Extraction of curcuminoids and ar-turmerone from turmeric (Curcuma longa L.) using hydrophobic deep eutectic solvents (HDESs) and application as HDES-based microemulsions.

The extraction of curcuminoids and aromatic (ar)-turmerone from Curcuma longa L. using organic solvents produces chemical waste, and is therefore incompatible with food applications. To address this issue, this study presents the design of hydrophobic deep eutectic solvents (HDESs) and HDES-based microemulsions. Using the response surface methodology (RSM), the optimal extraction conditions were identified as follows: HDES = OA:menthol (1:3.6 M ratio), solid-to-liquid ratio = 10:1 (mg/mL), and extraction duration = 90 min (prediction accuracy ≥ 85 %). Under these conditions, the HDES extraction yields of bisdemethoxycurcumin, demethoxycurcumin, curcumin, and ar-turmerone were 2.49 ± 0.25, 5.61 ± 0.45, 9.40 ± 0.86, and 3.83 ± 0.19 % (w/w, dry basis), respectively, while those obtained using the HDES-based microemulsion were 2.10 ± 0.18, 6.31 ± 0.48, 12.6 ± 1.20, and 2.58 ± 0.19 % (w/w, dry basis), respectively. The HDES and its microemulsions are more effective and environmentally friendly than conventional organic solvents for the extraction of curcuminoids and ar-turmerone, and these solvents are also compatible with food and pharmaceutical formulations.

Therapeutic hydrophobic deep eutectic solvents of menthol and fatty acid for enhancing anti-inflammation effects of curcuminoids and curcumin on RAW264.7 murine macrophage cells.

Owing to their water insolubility, low stability, and poor absorption, anti-inflammatory curcuminoids (CUN) are difficult to be extracted and delivered to the action site. As a result, therapeutic hydrophobic deep eutectic solvents (HDESs), containing menthol and fatty acids (capric, caprylic, and oleic acids), are being developed for CUN solubilization and delivery. In this study, the anti-inflammatory effects of various combinations of HDESs with CUN and curcumin (CUR) were investigated on RAW264.7 macrophage cells. The results showed that CUN can be solubilized using the HDESs. The HDESs of oleic acid (OLA) : menthol (1 : 2, 1 : 1, and 2 : 1 molar ratios) exhibited anti-inflammatory effects, and OLA : menthol (1 : 1 molar ratio) increased the anti-inflammatory effects of CUR. The cytotoxicity of CUN and CUR was also lowered when combined with some OLA : menthol HDESs. The combination of OLA, menthol, and CUR entirely suppressed NO secretion without significant cytotoxicity. These results clearly indicate the potential of HDESs to solubilize CUN and impart anti-inflammatory properties. Furthermore, these solvents could replace organic solvents for CUN extraction, with the added benefit of being therapeutic, biodegradable, and safe for human consumption.

PPARß/δ priming enhances the anti-apoptotic and therapeutic properties of mesenchymal stromal cells in myocardial ischemia-reperfusion injury.

