In silico ADMET and anti-inflammatory profiles of the stigmast-4-en-3-one and isolation of isovanillin as the antioxidant principle of Imperata cylindrica (L.) P. Beauv.

Imperata cylindrica (L.) P. Beauv. is an invasive species widely used in treatment of several diseases associated with pain and inflammation in different countries including Madagascar. This work aims to report the isolation of the antioxidant, analgesic and anti-nflammatory compounds from the methanol extract of I. cylindrica. The bio-guided method was used to isolate its bioactive compounds by combining chromatographic methods, writhing test in mice and antioxidant assays. Stigmast-4-en-3-one was isolated as one among the compounds responsible for the analgesic and anti-inflammatory properties and isovanillin as one among the antioxidant compounds from the extract. Stigmast-4-en-3-one showed a good oral pharmacokinetic profile and good binding affinities with some pro-inflammatory targets. It did not show any mutagenic effect, nor a carcinogenic one and had a low risk to be a cardiotoxic agent. All of our results provide scientific justification for its traditional medicinal use in the management of pain and inflammatory related diseases.

Cytotoxic and Pro-Apoptotic Effects of Leaves Extract of Antiaris africana Engler (Moraceae).

Antiaris africana Engler leaves have been used in Senegalese folk medicine to treat breast cancer. The present study aimed to investigate the anticancer potential of Antiaris africana Engler leaves using several human cancer cell lines. The leaves of Antiaris africana Engler were extracted in parallel with water or 70% ethanol and each extract divided into three parts by successive liquid-liquid extraction with ethyl acetate and butanol. The phytochemical components of the active extract were investigated using ultra-performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry (UPLC-DAD-QTOF-MS/MS). The cytotoxic and cytostatic effects of each extract, as well as their fractions, were evaluated in vitro via flow and image cytometry on different human cancer phenotypes, such as breast (MCF-7), pancreas (AsPC-1), colon (SW-620) and acute monocytic leukemia (THP-1). Both hydro-alcoholic and aqueous extracts induced strong apoptosis in MCF-7 cells. The water fraction of the hydro-alcoholic extract was found to be the most active, suppressing the cell growth of MCF-7 in a dose-dependent manner. The half maximum effective concentration (EC50) of this fraction was 64.6 ± 13.7 µg/mL for MCF-7, with equivalent values for all tested phenotypes. In parallel, the apoptotic induction by this fraction resulted in a EC50 of 63.5 ± 1.8 µg/mL for MCF-7, with again equivalent values for all other cellular tested phenotypes. Analysis of this fraction by UPLC-DAD-QTOF-MS/MS led to the identification of hydroxycinnamates as major components, one rutin isomer, and three cardiac glycosides previously isolated from seeds and bark of Antiaris africana Engler and described as cytotoxic in human cancer models. These results provide supportive data for the use of Antiaris africana Engler leaves in Senegal.

Cannabis sativa Extract Induces Apoptosis in Human Pancreatic 3D Cancer Models: Importance of Major Antioxidant Molecules Present Therein.

In recent years, interest in Cannabis sativa L. has been rising, as legislation is moving in the right direction. This plant has been known and used for thousands of years for its many active ingredients that lead to various therapeutic effects (pain management, anti-inflammatory, antioxidant, etc.). In this report, our objective was to optimize a method for the extraction of cannabinoids from a clone of Cannabis sativa L. #138 resulting from an agronomic test (LaFleur, Angers, FR). Thus, we wished to identify compounds with anticancer activity on human pancreatic tumor cell lines. Three static maceration procedures, with different extraction parameters, were compared based on their median inhibitory concentration (IC50) values and cannabinoid extraction yield. As CBD emerged as the molecule responsible for inducing apoptosis in the human pancreatic cancer cell line, a CBD-rich cannabis strain remains attractive for therapeutic applications. Additionally, while gemcitabine, a gold standard drug in the treatment of pancreatic cancer, only triggers cell cycle arrest in G0/G1, CBD also activates the cell signaling cascade to lead to programmed cell death. Our results emphasize the potential of natural products issued from medicinal hemp for pancreatic cancer therapy, as they lead to an accumulation of intracellular superoxide ions, affect the mitochondrial membrane potential, induce G1 cell cycle arrest, and ultimately drive the pancreatic cancer cell to lethal apoptosis.

Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis.

BACKGROUND: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and 2 lysosomal proteins, the NPC1 (Niemann-Pick C1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. METHODS: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells. To establish whether Wnt5a also protects against cholesterol accumulation in human vascular smooth muscle cells, we used a CRISPR/Cas9 guided nuclease approach to generate human vascular smooth muscle cells knockout for Wnt5a. RESULTS: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 (mechanistic target of rapamycin complex 1) kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum. Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the endoplasmic reticulum, and promoted atherosclerosis. CONCLUSIONS: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.

Influence of cell-cell contact between L. thermotolerans and S. cerevisiae on yeast interactions and the exo-metabolome.

