Revealing metabolic and biochemical variations via 1H NMR metabolomics in streptozotocin-nicotinamide-induced diabetic rats treated with metformin.

The increasing prevalence of lean diabetes has prompted the generation of animal models that mimic metabolic disease in humans. This study aimed to determine the optimum streptozotocin-nicotinamide (STZ-NA) dosage ratio to elicit lean diabetic features in a rat model. It also used a proton nuclear magnetic resonance (1H NMR) urinary metabolomics approach to identify the metabolic effect of metformin treatment on this novel rat model. Three different STZ-NA dosage regimens (by body weight: Group A: 110 mg/kg NA and 45 mg/kg STZ; Group B: 180 mg/kg NA and 65 mg/kg STZ and Group C: 120 mg/kg NA and 60 mg/kg STZ) were administered to Sprague-Dawley rats along with oral metformin. Group A diabetic rats (A-DC) showed favorable serum biochemical analyses and a more positive response toward oral metformin administration relative to the other STZ-NA dosage ratio groups. Orthogonal partial least squares-discriminant analysis (OPLS-DA) revealed that glucose, citrate, pyruvate, hippurate, and methylnicotinamide differentiating the OPLS-DA of A-MTF rats (Group A diabetic rats treated with metformin) and A-DC model rats. Subsequent metabolic pathway analyses revealed that metformin treatment was associated with improvement in dysfunctions caused by STZ-NA induction, including carbohydrate metabolism, cofactor metabolism, and vitamin and amino acid metabolism. In conclusion, our results identify the best STZ-NA dosage ratio for a rat model to exhibit lean type 2 diabetic features with optimum sensitivity to metformin treatment. The data presented here could be informative to improve our understanding of non-obese diabetes in humans through the identification of possible activated metabolic pathways in the STZ-NA-induced diabetic rats model.

Phytoconstituents of Endiandra kingiana; antidiabetic effects and molecular docking studies on alpha-amylase and alpha-glucosidase.

Phytochemical investigation on the bark of E. kingiana plant afforded ten compounds, including six polyketides namely kingianin A 1, kingianin B 2, kingianin E 3, kingianin F 4, kingianin K 5 and kingianin L 6, three endiandric acids; kingianic acid A 7, tsangibeilin B 8 and endiandric acid M 9, and one sesquiterpene; daibuoxide 10. All compounds were separated as racemic mixture by recycling high-performance liquid chromatography (RHPLC), except for daibuoxide. Their structures were elucidated by detailed spectroscopic and comparative literature data analysis. This is the first report on the presence of the sesquiterpene; daibuoxide in Endiandra genus. In vitro enzymatic bio-evaluation of the isolated compounds against α-amylase and α-glucosidase showed that 4 demonstrated the best α-amylase and α-glucosidase inhibitory activity with IC50 values of 181.54 ± 6.27 µg/mL and 237.87 ± 0.07 µg/mL, respectively. In addition, molecular docking analysis confirmed the α-amylase and α-glucosidase inhibitory activities demonstrated by 4.

Metabolic Alterations in Streptozotocin-nicotinamide-induced Diabetic Rats Treated with Muntingia calabura Extract via 1H-NMR-based Metabolomics.

