Validation of the traditional antimicrobial use of the Iranian medicinal plant Thymus daenensis Celak through phytochemical characterization of its bioactive constituents.

Thymus daenensis Celak (Lamiaceae family), known as denaian thyme, is an Iranian endemic plant, commonly used for its carminative, expectorant, antibacterial and antifungal properties. Previous studies report the chemical profile of the essential oil of T. daenensis aerial parts, but little is known about its non volatile constituents. Herein, phytochemical and biological investigation of the polar extract of T. daenensis aerial parts to provide further insight into traditional use of this plant has been accomplished. High-performance liquid chromatography coupled to linear ion-trap and orbitrap high-resolution mass spectrometry (LC-ESI/LTQOrbitrap/MS/MS) analysis of MeOH extract was performed to guide the isolation of specialized metabolites and successive characterization by NMR analysis. MeOH extract displayed antioxidant activity evaluated by DPPH (EC50 = 48.99 ± 1.47 µg/mL) and TEAC assay (1.37 mg/mL). Successively, the biofilm inhibitory activity of extract and isolated compounds against mature biofilms of Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Listeria monocytogenes, and Staphylococcus aureus, and their influence on the metabolism of sessile bacterial cells were evaluated. Two previously unreported thymol derivatives have been identified. The biofilm inhibitory activity of isolated compounds highlighted a promising antimicrobial action for the tested compounds. In particular, vanillic acid, (3S,5R,6R,7E,9S)-megastigm-7-ene-3,5,6,9-tetrol 3-O-ß-D-glucopyranoside, thymoquinol-2-O-ß-D-glucopyranoside and the never reported compound daenol resulted capable of exerting inhibitory activity vs all pathogenic strains. All compounds tested at a concentration of 10 µg/mL inhibited the metabolism of the sessile cells of E. coli at a percentage ranging between 37.7% and 77.39%, and of L.monocytogenes at a percentage ranging between 21.79% and 71.17%.

Development of a New Extraction Method for Pomegranate and Metabolite Profiling by a LC-MS and 1H NMR Combined Approach.

The pomegranate (Punica granatum L.) market has steadily grown due to the increasing demand of health-conscious consumers of products with health-promoting effects. Recently, a growing interest in developing ecological and environmentally friendly extraction methods has led to investigating extraction procedures to obtain a higher extraction yield using a lower amount of solvents and energy. Herein, a new extraction procedure was developed to obtain an enriched pomegranate food supplement by using the peels of pomegranate, cultivar "Dente di Cavallo" and its juice. The extraction was performed through a non-conventional extraction technique like SLDE-Naviglio using ethanol and pomegranate juice as a solvent, and peels as a matrix. The extract was analysed by a combined approach based on LCESI/QExactive/MS/MS and NMR analysis, and its chemical profile was compared with those of pomegranate juice and of the extract obtained from peels by SLDE-Naviglio by using ethanol:H2O. The LC-MS analysis highlighted the presence of hydrolysable tannins, flavonoids, ellagic acid and phenol glucoside derivatives, while 1H NMR analysis completed the profile by detecting the primary metabolites. The LC-MS and 1H NMR analysis indicated that the extract obtained by SLDE-Naviglio using ethanol and pomegranate juice was enriched in the bioactives as confirmed by the highest phenolic, tannin and flavonoid content.

Demethylcalabaxanthone from Garcinia mangostana Exerts Antioxidant Effects through the Activation of the Nrf2 Pathway as Assessed via Molecular Docking and Biological Evaluation.

