Complementary Analytical Platforms of NMR Spectroscopy and LCMS Analysis in the Metabolite Profiling of Isochrysis galbana.

This study was designed to profile the metabolites of Isochrysis galbana, an indigenous and less explored microalgae species. 1H Nuclear Magnetic Resonance (NMR) spectroscopy and Liquid Chromatography-Mass Spectrometry (LCMS) were used to establish the metabolite profiles of five different extracts of this microalga, which are hexane (Hex), ethyl acetate (EtOAc), absolute ethanol (EtOH), EtOH:water 1:1 (AqE), and 100% water (Aq). Partial least square discriminant analysis (PLS-DA) of the generated profiles revealed that EtOAc and Aq extracts contain a diverse range of metabolites as compared to the other extracts with a total of twenty-one metabolites, comprising carotenoids, polyunsaturated fatty acids, and amino acids, that were putatively identified from the NMR spectra. Meanwhile, thirty-two metabolites were successfully annotated from the LCMS/MS data, ten of which (palmitic acid, oleic acid, α-linolenic acid, arachidic acid, cholesterol, DHA, DPA, fucoxanthin, astaxanthin, and pheophytin) were similar to those present in the NMR profile. Another eleven glycerophospholipids were discovered using MS/MS-based molecular network (MN) platform. The results of this study, besides providing a better understanding of I.galbana's chemical make-up, will be of importance in exploring this species potential as a feed ingredient in the aquaculture industry.

Metabolite variations and antioxidant activity of Muntingia calabura leaves in response to different drying methods and ethanol ratios elucidated by NMR-based metabolomics.

INTRODUCTION: Muntingia calabura from the Muntingiaceae family has been documented for several medicinal uses. The combinations of drying treatment and extracting solvents for a plant species need to be determined and optimised to ensure that the extracts contain adequate amounts of the bioactive metabolites. OBJECTIVE: Evaluate the metabolite variations and antioxidant activity among M. calabura leaves subjected to different drying methods and extracted with different ethanol ratios using proton nuclear magnetic resonance (1 H-NMR)-based metabolomics. Methodology The antioxidant activity of M. calabura leaves dried with three different drying methods and extracted with three different ethanol ratios was determined by using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) scavenging assays. The metabolites variation among the extracts and correlation with antioxidant activity were analysed by 1 H-NMR-based metabolomics. RESULTS: Muntingia calabura leaves extracted with 50% and 100% ethanol from air-drying and freeze-drying methods had the highest total phenolic content and the lowest IC50 value for the DPPH scavenging activity. Meanwhile, oven-dried leaves extracted with 100% ethanol had the lowest IC50 value for the NO scavenging activity. A total of 43 metabolites, including sugars, organic acids, amino acids, phytosterols, phenolics and terpene glycoside were tentatively identified. A noticeable discrimination was observed in the different ethanol ratios by the principal component analysis. The partial least-squares analysis suggested that 32 compounds out of 43 compounds identified were the contributors to the bioactivities. CONCLUSION: The results established set the preliminary steps towards developing this plant into a high value product for phytomedicinal preparations.

Clitorienolactones and Isoflavonoids of Clitorea ternatea Roots Alleviate Stress-Like Symptoms in a Reserpine-Induced Zebrafish Model.

Clitorea ternatea has been used in Ayurvedic medicine as a brain stimulant to treat mental illnesses and mental functional disorders. In this study, the metabolite profiles of crude C. ternatea root extract (CTRE), ethyl acetate (EA), and 50% aqueous methanol (50% MeOH) fractions were investigated using ultrahigh-performance liquid chromatography-diode array detector-tandem mass spectrometry (UHPLC-DAD-MS/MS), while their effect on the stress-like behavior of zebrafish, pharmacologically induced with reserpine, was investigated. A total of 32 compounds were putatively identified, among which, a series of norneolignans, clitorienolactones, and various flavonoids (flavone, flavonol, isoflavone, and isoflavanone) was found to comprise the major constituents, particularly in the EA and 50% MeOH fractions. The clitorienolactones, presently unique to the species, were present in both the free and glycosylated forms in the roots. Both the EA and 50% MeOH fractions displayed moderate effects on the stress-induced zebrafish model, significantly decreasing freezing duration and elevating the total distance travelled and average velocity, 72 h post-treatment. The results of the present study provide further evidence that the basis for the use of C. ternatea roots in traditional medicine to alleviate brain-related conditions, such as stress and depression, is attributable to the presence of clitorienolactones and the isoflavonoidal constituents.

