Preclinical evaluation of dalbergin loaded PLGA-galactose-modified nanoparticles against hepatocellular carcinoma via inhibition of the AKT/NF-κB signaling pathway.

Prior research has shown the effectiveness of dalbergin (DL), dalbergin nanoformulation (DLF), and dalbergin-loaded PLGA-galactose-modified nanoparticles (DLMF) in treating hepatocellular carcinoma (HCC) cells. The present investigation constructs upon our previous research and delves into the molecular mechanisms contributing to the anticancer effects of DLF and DLMF. This study examined the anti-cancer effects of DL, DLF, and DLMF by diethyl nitrosamine (DEN)-induced HCC model in albino Wistar rats. In addition, we performed biochemical, antioxidant, lipid profile tests, and histological studies of liver tissue. The anticancer efficacy of DLMF is equivalent to that of 5-fluorouracil, a commercially available therapy for HCC. Immunoblotting studies revealed a reduction in the expression of many apoptotic markers, such as p53, BAX, and Cyt-C, in HCC. Conversely, the expression of Bcl-2, TNF-α, NFκB, p-AKT, and STAT-3 was elevated. Nevertheless, the administration of DL, DLF, and DLMF effectively controlled the levels of these apoptotic markers, resulting in a considerable decrease in the expression of Bcl-2, TNF-α, NFκB, p-AKT, and STAT-3. Specifically, the activation of TNF-alpha and STAT-3 triggers the signalling pathways that include the Bcl-2 family of proteins, Cyt-C, caspase 3, and 9. This ultimately leads to apoptosis and the suppression of cell growth. Furthermore, metabolomic analysis using 1H NMR indicated that the metabolites of animals reverted to normal levels after the treatment.

1H NMR analysis of metabolites from leaf tissue of resistant and susceptible oil palm breeding materials against Ganoderma boninense.

INTRODUCTION: Breeding for oil palm resistance against basal stem rot caused by Ganoderma boninense is challenging and time-consuming. Advanced oil palm gene pools are very limited, hence it is assumed that parental palms have experienced genetic drift and lost their resistance genes against Ganoderma. High-throughput selection criteria should be developed. Metabolomic analysis using 1H nuclear magnetic resonance (NMR) spectroscopy is easy, and the resulting metabolite can be used as a diagnostic tool for detecting disease in various host-pathogen combinations. OBJECTIVES: The objective of this study was to identify metabolite variations in Dura (D) and Pisifera (P) parental palms with different resistance levels against Ganoderma and moderately resistant DxP using 1H NMR analysis. METHODS: Leaf tissues of seven different oil palm categories consisting of: resistant, moderate, and susceptible Dura (D); moderate and susceptible Pisifera (P); resistant Tenera/Pisifera (T/P) parental palms; and moderately resistant DxP variety progenies, were sampled and their metabolites were determined using NMR spectroscopy. RESULTS: Twenty-nine types of metabolites were identified, and most of the metabolites fall in the monosaccharides, amino acids, and fatty acids compound classes. The PCA, PLS-DA, and heatmap multivariate analysis indicated two identified groups of resistance based on their metabolites. The first group consisted of resistant T/P, moderate P, resistant D, and moderately resistant DxP. In contrast, the second group consisted of susceptible P, moderate D, and susceptible D. Glycerol and ascorbic acid were detected as biomarker candidates by OPLS-DA to differentiate moderately resistant DxP from susceptible D and P. The pathway analysis suggested that glycine, serine, and threonine metabolism and taurine and hypotaurine metabolism were involved in the oil palm defense mechanism against Ganoderma. CONCLUSION: A metabolomic study with 1H NMR was able to describe the metabolite composition that could differentiate the characteristics of oil palm resistance against basal stem rot (BSR) caused by G. boninense. These metabolites revealed in this study have enormous potential to become support tools for breeding new oil palm varieties with higher resistance against BSR.

Development of a determination method for quality control markers utilizing metabolic profiling and its application on processed Zingiber officinale Roscoe rhizome.

