Metabolomic Investigation of Brain and Liver in Rats Fed Docosahexaenoic Acid in Regio- and Enantiopure Triacylglycerols.

SCOPE: N-3 polyunsaturated fatty acids (n-3 PUFAs) play important roles in cognitive functions. However, there is a lack of knowledge on the metabolic impact of regio- and stereo-specific positioning of n-3 PUFAs in dietary triacylglycerols. METHODS AND RESULTS: Rats in a state of mild n-3 PUFA deficiency are fed daily with 360 mg triacylglycerols containing DHA (docosahexaenoic acid) at sn (stereospecific numbering)-1, 2, or 3 positions and 18:0 at remaining positions, or an equal amount of tristearin for 5 days. Groups fed with n-3 deficient diet and normal n-3 adequate diet are included as controls. The metabolic profiles of the brain and liver are studied using NMR (nuclear magnetic resonance)-based metabolomics. Several metabolites of significance in membrane integrity and neurotransmission, and glutamate, in particular, are significantly lower in the brain of the groups fed with sn-1 and sn-3 DHA compared to the sn-2 DHA group. Further, the tristearin and DHA groups show a lower lactate level compared to the groups fed on normal or n-3 deficient diet, suggesting a prominent role of C18:0 in regulating energy metabolism. CONCLUSION: This study sheds light on the impact of stereospecific positioning of DHA in triacylglycerols and the role of dietary stearic acid on metabolism in the brain and liver.

Effects of acylated and nonacylated anthocyanins extracts on gut metabolites and microbiota in diabetic Zucker rats: A metabolomic and metagenomic study.

Anthocyanins have been shown to have prebiotic properties. This study investigated the impact of nonacylated anthocyanins and acylated anthocyanins on fecal and cecal metabolites and colonic gut microbiota in diabetic state using 1H nuclear magnetic resonance (NMR) metabolomics and metagenomic sequencing. Zucker diabetic fatty rats fed with high-fat diet were gavaged with nonacylated anthocyanins extracted from bilberries (NAAB) or acylated anthocyanins extracted from purple potatoes (AAPP) at daily doses of 25 and 50 mg/kg body weight for 8 weeks. Lean Zucker rats fed with normal diet (ND) and high-fat diet (Con) were used as healthy controls groups. Binned NMR spectra and sequenced gene abundance were used for data analysis. Dysbiosis of colonic microbiota and gut metabolites in the diabetic rats were observed compared to the lean Zucker rats. Both anthocyanin extracts increased cecal sugar levels and the abundance of Peptostreptococcaceae sp. and decreased the abundance of Parabacteroides spp. in colon. In addition to the increased fecal short-chain fatty acids, AAPP decreased colonic Ruminococcus torques and Lachnospiraceae bacterium 4_1_37FAA abundances and increased oxidative phosphorylation. The anthocyanin extracts modulated the gut metabolism and microbiota in diabetes, with AAPP showing more regulatory and beneficial effects on diabetes.

1H NMR Metabolomics and Full-Length RNA-Seq Reveal Effects of Acylated and Nonacylated Anthocyanins on Hepatic Metabolites and Gene Expression in Zucker Diabetic Fatty Rats.

Anthocyanins have been reported to possess antidiabetic effects. Recent studies indicate acylated anthocyanins have better stability and antioxidative activity compared to their nonacylated counterparts. This study compared the effects of nonacylated and acylated anthocyanins on hepatic gene expression and metabolic profile in diabetic rats, using full-length transcriptomics and 1H NMR metabolomics. Zucker diabetic fatty (ZDF) rats were fed with nonacylated anthocyanin extract from bilberries (NAAB) or acylated anthocyanin extract from purple potatoes (AAPP) at daily doses of 25 and 50 mg/kg body weight for 8 weeks. Both anthocyanin extracts restored the levels of multiple metabolites (glucose, lactate, alanine, and pyruvate) and expression of genes (G6pac, Pck1, Pklr, and Gck) involved in glycolysis and gluconeogenesis. AAPP decreased the hepatic glutamine level. NAAB regulated the expression of Mgat4a, Gstm6, and Lpl, whereas AAPP modified the expression of Mgat4a, Jun, Fos, and Egr1. This study indicated different effects of AAPP and NAAB on the hepatic transcriptomic and metabolic profiles of diabetic rats.

Prebiotic Xylo-Oligosaccharides Ameliorate High-Fat-Diet-Induced Hepatic Steatosis in Rats.

