Combining chemical profiles and biological abilities of different extracts from Tanacetum nitens ( Boiss. & Noë) Grierson using network pharmacology.

Tanacetum nitens ( Boiss. & Noë)  Grierson is an aromatic perennial herb used in Turkish traditional medicine to treat headache, fever, and skin diseases. This study aimed to investigate the chemical composition, antioxidant, enzyme inhibition, and cytotoxic properties of T. nitens aerial parts. Organic solvent extracts were prepared by sequential maceration in hexane, dichloromethane, ethyl acetate, and methanol while aqueous extracts were obtained by maceration or infusion. Nuclear magnetic resonance (NMR) and LC-DAD-MS analysis allowed the identification and quantification of different phytoconstituents including parthenolide, tanacetol B, tatridin B, quinic acid derivatives, ß-sitosterol, and glycoside derivatives of quercetin and luteolin. The type and amount of these phytochemicals recovered by each solvent were variable and significant enough to impact the biological activities of the plant. Methanolic and aqueous extracts displayed the highest scavenging and ions-reducing properties while the dichloromethane and ethyl acetate extracts exerted the best total antioxidant activity and metal chelating power. Results of enzyme inhibition activity showed that the hexane, ethyl acetate, and dichloromethane extracts had comparable anti-acetylcholinesterase activity and the latter extract revealed the highest anti-butyrylcholinesterase activity. The best α-amylase and α-glucosidase inhibition activities were obtained from the hexane extract. The dichloromethane and ethyl acetate extracts exhibited the highest cytotoxic effect against the prostate carcinoma DU-145 cells. In conclusion, these findings indicated that T. nitens can be a promising source of biomolecules with potential therapeutic applications.

Bioactive agents from Parkia biglobosa (Jacq.) R.Br. ex G. Don bark extracts for health promotion and nutraceutical uses.

BACKGROUND: Parkia biglobosa stem bark extracts were prepared using methanol, methanol 80%, water and ethyl acetate to investigate their phytochemical contents, as well as antioxidant and enzyme inhibitory properties. RESULTS: Liquid chromatography (LC) quadrupole time-of-flight mass spectrometry (MS) and LC-MSn revealed the presence of flavonoids, hydroxycinnamic acid derivatives and gallotannins. Particularly, the water extract contained rutin (480 µg per 100 mg) and 3-caffeoylquinic acid (1109 µg per 100 mg) in higher amounts, whereas the 80% methanol extract contains methoxyluteolin-7-O-rutinoside and catechin derivatives as major compounds. Total phenolic and flavonoid contents of the extracts were yielded in the range of 32.26-119.88 mg gallic acid equivalents g-1 and 0.60-2.39 mg rutin equivalents g-1 , respectively. Total antioxidant capacity was also displayed in the range of 0.53-6.34 mmol Trolox equivalents (TE) g-1 . Both the methanolic extracts showed higher total antioxidant capacity that could be related to the total phenolic contents. Radical scavenging capacity in DPPH (2,2-diphenyl-2-picryl-hydrazyl) (37.21-508.30 mg TE g-1 ) and ABTS [2,2-azinobis(3-ethylbenzothiazoline- 6-sulfonic acid)] (60.95-1068.06 mg TE g-1 ) assays, reducing power in cupric ion reducing antioxidant capacity (54.23-1002.78 mg TE g-1 ) and ferric ion reducing antioxidant power (33.18-558.68 mg TE g-1 ) assays, as well as metal chelating activity (2.45-11.28 mg EDTA equivalents g-1 ), were exhibited by all extracts. All extracts were found to inhibit acetylcholinesterase [0.23-2.47 mg galanthamine equivalents (GALAE) g-1 ], tyrosinase [27.20-83.33 mg kojic acid equivalents g-1 ], amylase [mmol acarbose equivalents (ACAE) g-1 ]. On the other hand, all extracts, except the water extract, inhibited butyrylcholinesterase (5.38-6.56 mg GALAE g-1 ), whereas only the water and ethyl acetate extract showed glucosidase inhibitory potential (1.96 and 1.82 mmol ACAE g-1 ). In general, the water extract was found to be a weaker enzyme inhibitor suggesting that water is not the preferrable extraction solvent to obtain active products. CONCLUSION: The present study demonstrated that the stem bark extracts of P. biglobosa contains good amount of phytochemical and extracts present significant antioxidant, as well as reasonable enzyme inhibitory effects. Hence, these findings suggest that further studies can be performed on more specific biological targets and models of bioactivity to determine their safe usage as a nutraceutical or for the preparation functional foods. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Melatonin MT1 receptors as a target for the psychopharmacology of bipolar disorder: A translational study.