Diabetes mellitus (DM) is a metabolic endocrine disorder caused by decreased insulin concentration or poor insulin response. Muntingia calabura (MC) has been used traditionally to reduce blood glucose levels. This study aims to support the traditional claim of MC as a functional food and blood-glucose-lowering regimen. The antidiabetic potential of MC is tested on a streptozotocin-nicotinamide (STZ-NA)-induced diabetic rat model by using the 1H-NMR-based metabolomic approach. Serum biochemical analyses reveal that treatment with 250 mg/kg body weight (bw) standardized freeze-dried (FD) 50% ethanolic MC extract (MCE 250) shows favorable serum creatinine (37.77 ± 3.53 µM), urea (5.98 ± 0.84 mM) and glucose (7.36 ± 0.57 mM) lowering capacity, which was comparable to the standard drug, metformin. The clear separation between diabetic control (DC) and normal group in principal component analysis indicates the successful induction of diabetes in the STZ-NA-induced type 2 diabetic rat model. A total of nine biomarkers, including allantoin, glucose, methylnicotinamide, lactate, hippurate, creatine, dimethylamine, citrate and pyruvate are identified in rats' urinary profile, discriminating DC and normal groups through orthogonal partial least squares-discriminant analysis. Induction of diabetes by STZ-NA is due to alteration in the tricarboxylic acid (TCA) cycle, gluconeogenesis pathway, pyruvate metabolism and nicotinate and nicotinamide metabolism. Oral treatment with MCE 250 in STZ-NA-induced diabetic rats shows improvement in the altered carbohydrate metabolism, cofactor and vitamin metabolic pathway, as well as purine and homocysteine metabolism.

Cytotoxicity and 1H NMR metabolomics analyses of microalgal extracts for synergistic application with Tamoxifen on breast cancer cells with reduced toxicity against Vero cells.

This study evaluated the cytotoxic activity of Tamoxifen (TMX), an anti-estrogen drug, with microalgal crude extracts (MCEs) in single and synergistic application (TMX-MCEs) on MCF-7 and 4T1 breast cancer cells, and non-cancerous Vero cells. The MCEs of Nannochloropsis oculata, Tetraselmis suecica and Chlorella sp. from five different solvents (methanol, MET; ethanol, ETH; water, W; chloroform, CHL; and hexane, HEX) were developed. The TMX-MCEs-ETH and W at the 1:2 and 1:3 ratios, attained IC50 of 15.84-29.51 µg/mL against MCF-7; 13.8-31.62 µg/mL against 4T1; and 24.54-85.11 µg/mL against Vero cells. Higher late apoptosis was exhibited against MCF-7 by the TMX-N. oculata-ETH (41.15 %); and by the TMX-T. suecica-ETH (65.69 %) against 4T1 cells. The TMX-T. suecica-ETH also showed higher ADP/ATP ratios, but comparable Caspase activities to control. For Vero cells, overall apoptotic effects were lowered with synergistic application, and only early apoptosis was higher with TMX-T. suecica-ETH but at lower levels (29.84 %). The MCEs-W showed the presence of alanine, oleic acid, linoleic acid, lactic acid, and fumaric acid. Based on Principal Component Analysis (PCA), the spectral signals for polar solvents such as MET and ETH, were found in the same cluster, while the non-polar solvent CHL was with HEX, suggesting similar chemical profiles clustered for the same polarity. The CHL and HEX were more effective with N. oculata and T. suecica which were of the marine origin, while the ETH and MET were more effective with Chlorella sp., which was of the freshwater origin. The synergistic application of microalgal bioactive compounds with TMX can maintain the cytotoxicity against breast cancer cells whilst reducing the toxicity against non-cancerous Vero cells. These findings will benefit the biopharmaceutical, and functional and healthy food industries.

Overcoming Methicillin-Resistance Staphylococcus aureus (MRSA) Using Antimicrobial Peptides-Silver Nanoparticles.

Antibiotics are regarded as a miracle in the medical field as it prevents disease caused by pathogenic bacteria. Since the discovery of penicillin, antibiotics have become the foundation for modern medical discoveries. However, bacteria soon became resistant to antibiotics, which puts a burden on the healthcare system. Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prominent antibiotic-resistant bacteria in the world since 1961. MRSA primarily developed resistance to beta-lactamases antibiotics and can be easily spread in the healthcare system. Thus, alternatives to combat MRSA are urgently required. Antimicrobial peptides (AMPs), an innate host immune agent and silver nanoparticles (AgNPs), are gaining interest as alternative treatments against MRSA. Both agents have broad-spectrum properties which are suitable candidates for controlling MRSA. Although both agents can exhibit antimicrobial effects independently, the combination of both can be synergistic and complementary to each other to exhibit stronger antimicrobial activity. The combination of AMPs and AgNPs also reduces their own weaknesses as their own, which can be developed as a potential agent to combat antibiotic resistance especially towards MRSA. Thus, this review aims to discuss the potential of antimicrobial peptides and silver nanoparticles towards controlling MRSA pathogen growth.

