Pressurized Hot Water Extraction of Mangosteen Pericarp and Its Associated Molecular Signatures in Endothelial Cells.

The mangosteen (Garcinia mangostana L.) pericarp is known to be rich in potent bioactive phytochemical compounds such as xanthones, which possess pharmacologically important antioxidant activity and beneficial cardiometabolic properties. Mangosteen pericarp is typically classified as unavoidable food waste and discarded, despite being rich in bioactive phytochemical compounds that therefore present an exciting opportunity for valorization. Thus, this study aims to extract phytochemical compounds from mangosteen pericarp using pressurized hot water extraction (PHWE) and determine its biological effects in endothelial cells using RNA sequencing. Liquid chromatography with MS/MS (LC/MSMS) and UV detection (LC/UV) was subsequently used to identify three key phytochemical compounds extracted from the mangosteen pericarp: α-Mangostin, γ-Mangostin, and Gartanin. Within the tested range of extraction temperatures by PHWE, our results demonstrated that an extraction temperature of 120 °C yielded the highest concentrations of α-Mangostin, γ-Mangostin, and Gartanin with a concomitant improvement in antioxidant capacity compared to other extraction temperatures. Using global transcriptomic profiling and bioinformatic analysis, the treatment of endothelial cells with mangosteen pericarp extracts (120 °C PHWE) for 48 h caused 408 genes to be differentially expressed. Furthermore, our results demonstrated that key biological processes related to "steroid biosynthesis and metabolism", likely involving the activation of the AMPK signaling pathway, were upregulated by mangosteen pericarp extract treatment. In conclusion, our study suggests a green extraction method to valorize phytochemical compounds from mangosteen pericarp as a natural product with potential beneficial effects on cardiometabolic health.

Time-dependent specific molecular signatures of inflammation and remodelling are associated with trimethylamine-N-oxide (TMAO)-induced endothelial cell dysfunction.

Endothelial dysfunction is a critical initiating factor contributing to cardiovascular diseases, involving the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO). This study aims to clarify the time-dependent molecular pathways by which TMAO mediates endothelial dysfunction through transcriptomics and metabolomics analyses in human microvascular endothelial cells (HMEC-1). Cell viability and reactive oxygen species (ROS) generation were also evaluated. TMAO treatment for either 24H or 48H induces reduced cell viability and enhanced oxidative stress. Interestingly, the molecular signatures were distinct between the two time-points. Specifically, few Gene Ontology biological processes (BPs) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were modulated after a short (24H) compared to a long (48H) treatment. However, the KEGG signalling pathways namely "tumour necrosis factor (TNF)" and "cytokine-cytokine receptor interaction" were downregulated at 24H but activated at 48H. In addition, at 48H, BPs linked to inflammatory phenotypes were activated (confirming KEGG results), while BPs linked to extracellular matrix (ECM) structural organisation, endothelial cell proliferation, and collagen metabolism were repressed. Lastly, metabolic profiling showed that arachidonic acid, prostaglandins, and palmitic acid were enriched at 48H. This study demonstrates that TMAO induces distinct time-dependent molecular signatures involving inflammation and remodelling pathways, while pathways such as oxidative stress are also modulated, but in a non-time-dependent manner.

Recent advances in the combination of organic solvent-free extraction, chemical standardization, antioxidant assay, and cell culture metabolomics for functional food and its by-product.

Functional foods and their by-products contain a wide range of bioactive components with an array of health benefits and were proposed to improve public health, well-being, and others. To achieve a circular economy, the processing and extraction of flavonoids, phenolic compounds, and others from functional food and agri-food wastes will require the use of environmentally friendly, sustainable, and a low-cost solution. Extraction methods that can eliminate the use of organic solvents, suitable for use in the laboratory and production of extracts will be covered. This will include subcritical water extraction (SBE), pressurized hot water extraction (PHWE), supercritical fluid extraction (SFE), and others. Based on the selected analytical methods, the determination of the marker or bioactive compounds and chemical fingerprints will provide the control measures to identify the batch-to-batch variation of the composition of the functional food products obtained. The combination of chemical standardization with antioxidant assay, such as DPPH and ABTS+ will provide further information on the quality of the extracts. Lastly, to ascertain the biological and physiological relevance of the antioxidant properties of the target sample, treatment of the antioxidant compounds or extracts was carried out using cellular models, and validated using other experimental endpoints, such as metabolomics.