1 H-NMR metabolomics for evaluating the protective effect of Clinacanthus nutans (Burm. f) Lindau water extract against nitric oxide production in LPS-IFN-γ activated RAW 264.7 macrophages.

INTRODUCTION: Clinacanthus nutans, a small shrub that is native to Southeast Asia, is commonly used in traditional herbal medicine and as a food source. Its anti-inflammation properties is influenced by the metabolites composition, which can be determined by different binary extraction solvent ratio and extraction methods used during plant post-harvesting stage. OBJECTIVE: Evaluate the relationship between the chemical composition of C. nutans and its anti-inflammatory properties using nuclear magnetic resonance (NMR) metabolomics approach. METHODOLOGY: The anti-inflammatory effect of C. nutans air-dried leaves extracted using five different binary extraction solvent ratio and two extraction methods was determined based on their nitric oxide (NO) inhibition effect in lipopolysaccharide-interferon-gamma (LPS-IFN-γ) activated RAW 264.7 macrophages. The relationship between extract bioactivity and metabolite profiles and quantifications were established using 1 H-NMR metabolomics and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The possible metabolite biosynthesis pathway was constructed to further strengthen the findings. RESULTS: Water and sonication prepared air-dried leaves possessed the highest NO inhibition activity (IC50  = 190.43 ± 12.26 µg/mL, P < 0.05). A total of 56 metabolites were tentatively identified using 1 H-NMR metabolomics. A partial least square (PLS) biplot suggested that sulphur containing glucoside, sulphur containing compounds, phytosterols, triterpenoids, flavones and some organic and amino acids were among the potential NO inhibitors. LC-MS/MS targeted quantification further supported sonicated water extract was among the extract that possessed the most abundant C-glycosyl flavones. CONCLUSION: The present study may serve as a preliminary reference for the selection of optimum extract in further C. nutans in vivo anti-inflammatory study.

Comparison of partial least squares and random forests for evaluating relationship between phenolics and bioactivities of Neptunia oleracea.

BACKGROUND: Neptunia oleracea is a plant consumed as a vegetable and which has been used as a folk remedy for several diseases. Herein, two regression models (partial least squares, PLS; and random forest, RF) in a metabolomics approach were compared and applied to the evaluation of the relationship between phenolics and bioactivities of N. oleracea. In addition, the effects of different extraction conditions on the phenolic constituents were assessed by pattern recognition analysis. RESULTS: Comparison of the PLS and RF showed that RF exhibited poorer generalization and hence poorer predictive performance. Both the regression coefficient of PLS and the variable importance of RF revealed that quercetin and kaempferol derivatives, caffeic acid and vitexin-2-O-rhamnoside were significant towards the tested bioactivities. Furthermore, principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) results showed that sonication and absolute ethanol are the preferable extraction method and ethanol ratio, respectively, to produce N. oleracea extracts with high phenolic levels and therefore high DPPH scavenging and α-glucosidase inhibitory activities. CONCLUSION: Both PLS and RF are useful regression models in metabolomics studies. This work provides insight into the performance of different multivariate data analysis tools and the effects of different extraction conditions on the extraction of desired phenolics from plants. © 2017 Society of Chemical Industry.

Multivariate modelling analysis for prediction of glycidyl esters and 3-monochloropropane-1,2-diol (3-MCPD) formation in periodically heated palm oil.