Nuclear factor erythroid 2-related factor 2 (Nrf2) pathway activation promotes the expression of antioxidant enzymes in response to rising oxidative stress, resulting in reactive oxygen species (ROS) detoxification and playing a central role in the maintenance of intracellular redox homeostasis and regulation of inflammation. Moreover, the biological effects of Nrf2 pathway activation contribute to reducing apoptosis and enhancing cell survival. The activity of Nrf2 is negatively regulated by Kelch-like ECH-associated protein 1 (Keap1). Prompted by the recent results reporting the impact of xanthone metabolites on oxidative stress, cancer, and inflammation, the antioxidant properties of xanthones isolated from Garcinia mangostana (γ-mangostin, α-mangostin, 8-deoxygartanin, demethylcalabaxanthone, garcinone D) were assessed. In particular, the capability of these natural products to disrupt the interaction between Kelch-like ECH-associated protein 1 (Keap1) and nuclear factor erythroid 2-related factor 2 (Nrf2), triggering the activation of the Nrf2-mediated pathway, was evaluated using molecular docking experiments and in vitro tests. The modulation of some key Nrf2-related mediators like glutathione (GSH) and lactate dehydrogenase (LDH) to highlight a possible direct antioxidant effect was investigated. Among the tested compounds, demethylcalabaxanthone showed an indirect antioxidant effect, as corroborated by a Western blot assay, displaying a significant increase in the translocated protein upon its administration.

LC-MS and NMR Based Plant Metabolomics: A Comprehensive Phytochemical Investigation of Symphytum anatolicum.

The application of metabolomics to the study of plants is growing because of the current development of analytical techniques. The most commonly used analytical technology driving plant metabolomics studies is Mass Spectrometry (MS) coupled to liquid chromatography (LC). In recent years, Nuclear Magnetic Resonance (NMR) spectroscopy, not requiring a previous chromatographic separation, has been receiving growing attention for metabolite fingerprinting of natural extracts. Herein, an integrated LC-MS and 1H NMR metabolomic approach provided a comprehensive phytochemical characterization of Symphytum anatolicum whole plant, taking into account both primary and specialized metabolites. Moreover, the NMR analyses provided direct quantitative information. Species belonging to the Symphytum genus, known as comfrey, have shown several biological activities including anti-inflammatory, analgesic, hepatoprotective, antifungal, and antibacterial. The LC-MS profile showed the presence of 21 main specialized metabolites, belonging to the classes of flavonoids, phenylpropanoids, salvianols, and oxylipins. The 1H NMR spectrum revealed the occurrence of metabolites including organic acids, phenolics, flavonoids, sugars, and amino acids. A quantitative analysis of these metabolites was performed and their concentration was obtained with respect to the known concentration of TSP, by means of the software package Chenomx which allows quantification of individual components in the NMR spectra. Furthermore, the phenolic content, antioxidant activity, glucosidase, and tyrosinase inhibitory activity of S. anatolicum extract were evaluated. The resulting bioactivity profile suggests how S. anatolicum represents a source of metabolites with health-promoting activity.

Corylus avellana "Nocciola Piemonte": metabolomics focused on polar lipids and phenolic compounds in fresh and roasted hazelnuts.

The common hazel plant (Corylus avellana L., Betulaceae) is one of the most popular tree nuts widespread in Europe and Asia. In Italy, there are different cultivars among which the cultivar affording the valuable hazelnut "Tonda Gentile Trilobata," also known as "Tonda Gentile delle Langhe," covered by the Protected Geographical Indication (PGI) label "Nocciola Piemonte" (NP), known for its sweetness, cooked-bread aroma, and the low intensity of the burnt aroma. In order to obtain a detailed and in-depth characterization of the polar fraction of fresh (NPF) and roasted (NPR) kernels of NP the analysis of the n-butanol extracts by liquid chromatography coupled to electrospray ionization and high-resolution mass spectrometry (LC-ESI/HRMS) was carried out. Moreover, to evaluate the quantitative distribution of the most representative polar lipids in NPF and NPR, the analysis by liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) was performed. To unambiguously identify the phenolic compounds highlighted by the LC-ESI/HRMS profiles, they were isolated from the n-butanol extract and characterized by Nuclear Magnetic Resonance (NMR) experiments. Finally, the ability of the isolated compounds to exert radical scavenging activity and to inhibit the lipid peroxidation induced by H2O2 or H2O2/Fe2+ was tested by Trolox Equivalent Antioxidant Capacity (TEAC) and thiobarbituric acid reactive substances (TBARS) assays, respectively. The LC-ESI/HRMS allowed to ascertain the presence of phenolic compounds and multiple classes of polar lipids including phospholipids, glycolipids, sphingolipids, and oxylipins. The quantitative analysis highlighted in NPR fraction a lipid content three times higher than in NPF, evidencing lyso-phospholipids and phospholipids as the most represented lipid classes in both NPF and NPR, together accounting for 94 and 97% of the considered lipids, respectively. Furthermore, phytochemical analysis permitted to identify flavonoid and diarylheptanoid derivatives. In particular, quercetin 3-O-ß-D-galactopyranosyl-(1→2)-ß-D-glucopyranoside and myricetin-3-O-α-L-rhamnopyranoside showed the highest antioxidant activity, exhibiting TEAC values similar to that of quercetin, used as reference compound (2.00 ± 0.03 and 2.06 ± 0.03 mM vs 2.03 ± 0.03 mM, respectively). Moreover, most of the tested compounds were found to reduce lipid peroxidation induced by H2O2 and H2O2/Fe2+ more than curcumin used as positive control, with myricetin-3-O-α-L-rhamnopyranoside determining 44.4 % and 34.1 % inhibition percentage, respectively.