Acetylcholinesterase and α-glucosidase inhibitory compounds from Callicarpa maingayi.

Phytochemical investigation on the soluble fractions of n-hexane and dichloromethane of methanolic leaves extract of the Callicarpa maingayi K. & G. led to the isolation of three triterpenoids [euscaphic acid (1), arjunic acid (2), and ursolic acid (3)] together with two flavones [apigenin (4) and acacetin (5)], two phytosterols [stigmasterol 3-O-ß-glycopyranoside (6) and sitosterol 3-O-ß-glycopyranoside (7)], and a fatty acid [n-hexacosanoic acid (8)]. Six (6) compounds (1, 2, 3, 4, 5, and 8) are reported for the first time from this species. Their structures were elucidated and identified by extensive NMR techniques, GC-MS and comparison with the previously reported literature. Compound 3 was found to displayed good inhibition against acetylcholinesterase with an IC50 value of 21.5 ± 0.022 µM, while 1 and 2 exhibited pronounced α-glucosidase inhibitory activity with IC50 values of 22.4 ± 0.016 µM and 24.9 ± 0.012 µM, respectively.

Effectiveness of Aqueous Extract of Marine Baitworm Marphysa moribidii Idris, Hutchings and Arshad, 2014 (Annelida, Polychaeta), on Acute Wound Healing Using Sprague Dawley Rats.

Wound healing is a well-coordinated process that restores skin integrity upon injury. However, some wound treatment poses harmful effects on the skin, which delay the normal wound healing process. Marphysa moribidii, a marine baitworm or polychaete, represents unique ability to regenerate posterior segment after injury, which may be beneficial in the wound healing treatment. The effectiveness of the polychaete as wound healing treatment was discovered through skin irritation, microbial testing, animal wound model, and chemical identifications. Three polychaete extracts (PE) emulsifying ointment (0.1%, 0.5%, and 1.0%) were topically applied to the full thickness wound model once daily for 14 days. Interestingly, PE 1.0% revealed the most rapid wound healing effects as compared to other treatments, including gamat (sea cucumber) oil (15% w/v) and acriflavine (0.1% w/v). Histopathological analysis using Masson's trichrome staining further confirms that PE treated wound exhibited minimal scar, high collagen deposition, and the emergence of neovascularisation. The extract also displayed a minimum inhibitory concentration (MIC) of 0.4 g/ml against Escherichia coli and absence of skin irritation, infectious bacteria, and heavy metals from the extract. Moreover, chemical compounds such as alkaloid, flavonoid, amino acids, and organic acid were detected in M. moribidii extracts, which could contribute to wound healing activity. In conclusion, this study further justifies the beneficial use of polychaete in treating wound healing and could be developed as a novel bioactive agent in nutraceuticals and pharmaceutical drugs.

Microalgal metabolites as anti-cancer/anti-oxidant agents reduce cytotoxicity of elevated silver nanoparticle levels against non-cancerous vero cells.

