Ullah, Hammad (2023) Preclinical assessment of the nutraceutical benefits of Prunus domestica L. regarding the prevention of metabolic syndrome risk factors. [Tesi di dottorato]

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Abstract

High consumption of fruit and vegetables has an inverse association with cardiometabolic risk factors. Plums (Prunus domestica L.) are fruits with low glycemic index, with high concentration of polyphenols and dietary fibers, thus their consumption in adequate amount and on a regular basis could be a potential preventive strategy against metabolic syndrome (MetS). The present study aimed to (1) characterize the metabolite profile of a hydroethanolic extract of P. domestica, using a multimethodological approach involving two analytical techniques i.e., targeted ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) and untargeted nuclear magnetic resonance (NMR) spectroscopy, and (2) study the biological effects of hydroethanolic extract of P. domestica involving in vitro and in vivo experimental models of MetS risk factors. Initially, two varieties of P. domestica fruit (P. domestica subsp. domestica and P. domestica subsp. syriaca) were selected, the skin and pulp of which were separately extracted with hydroethanolic solution of various strengths (99%, 70% and 50%), followed by the determination of total phenolic contents (TPC) using Folin-Ciocalteu method and in vitro antioxidant activity using ABTS assay. Based on the higher TPC, higher antioxidant activity, and the lower concentration of ethanol used for extraction, making the extraction method more sustainable from an economic and environmental point of view, the P. domestica subsp. syriaca fruit pulp extract obtained with 50% hydroethanolic solution was selected for the chemical characterization and subsequent biological activities. The UHPLC-HRMS analysis showed the presence of hydroxycinnamic and quinic acid derivatives (46.7% of total peak area), procyanidins (37%), and flavonol glycosides (7.9%). The NMR analysis showed, among saccharides, an abundant presence of glucose (106.59 µg/mg dry weight) followed by sucrose (31.59 µg/mg) and xylose (0.56 µg/mg). Considering the fact of being used in MetS risk factors, the sugar contents of fruit pulp extract were precipitated out with absolute ethanol and ultra-freezing temperature, leaving behind the extract (with possible minimal sugar contents) for assessment in biological activities. Inhibition of the activities of different enzymes associated with the MetS by P. domestica fruit extract was assessed and the half minimal inhibitory concentration (IC50) for each enzyme was determined by nonlinear regression analysis. The fruit extract inhibited α-amylase, α-glucosidase, HMG CoA reductase, and pancreatic lipase enzyme activities, with IC50 values of 7.01 mg/mL, 6.4 mg/mL, 2.5 mg/mL, and 6.0 mg/mL, respectively. The in vitro anti-inflammatory potential of P. domestica fruit extract was evaluated using Murine monocyte/macrophage J774 cell lines. The fruit pulp extract significantly inhibited lipopolysaccharide-induced production of pro-inflammatory mediators (nitrite, IL-1β and PGE2) in activated J774 macrophages in dose dependent fashion, while no such effect was seen in unstimulated cell lines. In vivo studies revealed considerable hypoglycemic and hypoinsulinemic activities of P. domestica fruit pulp extract in healthy mice. The BALB/c mice were challenged with oral sugars load (sucrose/glucose, 1 mg/6375 mg for 50 ml of solution) followed by the supplementation of P. domestica fruit pulp extract (75 mg/mL, 300 μL/mouse). The blood glucose and insulin levels were measured at time intervals T0, T10 min, T20 min, and T 1 h. P. domestica fruit extract showed significant effects at T10 min and T20 min in reducing blood glucose and insulin levels, when compared with control mice. The P. domestica fruit pulp extract (without sugar contents) was analyzed with HPLC-MS/MS technique, to determine the change in metabolic profile of the extract during chemical precipitation of sugar contents. HPLC-MS/MS analysis revealed the presence of 20 polyphenolic compounds representing the polyphenol classes of phenolic acids, flavan-3-ols, and procyanidins, while the presence of flavonol glycosides was not detected, that might be precipitated out with sugar precipitation. The in vitro bioaccessibility of P. domestica fruit pulp extract using in vitro static digestion model revealed the significant decrease in TPC in the extract (in particular at duodenal stage of digestion), where hydroxycinnamic acids turned out to be the only bioaccessible polyphenols class following oro-gastro-duodenal digestion. The present study investigated the nutraceutical benefits of P. domestica fruit pulp extract regards the MetS risk factors reduction. It resulted in the in vitro modulation of the activities of metabolic enzymes and significant reduction of pro-inflammatory mediators. The in vitro results were strongly supported by in vivo experiments, where the fruit pulp extract significantly reduced the blood glucose and insulin levels in healthy mice challenged with oral sugars load. In view of a possible application of this extract as food supplement ingredient, as the in vitro bioaccessibility study revealed only the presence of hydroxycinnamic acid derivatives following oro-gastric-duodenal digestion, the use of gastro resistant oral dosage forms is suggested to partially prevent the degradation of the bioactive extract and to maintain the phytocomplex. In conclusion, this extract could be used to reduce MetS risk factors, and in turn to prevent the cardio-metabolic disorders.

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