LC-MS- and 1H NMR-Based Metabolomics to Highlight the Impact of Extraction Solvents on Chemical Profile and Antioxidant Activity of Daikon Sprouts (Raphanus sativus L.).

Currently, the interest of consumers towards functional foods as source of bioactive compounds is increasing. The sprouts of Raphanus sativus var longipinnatus (Brassicaceae) are "microgreens" popular, especially in gourmet cuisine, for their appealing aspect and piquant flavour. They represent a functional food due to their high nutritional value and health-promoting effects. Herein, the sprouts of daikon were extracted by different solvent mixtures to highlight how this process can affect the chemical profile and the antioxidant activity. An in-depth investigation based on a preliminary LC-ESI/LTQOrbitrap/MS profiling was carried out, leading to the identification of nineteen compounds, including glucosinolates and hydroxycinnamic acid derivatives. An undescribed compound, 1-O-feruloyl-2-O-sinapoyl-ß-D-glucopyranoside, was isolated, and its structure was elucidated by NMR spectroscopy. The phenolic content and radical scavenging activity (DPPH and TEAC assays), along with the ability to activate Nrf2 (Nrf2-mediated luciferase reporter gene assay) of polar extracts, were evaluated. The results showed the highest antioxidant activity for the 70% EtOH/H2O extract with a TEAC value of 1.95 mM and IC50 = 93.97 µg/mL in the DPPH assay. Some 50% and 70% EtOH/H2O extracts showed a pronounced concentration-dependent induction of Nrf2 activity. The extracts of daikon sprouts were submitted to 1H NMR experiments and then analyzed by untargeted and targeted approaches of multivariate data analysis to highlight differences related to extraction solvents.

Anti-inflammatory Activity of Tanshinone-Related Diterpenes from Perovskia artemisioides Roots.

Perovskia artemisioides is a perennial and aromatic plant widely distributed in the Baluchestan region of Iran. Phytochemical analysis of a n-hexane extract of P. artemisioides roots, guided by an analytical approach based on LC-ESI/LTQOrbitrap/MS/MS, yielded six previously undescribed diterpenoid compounds (2, 9-11, 16, and 20), and 19 known diterpenoids, for which the structures were elucidated by 1D and 2D NMR experiments. Some of the isolated compounds showed significant anti-inflammatory activity using J774A.1 macrophage cells stimulated with Escherichia coli lipopolysaccharide. In particular, compounds 6, 8, 17, 18, 20, and 22 significantly inhibited the release of nitric oxide and the expression of related pro-inflammatory enzymes, such as inducible nitric oxide synthase and cycloxygenase-2. Moreover, two compounds that showed the highest activity in reducing nitric oxide release (6 and 18) were tested to evaluate their effects on nitrotyrosine formation and reactive oxygen species release. Both compounds inhibited ROS release and, in particular, compound 6 also inhibited nitrotyrosine formation at all tested concentrations, thus indicating a significant antioxidant potential.

Scabiosa atropurpurea: A rich source of iridoids with α-glucosidase inhibitory activity evaluated by in vitro and in silico studies.