The treatment of bipolar disorder (BD) still remains a challenge. Melatonin (MLT), acting through its two receptors MT1 and MT2, plays a key role in regulating circadian rhythms which are dysfunctional in BD. Using a translational approach, we examined the implication and potential of MT1 receptors in the pathophysiology and psychopharmacology of BD. We employed a murine model of the manic phase of BD (Clock mutant (ClockΔ19) mice) to study the activation of MT1 receptors by UCM871, a selective partial agonist, in behavioral pharmacology tests and in-vivo electrophysiology. We then performed a high-resolution Nuclear Magnetic Resonance study on isolated membranes to characterize the molecular mechanism of interaction of UCM871. Finally, in a cohort of BD patients, we investigated the link between clinical measures of BD and genetic variants located in the MT1 receptor and CLOCK genes. We demonstrated that: 1) UCM871 can revert behavioral and electrophysiological abnormalities of ClockΔ19 mice; 2) UCM871 promotes the activation state of MT1 receptors; 3) there is a significant association between the number of severe manic episodes and MLT levels, depending on the genetic configuration of the MT1 rs2165666 variant. Overall, this work lends support to the potentiality of MT1 receptors as target for the treatment of BD.

Structural Manipulations of Marine Natural Products Inspire a New Library of 3-Amino-1,2,4-Triazine PDK Inhibitors Endowed with Antitumor Activity in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the main aggressive types of cancer, characterized by late prognosis and drug resistance. Among the main factors sustaining PDAC progression, the alteration of cell metabolism has emerged to have a key role in PDAC cell proliferation, invasion, and resistance to standard chemotherapeutic agents. Taking into account all these factors and the urgency in evaluating novel options to treat PDAC, in the present work we reported the synthesis of a new series of indolyl-7-azaindolyl triazine compounds inspired by marine bis-indolyl alkaloids. We first assessed the ability of the new triazine compounds to inhibit the enzymatic activity of pyruvate dehydrogenase kinases (PDKs). The results showed that most of derivatives totally inhibit PDK1 and PDK4. Molecular docking analysis was executed to predict the possible binding mode of these derivatives using ligand-based homology modeling technique. Evaluation of the capability of new triazines to inhibit the cell growth in 2D and 3D KRAS-wild-type (BxPC-3) and KRAS-mutant (PSN-1) PDAC cell line, was carried out. The results showed the capacity of the new derivatives to reduce cell growth with a major selectivity against KRAS-mutant PDAC PSN-1 on both cell models. These data demonstrated that the new triazine derivatives target PDK1 enzymatic activity and exhibit cytotoxic effects on 2D and 3D PDAC cell models, thus encouraging further structure manipulation for analogs development against PDAC.

Nutraceutical formulation for immune system modulation: Active constituents, in vitro antibacterial and immunomodulatory activity, and metabolomics analysis.

