Molecular Characterization of Prunus lusitanica L. Fruit Extracts and Their Health-Promoting Potential in Inflammation, Diabetes, and Neurodegenerative Diseases.

Prunus lusitanica L. is a shrub belonging to the genus Prunus L. (Rosaceae family) that produces small fruits with none known application. Thus, the aim of this study was to determine the phenolic profile and some health-promoting activities of hydroethanolic (HE) extracts obtained from P. lusitanica fruits, harvested from three different locations. Qualitative and quantitative analysis of extracts was performed using HPLC/DAD-ESI-MS and antioxidant activity was assessed by in vitro methods. Antiproliferative/cytotoxic activity was determined on Caco-2, HepG2, and RAW 264.7 cells, anti-inflammatory activity was assessed using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, and the antidiabetic, antiaging, and neurobiological action of extracts was determined in vitro by assessing their inhibitory effect against the activity of α-amylase, α-glucosidase, elastase, tyrosinase, and acetylcholinesterase (AChE). Results showed that P. lusitanica fruit HE extracts from the three different locations showed identical phytochemical profile and bioactivities, although small differences were observed regarding the quantities of some compounds. Extracts of P. lusitanica fruits contain high levels in total phenolic compounds, namely, hydroxycinnamic acids, as well as flavan-3-ols and anthocyanins, primarily cyanidin-3-(6-trans-p-coumaroyl)glucoside. P. lusitanica fruit extracts have a low cytotoxic/antiproliferative effect, with the lowest IC50 value obtained in HepG2 cells (352.6 ± 10.0 µg/mL, at 48 h exposure), but high anti-inflammatory activity (50-60% NO release inhibition, at 100 µg/mL extract) and neuroprotective potential (35-39% AChE inhibition, at 1 mg/mL), and moderate antiaging (9-15% tyrosinase inhibition, at 1 mg/mL) and antidiabetic (9-15% α-glucosidase inhibition, at 1 mg/mL) effects. The bioactive molecules present in the fruits of P. lusitanica deserve to be further explored for the development of new drugs of interest to the pharmaceutical and cosmetic industry.

Natural Products as a Tool to Modulate the Activity and Expression of Multidrug Resistance Proteins of Intestinal Barrier.

The role of intestinal barrier homeostasis in an individual's general well-being has been widely addressed by the scientific community. Colorectal cancer is among the illnesses that most affect this biological barrier. While chemotherapy is the first choice to treat this type of cancer, multidrug resistance (MDR) is the major setback against the commonly used drugs, with the ATP-binding cassette transporters (ABC transporters) being the major players. The role of P-glycoprotein (P-gp), multidrug resistance protein 1 (MRP1), or breast cancer resistance protein (ABCG2) in the efflux of chemotherapeutic drugs is well described in cancer cells, highlighting these proteins as interesting druggable targets to reverse MDR, decrease drug dosage, and consequently undesired toxicity. Natural products, especially phytochemicals, have a wide diversity of chemical structures, and some particular classes, such as phenolic acids, flavonoids, or pentacyclic triterpenoids, have been reported as inhibitors of P-gp, MRP1, and ABCG2, being able to sensitize cancer cells to chemotherapy drugs. Nevertheless, ABC transporters play a vital role in the cell's defense against xenobiotics, and some phytochemicals have also been shown to induce the transporters' activity. A balance must be obtained between xenobiotic efflux in non-tumor cells and bioaccumulation of chemotherapy drugs in cancer cells, in which ABC transporters are essential and natural products play a pivotal role that must be further analyzed. This review summarizes the knowledge concerning the nomenclature and function of ABC-transporters, emphasizing their role in the intestinal barrier cells. In addition, it also focuses on the role of natural products commonly found in food products, e.g., phytochemicals, as modulators of ABC-transporter activity and expression, which are promising nutraceutical molecules to formulate new drug combinations to overcome multidrug resistance.

Modulation of Cell Death Pathways for Cellular Protection and Anti-Tumoral Activity: The Role of Thymus spp. Extracts and Their Bioactive Molecules.