Scabiosa atropurpurea L. subsp. maritima (L.) Arc. (Caprifoliaceae) is a plant widely distributed in the Mediterranean region and represented by 32 taxa in the flora of Turkey. In the present study, an in-depth phytochemical investigation of S. atropurpurea methanol extract of the whole plant was carried out using a combination of LC-ESI-FT-MS and NMR analysis. This approach allowed the isolation and structural elucidation of 28 compounds: 17 iridoids, 7 flavonoids, and 4 phenolic acids. Among these compounds, three previously unreported iridoids named secologanin-methyl-hemiacetal, atropurpurin A, and atropurpurin B were identified. The methanol extract of S. atropurpurea was assayed for its antioxidant and antihyperglycemic activity, showing a potent α-glucosidase inhibitory activity (IC50 = 100 µg/mL), higher than that exerted by acarbose (IC50 = 196 µg/mL), used as the positive control. Thus, the most abundant iridoids were selected to be tested for their antihyperglycemic activity, and molecular docking experiments were carried out to assess the possibility for selected compounds to form complexes with α-glucosidase enzyme active site.

Phytochemical Investigation and Biofilm-Inhibitory Activity of Bachtiari Savory (Satureja bachtiarica Bunge) Aerial Parts.

Satureja bachtiarica is an endemic plant from the Lamiaceae family, growing in the Zagros mountain range in Iran. Even if S. bachtiarica is reported to possess many biological activities, little is known about its chemical composition. For this reason, in the present research, a phytochemical investigation of this species was carried out. To have a preliminary metabolite profile of S. bachtiarica, the n-BuOH extract was analyzed using LC-ESI/LTQOrbitrap/MS/MS in negative ion mode, allowing the identification of specialized metabolites belonging to flavonoid, monoterpene, indol, phenylpropanoid, phenolic, lignan, coumarin, biphenyl, and triterpene classes. The LC-MS/MS analysis guided the isolation of compounds, and their structures were characterized using spectroscopic methods including 1D- and 2D-NMR experiments and HRMSn analysis. In this way, a compound never reported before belonging to the biphenyl class was identified. Total flavonoid content of the extract along with the antioxidant activity were assessed. Based on the traditional uses of S. bachtiarica suggesting potential antibacterial properties, an evaluation of the biofilm inhibitory activity of the extract and isolated compounds against mature biofilms of Acinetobacter baumannii, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, and Staphylococcus aureus, as well as their influence on the metabolism of sessile bacterial cells, was conducted. The results evidenced that some compounds including parmentin B, biphenyls, and 1-(1H-indole-3-carboxylate)-ß-D-glucopyranoside might inhibit some changes occurring in the bacterial cells, which increases their virulence. In particular, biphenyl derivatives at a concentration of 80 µg/mL were capable of limiting remarkably the mature biofilms of A. baumannii and L. monocytogenes remarkably at a percentage ranging between 52.76% and 75.02%, and they reached an inhibition percentage of 69.28 % against E. coli. Biphenyl derivatives were also effective in exerting an inhibitory action against the mature biofilm of P. aeruginosa (inhibition ranging from 59.38% to 81.08%) and Staphylococcus aureus (inhibition percentage reached 82.94%). Of note, the biphenyl derivatives resulted in being capable of acting on the metabolism of the cells within the biofilm of all five pathogens.

Comparative Volatilomic Profile of Three Finger Lime (Citrus australasica) Cultivars Based on Chemometrics Analysis of HS-SPME/GC-MS Data.

Finger lime is receiving growing attention as an ingredient of gastronomic preparations of haute cuisine for its delicious flavor and fragrance and for its appealing aspect. Volatile compounds play a crucial role in determining the organoleptic characteristics of the fruit and its pleasantness for consumers. The aim of the present study was to investigate the volatile profiles by headspace solid phase micro-extraction (HS-SPME) coupled to gas chromatography-mass spectrometry (GC-MS) in the peel and, for the first time, in the pulp of three Australian finger lime cultivars grown in Sicily (southern Italy): Pink Pearl, Sanguinea, and Faustrime, allowing to overall identify 84 volatile organic compounds (VOCs). The analytical data showed that the three cultivars were characterized by distinct volatile chemotypes: limonene/sabinene/bicyclogermacrene in the Pink Pearl, limonene/γ-terpinene/bicyclogermacrene in the Sanguinea, and limonene/ß-phellandrene/γ-terpinene in the Faustrime. Moreover, some volatiles, found exclusively in one cultivar, could be considered potential markers of the individual cultivar. PCA allowed us to clearly discriminate the samples into three clusters: the first related to the Sanguinea peel, the second to the Faustrime peel, and a third group associated with the Pink Pearl peel along with the pulp of the three cultivars. Accordingly, the VOCs that mostly contributed to the differentiation of the three finger lime cultivars were also identified. Among them, D-limonene, sabinene γ-terpinene, α-pinene, α-phellandrene, ß-myrcene, p-cymene, linalool, δ-elemene, ledene, bicyclogermacrene ß-citronellol, α-bergamotene, α-caryophillene, and ß-bisabolene, have been previously reported to exhibit important biological activities, suggesting that these cultivars, in addition to possessing unique volatile profiles, can show promise for several applications in pharmaceutical and food industry, namely for development of functional foods.