Heavy metal pollution has become a major concern globally as it contaminates eco-system, water networks and as finely suspended particles in air. In this study, the effects of elevated silver nanoparticle (AgNPs) levels as a model system of heavy metals, in the presence of microalgal crude extracts (MCEs) at different ratios, were evaluated against the non-cancerous Vero cells, and the cancerous MCF-7 and 4T1 cells. The MCEs were developed from water (W) and ethanol (ETH) as green solvents. The AgNPs-MCEs-W at the 4:1 and 5:1 ratios (v/v) after 48 and 72 h treatment, respectively, showed the IC50 values of 83.17-95.49 and 70.79-91.20 µg/ml on Vero cells, 13.18-28.18 and 12.58-25.7 µg/ml on MCF-7; and 16.21-33.88 and 14.79-26.91 µg/ml on 4T1 cells. In comparison, the AgNPs-MCEs-ETH formulation achieved the IC50 values of 56.23-89.12 and 63.09-91.2 µg/ml on Vero cells, 10.47-19.95 and 13.48-26.61 µg/ml on MCF-7; 14.12-50.11 and 15.13-58.88 µg/ml on 4T1 cells, respectively. After 48 and 72 h treatment, the AgNPs-MCE-CHL at the 4:1 and 5:1 ratios exhibited the IC50 of 51.28-75.85 and 48.97-69.18 µg/ml on Vero cells, and higher cytotoxicity at 10.47-16.98 and 6.19-14.45 µg/ml against MCF-7 cells, and 15.84-31.62 and 12.58-24.54 µg/ml on 4T1 cells, respectively. The AgNPs-MCEs-W and ETH resulted in low apoptotic events in the Vero cells after 24 h, but very high early and late apoptotic events in the cancerous cells. The Liquid Chromatography-Mass Spectrometry-Electrospray Ionization (LC-MS-ESI) metabolite profiling of the MCEs exhibited 64 metabolites in negative ion and 56 metabolites in positive ion mode, belonging to different classes. The microalgal metabolites, principally the anti-oxidative components, could have reduced the toxicity of the AgNPs against Vero cells, whilst retaining the cytotoxicity against the cancerous cells.

Mass Spectrometry-Based Metabolomics Combined with Quantitative Analysis of the Microalgal Diatom (Chaetoceros calcitrans).

Although many metabolomics studies of higher land plant species have been conducted, similar studies of lower nonland plant species, which include microalgae, are still developing. The present study represents an attempt to characterize the metabolic profile of a microalgal diatom Chaetoceros calcitrans, by applying high-resolution mass spectrometry detection, via Q-ExactiveTM Plus Orbitrap mass spectrometry. The results showed that 54 metabolites of various classes were tentatively identified. Experimentally, the chloroform and acetone extracts were clearly distinguished from other solvent extracts in chemometric regression analysis using PLS, showing the differences in the C. calcitrans metabolome between the groups. In addition, specific metabolites were evaluated, which supported the finding of antioxidant and anti-inflammatory activities. This study also provides data on the quantitative analysis of four carotenoids based on the identification results. Therefore, these findings could serve as a reliable tool for identifying and quantifying the metabolome that could reflect the metabolic activities of C. calcitrans.

Complementary NMR- and MS-based metabolomics approaches reveal the correlations of phytochemicals and biological activities in Phyllanthus acidus leaf extracts.

Proton nuclear magnetic resonance (1H NMR)- and ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS)-based analytical tools are frequently used in metabolomics studies. These complementary metabolomics platforms were applied to identify and quantify the metabolites in Phyllanthus acidus extracted with different ethanol concentrations. In total, 38 metabolites were tentatively identified by 1H NMR and 39 via UHPLC-MS, including 30 compounds are reported for the first time from this plant. The partial least square analysis (PLS) revealed the metabolites that contributed to α-glucosidase and nitric oxide (NO) inhibitory activities, including kaempferol, quercetin, myricetin, phyllanthusol A, phyllanthusol B, chlorogenic, catechin, cinnamic coumaric, caffeic, quinic, citric, ellagic and malic acids. This study shows the significance of combining 1H NMR- and UHPLC-MS-based metabolomics as the best strategies in identifying metabolites in P. acidus extracts and establishing an extract with potent antioxidant, anti-diabetic, and anti-inflammatory properties.

Antioxidant, α-Glucosidase, and Nitric Oxide Inhibitory Activities of Six Algerian Traditional Medicinal Plant Extracts and 1H-NMR-Based Metabolomics Study of the Active Extract.

