Nuclear magnetic resonance metabolomics approach in chemical and protective evaluations of Orthosiphon stamineus Benth. leaf extracts on cisplatin-induced nephrotoxicity

Orthosiphon stamineus (OS), locally known in Malaysia as ‘Misai Kucing’, is a herbaceous shrub belonging to the family Lamiaceae. Dried leaves of OS is gaining wide acceptance and marketed in the form of herbal tea, known as Java tea, owing to its traditional and scientific claims on various health benefits. OS has been a wellknown renoprotective agent primarily due to its diuretic potential. This research investigated the effects of commonly employed drying methods of OS leaves on their chemical constituent profile, and in vivo biological properties of the protective role in cisplatin induced nephrotoxicity using rats, through Nuclear Magnetic Resonance (NMR) metabolomics approach. The NMR spectra of rat urine and the OS leaf extracts were analysed and correlated using multivariate data analysis techniques employing metabolomics platform. The 1H NMR metabolite profiling of aqueous extract of OS leaves resulted in the identification of 31 metabolites. The presence of biologically active secondary metabolites including phenylpropanoids such as caffeic acid, protocatechuic acid, chlorogenic acid, flavonoids such as luteolin and apigenin, gallic acid and orthosiphol derivatives were confirmed by J resolved NMR technique. The HPLC - MS/MS analysis further confirmed the presence of these secondary metabolites. Metabolite fingerprinting in combination with multivariate analysis has successfully differentiated the three differently dried (Freeze, microwave and shade) OS leaves and established that the levels of 15 metabolites were varied significantly between the samples. The shade drying method retained maximum secondary metabolites followed by the microwave, while freeze drying retained the least. Assessment of the main beneficial properties, such as antioxidant, total phenolic and flavonoid contents of any tea preparation, confirmed that all the differently dried Java tea leaves gave good antioxidant activity, with the shade dried leaves recorded the highest level with an IC50 of 48.09 μg/mL. The chemical constituents correlated to the high antioxidant activity of the shade dried leaves were extracted from a Partial Least Square regression (PLS) model. In addition, the toxicity profile of the microwave dried OS leaves was investigated through acute oral toxicity test in Sprague Dawley (SD) rats of both sexes, whereby, the no-observed-adverse-effect level (NOAEL) of aqueous, 50% ethanolic and ethanolic extracts of the microwave dried OS was determined as 5000 mg/kg body weight/day. Thus, it is presumed that the microwave dried leaves are safe to be used as an oral health supplement. Cisplatin is an anticancer drug, which induces nephrotoxicity in a long term use. Metabolomic analysis of the rats’ urine revealed the involvement of a total of 17 biochemical markers from TCA cycle, carbohydrate, amino acid, and polyamine metabolic pathways in cisplatin nephrotoxicity. To the best of knowledge, 6 of the 17 involved metabolites are newly established in this study. In order to evaluate the protective efficacy of OS in cisplatin nephrotoxicity, shade and microwave dried OS extracts were administered at doses of 100, 200 and 400 mg/kg body weight to rats. The results suggested the dose independency of the extracts. Treatment with 50% aqueous ethanolic extract of shade dried OS leaves (OSFS) exhibited moderate ameliorative effect observed through a statistically significant reduction in the levels of 8 biomarkers. It was also revealed that the aqueous extract of the shade dried leaves (OSAS) exhibited slightly deteriorative activity via disturbance in the energy metabolism and gut microflora. The higher concentration of the secondary metabolites such as caffeic acid, chlorogenic acid, protocatechuic acid and orthosiphol in OSFS could be correlated to the ameliorative activity as revealed from a Principal Component Analysis (PCA) between OSAS and OSFS. A prediction model on nephroprotective effect of OS was constructed through PLS regression analysis. Thus, the impact of different drying techniques on chemical constituents of OS leaves was established. The metabolomics approach has proved to be successful in shedding light to the even minute variations in the biological profiles of the low intensity metabolites involved in the renal toxicity caused by cisplatin. A global comprehensive view of the OS effect in cisplatin toxicity was successfully profiled and correlated

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 ApplicationGlobally, 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.</p

NMR-based metabolomics approach on optimization of malolactic fermentation of sea buckthorn juice with Lactiplantibacillus plantarum

This work investigated the impact of malolactic fermentation on the metabolomic profile of sea buckthorn juice to optimize the fermentation process for flavor modification. Six strains of L. plantarum were used with varied pH of the juice, cell acclimation, and fermentation time. 1H-NOESY spectra were acquired from fresh and fermented juices with a total of 46 metabolites identified. Less sugars and quinic acid were metabolized at pH 2.7 while oxidation of ascorbic acid was reduced at pH 3.5. l-Malic acid, essential amino acids, and nucleosides were consumed early during fermentation while sugars in general were consumed later in the fermentation. If deacidification is the main target of fermentation, strains that produce less acids and ferment less sugars, shorter fermentation time, and lower starter pH should be used. Higher starter pH and longer fermentation time promote formation of antimicrobial compounds and potentially increase antioxidant stability. </p

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

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

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 400 mg/kg BW doses to rats which were then intraperitoneally injected with cisplatin at 5 mg/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

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 β-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

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 β-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. </p

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. prausnitziiprevented 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 β-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. </p