Phytochemical investigation of Volutaria lippii and evaluation of the antioxidant activity.

Volutaria lippii (L.) Cass. ex Maire, syn. Centaurea lippii (L.), (Asteraceae) is a plant from the central region of Algeria, considerably distributed in all Mediterranean areas. Herein, the antioxidant activity of the three derived fractions [chloroform (CHCl3), ethyl acetate (EtOAc) and n-butanol (n-BuOH)] of the 70% methanol extract of the aerial parts (leaves and flowers), was assessed by using CUPRAC, ABTS, DPPH free radical scavenging, and ß-carotene bleaching methods. The results obtained allowed to guide the fractionation of EtOAc and n-BuOH fractions by CC followed by purification by TLC and reverse phase HPLC. A guaianolide glucoside, 3ß-hydroxy-11ß,13-dihydrodehydrocostuslactone 8α-O-(6'-acetyl-ß-glucopyranoside) (1), never reported in the literature, was isolated together with other known compounds (2-14). Their structures were elucidated by the extensive use of 1 D- and 2 D-NMR experiments along with ESI-MS analyses and with comparison with literature data.

Pro-Apoptotic and Pro-Autophagic Properties of Cardenolides from Aerial Parts of Pergularia tomentosa

Pergularia tomentosa L., a milkweed tropical plant belonging to the family Asclepiadaceae, is a rich source of unusual cardiac glycosides, characterised by transfused A/B rings and a sugar moiety linked by a double link, generating a dioxanoid structure. In the present report, five cardenolides isolated from the aerial parts of the plant (calactin, calotropin, 12ß-hydroxycalactin, 12ß,6'-dihydroxycalotropin, and 16α-hydroxycalotropin) were investigated for their biological effects on a human hepatocarcinoma cell line. Cell viability was monitored by an MTT assay. The occurrence of apoptosis was evaluated by detecting caspase-3 activation and chromatin fragmentation. The ability of these compounds to induce autophagy was analysed by monitoring two markers of the autophagic process, LC3 and p62. Our results indicated that all cardenolides had cytotoxic effects, with IC50 ranging from 0.127 to 6.285 µM. All compounds were able to induce apoptosis and autophagy, calactin being the most active one. Some of them also caused a reduction in cell migration and a partial block of the cell cycle into the S-phase. The present study suggests that selected cardenolides from aerial parts of P. tomentosa, particularly calactin, possess potentially desirable properties for further investigation as anticancer agents.

Garcinia mangostana L. fruits and derived food supplements: Identification and quantitative determination of bioactive xanthones by NMR analysis.

Mangosteen (Garcinia mangostana L.), known as "the queen of fruits", is one of the most praised tropical fruit due to its delicious taste. In the last years, the use of mangosteen in functional products has been increasing, mainly in food beverages and nutraceutical formulations due to its biological activities related to the content of xanthones. The quantitative Nuclear Magnetic Resonance (qNMR) analysis, a rapid and accurate method used for simultaneous quantification of plant metabolites, was here employed to determine the amount of bioactive xanthones in the extracts of G. mangostana arils and shells obtained by using solvent of increasing polarity along with ''eco-friendly'' solvents like ethanol and ethanol-water. Furthermore, the content of xanthones was compared with that occurring in four selected commercial food supplements, among which tablets and capsules, and two fruit juices, based on mangosteen. Quantitative results highlighted a significant variability: the extracts of the shells displayed a higher amount of bioactive xanthones than those of the arils, in particular, of γ-mangostin and α-mangostin, while ß-mangostin, demethylcalabaxanthone, mangostanin, 8-deoxygartanin occurred in higher amounts in arils. A certain variability in the amount of biologically active xanthones (i.e. α-mangostin and γ-mangostin) could be observed in commercial food supplements.