3D printability and biochemical analysis of revalorized orange peel waste.

Orange peels are often discarded as food waste despite being a nutritious source of vitamins and antioxidants. These orange peel wastes (OPW) are produced in millions of tons globally every year; discarding them results in detrimental environmental and economical impacts. This paper discusses the application of 3D printing technology to effectively upcycle the OPW into edible, healthy snacks for consumption. We aimed to develop a method to enable OPW to formulate 3D-printable inks for direct ink writing (DIW). Using DIW 3D printing, we successfully created edible constructs of rheologically modified inks containing OPW. The formulated ink possessed an initial viscosity of 22.5 kPa.s, a yield stress of 377 Pa, and a storage modulus of 44.24 kPa. To validate the method, we conducted a biochemical analysis of the OPW at each stage of the fabrication process. This study suggested that our ink formulation and 3D printing process did not affect the content of bioflavonoids and antioxidants of the OPW. The cell viability test using human dermal microvascular endothelium (HMEC-1) suggested that the OPW did not exhibit cytotoxicity throughout the entire process of the ink manipulation. Overall, this study has highlighted a potential scenario to revalorize food waste into the food value chain using 3D printing toward more sustainable and circular food manufacturing and consumption.

Urine Metabolites and Bioactive Compounds from Functional Food: Applications of Liquid Chromatography Mass Spectrometry.

Bioactive compounds in functional foods, medicinal plants and others are considered attractive value-added molecules based on their wide range of bioactivity. It is clear that an important role is occupied by polyphenol, phenolic compounds and others. Urine is an effective biofluid to evaluate and monitor alterations in homeostasis and other processes related to metabolism. The current review provides a detailed description of the formation of urine in human body, various aspects relevant to sampling and analysis of urinary metabolites before presenting recent developments leveraging on metabolite profiling of urine. For the profiling of small molecules in urine, advancement of liquid chromatography mass tandem spectrometry (LC/MS/MS), establishment of standardized chemical fragmentation libraries, computational resources, data-analysis approaches with pattern recognition tools have made it an attractive option. The profiling of urinary metabolites gives an overview of the biomarkers associated with the diet and evaluates its biological effects. Metabolic pathways such as glycolysis, tricarboxylic acid cycle, amino acid metabolism, energy metabolism, purine metabolism and others can be evaluated. Finally, a combination of metabolite profiling with chemical standardization and bioassay in functional food and medicinal plants will likely lead to the identification of new biomarkers and novel biochemical insights.

Special Issue "Small Molecules, Influence of Molecular Pathways 2.0".

Small molecules play an important role in extracting energy from cells, synthesising new macromolecules, and indicating metabolic shift and other processes (Figure 1) [...].

Relaxin elicits renoprotective actions accompanied by increasing bile acid levels in streptozotocin-induced diabetic mice.

BACKGROUND: The peptide hormone relaxin has potent anti-fibrotic and anti-inflammatory properties in various organs, including the kidneys. However, the protective effects of relaxin in the context of diabetic kidney complications remain controversial. Here, we aimed to evaluate the effects of relaxin treatment on key markers of kidney fibrosis, oxidative stress, and inflammation and their subsequent impact on bile acid metabolism in the streptozotocin-induced diabetes mouse model. METHODS AND RESULTS: Male mice were randomly allocated to placebo-treated control, placebo-treated diabetes or relaxin-treated diabetes groups (0.5 mg/kg/d, final 2 weeks of diabetes). After 12 weeks of diabetes or sham, the kidney cortex was harvested for metabolomic and gene expression analyses. Diabetic mice exhibited significant hyperglycaemia and increased circulating levels of creatine, hypoxanthine and trimethylamine N-oxide in the plasma. This was accompanied by increased expression of key markers of oxidative stress (Txnip), inflammation (Ccl2 and Il6) and fibrosis (Col1a1, Mmp2 and Fn1) in the diabetic kidney cortex. Relaxin treatment for the final 2 weeks of diabetes significantly reduced these key markers of renal fibrosis, inflammation, and oxidative stress in diabetic mice. Furthermore, relaxin treatment significantly increased the levels of bile acid metabolites, deoxycholic acid and sodium glycodeoxycholic acid, which may in part contribute to the renoprotective action of relaxin in diabetes. CONCLUSION: In summary, this study shows the therapeutic potential of relaxin and that it may be used as an adjunctive treatment for diabetic kidney complications.