This study established an Orthogonal Partial Least Squares (OPLS) model combining 1H-NMR and GC-MS data to identify characteristic metabolites in complex extracts. Both in metabolomics studies, and natural product chemistry, the reliable identification of marker metabolites usually requires laborious isolation and purification steps, which remains a bottleneck in many studies. Both ginger (GR) and processed ginger (PGR) are listed in the Japanese pharmacopeia. The plant of origin, the rhizome of Zingiber officinale Roscoe, is differently processed for these crude drugs. Notably, the quality of crude drugs is affected by genetic and environmental factors, making it difficult to maintain a certain quality standard. Therefore, characteristic markers for the quality control of GR and PGR are required. Metabolomic analysis using 1H-NMR was able to discriminate between GR and PGR, but there were unidentified signals that were difficult to distinguish based on NMR data alone. Therefore, we combined 1H-NMR and GC-MS analytical data to identify them by OPLS. As a result, αr-curcumene was found to be a useful marker for these identifications. This new approach enabled rapid identification of characteristic marker compounds and reduced the labor involved in the isolation process.

Integrated metabolomics analysis of chill-stored rose shrimp (Parapenaeus longirostris) treated with different pressure levels of high hydrostatic pressure by 1H-NMR spectroscopy.

The antimicrobial effects of high hydrostatic pressure (HHP) treatments on chill-stored seafood are well-documented, while their impact on the metabolic profile of seafood, especially the metabolome of fish flesh, and remains underexplored. Addressing this gap, this study investigates the effects of HHP on the metabolome of chill-stored rose shrimp by conducting multivariate data analysis based on untargeted proton nuclear magnetic resonance observations. Vacuum-packed rose shrimp samples were subjected to HHP at 0, 400, 500, and 600 MPa for 10 min and then stored at 2-4°C. The microorganism analysis and metabolic analysis were carried out on days 1 and 14. HHP treatment effectively deactivated Lactobacillus spp., Escherichia coli, Pseudomonas spp., total Coliforms, and sulfite-reducing anaerobic bacteria. Consequently, HHP treatment significantly reduced the formation rate of decay-related metabolites, such as hypoxanthine, trimethylamine, and biogenic amines, which exhibited significant accumulation in untreated samples. Multivariate unsupervised analyses provided insights into the overall changes in the metabolite profile induced by HHP. Metabolic pathway analysis revealed several pathways underlying spoilage, including pyruvate metabolism, valine, leucine, and isoleucine biosynthesis, purine metabolism, methane metabolism, glycine, serine, and threonine metabolism, citrate cycle (TCA cycle), glycolysis/gluconeogenesis, alanine, aspartate, and glutamate metabolism, sulfur metabolism, pantothenate and CoA biosynthesis, glutathione metabolism, and glyoxylate and dicarboxylate metabolism. Importantly, these pathways underwent alterations due to the application of HHP, particularly at high-pressure levels. In summary, the results unveil the potential mechanisms of HHP effects on chill-stored rose shrimps.

Novel approach on the evaluation of enzyme-aided alkaline extraction of polysaccharide from Hordeum vulgare husk and molecular insight on the multifunctional scaffold.

Hordeum vulgare husk, a cereal grain, is rich in dietary fiber and prebiotics beneficial for the gut microbiota and host organism. This study investigates the effects of barley husk-derived water-soluble xylan (BH-WSX) on gut homeostasis and the microbiome. We enzymatically extracted BH-WSX and evaluated its prebiotic and antioxidant properties. A 40.0 % (w/v) xylan yield was achieved, with the extracted xylan having a molecular mass of 212.0885 and a xylose to glucuronic acid molar ratio of 6:1. Specialized optical rotation research indicated that the isolated xylan is composed of monomeric sugars such as D-xylose, glucose, and arabinose. Fourier Transform Infrared (FTIR) spectroscopy revealed that the xylan comprises ß (1 â†’ 4) linked xylose units, randomly substituted with glucose residues, α-arabinofuranose, and acetyl groups. Nuclear Magnetic Resonance (NMR) analysis showed that the barley husk extract's backbone is substituted with 4-O-methyl glucuronic acid at the O2 position. Thermogravimetric analysis indicated that WSX exhibits a single sharp peak at 266 °C on the Differential Thermal Gravimetry (DTG) curve. Furthermore, a combination of in vitro, in vivo models, and molecular docking analysis elaborated on the anti-adhesion properties of BH-WSX. This study presents a novel approach to utilizing barley husk as an efficient source of functional polysaccharides for food-related industrial applications.