Claims of effective therapy against diabetes using plants including Peganum harmala L., Zygophyllum album, Anacyclus valentinus L., Ammodaucus leucotrichus, Lupinus albus, and Marrubium vulgare in Algerian empirical medicine prompted our interest in evaluating their antidiabetic activity by screening their free radical scavenging (DPPH), α-glucosidase, and nitric oxide (NO) inhibitory activities as well as the total phenolic content (TPC). Extracts of the selected plants were prepared using different ratios of ethanol (0, 50, 80, and 100%). In this study, 100%, and 80% ethanol extracts of L. albus were found to be the most potent, in inhibiting α-glucosidase activity with IC50 values of 6.45 and 8.66 µg/mL, respectively. The 100% ethanol extract of A. leucotrichus exhibited the highest free radical scavenging activity with an IC50 value of 26.26 µg/mL. Moreover, the highest TPC of 612.84 µg GAE/mg extract was observed in M. vulgare, extracted with 80% ethanol. Metabolite profiling of the active extract was conducted using 1H-NMR metabolomics. Partial least square analysis (PLS) was used to assess the relationship between the α-glucosidase inhibitory activity of L. albus and the metabolites identified in the extract. Based on the PLS model, isoflavonoids (lupinoisoflavone G, lupisoflavone, lupinoisolone C), amino acids (asparagine and thiamine), and several fatty acids (stearic acid and oleic acid) were identified as metabolites that contributed to the inhibition of α-glucosidase activity. The results of this study have clearly strengthened the traditional claim of the antihyperglycemic effects of L. albus.

Potentially Bioactive Metabolites from Pineapple Waste Extracts and Their Antioxidant and α-Glucosidase Inhibitory Activities by 1H NMR.

Pineapple (Ananas comosus) waste is a promising source of metabolites for therapeutics, functional foods, and cosmeceutical applications. This study strives to characterize the complete metabolite profiles of a variety of MD2 pineapple waste extracts. Metabolomics strategies were utilized to identify bioactive metabolites of this variety prepared with different solvent ratios. Each pineapple waste extract was first screened for total phenolic content, 2,2-diphenyl-1-picrylhydrazyl free radical scavenging, nitric oxide scavenging, and α-glucosidase inhibitory activities. The highest TPC was found in all samples of the peel, crown, and core extracted using a 50% ethanol ratio, even though the results were fairly significant than those obtained for other ethanol ratios. Additionally, crown extracted with a 100% ethanol ratio demonstrated the highest potency in DPPH and NO scavenging activity, with IC50 values of 296.31 and 338.52 µg/mL, respectively. Peel extracted with 100% ethanol exhibited the highest α-glucosidase inhibitory activity with an IC50 value of 92.95 µg/mL. Then, the extracts were analyzed and the data from 1H NMR were processed using multivariate data analysis. A partial least squares and correlogram plot suggested that 3-methylglutaric acid, threonine, valine, and α-linolenic acid were the main contributors to the antioxidant activities, whereas epicatechin was responsible for the α-glucosidase inhibitory activity. Relative quantification further supported that 100% crown extract was among the extracts that possessed the most abundant potential metabolites. The present study demonstrated that the crown and peel parts of MD2 pineapple extracted with 100% ethanol are potentially natural sources of antioxidants and α-glucosidase inhibitors, respectively.

Acute Wound Healing Potential of Marine Worm, Diopatra claparedii Grube, 1878 Aqueous Extract on Sprague Dawley Rats.