Green Extraction of Orange Peel Waste Reduces TNFα-Induced Vascular Inflammation and Endothelial Dysfunction.

Orange peel waste (OPW) is known to contain an abundant amount of polyphenols compounds such as flavonoids, well-reported for their antioxidant and anti-inflammatory properties. While OPW is generally regarded as a food waste, the opportunity to extract bioactive compounds from these "wastes" arises due to their abundance, allowing the investigation of their potential effects on endothelial cells. Hence, this study aims to use a green extraction method and pressurized hot water extraction (PHWE) to extract bioactive compounds from OPW. Liquid chromatography with UV detection (LC/UV) and liquid chromatography mass spectrometry (LC/MS) were subsequently used to identify the bioactive compounds present. Through the optimization of the extraction temperature for PHWE, our results demonstrated that extraction temperatures of 60 °C and 80 °C yield distinct bioactive compounds and resulted in better antioxidant capacity compared to other extraction temperatures or organic solvent extraction. Despite having similar antioxidant capacity, their effects on endothelial cells were distinct. Specifically, treatment of endothelial cells with 60 °C OPW extracts inhibited TNFα-induced vascular inflammation and endothelial dysfunction in vitro, suggesting that OPW possess vasoprotective effects likely mediated by anti-inflammatory effects.

Valorization of avocado seeds with antioxidant capacity using pressurized hot water extraction.

The pulp of avocado (Persea Americana) is widely consumed as the primary food source, while the seed is often discarded as food waste. Increased consumption of avocado would inevitably results in production of waste by-products such as avocado seeds, hence the ability to extract phytochemicals from such waste, and upcycling to potential nutraceutical products is of great interest. The overall aim of this study is to explore avocado seeds as potential functional food through the combined use of a green extraction method, chemical standardization and pattern recognition tools, and biological characterization assays. Specifically, this study utilized an organic solvent-free extraction method, pressurized hot water extraction (PHWE) to extract phytochemicals from avocado seeds and liquid chromatography mass spectrometry (LCMS) was used to identify the phytochemicals present in the avocado seeds. Our results demonstrated that avocado seed extracts have antioxidant activity and inhibited oxidative stress-induced metabolomics changes in endothelial cells, suggesting that avocado seed extracts have vasoprotective actions.

Pressurized Hot Water Extraction of Okra Seeds Reveals Antioxidant, Antidiabetic and Vasoprotective Activities.

Abelmoschus esculentus L. Moench (okra) is a commonly consumed vegetable that consists of the seeds and peel component which are rich in polyphenolic compounds. The aim of this study is to utilize pressurized hot water extraction (PHWE) for the extraction of bioactive phytochemicals from different parts of okra. A single step PHWE was performed at various temperatures (60 °C, 80 °C, 100 °C and 120 °C) to determine which extraction temperature exhibits the optimum phytochemical profile, antioxidant and antidiabetic activities. The optimum temperature for PHWE extraction was determined at 80 °C and the biological activities of the different parts of okra (Inner Skin, Outer Skin and Seeds) were characterized using antioxidant (DPPH and ABTS), α-glucosidase and vasoprotective assays. Using PHWE, the different parts of okra displayed distinct phytochemical profiles, which consist of primarily polyphenolic compounds. The okra Seeds were shown to have the most antioxidant capacity and antidiabetic effects compared to other okra parts, likely to be attributed to their higher levels of polyphenolic compounds. Similarly, okra Seeds also reduced vascular inflammation by downregulating TNFα-stimulated VCAM-1 and SELE expression. Furthermore, metabolite profiling by LC/MS also provided evidence of the cytoprotective effect of okra Seeds in endothelial cells. Therefore, the use of PHWE may be an alternative approach for the environmentally friendly extraction and evaluation of plant extracts for functional food applications.

Antioxidant and Cytoprotective Effect of Quinoa (Chenopodium quinoa Willd.) with Pressurized Hot Water Extraction (PHWE).