There is a large demand for nutraceuticals in the market and studies related to their action are needed. In this paper, the antimicrobial activity and the immunomodulatory effect of a nutraceutical formulation containing 14.39% of ascorbic acid, 7.17% of coenzyme Q10, 1.33% of Echinacea polyphenols, 0.99% of pine flavan-3-ols, 0.69% of resveratrol and 0.023% of Echinacea alkylamides were studied using in vitro assays and cell-based metabolomics. Chromatographic analysis allowed us to study the nutraceutical composition. The antibacterial activity was evaluated on S. aureus, K. pneumoniae, P. aeruginosa, E. coli, H. influenzae, S. pyogenes, S. pneumoniae and M. catarrhalis. The immunomodulatory activity was assessed on human macrophages and dendritic cells. The production of IL-1ß, IL-12p70, IL-10 and IL-8 was evaluated on culture medium by ELISA and the activation/maturation of dendritic cells with cytofluorimetric analysis. Treated and untreated macrophages and dendritic cell lysates were analysed by liquid chromatography coupled with high-resolution mass spectrometry, and results were compared using multivariate data analysis to identify biological markers related to the treatment with the food supplement. The food supplement decreased K. pneumoniae, P. aeruginosa, E. coli, Methicillin-resistant Staphylococcus aureus (MRSA) and M. catharralis growth, reduced the inflammatory response in macrophages exposed to lipopolysaccharide (LPS) and modulated the activation and maturation of the dendritic cells. Oxidized phospholipids were identified as the main biological markers of treated cell lysates, compared with controls.

Chemical Characterization of Corydalis chaerophylla D.C. Extracts and Preliminary Evaluation of Their in Vitro and in Vivo Biological Properties.

Genus Corydalis is a rich source of isoquinoline alkaloids reported to having potential bioactivities. Corydalis chaerophylla collected from Nepal at an altitude of 2400-4800 m was extracted using hexane, methanol and chloroform as solvents. The resulting hexane, methanol and chloroform extracts were subjected to LC-DAD-MSn analysis to yield fifteen different alkaloids. To assess any potential pharmacological properties, antimicrobial activity against two Gram-positive, two Gram-negative bacterial strains and one fungal strain was assessed, revealing significant inhibitive action of the methanol and chloroform extracts. Of the extracts obtained using chloroform contained the highest content of phenolic compounds at 113 mg GAE/g, while the highest total flavonoid content was found for the hexane extract with a value of 46.45 mg QE/g. The chloroform extract also exhibited a considerable antioxidant activity at IC50 value, 261.5±3 µg/mL, for the DPPH assay. Conversely, the methanol extract exhibited the highest LC50 value for Brine Shrimp cytotoxicity at 196±3 µg/mL being least potential for the test. The methanol extract was found to be the most active against α-amylase inhibition with an IC50 of 51.52±2 µg/mL. In an in vivo acute oral toxicity study against mice, methanol and chloroform extracts presented harmful effects with 1000.36 mg/kg BW and 515 mg/kg BW for LD50 , respectively. By analyzing all the results of the solvents used, the chloroform extract was found to be the most active, a feature that will be used in future isolation procedures and other pharmacological tests.

LC-MS/MS and GC-MS profiling, antioxidant, enzyme inhibition, and antiproliferative activities of Thymus leucostomus H ausskn. & V elen. extracts.

