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.

Effect of different atmospheric and subatmospheric cooking techniques on physical and chemical qualitative properties of pork loin.

In this paper, the effects of four cooking procedures were evaluated, two occurring in atmospheric (in ventilated and steam oven) and two in subatmospheric (vacuum and sous vide cooking) conditions on pork Longissimus lumborum. The main objective of the study was to compare and evaluate the physical and chemical characteristics. Samples were cooked in four independent trials namely Oven (O), Steaming (ST), Vacuum Cooking (VC) and Sous Vide (SV). The analyses included temperature, cooking effect, percentage weight loss, texture (cutting and double compression tests), colour (superficially and inside the sample), microstructure (optical microscopy) and fibres shortening analysis. To assess cooking effects on significant nutritional constituents, the fatty acid composition and the content of B vitamins were analysed. Volatile profiles of samples were also compared using solid-phase microextraction. SV cooking resulted in the less favourable meat texture, presenting the highest hardness and chewiness. Moreover, high hardness values measured on SV samples is also related to the high weight loss. The technique of oven cooking (O) demonstrated superior results in terms of mechanical properties, which are closely associated with the cooking values. Specifically, the cook value C0 was significantly higher in the case of oven cooking compared to SV, VC, and ST. Mild temperature conditions and cooking times of the four considered cooking techniques did not induce significant variations in the fatty acid composition and volatile profile. Conversely, SV and VC allowed the highest amount of vitamin B retention in cooked meat. This work suggests that some differences emerged on the effects due to sub-atmospheric and atmospheric cooking compared to traditional ones.

"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.

In Vitro and In Vivo Sucrosomial® Berberine Activity on Insulin Resistance.

Background: Berberine is a natural alkaloid with hypoglycemic properties. However, its therapeutic use is limited by a very low oral bioavailability. Here we developed a new oral formulation of berberine based on Sucrosomial® technology and tested its effect on insulin resistance. Methods: Sucrosomial® berberine was first tested in vitro in the hepatoma cell line Huh7 to assess its effect on proteins involved in glucose homeostasis and insulin resistance. The pharmacokinetics and efficacy on insulin resistance were then studied in C57BL/6 mice fed with standard (SD) and high-fat diet (HFD) for 16 weeks and treated daily during the last 8 weeks with oral gavage of Sucrosomial® berberine or berberine. Results: Sucrosomial® berberine did not affect Huh7 cell viability at concentrations up to 40 µM. Incubation of Huh7 with 20 µM of Sucrosomial® and control berberine induced glucokinase (GK) and the phosphorylation of 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK), both known targets for the control of insulin resistance. In vivo, we observed an 8-fold higher plasma concentration after 3 weeks of oral administration of 50 mg/kg/day of Sucrosomial® formulation compared to berberine. HFD, compared to SD, induced insulin resistance in mice as determined by oral glucose tolerance test (OGTT). The treatment with a 6.25 mg/kg/daily dose of Sucrosomial® berberine significantly reduced the area under the curve (AUC) of OGTT (73,103 ± 8645 vs. 58,830 ± 5597 mg/dL × min), while control berberine produced the same effects at 50 mg/Kg/day (51518 ± 1984 mg/dL × min). Under these conditions, the two formulations resulted in similar berberine plasma concentration in mice. Nevertheless, a different tissue distribution of metabolites was observed with a significant accumulation of reduced, demethylated and glucuronide berberine in the brain after the oral administration of the Sucrosomial® form. Glucuronide berberine plasma concentration was higher with Sucrosomial® berberine compared to normal berberine. Finally, we observed similar increases of AMPK phosphorylation in the liver in response to the treatment with Sucrosomial® berberine and berberine. Conclusions: The Sucrosomial® formulation is an innovative and effective technology to improve berberine gastrointestinal (GI) absorption with proven in vitro and in vivo activity on insulin resistance.

Investigating the in vitro mode of action of okra (Abelmoschus esculentus) as hypocholesterolemic, anti-inflammatory, and antioxidant food.