Sequential fermentation of grape must inoculated with L. thermotolerans and then S. cerevisiae 24 h later (typical wine-making practice) was conducted with or without cell-cell contact between the two yeast species. We monitored cell viability of the two species throughout fermentation by flow cytometry. The cell viability of S. cerevisiae decreased under both conditions, but the decrease was greater if there was cell-cell contact. An investigation of the nature of the interactions showed competition between the two species for nitrogen compounds, oxygen, and must sterols. Volatile-compound analysis showed differences between sequential and pure fermentation and that cell-cell contact modifies yeast metabolism, as the volatile-compound profile was significantly different from that of sequential fermentation without cell-cell contact. We further confirmed that cell-cell contact modifies yeast metabolism by analyzing the exo-metabolome of all fermentations by FT-ICR-MS analysis. These analyses show specific metabolite production and quantitative metabolite changes associated with each fermentation condition. This study shows that cell-cell contact not only affects cell viability, as already reported, but markedly affects yeast metabolism.

Oral insulin delivery, the challenge to increase insulin bioavailability: Influence of surface charge in nanoparticle system.

Oral administration of insulin increases patient comfort and could improve glycemic control thanks to the hepatic first passage. However, challenges remain. The current approach uses poly (d, lactic-co-glycolic) acid (PLGA) nanoparticles (NPs), an effective drug carrier system with a long acting profile. However, this system presents a bioavailability of less than 20% for insulin encapsulation. In this context, physico-chemical parameters like surface charge could play a critical role in NP uptake by the intestinal barrier. Therefore, we developed a simple method to modulate NP surface charge to test its impact on uptake in vitro and finally on NP efficiency in vivo. Various NPs were prepared in the presence (+) or absence (-) of polyvinyl alcohol (PVA), sodium dodecyl sulfate (SDS), and/or coated with chitosan chloride. In vitro internalization was tested using epithelial culture of Caco-2 or using a co-culture (Caco-2/RevHT29MTX) by flow cytometry. NPs were then administered by oral route using a pharmaceutical complex vector (100 or 250 UI/kg) in a diabetic rat model. SDS-NPs (-42 ±â€¯2 mV) were more negatively charged than -PVA-NPs (-22 ±â€¯1 mV) and chitosan-coated NPs were highly positively charged (56 ±â€¯2 mV) compared to +PVA particles (-2 ±â€¯1 mV), which were uncharged. In the Caco-2 model, NP internalization was significantly improved by using negatively charged NPs (SDS NPs) compared to using classical NPs (+PVA NPs) and chitosan-coated NPs. Finally, the efficacy of insulin SDS-NPs was demonstrated in vivo (100 or 250 UI insulin/kg) with a reduction of blood glucose levels in diabetic rats. Formulation of negatively charged NPs represents a promising approach to improve NP uptake and insulin bioavailability for oral delivery.

Analysis of sitosteryl oleate esters in phytosterols esters enriched foods by HPLC-ESI-MS(2.).

Phytosteryl esters (PE)-enriched spreads are marketed for eating and cooking purposes. Temperature and also light exposure are the major factors leading to the formation of PE oxides in food matrix. In this study a high-speed HPLC-MS(2) method was developed to analyze the major PE present in PE-enriched spreads: sitosteryl oleate (SO) and its oxidation products, by using synthesized compounds as standards. This analytical method was used to quantify seven SO oxides formed in PE-enriched spreads after heating at different temperatures for varying time periods and after prolonged exposure to sunlight. Quantification of remaining native SO was also performed after these different treatments. It was found that under specific heating conditions the decrease of the SO amount was much more important compared to the formation of SO oxides showing that many other products are formed. In contrast to heating, sunlight radiation did not result in the degradation of SO and very few oxides were formed.

Relative bioavailability of soil-bound polychlorinated biphenyls in lactating goats.

Livestock may be exposed to organic pollutants via ingestion of contaminated matrices such as fodder or soil. The question on contribution of soil-bound polychlorinated biphenyls (PCB) to livestock exposure was not yet considered. The aim of this study was to assess the relative bioavailability of soil-bound PCB by assessing milk excretion of indicator PCB (I-PCB) after ingestion by goats of graded levels of PCB (mainly PCB forms 153, 180, and 138) in soil-contaminated feeds or in oil-contaminated feeds. Eight multiparous Alpine goats were grouped in 4 pairs on the basis of body weight and milk yield. In each pair, one goat was assigned to the soil feeds and the other one to the oil feeds. The experiment consisted of a 7-d adaptation period, followed by a 96-d exposure period. The exposure period was divided into 3 successive 32-d periods during which each goat received either 3 soil feeds or 3 oil feeds, distributed in increasing rank of contamination. During the last week of each 32-d period, milk from each goat was collected during 3 successive 24-h periods, stored at -20°C, and freeze dried before analysis (extraction by accelerated solvent extraction, followed by gas chromatography-mass spectrometry analysis). Bioavailability of I-PCB from soil or spiked oil feeds was estimated by means of the slope-ratio method from I-PCB concentration in milk in response to ingested I-PCB. Relative bioavailability was found to vary from 36 to 50% for PCB 118, 138, and 153 and it was 73% for PCB 180. When considered globally, the response obtained with the I-PCB was estimated to 51%. Relative bioavailability was not established for PCB 52 and 101, compounds known to be readily cleared and showing low concentrations in milk. For PCB 28, no significant interaction was found between matrix and dose. This experiment reveals that PCB bound to soil are potentially liberated from soil during the digestive process and may undergo absorption, distribution, metabolism, and excretion. Thus, soil has to be considered as a risk matrix for ruminants and rearing practices in contaminated areas should strictly reduce the risk of soil ingestion by the ruminants.