The chemical composition as well as antioxidant, antiproliferative, and enzyme inhibition activities of extracts from aerial parts of Thymus leucostomus H ausskn. & V elen. obtained with hexane, methanol, and water were evaluated. Results showed that the methanol extract had significantly (p < 0.05) the highest total phenolic content (TPC; 107.80 mg GAE/g) and total flavonoids content (TFC; 25.21 mg RE/g) followed by the aqueous extract (102.72 mg GAE/g and 20.88 mg RE/g, respectively). LC-MS/MS-guided profiling of the three extracts revealed that rosmarinic acid (34.8%), hesperetin (42.9%), and linoleic acid (18%) were the dominant compounds in the methanol, aqueous and hexane extracts, respectively. GC-MS analysis of the hexane extract showed that É£-sitosterol (29.9%) was the major constituent. The methanol extract displayed significantly (p < 0.05) the highest Cu++ , Fe+++ , and Mo(VI) ions scavenging and reducing properties while the aqueous extract exerted significantly (p < 0.05) the highest metal chelating power (42.51 mg EDTAE/g). Both the hexane and methanol extracts effectively inhibited the acetylcholinesterase enzyme (2.63 and 2.65 mg GALAE/g, respectively) while the former extract exerted significantly (p < 0.05) the highest butyrylcholinesterase (2.32 mg GALAE/g), tyrosinase (19.73 mg KAE/g), and amylase (1.16 mmol ACAE/g) inhibition capacity. The aqueous extract exhibited the best glucosidase inhibition property (0.49 mmol ACAE/g). The methanol and hexane extracts exerted a higher cytotoxic effect on HT-29 (IC50 : 8.12 µg/mL) and HeLa (IC50 = 8.08 µg/mL) cells, respectively. In conclusion, these results provide valuable insight into the potential use of T. leucostomus bioactive extracts in different pharmaceutical applications.

Lipidomic Profile of Human Sperm Membrane Identifies a Clustering of Lipids Associated with Semen Quality and Function.

Reduced sperm motility and/or count are among the major causes of reduced fertility in men, and sperm membranes play an important role in the spermatogenesis and fertilization processes. However, the impact of sperm lipid composition on male fertility remains under-investigated. The aim of the present study was to perform a lipidomic analysis of human sperm membranes: we performed an untargeted analysis of membrane lipid composition in fertile (N = 33) and infertile subjects (N = 29). In parallel, we evaluated their serum lipid levels. Twenty-one lipids were identified by their mass/charge ratio and post-source decay spectra. Sulfogalactosylglycerolipid (SGG, seminolipid) was the most abundant lipid component in the membranes. In addition, we observed a significant proportion of PUFAs. Important differences have emerged between the fertile and infertile groups, leading to the identification of a lipid cluster that was associated with semen parameters. Among these, cholesterol sulfate, SGG, and PUFAs represented the most important predictors of semen quality. No association was found between the serum and sperm lipids. Dietary PUFAs and SGG have acknowledged antioxidant functions and could, therefore, represent sensitive markers of sperm quality and testicular function. Altogether, these results underline the important role of sperm membrane lipids, which act independently of serum lipids levels and may rather represent an independent marker of reproductive function.

Chemical Characterization and Cytotoxic and Antioxidant Activity Evaluation of the Ethanol Extract from the Bulbs of Pancratium maritimun Collected in Sicily.

P. maritimum L., belonging to the Amaryllidaceae family, is a species that grows on beaches and coastal sand dunes mainly on both sides of the Mediterranean Sea and Black Sea, the Middle East, and up to the Caucasus region. It has been largely investigated due to its several interesting biological properties. With the aim of providing new insights into the phytochemistry and pharmacology of this species, the ethanolic extract of the bulbs from a local accession, not previously studied, growing in Sicily (Italy), was investigated. This chemical analysis, performed by mono- and bi-dimensional NMR spectroscopy, as well as LC-DAD-MSn, allowed to identify several alkaloids, three of which were never detected in the genus Pancratium. Furthermore, the cytotoxicity of the preparation was assessed in differentiated human Caco-2 intestinal cells by trypan blue exclusion assay, and its antioxidant potential was evaluated using the DCFH-DA radical scavenging method. The results obtained demonstrate that P. maritimum bulbs' extract exerts no cytotoxic effect and is able to remove free radicals at all the concentrations tested.

Evaluation of the effects of natural isoquinoline alkaloids on low density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) in hepatocytes, as new potential hypocholesterolemic agents.