Diopatra claparedii which is colloquially known as Ruat Sarung can be found along the west coast of Peninsular Malaysia. The species has a unique ability to regenerate anterior and posterior segments upon self-amputation or injury, thus having potential as a wound healing promoter. In this study, the wound healing potential of D. claparedii aqueous extract on acute wound model in rats was revealed for the first time. Various concentrations (0.1%, 0.5%, and 1.0% w/w) of D. claparedii ointment were formulated and tested on Sprague Dawley rats through topical application on full-thickness skin wounds for 14 days. The wound healing effects were investigated via behaviour observation, wound contraction, and histopathological analysis. Quality assessment was performed via skin irritation test, microbial contamination test (MCT), and heavy metal detection. The study also included test for antibacterial activities and detection of bioactive compounds in D. claparedii. One percent of D. claparedii ointment showed rapid wound healing potential with good soothing effects and more collagen deposition in comparison to the commercial wound healing ointments such as acriflavine (0.1% w/v) and traditional ointment gamat (sea cucumber extract) (15.0% w/v). No local skin irritation, microbial contamination, and insignificant concentration of heavy metals were observed, which indicate its safe application. Moreover, the aqueous extract of D. claparedii exhibited antibacterial activities against Escherichia coli and Pseudomonas aeruginosa with minimum inhibitory concentration (MIC) value at 0.4 g/ml. 1H NMR analysis of the aqueous extract of D. claparedii revealed some metabolites that might be responsible for its wound healing properties such as amino acids, halogenated aromatics, organic acids, vitamins, and others. Altogether, these results suggested that the aqueous extract of D. claparedii could be utilised as an alternative natural wound healing promoter.

1H-NMR-based metabolomics to investigate the effects of Phoenix dactylifera seed extracts in LPS-IFN-γ-induced RAW 264.7 cells.

Inflammation has been revealed to play a central role in the onset and progression of many illnesses. Nuclear magnetic resonance (NMR) based metabolomics method was adopted to evaluate the effects of Phoenix dactylifera seeds, in particular the Algerian date variety of Deglet on the metabolome of the LPS-IFN-γ-induced RAW 264.7 cells. Variations in the extracellular and intracellular profiles emphasized the differences in the presence of tyrosine, phenylalanine, alanine, proline, asparagine, isocitrate, inosine and lysine. Principal component analysis (PCA) revealed noticeable clustering patterns between the treated and induced RAW cells based on the metabolic profile of the extracellular metabolites. However, the effects of treatment on the intracellular metabolites appears to be less distinct as suggested by the PCA and heatmap analyses. A clear group segregation was observed for the intracellular metabolites from the treated and induced cells based on the orthogonal partial least squares-discriminant analysis (OPLS-DA) score plot. Likewise, 11 of the metabolites in the treated cells were significantly different from those in the induced groups, including amino acids and succinate. The enrichment analysis demonstrated that treatment with Deglet seed extracts interfered with the energy and of amino acids metabolism. Overall, the obtained data reinforced the possible application of Deglet seeds as a functional food with anti-inflammatory properties.

NMR metabolomics for evaluating passage number and harvesting effects on mammalian cell metabolome.

The variation in the extracellular metabolites of RAW 264.7 cells obtained from different passage numbers (passage 9, 12 and 14) was examined. The impact of different harvesting protocols (trypsinization and scraping) on recovery of intracellular metabolites was then assessed. The similarity and variation in the cell metabolome was investigated using 1H NMR metabolic profiling modeled using multivariate data analysis. The characterization and quantification of metabolites was performed to determine the passage-related and harvesting-dependent effects on impacted metabolic networks. The trypsinized RAW cells from lower passages gave higher intensities of most identified metabolites, including asparagine, serine and tryptophan. Principal component analysis revealed variation between cells from different passages and harvesting methods, as indicated by the formation of clusters in score plot. Analysis of S-plots revealed metabolites that acted as biomarkers in discriminating cells from different passages including acetate, serine, lactate and choline. Meanwhile lactate, glutamine and pyruvate served as biomarkers for differentiating trypsinized and scraped cells. In passage-dependent effects, glycolysis and TCA cycle were influential, whereas glycerophospholipid metabolism was affected by the harvesting method. Overall, it is proposed that typsinized RAW cells from lower passage numbers are more appropriate when conducting experiments related to NMR metabolomics.