Effects of oxidation on the hydrolysis by cholesterol esterase of sitosteryl esters as compared to a cholesteryl ester.

Phytosteryl esters (PE) are used as ingredients in functional food to decrease plasma concentration of low density lipoprotein-cholesterol (LDL-C). Effective impairment of cholesterol absorption by PE suggests that these esters are hydrolyzed by the pancreatic cholesterol esterase (CEase, EC 3.1.1.13) and the liberated sterol may interfere with cholesterol reducing its intestinal absorption. PE-enriched foods are marketed for cooking purposes, and temperature is one of the most important factors leading to the formation of oxidation products. Very little is known about the outcome of PE oxides during the digestive process. A new analytical method based on mass spectrometric detection directly after enzymatic reaction was developed to determine in vitro the activity of CEase on PE and their oxides present in functional food. Using this method, we identified a new inhibitor of CEase: sitosteryl 9,10-dihydroxystearate, which behaves as a non-competitive inhibitor of the hydrolysis of cholesteryl oleate and sitosteryl oleate.

Synthesis of highly pure oxyphytosterols and (oxy)phytosterol esters. Part II. (Oxy)-sitosterol esters derived from oleic acid and from 9,10-dihydroxystearic acid [1].

Several efficient synthetic routes giving readily access to (oxy)-sitosterol esters and (oxy)-cholesterol esters derived respectively from oleic acid and from 9,10-dihydroxystearic acid were developed for the first time. This approach allowed that sufficient amounts of the latter were available in order to carry out further biological studies.

Synthesis of highly pure oxyphytosterols and (oxy)phytosterol esters Part I. Regioselective hydrogenation of stigmasterol: an easy access to oxyphytosterols.

The synthesis of several oxyphytosterols is described starting from stigmasterol, the key step being the regioselective hydrogenation of the 22-23 double bond of the latter.

Quantitative analysis of beta-sitosterol oxides induced in vegetable oils by natural sunlight, artificially generated light, and irradiation.

UV radiation is able to induce lipid peroxidation. Photooxidation-induced beta-sitosterol oxides were monitored in four vegetable oils exposed to sunlight for 10, 20, and 30 days during May 2005 (northeastern France), exposed to artificial light generated by a high-pressure Hg lamp for 21, 42, and 63 h at room temperature, and exposed to a 10 MeV electron beam at 0.93, 2.69, and 9.30 kGy at 8 degrees C. Quantification was performed by capillary gas chromatography-mass spectrometry according to the total ion current mode and using a reconstructed ion trace chromatogram with specific ion fragments. Sunlight induced the formation of higher amounts of oxides than UV light, while no significant oxidizing effect was observed with electron beam irradiation. However, data suggested that the amount of the main oxides formed was strongly dependent on the dose rate (length of exposure). Accordingly, shorter but more intense treatments had lower oxidizing effects.

Identification and quantitative analysis of beta-sitosterol oxides in vegetable oils by capillary gas chromatography-mass spectrometry.

As vegetable oils and phytosterol-enriched spreads are marketed for frying food or cooking purposes, temperature is one of the most important factors leading to the formation of phytosterol oxides in food matrix. A methodology based on saponification, organic solvent extraction, solid-phase extraction (SPE), followed by mass spectrometric identification and quantitation of beta-sitosterol oxides using capillary gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring (SIM) mode was developed and characterized. Relative response factors of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were calculated against authentic standards of 19-hydroxycholesterol or cholestanol. Linear calibration data, limit of detection, and sample recoveries during analytical process. Recoveries of these oxidation compounds in spiked samples ranged from 88 to 115%, while relative standard derivation (R.S.D.) values were below 10% in most cases. The analytical method was applied to quantify beta-sitosterol oxides formed in thermal-oxidized vegetable oils which were heated at different temperatures and for varying time periods. Sitosterol oxidation is strikingly higher in sunflower oil relative to olive oil under all conditions of temperature and heating time.

Gram-scale chromatographic purification of beta-sitosterol. Synthesis and characterization of beta-sitosterol oxides.

An effective purification method for beta-sitosterol was developed starting from a commercial source of a phytosterol mixture using preparative adsorption column chromatography. beta-Sitosterol (> or = 95% purity) was obtained on a gram-scale. Thus, the synthesis of six beta-sitosterol oxides, including 7alpha-hydroxy, 7beta-hydroxy, 5,6alpha-epoxy, 5,6beta-epoxy, 7-keto, and 5alpha,6beta-dihydroxysitosterol, were successfully carried out. The spectral characteristics of all the synthetic intermediates and target compounds (approximately 95% purity) were well-documented.