Natural products used for their health-promoting properties have accompanied the evolution of humanity. Nowadays, as an effort to scientifically validate the health-promoting effects described by traditional medicine, an ever-growing number of bioactivities are being described for natural products and the phytochemicals that constitute them. Among them, medicinal plants and more specifically the Thymus genus spp., arise as products already present in the diet and with high acceptance, that are a source of phytochemicals with high pharmacological value. Phenolic acids, flavonoid glycoside derivatives, and terpenoids from Thymus spp. have been described for their ability to modulate cell death and survival pathways, much-valued bioactivities in the pharmaceutical industry, that continually sought-after new formulations to prevent undesired cell death or to control cell proliferation. Among these, wound treatment, protection from endogenous/exogenous toxic molecules, or the induction of selective cell death, such as the search for new anti-tumoral agents, arise as main objectives. This review summarizes and discusses studies on Thymus spp., as well as on compounds present in their extracts, with regard to their health-promoting effects involving the modulation of cell death or survival signaling pathways. In addition, studies regarding the main bioactive molecules and their cellular molecular targets were also reviewed. Concerning cell survival and proliferation, Thymus spp. present themselves as an option for new formulations designed for wound healing and protection against chemicals-induced toxicity. However, Thymus spp. extracts and some of their compounds regulate cell death, presenting anti-tumoral activity. Therefore Thymus spp. is a rich source of compounds with nutraceutical and pharmaceutical value.

Molecular Characterization of Thymus capitellatus Extracts and Their Antioxidant, Neuroprotective and Anti-Proliferative Activities.

Thymus capitellatus Hoffmanns & Link is an endemic species of the Iberian Peninsula listed as near-threatened, due to its restricted geographical distribution, occurring mainly in Portugal's mainland. In this work, we detail for the first time T. capitellatus extracts' phytochemical composition, as well as an evaluation of bioactivities to point out potential health benefits. Aqueous decoction (AD) and hydroethanolic (HE) extracts were obtained, both rich in flavonoids. However, quercetin-(?)-O-hexoside was identified as the main compound in T. capitellatus HE extract, while the phenolic acid rosmarinic acid was the main component of AD extracts. In addition, HE extract presents significant amounts of salvianolic acids and of the terpenoids oleanolic and ursolic acid. Both extracts showed antioxidant activity, evaluated by their capacity to scavenge ABTS and superoxide radicals, as well as an ability to prevent lipid peroxidation. AD extracts were also effective in scavenging hydroxyl and nitric oxide radicals. As potential functional foods, T. capitellatus extracts presented neuroprotective and anti-diabetic activity, in addition to time- and dose-dependent anti-proliferative activity against Caco-2 (colorectal adenocarcinoma) and HepG2 (hepatic carcinoma) cells. HE extract presented higher cytotoxicity than AD extract, and HepG2 cells were more resistant than Caco-2 cells. After 24 h exposure to HE extract, the IC50 values were 330 µg/mL and 447 µg/mL for Caco-2 and HepG2 cells, respectively. T. capitellatus has potential as a functional food or as a source of bioactive molecules. These results also highlight the need to preserve species with as yet unknown molecular compositions and potential medicinal applications.

Two- and Three-Dimensional Spectrofluorimetric Qualitative Analysis of Selected Vegetable Oils for Biomedical Applications.

Vegetable oils obtained from different plants are known for their beneficial effects on prophylaxis and supportive treatment of a great deal of inflammatory-mediated conditions. Their wide range of saturated and unsaturated fatty acids, and the presence of other ingredients (e.g., tocopherols, chlorophylls), provide them with anti-inflammatory, antioxidant and anticancer properties, which are worth being exploited. In this study, we have carried out the spectrofluorometric analysis of selected vegetable oils, namely apricot (Prunus armeniaca) kernel oil; blueberry (Vaccinium spp.) seed oil; argan (Argania spinosa) nut oil; kiwi (Actinidia deliciosa) seed oil; grape (Vitis vinifera) seed oil; evening primrose (Oenothera biennis) oil and meadowfoam (Limnanthes alba) seed oil, with the purpose to detect their fluorescent ingredients for further identification and bioactivity comparison. The obtained two- (2D) and three-dimensional (3D) emission spectra offered a complete description of the fluorescent components of the mixture and revealed different features for studied oils.