Okra (Abelmoschus esculentus) have been introduced as food relatively recently in Europe. It is native to India and one of the most important vegetables in Nigeria. The leaves can be consumed but also the fruit is rich in nutrients and bioactive compounds (i.e., dietary fiber, vitamins, oils, polysaccharides, polyphenols) and several health promoting actions have been ascribed, including a lipid-lowering properties. In this work the effects of fruit and leaf extracts on expression of key mediators of cholesterol metabolism, i.e., the low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), were investigated in human hepatoma cell line Huh7. Furthermore, effects on proinflammatory cytokines (IL-1ß, IL-6 and TNF-α) expressed by THP1-derived macrophages were studied to assess potential anti-inflammatory actions. Okra fruit extract significantly induced the mRNA and protein levels of the LDLR by 1.4 ± 0.3 and 4.8 ± 1.5-fold, respectively without any significant modification of PCSK9 expression. In addition, fruit extract showed a significant sequestering ability of cholic acid. Leaf butanol extract exerted similar action by inducing the expression of both the LDLR (+3.1 ± 1.6-fold vs control) and PCSK9 (+1.3 ± 0.4-fold vs control). The evaluation of the potential anti-inflammatory effect revealed a significant action of leaf butanol extract with reduced mRNA levels of IL-1ß (-28 ± 8 % vs control), IL-6 (-11 ± 1 % vs control) and TNF-α (-43 ± 8 % vs control), while fruit extract did not show any anti-inflammatory activity. Finally, leaf ethyl acetate extract showed a significant antioxidant capacity comparable to ascorbic acid. Taken together, we provided evidence that leaf butanol extract and, more effectively, fruit extract induced the LDLR expression, effect that may explain the previously reported hypocholesterolemic action of okra. In addition, okra's extracts reduced the expression of pro-inflammatory cytokines from THP1-derived macrophages, an effect that may suggest a vascular protective action of okra.

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.

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.

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.

An Integrated NMR, LC-DAD-MS, LC-QTOF Metabolomic Characterization of Sartoria hedysaroides: Correlation of Antioxidant and Enzyme Inhibitory Activity with Chemical Composition by Multivariate Data Analysis.

Sartoria hedysaroides Boiss and Heldr. (Fabaceae) is an endemic plant of Turkey that has received little scientific consideration so far. In the present study, the chemical profiles of extracts from the aerial part and roots of S. hedysaroides obtained using solvents with different polarities were analyzed combining integrated NMR, LC-DAD-MSn, and LC-QTOF methods. In vitro antioxidant and enzyme inhibitory activities were evaluated, and the results were combined with chemical data using multivariate approaches. Phenolic acids, flavonoids, ellagitannins, and coumarins were identified and quantified in the extracts of aerial part and roots. Methanolic extract of S. hedysaroides aerial part showed the highest phenolic content and the highest antioxidant activity and cupric ion reducing antioxidant capacity. Dichloromethane extract of S. hedysaroides roots showed the highest inhibition of butyryl cholinesterase, while methanolic extract of S. hedysaroides aerial part was the most active tyrosinase inhibitor. Multivariate data analysis allowed us to observe a good correlation between phenolic compounds, especially caffeoylquinic derivatives and flavonoids and the antioxidant activity of extracts. Acetylcholinesterase inhibition was correlated with the presence of caffeoylquinic acids and coumarins. Overall, the present study appraised the biological potential of understudied S. hedysaroides, and provided a comprehensive approach combining metabolomic characterization of plant material and multivariate data analysis for the correlation of chemical data with results from multi-target biological assays.

LC-MS Based Analysis and Biological Properties of Pseudocedrela kotschyi (Schweinf.) Harms Extracts: A Valuable Source of Antioxidant, Antifungal, and Antibacterial Compounds.

The impact of two extraction solvents on the phenolic composition, antioxidant, and enzymes inhibitory and antimicrobial activities of two parts (leaves and stem bark) of P. kotschyi was studied. Two different LC-DAD-MSn approaches were used to identify and quantify the bioactive compounds in the different extracts. A total of thirty-two compounds were quantified, being the procyanidin the most abundant in stem bark while catechin and flavonoids are most abundant in leaves. Overall, the stem bark extraction using methanol showed higher amounts of total phenolic (131.83 ± 1.81 mg GAE/g) and flavanol (14.14 ± 0.11 mg CE/g) while the leaves extraction using water exhibited stronger levels of total flavonoid (44.95 ± 0.38 mg RE/g) and phenolic acid (63.58 ± 2.00 mg CAE/g). As regards the antioxidant assays, methanol stem bark extracts were characterized by the highest antioxidant activities (DPPH: 1.94 ± 0.01 mmol TE/g, ABTS: 3.31 ± 0.01 mmol TE/g, FRAP: 2.86 ± 0.02 mmol TE/g, CUPRAC: 5.09 ± 0.08 mmol TE/g, phosphomolybdenum: 5.16 ± 0.23 mmol TE/g and metal chelating: 17.12 ± 0.46 mg EDTAE/g). In addition, the methanolic extracts of stem bark had highest impact on acetylcholinesterase (2.54 mg GALAE/g), butyrylcholinesterase (5.48 mg GALAE/g). In contrast, the methanolic extracts of leaves was potent against tyrosinase (77.39 ± 0.21 mg KAE/g) and α-glucosidase (0.97 ± 0.01 mmol ACAE/g), while a higher anti-α-amylase (0.97 ± 0.01 mmol ACAE/g) was observed for water extracts of the same part. All of the tested extracts showed inhibitory effects on elastase, except methanolic leaves extracts. Additionally, the extracts exhibited appreciable antifungal toward A. ochraceus, A. fumigatus, P. ochrochloron, T. viride, and P. funiculosum and promising antibacterial activity against M. flavus, S. aureus, L. monocytogenes, E. coli, P. aeruginosa, E. cloacae, and S. typhimurium. Taken together, the outcomes demonstrated P. kotschyi as a novel source of bioactive molecules of interest with an evident therapeutic value.