BACKGROUND: Mesenchymal Stromal Cells (MSC) have been widely used for their therapeutic properties in many clinical applications including myocardial infarction. Despite promising preclinical results and evidences of safety and efficacy in phases I/ II, inconsistencies in phase III trials have been reported. In a previous study, we have shown using MSC derived from the bone marrow of PPARß/δ (Peroxisome proliferator-activated receptors ß/δ) knockout mice that the acute cardioprotective properties of MSC during the first hour of reperfusion are PPARß/δ-dependent but not related to the anti-inflammatory effect of MSC. However, the role of the modulation of PPARß/δ expression on MSC cardioprotective and anti-apoptotic properties has never been investigated. OBJECTIVES: The aim of this study was to investigate the role of PPARß/δ modulation (inhibition or activation) in MSC therapeutic properties in vitro and ex vivo in an experimental model of myocardial infarction. METHODS AND RESULTS: Naïve MSC and MSC pharmacologically activated or inhibited for PPARß/δ were challenged with H2O2. Through specific DNA fragmentation quantification and qRT-PCR experiments, we evidenced in vitro an increased resistance to oxidative stress in MSC pre-treated by the PPARß/δ agonist GW0742 versus naïve MSC. In addition, PPARß/δ-priming allowed to reveal the anti-apoptotic effect of MSC on cardiomyocytes and endothelial cells in vitro. When injected during reperfusion, in an ex vivo heart model of myocardial infarction, 3.75 × 105 PPARß/δ-primed MSC/heart provided the same cardioprotective efficiency than 7.5 × 105 naïve MSC, identified as the optimal dose in our experimental model. This enhanced short-term cardioprotective effect was associated with an increase in both anti-apoptotic effects and the number of MSC detected in the left ventricular wall at 1 h of reperfusion. By contrast, PPARß/δ inhibition in MSC before their administration in post-ischemic hearts during reperfusion decreased their cardioprotective effects. CONCLUSION: Altogether these results revealed that PPARß/δ-primed MSC exhibit an increased resistance to oxidative stress and enhanced anti-apoptotic properties on cardiac cells in vitro. PPARß/δ-priming appears as an innovative strategy to enhance the cardioprotective effects of MSC and to decrease the therapeutic injected doses. These results could be of major interest to improve MSC efficacy for the cardioprotection of injured myocardium in AMI patients.

Lyophilized cell-free supernatants of Lactobacillus isolates exhibited antibiofilm, antioxidant, and reduces nitric oxide activity in lipopolysaccharide-stimulated RAW 264.7 cells.

BACKGROUND: Probiotics can release bioactive substances known as postbiotics, which can inhibit pathogenic microorganisms, improve immunomodulation, reduce antioxidant production, and modulate the gut microbiota. METHODS: In this study, we evaluated the in vitro antimicrobial effects, antioxidant activity, and anti-inflammatory potential of 10 lyophilized cell-free supernatants (LCFS) of Lactobacillus isolates. LCFS was obtained via centrifugation and subsequent lyophilization of the supernatant collected from the culture medium ofeach isolate. The antibacterial and antibiofilm activities of the LCFS were determined using broth microdilution. The antioxidant potential was evaluated by measuring the total phenolic and flavonoid contents and 2,2-Diphennyl-1-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS+) radical scavenging activities. RESULTS: All the isolates were able to inhibit the four tested pathogens. The isolates exhibited strong antibiofilm activity and eradicated the biofilms formed by Acinetobacter buamannii and Escherichia coli. All the prepared Lactobacillus LCFS contained phenols and flavonoids and exhibited antioxidant activities in the DPPH and ABTS+ radical scavenging assays. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay revealed that LCFS was not cytotoxic to RAW 264.7 cells. In addition, the ten Lactobacillus LCFS decreased the production of nitric oxide. CONCLUSIONS: All the isolates have beneficial properties. This research sheds light on the role of postbiotics in functional fermented foods and pharmaceutical products. Further research to elucidate the precise molecular mechanisms of action of probiotics is warranted.

p38 MAPK Inhibitor (SB203580) and Metformin Reduces Aortic Protein Carbonyl and Inflammation in Non-obese Type 2 Diabetic Rats.

Microvascular and macrovascular diseases are the main causes of morbidity in type 2 diabetes patients through chronic hyperglycaemic condition via oxidative stress and inflammation. Reactive oxygen species (ROS) activate p38 MAPK phosphorylation and inflammation which enhances protein modification by carbonylation. The use of metformin and a p38 MAPK inhibitor is hypothesised to reduce ROS production and inflammation but effects of metformin and p38 MAPK inhibitor (SB203580) on ROS production and inflammation in vascular type 2 diabetes mellitus non-obese (T2DM) have not been investigated. The Goto-Kakizaki rat T2DM model was divided into three groups as T2DM, T2DM treated with 15 mg/kg bw metformin and T2DM treated with 2 mg/kg bw SB203580 for 4 weeks. Rat aortas were isolated and protein carbonyl (PC) contents were measured by spectrophotometric DNPH assay. Aortic IL-1ß level was determined by ELISA. Results showed that aortic PC contents in the T2DM group were significantly higher than in non-diabetic rats. Treatment with metformin or SB203580 significantly reduced PC contents while only metformin significantly reduced IL-1ß levels. Findings indicated that metformin reduced ROS production and inflammation in diabetic vessels and possibly reduce vascular complications in non-obese T2DM.