Metabolite Profiling of "Green" Extracts of Cynara cardunculus subsp. scolymus, Cultivar "Carciofo di Paestum" PGI by 1H NMR and HRMS-Based Metabolomics.

Globe artichoke (Cynara cardunculus L. var. scolymus L.), is a perennial plant widely cultivated in the Mediterranean area, known for its edible part named capitula or heads. Its functional properties are related to its high levels of polyphenolic compounds and inulin. "Carciofo di Paestum", an Italian traditional cultivar, is a labeled PGI (Protected Geographical Indication) product of the Campania region, representing an important economic resource. So far, a few chemical investigations were performed on this cultivar, mainly focused on the analysis of methanol extracts. Due to the increasing use of food supplements, in this study, a comprehensive analysis of green extracts of "Carciofo di Paestum" PGI heads was performed. EtOH, EtOH: H2O (80:20, 70:30, 60:40) extracts, as well as infusions and decoctions prepared according to Pharmacopeia XII were analyzed by LC-ESI/QExactive/MS/MS. A total of 17 compounds corresponding to caffeoylquinic acid derivatives, phenolics, flavonoids, and terpenoids were identified. The extracts were further submitted to NMR analysis to highlight the occurrence of primary metabolites. Both LCMS and NMR data were analyzed by Principal Component Analysis (PCA), showing significant differences among the extraction methods. Moreover, 5-caffeoylquinic acid and 1,5-dicaffeoylquinic acid were quantified in the extracts by LC-ESI/QTrap/MS/MS using the Multiple Reaction Monitoring (MRM) method. Furthermore, the phenolic content, antioxidant activity, and α-glucosidase inhibitory activity of C. cardunculus var. scolymus "Carciofo di Paestum" extracts were evaluated.

Corylus avellana: A Source of Diarylheptanoids With α-Glucosidase Inhibitory Activity Evaluated by in vitro and in silico Studies.

Corylus avellana hard shells, green leafy involucres, leaves, and male flowers have shown to be a source of diarylheptanoids, a class of natural products with promising biological activities. Cyclic diarylheptanoids, named giffonins, were isolated from the Italian cultivar "Tonda di Giffoni." Even if many efforts have been made to establish the chemistry of these compounds, little is known about their biological properties. Herein, the inhibitory effects of diarylheptanoids isolated from C. avellana byproducts against α-glucosidase enzyme were evaluated. Molecular docking experiments disclosed the establishment of several key interactions between all the screened diarylheptanoids and the protein counterpart, whose model was built through homology modeling procedure, thus rationalizing the detected inhibitory activities. Specifically, the most active compounds giffonin J (10), K (11), and P (16) were able to make both H-bonds and π-π stacking contacts with different residues belonging to the binding site responsible for the catalytic activity of the investigated enzyme. To highlight the occurrence of the bioactive diarylheptanoids in the extracts of C. avellana byproducts obtained by eco-friendly extractions, their LC-MS profiles were analyzed. LC-MS analysis showed how giffonin J (10), K (11), and P (16) occurred in the ethanol extract of the leaves, while in the extracts of shells and green leafy involucres only giffonin P (16) was evident. Moreover, the quantitative analysis of giffonin J (10), K (11), and P (16) in C. avellana byproducts was carried out by an analytical approach based on LC-ESI/QTrap/MS, using the Multiple Reaction Monitoring (MRM) experiment. These results prompt to evaluate C. avellana byproducts, especially the leaves, as a prospective source of bioactive diarylheptanoids for the development of functional ingredients for the treatment of diabetes.

Salviolone from Salvia miltiorrhiza Roots Impairs Cell Cycle Progression, Colony Formation, and Metalloproteinase-2 Activity in A375 Melanoma Cells: Involvement of P21(Cip1/Waf1) Expression and STAT3 Phosphorylation.