The Bark of Picea abies L., a Waste from Sawmill, as a Source of Valuable Compounds: Phytochemical Investigations and Isolation of a Novel Pimarane and a Stilbene Derivative.

In this work, the sawmill waste from Picea abies debarking was considered as source of valuable phytoconstituents. The extraction was performed using different ethanol/water mixtures, and characterization was obtained by LC-MSn. This latter revealed flavonoid glycosides, lignans, and procyanidins. Extraction with organic solvents (dichloromethane and methanol) and chromatographic separations of the obtained extracts by silica column followed by semi-preparative HPLC led to the isolation of polyphenols and terpenoids such as 21α-metoxy-serrat-14-en-3-one, 21α-hydroxy-serrat-14-en-3-one, pinoresinol, dehydroabietic acid, 15-hydroxy-dehydroabietic acid, 7-oxo-dehydroabietic acid, pimaric acid, 9ß-pimara-7,15-dien-19-ol, 13-epi-manoyl oxide, taxifolin-3'-O-glucopyranoside, trans-astringin, and piceasides. Piceaside V and 9ß-pimara-7-keto-19ß-olide, two novel compounds identified for the first time in P. abies bark, were isolated, and their structures were elucidated using 1D and 2D NMR and MS techniques. The polyphenolic composition of the methanolic portion was also investigated using LC-MSn, and the piceaside content was estimated. To assess the antioxidant activity of main constituents, semi-preparative HPLC was performed on the methanolic extract, and the obtained fractions were assayed by using the DPPH test. Overall, this work shows the potential usefulness of P. abies bark as a source of valuable phytochemicals.

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.

NMR and LC-MSn coupled with pharmacological network analysis for the assessment of phytochemical content and biopharmaceutical potential of Carapa procera extracts.

The methanolic and water extracts of Carapa procera leaves and stem barks were screened for their phytochemical content using a multi-technique approach. The extracts were also assessed for their in vitro antioxidant capacity along with their anti-diabetic (α-amylase, α-glucosidase), anti-tyrosinase, anti-elastase and anti-cholinesterase (AChE, BChE) activities. Furthermore, antibacterial and antifungal effects were determined against several bacterial and fungal strains. Data Integration Analysis for Biomarker discovery using Latent components (DIABLO) integrative analysis was conducted on collected data to determine the influence of extraction solvents and plant parts on phytochemical content, antioxidant properties and enzyme inhibitory properties of C. procera samples. Additionally, the major identified compounds were screened as modulators of multiple pathways involved in human diseases via Gene Ontology (GO) enrichment analysis. Results showed that methanolic stem bark extract exhibited the most potent ABTS scavenging, Cu2+ and Fe3+ reducing power, total antioxidant capacity and Fe2+ chelating power and displayed the highest total flavanol content. Methanolic extracts of leaves and stem barks were abounded with phenolics and had the greatest anti-AChE, anti-BChE, anti-tyrosinase and anti-elastase activities. A significant antifungal activity was observed, with the lowest minimum inhibitory concentration and minimum fungicidal values of 0.07 and 0.15 mg/mL, respectively. DIABLO integrative analysis suggested that the phytochemical content and biological activities varied significantly within the plant parts and were influenced by types of solvent used. GO enrichment analysis on the main bioactive compounds showed modulation of multiple pathways associated with cancer. Obtained results demonstrated that stem bark and leaves of C. procera can be considered as promising sources of bioactive molecules with high pharmacological values.

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.

Hypericum triquetrifolium and H. neurocalycinum as Sources of Antioxidants and Multi-Target Bioactive Compounds: A Comprehensive Characterization Combining In Vitro Bioassays and Integrated NMR and LC-MS Characterization by Using a Multivariate Approach.