Nine different isoquinoline alkaloids, berberine, govaniadine, stylopine, adlumine, adlumidine, bicuculline, sanguinarine, protopine and californidine have been evaluated for their effects on a cellular model of hepatocyte for their effect on low density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression compared to simvastatin. Berberine, californidine and govaniadine induced LDLR with an effect similar to 2.5 µM simvastatin. Californidine and berberine at tested doses reduced the expression of PCSK9, with an opposite behaviour to simvastatin on this target. Govaniadine, on the other hand, showed a statin-like effect, although less potently, by increasing both LDLR and PCSK9 levels. Berberine californidine and govaniadine were then tested on the same cellular model to assess possible effect of reduction of total cholesterol, compared to simvastatin. All compounds were able to reduce total cholesterol level in the hepatocytes.

Development of an LC-DAD-MS-Based Method for the Analysis of Hydroxyanthracene Derivatives in Food Supplements and Plant Materials.

Products based on plants containing hydroxyanthracene derivatives (HADs)­such as Rheum, Cassia, and Aloe species­are widely used in food supplements or nutraceuticals due to their laxative effects. A more restricted control of HAD contents in food supplements has been implemented by EU Regulation 2021/468, in order to increase the safety of these preparations. Due to their toxicity, aloin A, aloin B, aloe emodin, emodin, and the synthetic derivative danthron have been listed as prohibited substances in food supplements, being tolerated in amounts < 1 mg kg−1 in marketed products. In this work, we report the development of a sensitive and fast LC−DAD−MS-based procedure for the determination of these five compounds in food supplements and plant materials or extracts. The entire procedure includes a simple sample preparation step, where target analytes are concentrated by means of solvent extraction and evaporative concentration (solid samples), or by lyophilisation (liquid samples). The average LOQ of 0.10 mg/L, LOD of 0.03 mg/L, accuracy, and precision with CVs below 12.72 were obtained for the studied analytes. This method is suitable for assessing the compliance of commercial products and raw materials with EU Regulation 2021/468. Furthermore, the proposed method can represent a starting point for the development of a unique and standardised analytical approach for the determination of other HADs under the attention of EU authorities.

NMR, LC-MS Characterization of Rydingia michauxii Extracts, Identification of Natural Products Acting as Modulators of LDLR and PCSK9.

Rydingia michauxii (Briq.) Scheen and V.A.Albert (Lamiaceae) is used in Iranian traditional medicine to treat malaria, diabetes, hyperlipidemia, rheumatism and cardiovascular diseases. NMR and LC-DAD-MSn analyses were used to establish extract composition and phenylethanoid, flavonoid glycosides, lignans, labdane diterpenes and iridoids were identified and quantified. The main constituents were isolated, and structures were elucidated based on NMR, polarimetric and MS measurements. A new natural compound, ent-labda-8(17),13-dien-18-glucopyranosyl ester-15,16-olide is described here. The effects of ent-labda-8(17),13-dien-18-oic acid-15,16-olide (1), ent-labda-8(17),13-dien-18-glucopyranosyl es-ter-15,16-olide (2), antirrhinoside (3), echinacoside (4), verbascoside (5), and apigenin 6,8-di-C-glucoside (6), on the low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), were studied in the human hepatocarcinoma cell line Huh7. Among the six constituents, (3) showed the strongest induction of the LDLR (3.7 ± 2.2 fold vs. control) and PCSK9 (3.2 ± 1.5 fold vs. control) at a concentration of 50 µM. The in vitro observations indicated a potential lipid lowering activity of (3) with a statin-like mechanism of action.

"Smart Extraction Chain" with Green Solvents: Extraction of Bioactive Compounds from Picea abies Bark Waste for Pharmaceutical, Nutraceutical and Cosmetic Uses.