Palm oil is a vegetable oil that is widely used for cooking and deep-frying because of its affordability. However, repeatedly heated palm oil is also prone to oxidation due to its significant content of unsaturated fatty acids and other chemical toxicants such as glycidyl esters and 3-monochloropropane-1,2-diol (3-MCPD). Initially, the physicochemical properties such as colour, viscosity, peroxide, p-anisidine and total oxidation (TOTOX) of periodically heated palm oil were investigated. Chemical profiling and fingerprinting of six different brands of palm cooking oil during heating cycles between 90 and 360 min were conducted using Fourier transform infrared (FTIR) and 1H Nuclear Magnetic Resonance (NMR) metabolomics. In addition, the multivariate analysis was employed to evaluate the 1H NMR spectroscopic pattern of repeatedly heated palm oil with the corresponding physicochemical properties. The FTIR metabolomics showed significant different of the chemical fingerprinting subjected to heating duration, which in agreement with the result of 1H NMR metabolomics. Partial least squares (PLS) model revealed that most of the physicochemical properties of periodically heated palm oil are positively correlated (R2 values of 0.98-0.99) to their spectroscopic pattern. Based on the findings, the color of the oils darkened with increased heating time. The peroxide value (PV), p-anisidine value (p-AnV), and total oxidation (TOTOX) values increased significantly due to degradation of unsaturated compounds and oxidation products formed. We identified targeted metabolites (probable carcinogens) such as 3-monochloropropane-1,2-diol (3-MCPD) and glycidyl ester (GE), indicating the conversion of 3-MCPD to GE in repeatedly heated oils based on PCA and OPLSDA models. Our correlation analysis of NMR and physicochemical properties has shown that the conversion of 3-MCPD to GE was significantly increased from 180 to 360 min cooking time. The combination spectroscopic techniques with physicochemical properties are a reliable and robust methods to evaluate the characteristics, stability and chemical's structure changes of periodically heated palm oil, which may contribute to probable carcinogens development. This study has proven that combination of NMR and physicochemical analysis may predict the formation of the probable carcinogens of heated cooking oil over time which emphasizing the need to avoid certain heating cycles to mitigate formation of probable carcinogens during cooking process.

A Preliminary Nuclear Magnetic Resonance Metabolomics Study Identifies Metabolites that Could Serve as Diagnostic Markers of Major Depressive Disorder.

BACKGROUND: The evaluation of metabolites that are directly involved in the physiological process, few steps short of phenotypical manifestation, remains vital for unravelling the biological moieties involved in the development of the (MDD) and in predicting its treatment outcome. METHODOLOGY: Eight (8) urine and serum samples each obtained from consenting healthy controls (HC), twenty-five (25) urine and serum samples each from first episode treatment naïve MDD (TNMDD) patients, and twenty (22) urine and serum samples each s from treatment naïve MDD patients 2 weeks after SSRI treatment (TWMDD) were analysed for metabolites using proton nuclear magnetic resonance (1HNMR) spectroscopy. The evaluation of patients' samples was carried out using Partial Least Squares Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Square- Discriminant Analysis (OPLSDA) models. RESULTS: In the serum, decreased levels of lactate, glucose, glutamine, creatinine, acetate, valine, alanine, and fatty acid and an increased level of acetone and choline in TNMDD or TWMDD irrespective of whether an OPLSDA or PLSDA evaluation was used were identified. A test for statistical validations of these models was successful. CONCLUSION: Only some changes in serum metabolite levels between HC and TNMDD identified in this study have potential values in the diagnosis of MDD. These changes included decreased levels of lactate, glutamine, creatinine, valine, alanine, and fatty acid, as well as an increased level of acetone and choline in TNMDD. The diagnostic value of these changes in metabolites was maintained in samples from TWMDD patients, thus reaffirming the diagnostic nature of these metabolites for MDD.

Perturbations in Amino Acid Metabolism in Reserpine-Treated Zebrafish Brain Detected by 1H Nuclear Magnetic Resonance-Based Metabolomics.