Hypericum triquetrifolium and H. neurocalycinum were evaluated for their phytochemical content and in vitro bioactivity. NMR analyses were performed on the methanol extract of the aerial parts of H. triquetrifolium to establish the main classes of phytoconstituents. Then, LC-DAD-MSn analyses were performed in order to compare the composition of aerial parts and roots extracts of both Hypericum species, obtained using either methanol or water as solvents. Results, processed using multivariate data analysis, showed a significantly higher phenolic content of methanol extracts compared to water extracts, while minor qualitative differences were observed between the two. Distinctive flavonoid and PAC patterns were observed for H. triquetrifolium and H. neurocalycinum, and specific compounds were exclusively detected in one or the other species. Specifically, the phloroglucinols 7-epiclusianone, hyperfirin and hyperforin were present only in H. neurocalycinum, while hyperforin was detected only in H. triquetrifolium. Extracts were assayed using different in vitro tests to evaluate their antioxidant properties and their inhibitory activity against several enzymes, showing significant antioxidant and metal chelating activities. Furthermore, inhibitory properties against acetylcholinesterase, butyrylcholinesterase and tyrosinase were observed. Multivariate approaches were used to correlate biological data with the phytochemical composition of the different extracts. The results, showing positive correlations between specific chemical constituents and the measured bioactivities, represent preliminary data that could guide future studies aimed at isolating bioactive constituents from H. neurocalycinum and H. triquetrifolium for further pharmacological evaluations.

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.

Chemical characterization, antioxidant and enzyme inhibitory effects of Mitracarpus hirtus extracts.

Mitracarpus hirtus (L.) DC. (Family: Rubiaceae) is a tropical annual herb commonly found in America and Mexico. In the present study, the methanol, ethyl acetate, dichloromethane and aqueous extracts of the plant were tested for total phenolic (TPC) and flavonoid content (TFC) and antioxidant activities were evaluated using a battery of assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity, total antioxidant capacity (TAC) (phosphomolybdenum) and metal chelating. Enzyme inhibitory effects were investigated using acetylcholinesterase (AChE), tyrosinase, α-amylase and α-glucosidase as target enzymes. The phytochemical profile was obtained using liquid chromatography-diode array detection-electrospray ionization-mass spectrometry (LC-DAD-ESI-MSn), liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MSn) and nuclear magnetic resonance (NMR) experiments. Results showed that the dichloromethane and ethyl acetate extracts yielded the highest TPC (29.10 ± 0.07 mg gallic acid equivalent/g) and TFC (38.14 ± 0.91 mg rutin equivalent/g), respectively. Aqueous extract showed weak activity against tested enzymes but demonstrated the strongest ABTS scavenging activity (59.39 ± 1.19 mg trolox equivalent/g) and is the strongest Fe3+ reducer (59.42 ± 0.59 mg trolox equivalent/g). Phytochemical analysis revealed the presence of phenolics, pyrrolizidine alkaloids and triterpene acid. This is the first report gathering scientific data on antioxidant, enzyme inhibitory activities and phytochemical composition of M. hirtus and the obtained results can be used as starting point for further investigation on this traditional medicinal herb.

Chromatographic Separation of Breynia retusa (Dennst.) Alston Bark, Fruit and Leaf Constituents from Bioactive Extracts.

Breynia retusa (Dennst.) Alston (also known as Cup Saucer plant) is a food plant with wide applications in traditional medicine, particularly in Ayurveda. Extracts obtained with four solvents (dichloromethane, methanol, ethyl acetate and water), from three plant parts, (fruit, leaf and bark) were obtained. Extracts were tested for total phenolic, flavonoid content and antioxidant activities using a battery of assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), total antioxidant capacity (TAC) (phosphomolybdenum) and metal chelating. Enzyme inhibitory effects were investigated using acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase, α-amylase and α-glucosidase as target enzymes. Results showed that the methanolic bark extract exhibited significant radical scavenging activity (DPPH: 202.09 ± 0.15; ABTS: 490.12 ± 0.18 mg Trolox equivalent (TE)/g), reducing potential (FRAP: 325.86 ± 4.36: CUPRAC: 661.82 ± 0.40 mg TE/g) and possessed the highest TAC (3.33 ± 0.13 mmol TE/g). The methanolic extracts were subjected to LC-DAD-MSn and NMR analysis. A two-column LC method was developed to separate constituents, allowing to identify and quantify forty-four and fifteen constituents in bark and fruits, respectively. Main compound in bark was epicatechin-3-O-sulphate and isolation of compound was performed to confirm its identity. Bark extract contained catechins, procyanidins, gallic acid derivatives and the sulfur containing spiroketal named breynins. Aerial parts mostly contained flavonoid glycosides. Considering the bioassays, the methanolic bark extract resulted a potent tyrosinase (152.79 ± 0.27 mg kojic acid equivalent/g), α-amylase (0.99 ± 0.01 mmol acarbose equivalent ACAE/g) and α-glucosidase (2.16 ± 0.01 mmol ACAE/g) inhibitor. In conclusion, methanol is able to extract the efficiently the phytoconstituents of B. retusa and the bark is the most valuable source of compounds.