Understanding the importance of the gut microbiota (GM) in non-alcoholic fatty liver disease (NAFLD) has raised the hope for therapeutic microbes. We have shown that high hepatic fat content associated with low abundance of Faecalibacterium prausnitzii in humans and, further, the administration of F. prausnitzii prevented NAFLD in mice. Here, we aimed at targeting F. prausnitzii by prebiotic xylo-oligosaccharides (XOS) to treat NAFLD. First, the effect of XOS on F. prausnitzii growth was assessed in vitro. Then, XOS was supplemented or not with high (HFD, 60% of energy from fat) or low (LFD) fat diet for 12 weeks in Wistar rats (n = 10/group). XOS increased F. prausnitzii growth, having only a minor impact on the GM composition. When supplemented with HFD, XOS ameliorated hepatic steatosis. The underlying mechanisms involved enhanced hepatic ß-oxidation and mitochondrial respiration. Nuclear magnetic resonance (1H-NMR) analysis of cecal metabolites showed that, compared to the HFD, the LFD group had a healthier cecal short-chain fatty acid profile and on the HFD, XOS reduced cecal isovalerate and tyrosine, metabolites previously linked to NAFLD. Cecal branched-chain fatty acids associated positively and butyrate negatively with hepatic triglycerides. In conclusion, XOS supplementation can ameliorate NAFLD by improving hepatic oxidative metabolism and affecting GM.

Effects of Anthocyanin Extracts from Bilberry (Vaccinium myrtillus L.) and Purple Potato (Solanum tuberosum L. var. 'Synkeä Sakari') on the Plasma Metabolomic Profile of Zucker Diabetic Fatty Rats.

This study compared the effects of the nonacylated and acylated anthocyanin-rich extracts on plasma metabolic profiles of Zucker diabetic fatty rats. The rats were fed with the nonacylated anthocyanin extract from bilberries (NAAB) or the acylated anthocyanin extract from purple potatoes (AAPP) at daily doses of 25 and 50 mg/kg body weight for 8 weeks. 1H NMR metabolomics was used to study the changes in plasma metabolites. A reduced fasting plasma glucose level was seen in all anthocyanin-fed groups, especially in the groups fed with NAAB. Both NAAB and AAPP decreased the levels of branched-chain amino acids and improved lipid profiles. AAPP increased the glutamine/glutamate ratio and decreased the levels of glycerol and metabolites involved in glycolysis, suggesting improved insulin sensitivity, gluconeogenesis, and glycolysis. AAPP decreased the hepatic TBC1D1 and G6PC messenger RNA level, suggesting regulation of gluconeogenesis and lipogenesis. This study indicated that AAPP and NAAB affected the plasma metabolic profile of diabetic rats differently.

Untargeted metabolic fingerprinting reveals impact of growth stage and location on composition of sea buckthorn (Hippophaë rhamnoides) leaves.

Sea buckthorn (Hippophaë rhamnoides) is increasingly cultivated to produce raw materials for food and nutraceuticals. There is little knowledge on composition of sea buckthorn leaves (SBLs) and the key factors influencing the composition. This research aims to unravel the metabolic profile of SBLs and the effects of cultivar, location and stage of growth, and climatic conditions on the metabolic profile of SBLs. Leaves of two sea buckthorn cultivars grown in the south and north of Finland during two consecutive growth seasons were studied using untargeted nuclear magnetic resonance (NMR) metabolomics. The highest variance in the metabolic profile was linked to the growth stage, wherein leaves from the first 7 weeks of harvest were characterized with higher abundance of polyphenols, while relatively higher abundance of carbohydrates and sugars was observed in the later weeks. The growth location attributed for the second highest variation, wherein the north-south comparison identified fatty acids and sugars as discriminatory metabolites, and the potential association of metabolome to natural abiotic stressors was revealed. An inverse correlation between carbohydrate/sugar content as well as fatty acids of higher carbon chain length with the temperature variables was evident. The supervised chemometric models with high sensitivity and specificity classified and predicted the samples based on growth stage and location, and cultivar. Nontargeted NMR-metabolomics revealed the metabolic profile of SBLs and their variation associated with various biotic and abiotic factors. Cultivar and growth stage are key factors to consider when harvesting SBLs for use in food and nutraceuticals. PRACTICAL APPLICATION: Globally, sea buckthorn cultivation has been rapidly increasing due to the known health-promoting benefits of the berries and leaves of the plant. The current research obtained new comprehensive information on the compositional profile of sea buckthorn leaves as well as the impact of major contributory factors, such as cultivars, the advancement of growth stage, geographical location, and weather parameters. The findings of this research provide new knowledge and guidance for plant breeding, cultivation and commercial utilization of sea buckthorn leaves as raw materials for food, feed, and nutraceuticals.

Urinary metabolomics study on the protective role of Orthosiphon stamineus in Streptozotocin induced diabetes mellitus in rats via 1H NMR spectroscopy.

BACKGROUND: Orthosiphon stamineus (OS) is a herb known in ethnomedicine for treating diabetes mellitus (DM). In this study, a 1H NMR based urine metabolomics tool has been used for the first time to identify the metabolic protective mechanism of OS in DM using Streptozotocin (STZ) induced experimental model in rats. METHODS: Four different solvent extracts of OS, namely aqueous, ethanolic, 50% aqueous ethanolic and methanolic, at a dose of 500 mg/kg body weight (bw) were orally administered for 14 days to diabetic rats induced via intraperitoneal injection of 60 mg/kg bw STZ. NMR metabolomics approach using pattern recognition combined with multivariate statistical analysis was applied in the rat urine to study the resulted metabolic perturbations. RESULTS: OS aqueous extract (OSAE) caused a reversal of DM comparable to that of 10 mg/kg bw glibenclamide. A total of 15 urinary metabolites, which levels changed significantly upon treatment were identified as the biomarkers of OSAE in diabetes. A systematic metabolic pathways analysis identified that OSAE contributed to the antidiabetic activity mainly through regulating the tricarboxylic acid cycle, glycolysis/gluconeogenesis, lipid and amino acid metabolism. CONCLUSIONS: The results of this study validated the ethnopharmacological use of OS in diabetes and unveiled the biochemical and metabolic mechanisms involved.