Quinoa is widely noted for its nutritional value. The seed is the main edible part of the plant and exists in at least three different colors: white, red and black. This study utilized a pressurized hot water extraction (PHWE) for the extraction of phytochemicals from quinoa. Chemical fingerprints with LC/UV and LC/MS using a targeted approach and pattern recognition tools were used to evaluate the quinoa extracts. The antioxidant properties for various types of quinoa were evaluated using DPPH assay, ABTS assay and the cytoprotective effect of quinoa extracts were investigated in HMEC-1 cell line. Distinctive chemical profiles obtained from black and red quinoa were well correlated with the antioxidant activities and cytoprotective effects. The combination of PHWE, chemical standardization with LC/UV and LC/MS, pattern recognition tools and biological assay provided an approach for the evaluation and eventual production of quinoa extracts for functional food.

Distribution of Alox15 in the Rat Brain and Its Role in Prefrontal Cortical Resolvin D1 Formation and Spatial Working Memory.

Docosahexaenoic acid (DHA) is enriched in membrane phospholipids of the central nervous system (CNS) and has a role in aging and neuropsychiatric disorders. DHA is metabolized by the enzyme Alox15 to 17S-hydroxy-DHA, which is then converted to 7S-hydroperoxy,17S-hydroxy-DHA by a 5-lipoxygenase, and thence via epoxy intermediates to the anti-inflammatory molecule, resolvin D1 (RvD1 or 7S,8R,17S-trihydroxy-docosa-Z,9E,11E,13Z,15E,19Z-hexaenoic acid). In this study, we investigated the distribution and function of Alox15 in the CNS. RT-PCR of the CNS showed that the prefrontal cortex exhibits the highest Alox15 mRNA expression level, followed by the parietal association cortex and secondary auditory cortex, olfactory bulb, motor and somatosensory cortices, and the hippocampus. Western blot analysis was consistent with RT-PCR data, in that the prefrontal cortex, cerebral cortex, hippocampus, and olfactory bulb had high Alox15 protein expression. Immunohistochemistry showed moderate staining in the olfactory bulb, cerebral cortex, septum, striatum, cerebellar cortex, cochlear nuclei, spinal trigeminal nucleus, and dorsal horn of the spinal cord. Immuno-electron microscopy showed localization of Alox15 in dendrites, in the prefrontal cortex. Liquid chromatography mass spectrometry analysis showed significant decrease in resolvin D1 levels in the prefrontal cortex after inhibition or antisense knockdown of Alox15. Alox15 inhibition or antisense knockdown in the prefrontal cortex also blocked long-term potentiation of the hippocampo-prefrontal cortex pathway and increased errors in alternation, in the T-maze test. They indicate that Alox15 processing of DHA contributes to production of resolvin D1 and LTP at hippocampo-prefrontal cortical synapses and associated spatial working memory performance. Together, results provide evidence for a key role of anti-inflammatory molecules generated by Alox15 and DHA, such as resolvin D1, in memory. They suggest that neuroinflammatory brain disorders and chronic neurodegeneration may 'drain' anti-inflammatory molecules that are necessary for normal neuronal signaling, and compromise cognition.

Solventless extraction methods for immature fruits: Evaluation of their antioxidant and cytoprotective activities.

In this study, extraction of immature fruits using an environmentally friendly pressurized hot water extraction (PHWE) method was compared with the traditional reflux method. Extracts were tested for their polyphenol content using the Folin-Ciocalteu assay and for their antioxidant activity using the oxygen radical absorbance capacity (ORAC) assay. The highest amount of polyphenol was extracted from grape (stem) using PHWE at 100°C, or reflux extraction. This was followed by reflux extraction of grape (fruit). The results were similar for the ORAC assay. All samples extracted using PHWE at 100°C showed cytoprotective activity against H2O2-induced oxidative stress in Crandell-Reese feline kidney (CRFK) cells. This study demonstrated that beneficial compounds can be extracted from immature fruits without the use of organic solvents. The utilization of beneficial compounds present in immature fruits can also contribute to the reduction in agriculture waste generated.

Surfactant-Assisted Pressurized Liquid Extraction at Room Temperature for Radix glycyrrhizae by a New Class of Surfactants.