Comparison of Biostimulant Treatments in Acmella oleracea Cultivation for Alkylamides Production.

Acmella oleracea is a promising cosmetic, nutraceutical, and pharmaceutical ingredient, and plants with high levels of active compounds are needed in the market. Cultivation can be valuable if sufficient levels of alkylamides are present in plant material. In this regard the application of biostimulants can be an innovative approach to increase yield of cultivation or bioactive compound levels. A. oleracea plants were cultivated in Northern Italy in an experimental site using three different types of biostimulants, triacontanol-based mixture (Tria), an extract from plant tissues (LL017), and seaweed extract (Swe). Plants were grown in the field in two different growing seasons (2018 and 2019). After treatments inflorescences were harvested and the quali-quantitative analysis of alkylamides and polyphenols was performed. Treated and control plants were compared for yields, morphometric measurements, quali-quantitative composition in secondary metabolites. Overall results show that both triacontanol-based mixture and the LL017 positively influenced plant growth (Tria >+ 22%; LL017 >+ 25%) and flower production (Tria >+ 34%; LL017 >+ 56%). The amount of alkylamides and polyphenols in flowers were between 2.0-5.2% and 0.03-0.50%, respectively. Biostimulant treatments ensure higher cultivation yields and allow maintenance of the alkylamide and polyphenol levels based on % (w/w), thus offering an advantage in the final quantity of extractable chemicals. Furthermore, data revealed that samples harvested in late season show a decrease of polyphenols.

Phenolic compounds analysis of three Euphorbia species by LC-DAD-MSn and their biological properties.

Members of the genus Euphorbia have attracted much interest as potential sources of bioactive compounds. In this study, the ethyl acetate, methanolic and aqueous extracts of aerial parts of three Euphorbia species namely Euphorbia hirta L., Euphorbia heterophylla L. and Euphorbia convolvuloides Hochst. ex Benth. were screened for their antioxidant [2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), cupric antioxidant reducing capacity (CUPRAC), metal chelating, total antioxidant capacity (phosphomolybdenum)], and enzymatic properties [α-amylase, α-glucosidase, tyrosinase, acetyl- (AChE), butyryl-cholinesterase (BChE)]. Total phenolic (TPC), total flavonoid content (TFC) and phytochemical profile were established via LC-DAD-MSn. Results showed that the methanolic and aqueous extracts of the three plants yielded the highest TPC with values ranging from 35.84-141.90 mg gallic acid equivalent/g. E. hirta was the most potent DPPH scavenger and chelator reporting the highest equivalent [DPPH: 195.22 mg Trolox equivalent (TE)/g, chelator: 30.73 mg ethylenediaminetetraacetic acid equivalent (EDTAE)/g], while E. heterophylla demonstrated the most significant Fe3+ and Cu2+ reducing potential (FRAP: 194.28 mg TE/g; CUPRAC: 631.25 mg TE/g) and possessed the highest total antioxidant capacity (3.76 mmol TE/g). The AChE, BChE, α-amylase and α-glucosidase enzyme inhibitory effects of the three species were relatively the same. However, E. hirta exhibited the highest anti-tyrosinase activity (109.71 mg kojic acid equivalent /g). Euphorbia convolvuloides showed low antioxidant activities and enzymatic inhibitory effects compared to other. LC-DAD-MSn fingerprint of the phenolics of the three species showed the presence of different derivatives. Quantitative data revealed tannins and flavonoids (25 and 20 mg/g respectively) for E. hirta, high amount of tannins 63 and 21 mg/g of flavonoids for E. heterophylla, while high flavonoid contents for E. convolvuloides, 56 mg/g. Euphorbia hirta proved to the most promising among the three tested species and thus can be explored further by isolating and characterizing active bioactive agents that could be used to manage oxidative stress-related diseases and hyperpigmentation problems.