Secondary metabolites from the sawmill waste Picea abies bark were extracted using an innovative two-step extraction that includes a first step with supercritical CO2 (SCO2) and a second step using green solvents, namely ethanol, water, and water ethanol mixture. Maceration (M), ultrasound assisted extraction (UAE) and microwave assisted extraction (MAE) techniques were applied in the second step. A total of nineteen extract were obtained and yield were compared. Bark extracts were characterized by LC-DAD-MSn and classes of compounds were quantified as abietane derivatives, piceasides, flavonoids, and phenolics to compare different extractions. Obtained extracts were studied by in vitro assay to evaluate potential pharmaceutical, nutraceutical and cosmetic uses assessing the antioxidant activity as well as the inhibitory activity on target enzymes. Results show that the "smart extraction chain" is advantageous in term of yield of extraction and phytoconstituent concentration. SCO2 extract, presenting a unique composition with a large amount of abietane derivatives, exerted the best activity for amylase inhibition compared to the other extracts.

Phytochemical Characterization, Anti-Oxidant, Anti-Enzymatic and Cytotoxic Effects of Artemisia verlotiorum Lamotte Extracts: A New Source of Bioactive Agents.

Artemisia verlotiorum Lamotte is recognized medicinally given its long-standing ethnopharmacological uses in different parts of the world. Nonetheless, the pharmacological properties of the leaves of the plant have been poorly studied by the scientific community. Hence, this study aimed to decipher the phytochemicals; quantify through HPLC-ESI-MS analysis the plant's biosynthesis; and evaluate the antioxidant, anti-tyrosinase, amylase, glucosidase, cholinesterase, and cytotoxicity potential on normal (NIH 3T3) and human liver and human colon cancer (HepG2 and HT 29) cell lines of this plant species. The aqueous extract contained the highest content of phenolics and phenolic acid, methanol extracted the most flavonoid, and the most flavonol was extracted by ethyl acetate. The one-way ANOVA results demonstrated that all results obtained were statistically significant at p < 0.05. A total of 25 phytoconstituents were identified from the different extracts, with phenolic acids and flavonoids being the main metabolites. The highest antioxidant potential was recorded for the aqueous extract. The best anti-tyrosinase extract was the methanolic extract. The ethyl acetate extract of A. verlotiorum had the highest flavonol content and hence was most active against the cholinesterase enzymes. The ethyl acetate extract was the best α-glucosidase and α-amylase inhibitor. The samples of Artemisia verlotiorum Lamotte in both aqueous and methanolic extracts were found to be non-toxic after 48 h against NIH 3T3 cells. In HepG2 cells, the methanolic extract was nontoxic up to 125 µg/mL, and an IC50 value of 722.39 µg/mL was recorded. The IC50 value exhibited in methanolic extraction of A. verlotiorum was 792.91 µg/mL in HT29 cells. Methanolic extraction is capable of inducing cell cytotoxicity in human hepatocellular carcinoma without damaging normal cells. Hence, A. verlotiorum can be recommended for further evaluation of its phytochemical and medicinal properties.

Green extraction of hemp (Cannabis sativa L.) using microwave method for recovery of three valuable fractions (essential oil, phenolic compounds and cannabinoids): a central composite design optimization study.

BACKGROUND: Solvent-free microwave-assisted extraction (MAE) is a green extraction method capable of boosting the yield and quality profile of hemp essential oil when compared with other conventional extraction techniques. During this process, two by-products are produced, namely the aqueous residue containing bioactive phenolics and the residual deterpenated biomass, which can be used for further extraction and purification of phytocannabinoids. To date, the hemp industry has not utilized these products, although they can be valuable for the food, cosmetic, nutraceutical and pharmaceutical market. RESULTS: This study assessed and optimized the variables affecting MAE efficiency, namely microwave irradiation power, extraction time and added water, which were studied using a central composite design approach, and results were used to optimize the extraction process for recovering three valuable fractions: essential oil, polyphenols and phytocannabinoids. The products obtained using the optimized conditions were characterized in terms of yield, chemical profile and antioxidant potential. Moreover, the by-products obtained during the optimized run were further analyzed in terms of their biological activity using both enzymatic and non-enzymatic assays. The aqueous residue demonstrated a powerful α-glucosidase inhibition, a good activity in terms of superoxide radical scavenging activity, a modest efficacy in terms of inhibition of advanced glycation end products formation and no activity in terms of lipase inhibition. The residual deterpenated biomass did not possess significant biological activity. CONCLUSION: This work demonstrated valorization of industrial hemp essential oil and its by-products, obtained by a sustainable and eco-friendly extraction method, through an almost waste-free approach. Cannabinoids as well as other valuable bioactive compounds such as glycosidic flavones may be recovered from the residues of the essential oil extraction, representing interesting substances in the pharmaceutical, cosmetic and nutraceutical fields. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Actinidia arguta Pulp: Phytochemical Composition, Radical Scavenging Activity, and in†Vitro Cells Effects.