Spiny dogfish, Squalus suckleyi, shows a good tolerance for hypoxia but need long recovery times.

Pacific spiny dogfish, Squalus suckleyi, move to shallow coastal waters during critical reproductive life stages and are thus at risk of encountering hypoxic events which occur more frequently in these areas. For effective conservation management, we need to fully understand the consequences of hypoxia on marine key species such as elasmobranchs. Because of their benthic life style, we hypothesized that S. suckleyi are hypoxia tolerant and able to efficiently regulate oxygen consumption, and that anaerobic metabolism is supported by a broad range of metabolites including ketones, fatty acids and amino acids. Therefore, we studied oxygen consumption rates, ventilation frequency and amplitude, blood gasses, acid-base regulation, and changes in plasma and tissue metabolites during progressive hypoxia. Our results show that critical oxygen levels (P crit) where oxyregulation is lost were indeed low (18.1% air saturation or 28.5 Torr at 13°C). However, many dogfish behaved as oxyconformers rather than oxyregulators. Arterial blood PO2 levels mostly decreased linearly with decreasing environmental PO2. Blood gases and acid-base status were dependent on open versus closed respirometry but in both set-ups ventilation frequency increased. Hypoxia below Pcrit resulted in an up-regulation of anaerobic glycolysis, as evidenced by increased lactate levels in all tissues except brain. Elasmobranchs typically rely on ketone bodies as oxidative substrates, and decreased concentrations of acetoacetate and ß-hydroxybutyrate were observed in white muscle of hypoxic and/or recovering fish. Furthermore, reductions in isoleucine, glutamate, glutamine and other amino acids were observed. After 6 hours of normoxic recovery, changes persisted and only lactate returned to normal in most tissues. This emphasizes the importance of using suitable bioindicators adjusted to preferred metabolic pathways of the target species in conservation physiology. We conclude that Pacific spiny dogfish can tolerate severe transient hypoxic events, but recovery is slow and negative impacts can be expected when hypoxia persists.

HRMAS 1H NMR and CPMAS 13C NMR spectroscopies coupled with chemometrics for the metabolomic investigation of commercial teas.

In this study, the metabolome of different types of tea (i.e., black, green and earl grey) is explored by means of HRMAS 1H (i.e., semisolid state) NMR and CPMAS 13C (i.e., solid state) NMR spectroscopies. By elaborating the metabolomic data with unsupervised and supervised chemometric tools (PCA, PLS-DA), it was possible to set up classification models with the aim to discriminate the different types of tea as based on differences in their chemical composition. Both the applications of the NMR spectroscopies also allowed to obtain information about the metabolic biomarkers leading the differentiation among teas. These were mainly represented by phenolic compounds. Also, some non-phenolic compounds, such as amino acids, carbohydrates, and terpenoids, played important roles in shaping tea quality. The findings of this study provided useful insights into the application of solid and semisolid state NMR spectroscopies, in combination with chemometrics, in the context of food authentication and traceability.

Bidentate bromhexine drug coordination modes with various transition metal ions: Synthesis, characterization, and in vitro antibacterial and anti-breast cancer activity tests.