Polyphenol composition and biological activity of Thymus citriodorus and Thymus vulgaris: Comparison with endemic Iberian Thymus species.

The polyphenol compositions of Thymus × citriodorus and Thymus vulgaris extracts as obtained by exhaustive hydroethanolic (HE) extraction and aqueous decoction (AD) were compared. In addition, their compositions and bioactivities were compared to those of Thymus pulegioides and Thymus mastichina, grown under the same edaphoclimatic conditions, and Thymus carnosus. Rosmarinic acid was the most abundant polyphenol followed by luteolin-hexuronide, salvianolic acids I and K. Cluster analysis suggests a similarity of the polyphenol composition of T. citriodorus and T. vulgaris. A significant antioxidant activity was observed and correlated with their polyphenol levels. The same being observed for the higher anti-proliferative activity/cytotoxicity of HE extracts on Caco-2 and HepG2 cells as compared to AD extracts. Significant association between the total phenolic compounds with the anti-proliferative activity, for both cell lines, was observed. These results support the importance of salvianolic acids levels in Thymus extracts and their in vitro anti-proliferative/cytotoxic activities.

Thymus zygis subsp. zygis an Endemic Portuguese Plant: Phytochemical Profiling, Antioxidant, Anti-Proliferative and Anti-Inflammatory Activities.

Thymus zygis subsp. zygis is an endemic Portuguese plant belonging to the Thymus zygis species. Although T. zygis is commonly used as a condiment and as a medicinal herb, a detailed description of the polyphenol composition of hydroethanolic (HE) and aqueous decoction (AD) extracts is not available. In this work, we describe for the first time a detailed phenolic composition of Thymus zygis subsp. zygis HE and AD extracts, together with their antioxidant, anti-proliferative and anti-inflammatory activities. Unlike other Thymus species, T. zygis subsp. zygis extracts contain higher amounts of luteolin-(?)-O-hexoside. However, the major phenolic compound is rosmarinic acid, and high amounts of salvianolic acids K and I were also detected. T. zygis subsp. zygis extracts exhibited significant scavenging activity of ABTS+, hydroxyl (•OH), and nitric oxide (NO) radicals. Regarding the anti-proliferative/cytotoxic effect, tested against Caco-2 and HepG2 cells, the AD extract only slightly reduced cell viability at higher concentrations (IC50 > 600 µg/mL, 48 h exposure), denoting very low toxicity, while the HE extract showed a high anti-proliferative effect, especially at 48 h exposure (IC50 of 85.01 ± 15.10 µg/mL and 82.19 ± 2.46 µg/mL, for Caco-2 and HepG2, respectively). At non-cytotoxic concentrations, both extracts reduced the nitric oxide (NO) release by lipopolysaccharide (LPS)-stimulated RAW 264.7 cells (at 50 µg/mL, HE and AD extracts inhibited NO release in ~89% and 48%, respectively). In conclusion, the results highlight the non-toxic effect of aqueous extracts, both resembling the consumption of antioxidants in foodstuff or in functional food. Furthermore, the HE extract of T. zygis subsp. zygis is a source of promising molecules with antioxidant, anti-inflammatory and anticancer activities, highlighting its potential as a source of bioactive ingredients for nutraceutical and pharmaceutical industries.

The Nutraceutical Value of Carnitine and Its Use in Dietary Supplements.

Carnitine can be considered a conditionally essential nutrient for its importance in human physiology. This paper provides an updated picture of the main features of carnitine outlining its interest and possible use. Particular attention has been addressed to its beneficial properties, exploiting carnitine's properties and possible use by considering the main in vitro, in animal, and human studies. Moreover, the main aspects of carnitine-based dietary supplements have been indicated and defined with reference to their possible beneficial health properties.

In Vitro Characterization, Modelling, and Antioxidant Properties of Polyphenon-60 from Green Tea in Eudragit S100-2 Chitosan Microspheres.