Hardy kiwifruit (Actinidia arguta) is a highly appreciated exotic fruit endowed with outstanding bioactive compounds. The present work proposes to characterize the pulp from A. arguta organic fruits, emphasizing its radicals scavenging capacity and effects on intestinal cells (Caco-2 and HT29-MTX). The physicochemical properties and phenolic profile were also screened. The total phenolic and flavonoid contents (TPC and TFC, respectively) of pulp were 12.21 mg GAE/g on dry weight (DW) and 5.92 mg CE/g DW, respectively. A high antioxidant activity was observed (FRAP: 151.41 µmol FSE/g DW; DPPH: 12.17 mg TE/g DW). Furthermore, the pulp did not induce a toxic effect on Caco-2 and HT29-MTX cells viability up to 1000 µg/mL. Regarding in vitro scavenging capacity, the pulp revealed the highest scavenging power against NO. (IC50 =3.45 µg/mL) and HOCl (IC50 =12.77 µg/mL). These results emphasize the richness of A. arguta fruit pulp to be used in different food products.

Phytochemical Investigations and In Vitro Bioactivity Screening on Melia azedarach L. Leaves Extract from Nepal.

Melia azedarach is a common tree used in the traditional medicine of Nepal. In this work, leaves were considered as source of bioactive constituents and composition of methanol extract was evaluated and compared with starting plant material. Flavonoid glycosides and limonoids were identified and quantified by HPLC-DAD-MSn approaches in dried leaves and methanolic extract, while HPLC-APCI-MSn and GC/MS analysis were used to study phytosterol and lipid compositions. ß-Sitosterol and rutin were the most abundant constituents. HPLC-APCI-MSn and HPLC-DAD-MSn analysis revealed high levels of phytosterols and flavonoids in methanolic extract accounting 9.6 and 7.5 % on the dried weight, respectively. On the other hand, HPLC/MSn data revealed that limonoid constituents were in minor amount in the extract <0.1 %, compared with leaves (0.7 %) indicating that degradation occurred during extraction or concentration procedures. The methanol extract was subjected to different bioassays, and antioxidant activity was evaluated. Limited inhibitory activity on acetyl and butyryl cholinesterase, as well as on amylase were detected. Moreover, tyrosinase inhibition was significant resulting in 131.57±0.51 mg kojic acid equivalents/g of dried methanol extract, suggesting possible use of this M. azedarach extract in skin hyperpigmentation conditions. Moderate cytotoxic activity, with IC50 of 26.4 µg/mL was observed against human ovarian cancer cell lines (2008 cells). Our findings indicate that the Nepalese M. azedarach leaves can be considered as valuable starting material for the extraction of phenolics and phytosterols, yielding extracts with possible cosmetic and pharmaceutical applications.

Sperm Cholesterol Content Modifies Sperm Function and TRPV1-Mediated Sperm Migration.