Rapid assessment of total MCPD esters in palm-based cooking oil using ATR-FTIR application and chemometric analysis.

The technique of Fourier transform infrared spectroscopy is widely used to generate spectral data for use in the detection of food contaminants. Monochloropropanediol (MCPD) is a refining process-induced contaminant that is found in palm-based fats and oils. In this study, a chemometric approach was used to evaluate the relationship between the FTIR spectra and the total MCPD content of a palm-based cooking oil. A total of 156 samples were used to develop partial least squares regression (PLSR), artificial neural network (nnet), average artificial neural network (avNNET), random forest (RF) and cubist models. In addition, a consensus approach was used to generate fusion result consisted from all the model mentioned above. All the models were evaluated based on validation performed using training and testing datasets. In addition, the box plot of coefficient of determination (R2), root mean square error (RMSE), slopes and intercepts by 100 times randomization was also compared. Evaluation of performance based on the testing R2 and RMSE suggested that the cubist model predicted total MCPD content with the highest accuracy, followed by the RF, avNNET, nnet and PLSR models. The overfitting tendency was assessed based on differences in R2 and RMSE in the training and testing calibrations. The observations showed that the cubist and avNNET models possessed a certain degree of overfitting. However, the accuracy of these models in predicting the total MCPD content was high. Results of the consensus model showed that it slightly improved the accuracy of prediction as well as significantly reduced its uncertainty. The important variables derived from the cubist and RF models suggested that the wavenumbers corresponding to the MCPDs originated from the -CH=CH2 or CH=CH (990-900 cm-1) and C-Cl stretch (800-700 cm-1) regions of the FTIR spectrum data. In short, chemometrics in combination with FTIR analysis especially for the consensus model represent a potential and flexible technique for estimating the total MCPD content of refined vegetable oils.

Classification of Raw Stingless Bee Honeys by Bee Species Origins Using the NMR- and LC-MS-Based Metabolomics Approach.

The official standard for quality control of honey is currently based on physicochemical properties. However, this method is time-consuming, cost intensive, and does not lead to information on the originality of honey. This study aims to classify raw stingless bee honeys by bee species origins as a potential classifier using the NMR-LCMS-based metabolomics approach. Raw stingless bee honeys were analysed and classified by bee species origins using proton nuclear magnetic resonance (¹H-NMR) spectroscopy and an ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF MS) in combination with chemometrics tools. The honey samples were able to be classified into three different groups based on the bee species origins of Heterotrigona itama, Geniotrigona thoracica, and Tetrigona apicalis. d-Fructofuranose (H. itama honey), ß-d-Glucose, d-Xylose, α-d-Glucose (G. thoracica honey), and l-Lactic acid, Acetic acid, l-Alanine (T. apicalis honey) ident d-Fructofuranose identified via ¹H-NMR data and the diagnostic ions of UHPLC-QTOF MS were characterized as the discriminant metabolites or putative chemical markers. It could be suggested that the quality of honey in terms of originality and purity can be rapidly determined using the classification technique by bee species origins via the ¹H-NMR- and UHPLC-QTOF MS-based metabolomics approach.

Hematological, Biochemical, Histopathological and ¹H-NMR Metabolomics Application in Acute Toxicity Evaluation of Clinacanthus nutans Water Leaf Extract.

The present study aims for the first time to provide the in vivo acute toxicological profile of the highest dose of Clinacanthus nutans (Burm. f.) Lindau water leaf extract according to the Organization for economic co-operation and development (OECD) 423 guidelines through conventional toxicity and advanced proton nuclear magnetic resonance (¹H-NMR) serum and urinary metabolomics evaluation methods. A single dose of 5000 mg/kg bw of C. nutans water extract was administered to Sprague Dawley rats, and they were observed for 14 days. Conventional toxicity evaluation methods (physical observation, body and organ weight, food and water consumption, hematology, biochemical testing and histopathological analysis) suggested no abnormal toxicity signs. Serum ¹H-NMR metabolome revealed no significant metabolic difference between untreated and treated groups. Urinary ¹H-NMR analysis, on the other hand, revealed alteration in carbohydrate metabolism, energy metabolism and amino acid metabolism in extract-treated rats after 2 h of extract administration, but the metabolic expression collected after 24 h and at Day 5, Day 10 and Day 15 indicated that the extract-treated rats did not accumulate any toxicity biomarkers. Importantly, the outcomes further suggest that single oral administration of up to 5000 mg/kg bw of C. nutans water leaf extract is safe for consumption.