Eudragit S100-coated chitosan microspheres (S100Ch) are proposed as a new oral delivery system for green tea polyphenon-60 (PP60). PP60 is a mixture of polyphenolic compounds, known for its active role in decreasing oxidative stress and metabolic risk factors involved in diabetes and in other chronic diseases. Chitosan-PP60 microspheres prepared by an emulsion cross-linking method were coated with Eudragit S100 to ensure the release of PP60 in the terminal ileum. Different core-coat ratios of Eudragit and chitosan were tested. Optimized chitosan microspheres were obtained with a chitosan:PP60 ratio of 8:1 (Ch-PP608:1), rotation speed of 1500 rpm, and surfactant concentration of 1.0% (m/v) achieving a mean size of 7.16 µm. Their coating with the enteric polymer (S100Ch-PP60) increased the mean size significantly (51.4 µm). The in vitro modified-release of PP60 from S100Ch-PP60 was confirmed in simulated gastrointestinal conditions. Mathematical fitting models were used to characterize the release mechanism showing that both Ch-PP608:1 and S100Ch-PP60 fitted the Korsmeyers-Peppas model. The antioxidant activity of PP60 was kept in glutaraldehyde-crosslinked chitosan microspheres before and after their coating, showing an IC50 of 212.3 µg/mL and 154.4 µg/mL, respectively. The potential of chitosan microspheres for the delivery of catechins was illustrated, with limited risk of cytotoxicity as shown in Caco-2 cell lines using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The beneficial effects of green tea and its derivatives in the management of metabolic disorders can be exploited using mucoadhesive chitosan microspheres coated with enteric polymers for colonic delivery.

Effect of harvesting year and elderberry cultivar on the chemical composition and potential bioactivity: A three-year study.

Sambucus nigra is one of the richest sources of anthocyanins and other polyphenols being used industrially as a source of antioxidants, colorants, and bioactives. Although cultivars can influence elderberry composition, no study has addressed the effect of harvesting year on elderberries composition and bioactivity. The composition of the main Portuguese cultivars, "Sabugueiro", "Sabugueira" and "Bastardeira", were evaluated during three consecutive years. Harvesting year had a stronger influence on the chemical composition than cultivars, including total sugars, anthocyanins, and phenolic compounds, being related to the different climatic conditions, especially water status. "Bastardeira" was the best cultivar concerning total soluble solids, anthocyanins, polyphenols, and antioxidant activity, but containing a lower total free sugar content compared to "Sabugueiro". The results obtained in this study provide novel information from a nutritional perspective and for breeding programs aiming to select cultivars with enhanced levels of health-promoting compounds or for other industrial applications of elderberries.

Trends in Atopic Dermatitis-From Standard Pharmacotherapy to Novel Drug Delivery Systems.

Atopic dermatitis (AD) is a predominant and deteriorating chronic inflammation of the skin, categorized by robust burning and eczematous lacerations in diverse portions of the body. AD affects about 20% of both offspring and adults worldwide. The pathophysiology of AD combines environmental, hereditary, and immunological aspects, together with skin barrier dysfunction. The procedures used to prevent the disease are the everyday usage of creams to support the restoration of the epidermal barrier. The classical treatments include the use of topical corticosteroids as a first-line therapy, but also calcineurin inhibitors, antihistamines, antibiotics, phototherapy, and also immunosuppressant drugs in severe cases of AD. Topical drug delivery to deeper skin layers is a difficult task due to the skin anatomic barrier, which limits deeper penetration of drugs. Groundbreaking drug delivery systems, based on nanoparticles (NPs), have received much attention due to their ability to improve solubility, bioavailability, diffusion, targeting to specific types of cells, and limiting the secondary effects of the drugs employed in the treatment of AD. Even so, additional studies are still required to recognize the toxicological characteristics and long-term safety of NPs. This review discusses the current classical pharmacotherapy of AD against new nanoparticle skin delivery systems and their toxicologic risks.

Sugar-Lowering Drugs for Type 2 Diabetes Mellitus and Metabolic Syndrome-Review of Classical and New Compounds: Part-I.

Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia together with disturbances in the metabolism of carbohydrates, proteins and fat, which in general results from an insulin availability and need imbalance. In a great number of patients, marketed anti-glycemic agents have shown poor effectiveness in maintaining a long-term glycemic control, thus being associated with severe adverse effects and leading to an emerging interest in natural compounds (e.g., essential oils and other secondary plant metabolites, namely, flavonoid-rich compounds) as a novel approach for prevention, management and/or treatment of either non-insulin-dependent diabetes mellitus (T2DM, type 2 DM) and/or Metabolic Syndrome (MS). In this review, some of these promising glucose-lowering agents will be comprehensively discussed.

Clotrimazole-Loaded Mediterranean Essential Oils NLC: A Synergic Treatment of Candida Skin Infections.

The increasing development of resistance of Candida species to traditional drugs represents a great challenge to the medical field for the treatment of skin infections. Essential oils were recently proposed to increase drug effectiveness. Herein, we developed and optimized (23 full factorial design) Mediterranean essential oil (Rosmarinus officinalis, Lavandula x intermedia "Sumian", Origanum vulgare subsp. hirtum) lipid nanoparticles for clotrimazole delivery, exploring the potential synergistic effects against Candida spp. Small sized nanoparticles (<100 nm) with a very broad size distribution (PDI < 0.15) and long-term stability were successfully prepared. Results of the in vitro biosafety on HaCaT (normal cell line) and A431 (tumoral cell line), allowed us to select Lavandula and Rosmarinus as anti-proliferative agents with the potential to be used as co-adjuvants in the treatment of non-tumoral proliferative dermal diseases. Results of calorimetric studies on biomembrane models, confirmed the potential antimicrobial activity of the selected oils due to their interaction with membrane permeabilization. Nanoparticles provided a prolonged in vitro release of clotrimazole. In vitro studies against Candida albicans, Candida krusei and Candida parapsilosis, showed an increase of the antifungal activity of clotrimazole-loaded nanoparticles prepared with Lavandula or Rosmarinus, thus confirming nanostructured lipid carriers (NLC) containing Mediterranean essential oils represent a promising strategy to improve drug effectiveness against topical candidiasis.

Chemical Characterization and Bioactivity of Extracts from Thymus mastichina: A Thymus with a Distinct Salvianolic Acid Composition.

Thymus mastichina, also called mastic thyme or Spanish marjoram, is endemic to the Iberian Peninsula, where it is widely used in folk medicine especially for treating digestive and respiratory systems disorders, and as a condiment to season olives. This work describes for the first time the detailed phenolic composition of exhaustive hydroethanolic extracts and aqueous decoctions of Thymus mastichina. Unlike other species of the Thymus genera, Thymus mastichina extracts contain high amounts of salvianolic acid derivatives, with salvianolic acid A isomer being the main derivative. This isomer was identified in extracts from Thymus mastichina for the first time. Also, an undescribed salvianolic acid derivative in Thymus mastichina was identified and its structure was tentatively described. Extracts from Thymus mastichina showed significant scavenging activity of 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical cation, hydroxyl, and nitric oxide radicals. The anti-proliferative effect of both T. mastichina extracts were tested against Caco-2 and HepG2 cells; the hydroethanolic extract showed a high anti-proliferative activity against Caco-2 cells compared to HepG2 cells (at 24 h exposure, the concentration that inhibits 50% of proliferation, IC50, was 71.18 ± 1.05 µg/mL and 264.60 ± 11.78 µg/mL for Caco-2 and HepG2, respectively). Thus, these results make this species a promising candidate for further investigation of its anti-tumoral potential. Therefore, Thymus mastichina can be potentially used as a functional food (used as a decoction or herbal tea) or as a source of bioactive ingredients with antioxidant and anti-proliferative properties.

Thymus pulegioides L. as a rich source of antioxidant, anti-proliferative and neuroprotective phenolic compounds.