Endothelial-Cell-Derived Human Secretory Leukocyte Protease Inhibitor (SLPI) Protects Cardiomyocytes against Ischemia/Reperfusion Injury.

Vascular endothelial cell (EC)-derived factors play an important role in endothelial-cardiomyocyte crosstalk and could save cardiomyocytes (CMs) from injury. The manipulation of endothelial cells to secrete protective factors could enhance cardioprotection. Secretory leukocyte protease inhibitor (SLPI) has been known to protect the heart. The goal of this study was to evaluate the in vitro paracrine protective effect and mechanisms of EC-derived human SLPI on cardiomyocytes subjected to hypoxia/reoxygenation (H/R) injury. Stable endothelial cells overexpressing human SLPI were generated from an endothelial cell line (EA.hy926). The cytoprotective effect was determined by cell survival assay. The results showed that endothelial-derived recombinant human SLPI (rhSLPI) reduced simulated ischemia/reperfusion (I/R)-(81.75% ± 1.42% vs. 60.27% ± 2.52%, p < 0.05) and hypoxia/reoxygenation (H/R)-induced EC injury (83.57% ± 1.78% vs. 63.07% ± 1.93%, p < 0.05). Moreover, co-culture of ECs overexpressing rhSLPI with CMs at ratios 1:1 and 1:3 or treatment with conditioned medium enhanced cell viability by 10.51-16.7% (co-culture) and 15.25-20.45% (conditioned medium) by reducing intracellular reactive oxygen species (ROS) production, the Bax/Bcl-2 expression ratio, caspase-3, and caspase-8, and in preconditioned CMs by activation of p38 MAPK and Akt survival kinase. In conclusion, this study showed for the first time that EC-derived rhSLPI provided cardio-vasculoprotective effects against I/R injury as a possible alternative therapeutic strategy for cardioprotection.

Combination of metformin and p38 MAPK inhibitor, SB203580, reduced myocardial ischemia/reperfusion injury in non-obese type 2 diabetic Goto-Kakizaki rats.

Diabetic cardiomyopathy, especially myocardial ischemia reperfusion (I/R) injury, is a major cause of morbidity and mortality in type 2 diabetic patients. The increasing of basal p38 MAP Kinase (p38 MAPK) activation is a major factor that aggravates cardiac death on diabetic cardiomyopathy. In addition, metformin also shows cardio-protective effects on myocardial ischemia/reperfusion injury. In this study, we investigated the effect of the combination between metformin and p38 MAPK inhibitor (SB203580) in diabetic rats subjected to I/R injury. H9c2 cells were induced into a hyperglycemic condition and treated with metformin, SB203580 or the combination of metformin and SB203580. In addition, cells in both the presence and absence of drug treatment were subjected to simulated ischemia/reperfusion injury. Cell viability and cellular reactive oxygen species (ROS) were determined. Moreover, the Goto-Kakizaki (GK) rats were treated with metformin, SB203580, and the combination of metformin and SB203580 for 4 weeks. Diabetic parameters and cardiac functions were assessed. Finally, rat hearts were induced ischemia/reperfusion injury for the purpose of infarct size analysis and determination of signal transduction. A high-glucose condition did not reduce cell viability but significantly increased ROS production and significantly decreased cell viability after induced sI/R. Treatment using drugs was shown to reduce ROS generation and cardiac cell death. The GK rats displayed diabetic phenotype by increasing diabetic parameters and these parameters were significantly decreased when treated with drugs. Treatment with metformin or SB203580 could significantly reduce the infarct size. Interestingly, the combination of metformin and SB203580 could enhance cardio-protective ability. Myocardial I/R injury significantly increased p38 MAPK phosphorylation, Bax/Bcl-2 ratio and caspase-3 level. Treatment with drugs significantly decreased the p38 MAPK phosphorylation, Bax/Bcl-2 ratio, caspase-3 level and increased Akt phosphorylation. In conclusion, using the combination of metformin and SB203580 shows positive cardio-protective effects on diabetic ischemic cardiomyopathy.