Transient receptor potential channels-vanilloid receptor 1 (TRPV1) regulates thermotaxis in sperm-oriented motility. We investigated the role of membrane cholesterol (Chol) on TRPV1-mediated human sperm migration. Semen samples were obtained from five normozoospemic healthy volunteers. Sperm membrane Chol content, quantified by liquid chromatography-mass spectrometry, was modified by incubating cells with 2-hydroxypropyl-ß-cyclodextrin (CD) or the complex between CD and Chol (CD:Chol). The effect on sperm migration on a 10 µM capsaicin gradient (CPS), a TRPV1 agonist, was then investigated. Motility parameters were evaluated by Sperm Class Analyser. Intracellular calcium concentration and acrosome reaction were measured by staining with calcium orange and FITC-conjugated anti-CD46 antibody, respectively. TRPV1-Chol interaction was modelled by computational molecular-modelling (MM). CD and CD:Chol, respectively, reduced and increased membrane Chol content in a dose-dependent manner, resulting in a dose-dependent increase and reduction of sperm migration in a CPS gradient. MM confirmed a specific interaction of Chol with a TRPV1 domain that appeared precluded to the Chol epimer epicholesterol (Epi-Chol). Accordingly, CD:Epi-Chol was significantly less efficient than CD:Chol, in reducing sperm migration under CPS gradient. Chol inhibits TRPV1-mediated sperm function by directly interacting with a consensus sequence of the receptor.

Evaluation of Antioxidant and Enzyme Inhibition Properties of Croton hirtus L'Hér. Extracts Obtained with Different Solvents.

Croton hirtus L'Hér methanol extract was studied by NMR and two different LC-DAD-MSn using electrospray (ESI) and atmospheric pressure chemical ionization (APCI) sources to obtain a quali-quantitative fingerprint. Forty different phytochemicals were identified, and twenty of them were quantified, whereas the main constituents were dihydro α ionol-O-[arabinosil(1-6) glucoside] (133 mg/g), dihydro ß ionol-O-[arabinosil(1-6) glucoside] (80 mg/g), ß-sitosterol (49 mg/g), and isorhamnetin-3-O-rutinoside (26 mg/g). C. hirtus was extracted with different solvents-namely, water, methanol, dichloromethane, and ethyl acetate-and the extracts were assayed using different in vitro tests. The methanolic extracts presented the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), and ferric reducing antioxidant power (FRAP) values. All the tested extracts exhibited inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), with a higher activity observed for dichloromethane (AChE: 5.03 and BChE: 16.41 mgGALAE/g), while the methanolic extract showed highest impact against tyrosinase (49.83 mgKAE/g). Taken together, these findings suggest C. hirtus as a novel source of bioactive phytochemicals with potential for commercial development.

The Modulation of PCSK9 and LDLR by Supercritical CO2 Extracts of Mentha longifolia and Isolated Piperitone Oxide, an In Vitro Study.

In the present study the ability of supercritical carbon dioxide (SCO2) extracts of M. longifolia L. leaves to modulate low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression was evaluated in cultured human hepatoma cell lines Huh7 and HepG2. Two SCO2 extracts, one oil (ML-SCO2) and a semisolid (MW-SCO2), were subjected to detailed chemical characterization by mono- and bidimensional nuclear magnetic resonance (1D, 2D-NMR), gas chromatography coupled with mass spectrometry (GC-MS) and liquid chromatography coupled with mass spectrometry (LC-MS). Chemical analysis revealed significant amounts of fatty acids, phytosterols and terpenoids. ML-SCO2 was able to induce LDLR expression at a dose of 60 µg/mL in HuH7 and HepG2 cell lines. Furthermore, ML-SCO2 reduced PCSK9 secretion in a concentration-dependent manner in both cell lines. Piperitone oxide, the most abundant compound of the volatile constituent of ML-SCO2 (27% w/w), was isolated and tested for the same targets, showing a very effective reduction of PCSK9 expression. The overall results revealed the opportunity to obtain a new nutraceutical ingredient with a high amount of phytosterols and terpenoids using the SCO2 extraction of M. longifolia L., a very well-known botanical species used as food. Furthermore, for the first time we report the high activity of piperitone oxide in the reduction of PCSK9 expression.