Depression is a complex and disabling psychiatric disorder, which is expected to be a leading cause for disability by 2030. According to World Health Organization, about 350 million people are suffering with mental health disorders around the globe, especially depression. However, the mechanisms involved in stress-induced depression have not been fully elucidated. In this study, a stress-like state was pharmacologically induced in zebrafish using reserpine, a drug widely used to mediate depression in experimental animal models. Zebrafish received single intraperitoneal (i.p.) injections of 20, 40, and 80 mg/kg body weight reserpine doses and were subjected to open-field test at 2, 24, 48, 72, and 96 h after the treatment. Along with observed changes in behavior and measurement of cortisol levels, the fish were further examined for perturbations in their brain metabolites by 1H nuclear magnetic resonance (NMR)-based metabolomics. We found a significant increase in freezing duration, whereas total distance travelled was decreased 24 h after single intraperitoneal injection of reserpine. Cortisol level was also found to be higher after 48 h of reserpine treatment. The 1H NMR data showed that the levels of metabolites such as glutamate, glutamine, histamine, valine, leucine and histidine, lactate, l-fucose, betaine and γ-amino butyric acid (GABA), ß-hydroxyisovalerate, and glutathione were significantly decreased in the reserpine-treated group. This study provided some insights into the molecular nature of stress that could contribute toward a better understanding of depression disorder.

Metabolite profiling of Andrographis paniculata (Burm. f.) Nees. young and mature leaves at different harvest ages using 1H NMR-based metabolomics approach.

Andrographis paniculata (Burm. F.) Nees. is considered as the herb of the future due to its precious chemical compounds, andrographolide (ANDRO), neoandrographolide (NAG) and 14-deoxyandrographolide (DAG). This study aims to profile the metabolites in young and mature leaf at six different harvest ages using 1HNMR-based metabolomics combined with multivariate data analysis. Principal component analysis (PCA) indicated noticeable and clear discrimination between young and mature leaves. A comparison of the leaves stage indicated that young leaves were separated from mature leaves due to its larger quantity of ANDRO, NAG, DAG, glucose and sucrose. These similar metabolites are also responsible for the PCA separation into five clusters representing the harvest age at 14, 16, 18, 20, 22 weeks of leaves extract. Loading plots revealed that most of the ANDRO and NAG signals were present when the plant reached at the pre-flowering stage or 18 weeks after sowing (WAS). As a conclusion, A. paniculata young leaves at pre-flowering harvest age were found to be richer in ANDRO, NAG and DAG compared to mature leaves while glucose and choline increased with harvest age. Therefore, young leaves of A. paniculata should be harvested at 18 WAS in order to produce superior quality plant extracts for further applications by the herbal, nutraceutical and pharmaceutical industries.

Metabolite profiling, antioxidant, and α-glucosidase inhibitory activities of germinated rice: nuclear-magnetic-resonance-based metabolomics study.

In an attempt to profile the metabolites of three different varieties of germinated rice, specifically black (GBR), red, and white rice, a 1H-nuclear-magnetic-resonance-based metabolomics approach was conducted. Multivariate data analysis was applied to discriminate between the three different varieties using a partial least squares discriminant analysis (PLS-DA) model. The PLS model was used to evaluate the relationship between chemicals and biological activities of germinated rice. The PLS-DA score plot exhibited a noticeable separation between the three rice varieties into three clusters by PC1 and PC2. The PLS model indicated that α-linolenic acid, γ-oryzanol, α-tocopherol, γ-aminobutyric acid, 3-hydroxybutyric acid, fumaric acid, fatty acids, threonine, tryptophan, and vanillic acid were significantly correlated with the higher bioactivities demonstrated by GBR that was extracted in 100% ethanol. Subsequently, the proposed biosynthetic pathway analysis revealed that the increased quantities of secondary metabolites found in GBR may contribute to its nutritional value and health benefits.