of potential anti-inflammatory, neuroprotective, and anti-cancer agents. To this aim the Iwaoka's assay, relying on 1H NMR data recording, has been the most exploited test. In this short communication we propose an efficient and easy to perform methodology to accomplish the same process based on the application of hyphenated techniques like gas-chromatography coupled to mass spectrometry (GC-MS) and high-performance liquid chromatography with diode array detection (HPLC-DAD). The assay consisted in monitoring and quantifying the oxidation at different times of 1,4-dithiothreitol to 4,5-dihydroxy-1,2-dithiane in the presence of H2O2 and 2-phenyl-1,2-benzoselenazol-3(2H)-one (Ebselen) as the catalyst in EtOAc or MeOH as the solvents. The results we recorded by the application of both GC-MS and HPLC-DAD, showed that the GPx-like activity of Ebselen, used as the reference compound, can be effectively monitored in a time-course manner, and compare favorably to the NMR-based test.

An easy way for the hydrolysis, pre-concentration, and chemical stabilization of crocetin from saffron powder.

To date there are no methods in the literature leading to crocetin selective concentration from saffron powder aqueous solutions. To this aim, we decided to test the performance of its heterogeneous extraction by means of a panel of 21 synthetic clays, 4 of which demonstrated to selectively retain crocetin in the solid phase after hydrolysis of its digentiobyosil ester (crocin) (and its isomers) and to its chemical stabilization (e.g., oxidation) over time. The best adsorption yield was obtained with zinc hydroxy chloride (66.18 ± 0.06 µg/g dry powder). This phenomenon was assessed by HPLC-DAD analyses after desorption of crocetin from the respective support and assessing its degradation along a period of 30 days. The method we established could represent a good mean to provide pure crocetin from saffron powder, preserving in the meantime its chemical properties for a concrete future exploitation for food pharmaceutical, and cosmetic purposes.

Oxyprenylated Secondary Metabolites as Modulators of Lipid and Sugar Metabolism.

O-Prenylcoumarins (3,3-dimethylallyl, geranyl-, farnesyl- and related biosynthetic derivatives) represent a class of rarely occurring natural compounds. Most of these secondary metabolites have been obtained from plant species belonging to the Rutaceae, Apiaceae, and Fabaceae families, and from fungi, and bacteria. In the last two decades, prenyloxycoumarins have been found to possess great potential in terms of pharmacological activities. The aim of this comprehensive review is to make a survey of the so far reported literature citations about these valuable phytochemicals and structurally related compounds about their modulatory properties of lipid and sugar metabolism. Literature data have been acquired from the main Internet database. Several oxyprenylated secondary metabolites have been surveyed. Among these, prenyloxycoumarins represented the main group displaying valuable effects as modulators of lipid and sugar metabolism. The title phytochemicals have been found in common fruits and vegetables already known to have beneficial effects, thus enforcing the nutraceutical role of these food plants. All compounds outlined in the present review article have a great potential in future for the prevention and management of acute and chronic metabolic disorders.

Pro-Osteogenic Properties of Violina pumpkin (Cucurbita moschata) Leaf Extracts: Data from In Vitro Human Primary Cell Cultures.

Traditional medicines rely mainly on use of plant extracts to mitigate or treat a wide range of disorders, including those that affect skeletal homeostasis. In this study, we investigated for the first time the potential pro-osteogenic effects of hexane, acetone and methanol extracts of the leaves of Cucurbita moschata, a very popular pumpkin cultivar in Western countries. We found that in Cucurbita moschata leaves, there are acetone-extractable substances-in particular, fatty acids such as 13-OH-9Z,11E,15E-octadecatrienoic acid (PU-13OH-FA), which is capable of both stimulating the function of human primary osteoblasts, which are responsible for bone formation, and inhibiting the differentiation of human osteoclasts, which are responsible for bone resorption. This dual effect was monitored by analyzing Runx2 expression, deposition of mineralized matrix, ALP activity, TRAP and actin ring staining respectively. This study suggests that bioactive chemicals from Cucurbita moschata leaves are potentially suitable as therapeutics for managing metabolic bone disorders such as osteoporosis and rheumatoid arthritis, and promoting tissue healing and functional recovery after bone fractures. The data we obtained increase knowledge on the biological activities of Cucurbita moschata, and in particular underline the potential benefits of consuming leaves which are a part of the plant currently little considered in the Western world.

A Novel Auraptene-Enriched Citrus Peels-Based Blend with Enhanced Antioxidant Activity.

The aim of this study was to establish a convenient process to get auraptene-enriched blends from Citrus limon L. Osbeck (Rutaceae) peels powder with functional food and nutraceutical potential and antioxidant properties. The process consisted in the selective adsorption of auraptene by bentonite followed by desorption and evaporation of solvents to get the final product. HPLC analyses revealed an adsorption > 99% from lemon fruit peel powder and a 13.7-fold enrichment of the obtained composition respect to the parent food-derived sample. The antioxidant and radical scavenger activities of the Citrus peels based preparation were assayed by the DPPH and ABTS tests and reveal deffects higher than ascorbic acid and Trolox® used as the references. To the best of our knowledge, the findings described herein is the first example reported in the literature of solid phase adsorption experiments carried out on auraptene as a component of edible fruits.

Semisynthesis of Selenoauraptene.

Selenium-containing compounds are gaining more and more interest due to their valuable and promising pharmacological properties, mainly as anticancer and antioxidant agents. Ebselen, the up to now only approved drugs, is well known to possess very good glutathione peroxidase mimicking effects. To date, the most of efforts have been directed to build pure synthetic Se containing molecules, while less attention have been devoted to Se-based semisynthetic products resembling natural compounds like terpenes, polyphenols, and alkaloids. The aim of this short communication is to report the synthesis of the first example of a Se-phenylpropanoids, namely selenoauraptene, containing a selenogeranyl side chain in position 7 of the umbelliferone core. The key step was the Newman-Kwart rearrangement to obtain a selenocarbamate in which the Se atom was directly attached to umbelliferone (replacing its 7-OH function) followed by hydrolysis to get diumbelliferyl diselenide, which was finally easily converted to the desired Se-geranyl derivative in quite a good overall yield (28.5%). The synthesized adduct displayed a greater antioxidant and a radical scavenger in vitro activity than parent auraptene. The procedure we describe herein, to the best of our knowledge for the first time in the literature, represents an easy-to-handle method for the synthesis of a wide array of seleno analogues of naturally occurring biologically active oxyprenylated secondary metabolites.

An improved method for the isolation of amarogentin, the bitter principle of yellow gentian roots.

Amarogentin is well known to be among the most bitter naturally occurring compound. Either as an individual one or extracts, amarogentin is used as a food additive and as a dietary supplement. The aim of the present investigation is to set-up a convenient process to selectively isolate amarogentin from the ethanolic roots extract of Gentiana lutea. The process consisted in the treatment of an aqueous suspension of such an extract with a panel of 21 solid inorganic / organic sorbents followed by filtration, desorption, and high performance liquid chromatography (HPLC) analyses. Among the solid materials tested, those containing Mg+2 in the frame of a lamellar structure provided very good adsorption yields in the range 86.4% - 99.9% (p < 0.05 at Student's t-test). The method we set up could be in principle useful to obtain a pure nature-derived food additive to provide bitter taste to foods and beverages.