Metabolomic analysis and biochemical changes in the urine and serum of streptozotocin-induced normal- and obese-diabetic rats

Diabetes mellitus (DM) is a chronic disease that can affect metabolism of glucose and other metabolites. In this study, the normal- and obese-diabetic rats were compared to understand the diabetes disorders of type 1 and 2 diabetes mellitus. This was done by evaluating their urine metabolites using proton nuclear magnetic resonance (1H NMR)-based metabolomics and comparing with controls at different time points, considering the induction periods of obesity and diabetes. The biochemical parameters of the serum were also investigated. The obese-diabetic model was developed by feeding the rats a high-fat diet and inducing diabetic conditions with a low dose of streptozotocin (STZ) (25 mg/kg bw). However, the normal rats were induced by a high dose of STZ (55 mg/kg bw). A partial least squares discriminant analysis (PLS-DA) model showed the biomarkers of both DM types compared to control. The synthesis and degradation of ketone bodies, tricarboxylic (TCA) cycles, and amino acid pathways were the ones most involved in the variation with the highest impact. The diabetic groups also exhibited a noticeable increase in the plasma glucose level and lipid profile disorders compared to the control. There was also an increase in the plasma cholesterol and low-density lipoprotein (LDL) levels and a decline in the high-density lipoprotein (HDL) of diabetic rats. The normal-diabetic rats exhibited the highest effect of all parameters compared to the obese-diabetic rats in the advancement of the DM period. This finding can build a platform to understand the metabolic and biochemical complications of both types of DM and can generate ideas for finding targeted drugs

Solvent Extraction and Identification of Active Anticariogenic Metabolites in Piper cubeba L. through ¹H-NMR-Based Metabolomics Approach.

The aim of this study was to determine the effects of different solvents for extraction, liquid⁻liquid partition, and concentrations of extracts and fractions of Piper cubeba L. on anticariogenic; antibacterial and anti-inflammatory activity against oral bacteria. Furthermore, ¹H-Nuclear Magnetic Resonance (NMR) coupled with multivariate data analysis (MVDA) was applied to discriminate between the extracts and fractions and examine the metabolites that correlate to the bioactivities. All tested bacteria were susceptible to Piper cubeba L. extracts and fractions. Different solvents extraction, liquid⁻liquid partition and concentrations of extracts and fractions have partially influenced the antibacterial activity. MTT assay showed that P. cubeba L. extracts and fractions were not toxic to RAW 264.7 cells at selected concentrations. Anti-inflammatory activity evaluated by nitric oxide (NO) production in lipopolysaccharide (LPS) stimulated cells showed a reduction in NO production in cells treated with P. cubeba L. extracts and fractions, compared to those without treatment. Twelve putative metabolites have been identified, which are (1) cubebin, (2) yatein, (3) hinokinin, (4) dihydrocubebin, (5) dihydroclusin, (6) cubebinin, (7) magnosalin, (8) p-cymene, (9) piperidine, (10) cubebol, (11) d-germacrene and (12) ledol. Different extraction and liquid⁻liquid partition solvents caused separation in principal component analysis (PCA) models. The partial least squares (PLS) models showed that higher anticariogenic activity was related more to the polar solvents, despite some of the active metabolites also present in the non-polar solvents. Hence, P. cubeba L. extracts and fractions exhibited antibacterial and anti-inflammatory activity and have potential to be developed as the anticariogenic agent.