A laboratory-assembled surfactant-assisted pressurized liquid extraction system at room temperature was used for the extraction of glycyrrhizin (GLY) in Radix glycyrrhizae. Environmentally friendly saccharide fatty acid ester such as glucose oleic acid ester is proposed to replace chemical-based surfactants. As the chemical properties of the surfactant obtained were unknown initially, lipase-catalyzed synthesis and liquid chromatography with tandem mass spectrometry were used to ascertain the identity. Surfactant-assisted pressurized liquid extraction (PLE) was carried out dynamically and the extraction efficiencies of the proposed method using different concentration of glucose oleic acid ester were compared with sonication using an organic solvent (ethanol/water, 70:30). The extraction efficiencies of GLY in Radix glycyrrhizae using surfactant-assisted PLE was observed to be higher compared with sonication. The method precision was found to vary from 1.3 to 5.1% (relative standard deviation, RSD, n= 6) on different days. The new method demonstrated the possibility for the extraction to be carried out at room temperature for the production of botanical extracts.

A green and effective approach for characterisation and quality control of chrysanthemum by pressurized hot water extraction in combination with HPLC with UV absorbance detection.

Chrysanthemum is a ubiquitous plant with many species and wide uses, and it is usually consumed as functional food. The main aim of this paper is to demonstrate that chromatographic fingerprints obtained from the HPLC/UV analysis of the pressurized hot water extraction (PHWE) extracts together with the aid of principal component analysis (PCA), allowed for the clustering of various chrysanthemums of different species and provenance. In addition, a parallel study of pressurized fluid extraction (PFE) with methanol was carried out for comparison. From the results, a clearer separation and clustering was obtained with the environmentally-benign water extracts compared with methanol extracts. This study shows that PHWE in combination with HPLC/UV and PCA can be used successfully as a green and effective approach for characterisation and quality control of ubiquitous functional food such as chrysanthemum.

A combination of metabolomics and metallomics studies of urine and serum from hypercholesterolaemic rats after berberine injection.

Berberine, long used as a remedy in China and India for intestinal infections, has been discovered in recent years in western countries and is now being used to treat ailments ranging from urinary tract infections to diabetes and obesity. In order to study the effect of berberine more deeply, a combined metabolomic and metallomic approach was developed in this study using the hypercholesterolaemic rat model, which involved the use of proton nuclear magnetic resonance for the analysis of rat urine to achieve metabolic fingerprinting and inductively coupled plasma mass spectrometry for the analysis of rat blood serum to achieve metallomic fingerprinting. The results obtained indicated that major metabolic processes like Krebs cycle, cholesterol metabolism and osmoregulation in hypercholesterolaemic rats are perturbed upon berberine injection. In addition, the changes of some elements, such as V, Mn, Na and K, revealed in the metallomic study may contribute to the search of new biomarkers for hypercholesterolaemic disease. We concluded that both the metabolomic and metallomic profiles of berberine-treated hypercholesterolaemic rats were different from those of the control group and that the selected metabolites and elements could probably be applied as potential biomarkers for the understanding of the effect of berberine on biochemical process in the animal model. Such a multi-analytical approach will potentially provide an information-rich platform for the elucidation of effects of xenobiotics and drug efficacy studies.

Metabolic profiling in colorectal cancer reveals signature metabolic shifts during tumorigenesis.

Colorectal cancer (CRC) arises as the consequence of progressive changes from normal epithelial cells through polyp to tumor, and thus is an useful model for studying metabolic shift. In the present study, we studied the metabolomic profiles using high analyte specific gas chromatography/mass spectrometry (GC/MS) and liquid chromatography tandem mass spectrometry (LC/MS/MS) to attain a systems-level view of the shift in metabolism in cells progressing along the path to CRC. Colonic tissues including tumor, polyps and adjacent matched normal mucosa from 26 patients with sporadic CRC from freshly isolated resections were used for this study. The metabolic profiles were obtained using GC/MS and LC/MS/MS. Our data suggest there was a distinct profile change of a wide range of metabolites from mucosa to tumor tissues. Various amino acids and lipids in the polyps and tumors were elevated, suggesting higher energy needs for increased cellular proliferation. In contrast, significant depletion of glucose and inositol in polyps revealed that glycolysis may be critical in early tumorigenesis. In addition, the accumulation of hypoxanthine and xanthine, and the decrease of uric acid concentration, suggest that the purine biosynthesis pathway could have been substituted by the salvage pathway in CRC. Further, there was a step-wise reduction of deoxycholic acid concentration from mucosa to tumors. It appears that to gain a growth advantage, cancer cells may adopt alternate metabolic pathways in tumorigenesis and this flexibility allows them to adapt and thrive in harsh environment.