Melanoma is a highly malignant solid tumor characterized by an elevated growth and propagation rate. Since, often, melanoma treatment cannot prevent recurrences and the appearance of metastasis, new anti-melanoma agents need to be discovered. Salvia miltiorrhiza roots are a source of diterpenoid derivatives, natural compounds with several biological activities, including antiproliferative and anticancer effects. Seven diterpenoid derivatives were purified from S. miltiorrhiza roots and identified by NMR and MS analysis. Tanshinone IIA and cryptotanshinone were detected as the main components of S. miltiorrhiza root ethanol extract. Although their antitumor activity is already known, they have been confirmed to induce a reduction in A375 and MeWo melanoma cell growth. Likewise, salviolone has been shown to impair the viability of melanoma cells without affecting the growth of normal melanocytes. The underlying anticancer activity of salviolone has been investigated and compared to that of cryptotanshinone in A375 cells, showing an increased P21 protein expression in a P53-dependent manner. In that way, salviolone, even more than cryptotanshinone, displays a multitarget effect on cell-cycle-related proteins. Besides, it modulates the phosphorylation level of the signal transducer and activator of transcription (STAT)3. Unexpectedly, salviolone and cryptotanshinone induce sustained activation of the extracellular signal-regulated kinases (ERK)1/2 and the protein kinase B (Akt). However, the blockage of ERK1/2 or Akt activities suggests that kinase activation does not hinder their ability to inhibit A375 cell growth. Finally, salviolone and cryptotanshinone inhibit to a comparable extent some crucial malignancy features of A375 melanoma cells, such as colony formation in soft agar and metalloproteinase-2 activity. In conclusion, it has been shown for the first time that salviolone, harboring a different molecular structure than tanshinone IIA and cryptotanshinone, exhibits a pleiotropic effect against melanoma by hampering cell cycle progression, STAT3 signaling, and malignant phenotype of A375 melanoma cells.

Mangostanin, a Xanthone Derived from Garcinia mangostana Fruit, Exerts Protective and Reparative Effects on Oxidative Damage in Human Keratinocytes.

The fruit of Garcinia mangostana (mangosteen) is known in ancient traditional Asian medicine for its antioxidant, anti-inflammatory, immunomodulatory and anticancer activities. These effects are mainly due to the action of polyphenols known as xanthones, which are contained in the pericarp of the fruit. In recent years, there has been a growing interest from pharmaceutical companies in formulating new topicals based on mangosteen full extracts to prevent skin aging. However, the molecules responsible for these effects and the mechanisms involved have not been investigated so far. Here, the arils and shells of Garcinia mangostana were extracted with chloroform and methanol, and the extracts were further purified to yield 12 xanthone derivatives. Their effects were evaluated using in vitro cultures of human epidermal keratinocytes. After confirming the absence of cytotoxicity, we evaluated the antioxidant potential of these compounds, identifying mangostanin as capable of both protecting and restoring oxidative damage induced by H2O2. We showed how mangostanin, by reducing the generation of intracellular reactive oxygen species (ROS), prevents the activation of AKT (protein kinase B), ERK (extracellular signal-regulated kinase), p53, and other cellular pathways underlying cell damage and apoptosis activation. In conclusion, our study is the first to demonstrate that mangostanin is effective in protecting the skin from the action of free radicals, thus preventing skin aging, confirming a potential toward its development in the nutraceutical and cosmeceutical fields.

Metabolite Profiling of Helichrysum italicum Derived Food Supplements by 1H-NMR-Based Metabolomics.