The potential of Thymus pulegioides L. as an alternative and valuable source of functional ingredients has been assessed. For this purpose, the phenolic constituent profiles and the antioxidant, anti-proliferative, neuroprotective, anti-aging and anti-diabetic activities of both T. pulegioides aqueous decoctions (AD) and hydro-ethanolic extracts (HE) were studied and compared for the first time. Rosmarinic acid was the main phenolic compound, accounting for 35.2% or 47.8% of total identified phenols in AD or HE, respectively (58.5 and 151.9 mg g-1 extract). Furthermore, large amounts of luteolin-O-hexuronide (AD: 39.9 ± 2.5 mg g-1, HE: 60.8 ± 7.0 mg g-1), eriodictyol-O-hexuronide (AD: 19.9 ± 2.5 mg g-1, HE: 26.8 ± 7.0 mg g-1) and chrysoeriol hexoside (AD: 23.5 ± 0.7 mg g-1, HE: 16.0 ± 0.7 mg g-1) were found. Both extracts showed significant in vitro antioxidant activity and anti-proliferative activity against Caco-2 cells (IC50 82.25 µg mL-1 and 105.44 µg mL-1, AD and HE, respectively), and reduced hepatotoxicity (HepG2 cells). In general, both T. pulegioides extracts showed poor anti-diabetic activity, moderate anti-aging effects and high neuroprotective activity with both AD and HE extracts, at 0.5 mg mL-1, showing 80% inhibition of the acetylcholinesterase activity and 94% inhibition of the tyrosinase activity. The present study highlights the important potential of this herb as a functional food ingredient.

New grape stems' isolated phenolic compounds modulate reactive oxygen species, glutathione, and lipid peroxidation in vitro: Combined formulations with vitamins C and E.

The antioxidant potential of grape (Vitis vinífera L.) stems has been reported in the last decade although no identification of the individual compounds responsible for such action has been done. In this work, polyphenolic extract of grape stems was processed resorting to semi-preparative HPLC, allowing to obtain 5 purified polyphenols (caftaric acid, malvidin-3-O-glucoside, quercetin-3-O-glucuronide, mailvidin-3-O-(6-O-caffeoyl)-glucoside, and Σ-viniferin), which were fully characterized by HPLC-PDA-ESI-MSn. Isolated compounds were featured on their radical scavenging capacity (DPPH and ABTS), cell viability, anti-inflammatory activity, and capacity to modulate the level of reactive oxygen species, glutathione, lipid peroxidation, and overall oxidative stress in a biological model (human keratinocytes) in vitro, under basal and oxidative conditions. The results obtained noticed the combinations malvidin-3-O-glucoside+Vitamin E and quercetin-3-O-glucuronide+vitamin C as the most effective, allowing to improve the capacity of complete extracts or individual compounds, and being candidates to be used in the development of new functional products.

alpha-Latrotoxin increases spontaneous and depolarization-evoked exocytosis from pancreatic islet beta-cells.

alpha-Latrotoxin (alpha-LT), a potent excitatory neurotoxin, increases spontaneous, as well as action potential-evoked, quantal release at nerve terminals and increases hormone release from excitable endocrine cells. We have investigated the effects of alpha-LT on single human, mouse and canine beta-cells. In isolated and combined measurements, alpha-LT, at nanomolar concentrations, induces: (i) rises in cytosolic Ca(2+), into the micromolar range, that are dependent on extracellular Ca(2+); (ii) large conductance non-selective cation channels; and (iii) Ca(2+)-dependent insulin granule exocytosis, measured as increases in membrane capacitance and quantal release of preloaded serotonin. Furthermore, at picomolar concentrations, alpha-LT potentiates depolarization-induced exocytosis often without evidence of inducing channel activity or increasing cytosolic Ca(2+). These results strongly support the hypothesis that alpha-LT, after binding to specific receptors, has at least two complementary modes of action on excitable cells. (i) alpha-LT inserts into the plasma membrane to form Ca(2+) permeable channels and promote Ca(2+) entry thereby triggering Ca(2+)-dependent exocytosis in unstimulated cells. (ii) At lower concentrations, where its channel forming activity is hardly evident, alpha-LT augments depolarization-evoked exocytosis probably by second messenger-induced enhancement of the efficiency of the vesicle recruitment or vesicle fusion machinery. We suggest that both modes of action enhance exocytosis from a newly described highly Ca(2+)-sensitive pool of insulin granules activated by global cytosolic Ca(2+) concentrations in the range of approximately 1 microm.