Pressurized hot water extraction (PHWE).

Pressurized hot water extraction (PHWE) has become a popular green extraction method for different classes of compounds present in numerous kinds of matrices such as environmental, food and botanical samples. PHWE is also used in sample preparation to extract organic contaminants from foodstuff for food safety analysis and soils/sediments for environmental monitoring purposes. The main parameters which influence its extraction efficiency are namely the temperature, extraction time, flow rates and addition of modifiers/additives. Among these different parameters studied, temperature is described as the most important one. It is reported that the extraction of certain compounds is rather dependent on pressurized water with different applied temperature. Thus, the stability and reduced solubilities of certain compounds at elevated temperatures are highlighted in this review. With some modifications, a scaled-up PHWE could extract a higher amount of desirable compounds from solid and powdered samples such as plant and food materials. The PHWE extracts from plants are rich in chemical compounds or metabolites which can be a potential lead for drug discovery or development of disease-resistant food crops.

Determination of pyrrolizidine alkaloids in comfrey by liquid chromatography-electrospray ionization mass spectrometry.

Symphytum officinale L. (comfrey) is a medicinal plant commonly used in decoctions and aliments. Besides therapeutic bioactive compounds present in the herb, it is found to contain hepatotoxic pyrrolizidine alkaloids (PAs), such as lycopsamine and others. In the present study, PAs such as lycopsamine, echimidine and lasiocarpine were determined using electrospray liquid chromatography-mass spectrometry (LC-MS) with the method precision (relative standard deviation, RSD) <10%. Detection of lycopsamine, symviridine and their N-oxides could be confirmed with a newly developed method based on HPLC ion-trap and orbitrap MS with electrospray ionization interface. With LC-MS, quantitative analysis of lycopsamine in the botanical extract was carried out. The effect of extraction solvent was optimized by sonication and methanol: H(2)O (50:50) was selected. Then a rapid method based on pressurized hot water extraction (PHWE) was employed for the extraction of lycopsamine from comfrey followed by the comparison with heating under reflux with the RSD ranging from 2.49% to 19.32%. Our results showed a higher extraction efficiency for heating under reflux compared with PHWE. It was proposed that the lower extraction efficiency for PHWE was attributable to dissolved nitrogen from air which caused the reduction in the solubility of lycopsamine in the compressed hot solvent. In this study, quantitative analysis of PAs in comfrey was demonstrated. In addition, it was found that the use of subcritical water for extractions depended on the physical properties of the dissolved solutes and their tendency to degrade under the chosen extraction conditions.

Combination of 1H nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry with pattern recognition techniques for evaluation of metabolic profile associated with albuminuria.

A method using 1H NMR and LC/MS with pattern recognition tools such as principal component analysis (PCA) and orthogonal projection to latent structure discriminant analysis (O-PLS-DA) was used to study the urinary metabolic profiles associated with an increase in urinary albumin in a general population. The normalized peak intensities obtained from 1H NMR and LC/MS with nonparametric two-tailed Mann-Whitney analysis was used for the identification of network of potential biomarkers corresponding to the increase of albumin in urine. The specificity of detecting the stated metabolites by 1H NMR and LC/MS was demonstrated. Our preliminary data obtained demonstrated that LC/MS may produce more distinctive metabolic profiles. For the patient group, changes in alanine, kyneurnic acid, and xanthurenic acid might be associated with changes in the tryptophan metabolism. At the same time, other metabolites that were involved in citric acid cycle, amino acid metabolism, and cellular functions were affected in the patient group. The proposed approach provided a comprehensive picture of the metabolic changes induced by the increase of protein in urine and demonstrated the advantages of using multiple diagnostic biomarkers. At the same time, the current work was demonstrated as a potential cost-effective solution of high-throughput analysis with pattern recognition tools as applied here in a real clinical situation.