BACKGROUND: Bromhexine (BHX) is a mucolytic drug used in treatment the respiratory disorders which are associated with excessive or viscid mucus. Transition metal complexes have made tremendous progress in the treatment of a variety of human ailments, according to reported articles. Transition metal complexes are being developed as medications with a lot of effort. Metal complexes can form a variety of coordination geometries, giving them distinct forms. So, binary metal complexes of bromhexine drug have been prepared to enhance the biological activity and stability of the free drug. METHODS: A new series of binary complexes with bromhexine drug (BHX) has been prepared with some transition metal ions namely Cr(III), Mn(II), Fe(III), Co(II), Ni(II), Cu(II), Zn(II), and Cd(II). Elemental analyses, FT-IR, mass spectrometry, thermal studies and UV-Vis spectra have been used to characterize and structurally elucidate the produced metal complexes. Antibacterial activity has been tested for the ligand and metal complexes against a variety of pathogenic bacterial species (Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus). In addition, the ligand has been tested for anticancer efficacy against the MCF-7 breast cancer cell line, as opposed to binary metal complexes. The binding orientation or conformation of the free BHX ligand and Co(II) complex in the active region of the protein of crystal structure of Escherichia coli (PDB ID: 3T88) and Pseudomonas aeruginosa (PDB ID: 6NE0) has been performed using molecular docking studies. RESULTS: The BHX ligand coupled in neutral bidentate mode to the metal ions, according to FT-IR and 1H-NMR spectral results. The molar conductivity measurements of the complexes in DMF proved the electrolytic nature of all binary complexes. Co(II) complex showed the highest inhibition zone diameter against S. aureus, E. coli and P. aeruginosa. Zn(II) complex had the greatest inhibitory effect against P. aeruginosa and B. subtilis. Also, Cd(II) chelate appeared high efficacy as antibacterial agent against Pseudomonas aeruginosa and Staphylococcus aureus. CONCLUSION: All the output data conjugated to confirm the octahedral geometry of the metal complexes. The biological findings revealed that metal complexes can be more active than the free BHX ligand. Against MCF-7 cell line, Cd(II)-L complex is highly active complex (4.95 µg/mL) but BHX free drug is the most active compound (3.96 µg/mL).

Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides by enzyme-linked immunosorbent assay.

Capsular polysaccharides of Streptococcus pneumoniae are used in pneumococcal polysaccharide and protein-conjugate vaccines. Cell-wall polysaccharide (C-Ps) is a critical impurity that must be kept at low levels in purified polysaccharide preparations. Hence, accurate and precise methods for determining C-Ps are needed. Currently available methods include nuclear magnetic resonance (NMR) spectroscopy and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Both these methods suffer from their own limitations; therefore, we developed a simple and efficient enzyme-linked immunosorbent assay (ELISA) for accurate and precise quantification of C-Ps in samples of any serotype of pneumococcal capsular polysaccharide without interference. We quantified C-Ps in preparations of 14 serotype polysaccharides using newly developed ELISA method and compared the results with C-Ps values obtained using two previously reported methods, 1H NMR and HPAEC-PAD. The C-Ps value determined using 1H NMR for serotype 5 was 21.08%, whereas the values obtained using HPAEC-PAD and ELISA were 2.38% and 2.89% respectively, indicating some interference in 1H NMR method. The sensitivity of the ELISA method is higher because the sample is used directly unlike HPAEC-PAD method where sample is subjected to harsh treatment, such as acid digestion and quantify C-Ps based on peak area of ribitol or AAT. Furthermore, 1H NMR and HPAEC-PAD are expensive and laborious methods. Our work, underscores the simple and efficient ELISA that can be used for quantification of C-Ps in pneumococcal polysaccharide preparations.

The diagnostic potential of urine in paediatric patients undergoing initial treatment for tuberculous meningitis.

Tuberculous meningitis (TBM)-the extrapulmonary form of tuberculosis, is the most severe complication associated with tuberculosis, particularly in infants and children. The gold standard for the diagnosis of TBM requires cerebrospinal fluid (CSF) through lumbar puncture-an invasive sample collection method, and currently available CSF assays are often not sufficient for a definitive TBM diagnosis. Urine is metabolite-rich and relatively unexplored in terms of its potential to diagnose neuroinfectious diseases. We used an untargeted proton magnetic resonance (1H-NMR) metabolomics approach to compare the urine from 32 patients with TBM (stratified into stages 1, 2 and 3) against that from 39 controls in a South African paediatric cohort. Significant spectral bins had to satisfy three of our four strict cut-off quantitative statistical criteria. Five significant biological metabolites were identified-1-methylnicotinamide, 3-hydroxyisovaleric acid, 5-aminolevulinic acid, N-acetylglutamine and methanol-which had no correlation with medication metabolites. ROC analysis revealed that methanol lacked diagnostic sensitivity, but the other four metabolites showed good diagnostic potential. Furthermore, we compared mild (stage 1) TBM and severe (stages 2 and 3) TBM, and our multivariate metabolic model could successfully classify severe but not mild TBM. Our results show that urine can potentially be used to diagnose severe TBM.