Helichrysum italicum is a medicinal plant from the Mediterranean area, widely used in traditional medicine for its anti-inflammatory, antibacterial and antioxidant properties and for its preventive effects on microcirculation diseases. Due to these properties, it finds large applications in cosmetic, food and pharmaceutical fields. Additionally, hydroalcoholic extracts and mother tinctures based on H. italicum represent products with a high commercial value, widely distributed not only in drug stores but also on on-line markets. The different extraction procedures used can greatly affect the fingerprints of the extracts, resulting in a different qualitative or quantitative profile of the chemical constituents responsible for biological activity. The aim of the present study was to characterize the composition of bioactive compounds present in water-ethanol and glycerol extracts of H. italicum derived food supplements. Metabolite profiles of the extracts were obtained by 1H NMR experiments and data were processed by multivariate statistical analysis to highlight differences in the extracts and to evidence the extracts with the highest concentrations of bioactive metabolites. In detail, this work highlights how derived food supplements of H. italicum obtained using ethanol-water mixtures ranging from 45% to 20% of ethanol represent the products with the highest amount of both primary (amino acids) and secondary metabolites including 3,4-dicaffeoylquinic acid (9), chlorogenic acid (10), 3,5-dicaffeoylquinic acid (11), and kaempferol 3-O-glucopyranoside (12). Moreover, it is evident that the use of an ethanol-water mixture 20:80 is the most suitable method to afford the highest number of phenolic compounds, while food-derived supplements obtained by glycerol extraction are characterized by a high amount of ß-glucose and α-glucose and a low content of phenolic compounds.

Pouteria lucuma Pulp and Skin: In Depth Chemical Profile and Evaluation of Antioxidant Activity.

Pouteria lucuma Ruiz and Pav., known as the 'Gold of the Incas' or 'lucuma', is a subtropical fruit belonging to the Sapotaceae family, with a very sweet flavor, used to prepare cakes, ice creams as well as in the baking and dairy industries. To date, the content of primary metabolites is known, but little information is reported about the composition in specialized metabolites. Moreover, no study is reported on skin which represent an important agricultural waste due to the high demand for lucuma. In order to have a preliminary metabolite profile of Pouteria lucuma, the extracts of pulp and skin have been analyzed by LC-ESI/LTQOrbitrap/MS/MS in negative ion mode. The careful analysis of the accurate masses, of the molecular formulas and of the ESI/MS spectra allowed to identify specialized metabolites belonging to phenolic, flavonoid and polar lipid classes. The LC-MS/MS analysis guided the isolation of compounds occurring in the pulp extract whose structures have been characterized by spectroscopic methods including 1D- and 2D-NMR experiments and ESI-MS analysis. Furthermore, the phenolic content of the extracts along with the antioxidant activity of extracts and isolated compounds was evaluated.

Antioxidant and In Vitro Preliminary Anti-Inflammatory Activity of Castanea sativa (Italian Cultivar "Marrone di Roccadaspide" PGI) Burs, Leaves, and Chestnuts Extracts and Their Metabolite Profiles by LC-ESI/LTQOrbitrap/MS/MS.

The Italian "Marrone di Roccadaspide" (Castanea sativa), a labeled Protected Geographical Indication (PGI) product, represents an important economic resource for the Italian market. With the aim to give an interesting opportunity to use chestnuts by-products for the development of nutraceutical and/or cosmetic formulations, the investigation of burs and leaves along with chestnuts of C. sativa, cultivar "Marrone di Roccadaspide", has been performed. The phenolic, tannin, and flavonoid content of the MeOH extracts of "Marrone di Roccadaspide" burs, leaves, and chestnuts as well as their antioxidant activity by spectrophotometric methods (1,1-diphenyl-2-picrylhydrazyl (DPPH), Trolox Equivalent Antioxidant Capacity (TEAC), and Ferric Reducing Antioxidant Power (FRAP) have been evaluated. Furthermore, a cell-based antioxidant in vitro test along with in vitro assays for the evaluation of the ability to reduce nuclear factor-kappa B (NF-κB) activation and nitric oxide (NO) production have been carried out. In order to identify the secondary metabolites responsible for the high phenolic content and the strong antioxidant activity shown by leaves and burs extracts, and to highlight the differences between their chemical composition, the analysis of the metabolite profile of the MeOH extracts obtained from both by-products and chestnuts by liquid chromatography coupled to electrospray ionization and multiple-stage linear ion-trap and Orbitrap high-resolution mass spectrometry (LC-(-)ESI/LTQOrbitrap/MS/MS) has been performed. LC-MS analysis allowed the identification of different classes of specialized metabolites including hydrolyzable tannins, flavonoids, ellagic acid and phenol glucoside derivatives, and triterpenoids as well as polar lipids. Our results show how the antioxidant activity of the extracts can be correlated to their high tannins and flavonoids content while polar lipids occurring in the MeOH extract of the leaves could contribute to determining its higher anti-inflammatory activity.