Identification of the compositional changes in Orthosiphon stamineus leaves triggered by different drying techniques using 1 H NMR metabolomics.

BACKGROUND: Java tea is a well-known herbal infusion prepared from the leaves of Orthosiphon stamineus (OS). The biological properties of tea are in direct correlation with the primary and secondary metabolite composition, which in turn largely depends on the choice of drying method. Herein, the impact of three commonly used drying methods, i.e. shade, microwave and freeze drying, on the metabolite composition and antioxidant activity of OS leaves was investigated using proton nuclear magnetic resonance (1 H NMR) spectroscopy combined with multivariate classification and regression analysis tools. RESULTS: A total of 31 constituents comprising primary and secondary metabolites belonging to the chemical classes of fatty acids, amino acids, sugars, terpenoids and phenolic compounds were identified. Shade-dried leaves were identified to possess the highest concentrations of bioactive secondary metabolites such as chlorogenic acid, caffeic acid, luteolin, orthosiphol and apigenin, followed by microwave-dried samples. Freeze-dried leaves had higher concentrations of choline, amino acids leucine, alanine and glutamine and sugars such as fructose and α-glucose, but contained the lowest levels of secondary metabolites. CONCLUSION: Metabolite profiling coupled with multivariate analysis identified shade drying as the best method to prepare OS leaves as Java tea or to include in traditional medicine preparation. © 2017 Society of Chemical Industry.

Urinary metabolic profiling of cisplatin nephrotoxicity and nephroprotective effects of Orthosiphon stamineus leaves elucidated by 1H NMR spectroscopy.

Orthosiphon stamineus (OS) is a popular medicinal herb used in traditional Chinese medicine as a diuretic agent and for renal system disorders. This study employed 1H NMR based metabolomics approach to investigate the possible protective activity of OS in cisplatin induced nephrotoxicity owing to its diuretic and antioxidant activities. Aqueous (OSAE) and 50% aqueous ethanolic (OSFE) extracts of OS leaves were orally administered at 400mg/kg BW doses to rats which were then intraperitoneally injected with cisplatin at 5mg/kg BW dose. The 1H NMR profile of the urine samples collected on day 5 after cisplatin administration were analyzed by multivariate pattern recognition techniques, whereby 19 marker metabolites suggestive in the involvement of TCA cycle, disturbed energy metabolism, altered gut microflora and BCAA metabolism pathways were identified. It was observed that OSFE caused significant changes (p<0.05) in the levels of 8 markers namely leucine, acetate, hippurate, lysine, valine, 2-oxoglutarate, 3-HBT and acetoacetate resulting in a moderate ameliorative effect, however, it did not completely protect from nephrotoxicity. OSAE did not demonstrate significant down regulatory effects on any markers, albeit, it potentiated the cisplatin nephrotoxicity by inducing significant increase in glucose, glycine, creatinine, citrate, TMAO, acetate and creatine levels. A Principal Component Analysis (PCA) of the 1H NMR spectra of OS extracts identified that OSFE had higher concentrations of the secondary metabolites such as caffeic acid, chlorogenic acid, protocatechuic acid and orthosiphol, among others. Whereas, OSAE was characterized by higher concentrations of acetate, lactate, succinic acid, valine and phosphatidylcholine. This research denotes the first comprehensive analysis to identify the effects of OS extracts on cisplatin nephrotoxicity.

Phytochemical Screening and Acute Oral Toxicity Study of Java Tea Leaf Extracts.

The term Java tea refers to the decoction of Orthosiphon stamineus (OS) Benth (Lamiaceae) leaves, which are widely consumed by the people in Europe and South East Asian countries. The OS leaves are known for their use in traditional medicinal systems as a prophylactic and curative agent for urinary stone, diabetes, and hypertension and also as a diuretic agent. The present study was aimed at evaluating its possible toxicity. Herein, the major phytochemical constituents of microwave dried OS leaf, which is the common drying process for tea sachets in the market, were also identified. The acute oral toxicity test of aqueous, 50% aqueous ethanolic, and ethanolic extracts of OS was performed at a dose of 5000 mg/Kg body weight of Sprague-Dawley rats. During the 14-day study, the animals were observed for any mortality, behavioral, motor-neuronal abnormalities, body weight, and feed-water consumption pattern. The hematological and serum biochemical parameters to assess the kidney and liver functions were carried out, along with the histological analysis of these organs. It was found that all microwave dried OS leaf extracts did not cause any toxic effects or mortality at the administered dose. No abnormality was noticed in all selected parameters in rats of both sexes as compared with their respective control groups. Thus, the possible oral lethal dose for microwave dried Java tea leaves is more than 5000 mg/Kg body weight.