Screening of postoperative adjuvant chemotherapy-related serum metabolic markers in breast cancer patients based on 1H NMR metabonomics.

Background: Breast cancer (BC) has the highest incidence rate among female malignant tumors. Adjuvant chemotherapy is commonly used to reduce micrometastasis in postoperative patients. However, monitoring the efficacy of chemotherapy in BC is a major challenge in clinical practice. In this study, 1H nuclear magnetic resonance (NMR) metabonomics was performed to explore the serum metabolic characteristics of BC patients before and after adjuvant chemotherapy. Methods: In this study, we collected serum samples from 51 healthy controls and 61 BC patients before and after chemotherapy for 1H NMR metabolomic analysis, and tested the performance of each metabolite and combination segment by the receiver operating characteristic (ROC) curves. Results: Nine metabolites, namely glutamine, citrate, creatine, glycerophosphatidylcholine/phosphatidylcholine, glycine, 1-methylhistidine, lactate, pyruvate and formate had significant changes in BC patients before chemotherapy compared with healthy controls. Lactate, pyruvate, 1-methylhistidine and formate were found to be inversely regulated by chemotherapy. ROC analysis showed that a combination of the four metabolites had good prediction for chemotherapy efficacy with area under the curve of 0.958, sensitivity of 98.36% and specificity of 91.30%. There was no significant correlation between chemotherapy-related metabolites and clinical indicators of cancer patients, indicating that they can be used to evaluate the chemotherapy efficacy of patients with different clinical indicators. Conclusions: Effectively, dynamic and non-invasive metabolic markers for the evaluation of the efficacy of chemotherapy were identified in this study.

Metabolomics analysis of human spermatozoa reveals impaired metabolic pathways in asthenozoospermia.

BACKGROUND: Infertility is a major health issue, affecting 15% of reproductive-age couples with male factors contributing to 50% of cases. Asthenozoospermia (AS), or low sperm motility, is a common cause of male infertility with complex aetiology, involving genetic and metabolic alterations, inflammation and oxidative stress. However, the molecular mechanisms behind low motility are unclear. In this study, we used a metabolomics approach to identify metabolic biomarkers and pathways involved in sperm motility. METHODS: We compared the metabolome and lipidome of spermatozoa of men with normozoospermia (n = 44) and AS (n = 22) using untargeted LC-MS and the metabolome of seminal fluid using 1H-NMR. Additionally, we evaluated the seminal fluid redox status to assess the oxidative stress in the ejaculate. RESULTS: We identified 112 metabolites and 209 lipids in spermatozoa and 27 metabolites in the seminal fluid of normozoospermic and asthenozoospermic men. PCA analysis of the spermatozoa's metabolomics and lipidomics data showed a clear separation between groups. Spermatozoa of asthenozoospermic men presented lower levels of several amino acids, and increased levels of energetic substrates and lysophospholipids. However, the metabolome and redox status of the seminal fluid was not altered inAS. CONCLUSIONS: Our results indicate impaired metabolic pathways associated with redox homeostasis and amino acid, energy and lipid metabolism in AS. Taken together, these findings suggest that the metabolome and lipidome of human spermatozoa are key factors influencing their motility and that oxidative stress exposure during spermatogenesis or sperm maturation may be in the aetiology of decreased motility in AS.

Integration of LC-HRMS and 1H NMR metabolomics data fusion approaches for classification of Amarone wine based on withering time and yeast strain.

Two untargeted metabolomics approaches (LC-HRMS and 1H NMR) were combined to classify Amarone wines based on grape withering time and yeast strain. The study employed a multi-omics data integration approach, combining unsupervised data exploration (MCIA) and supervised statistical analysis (sPLS-DA). The results revealed that the multi-omics pseudo-eigenvalue space highlighted a limited correlation between the datasets (RV-score = 16.4%), suggesting the complementarity of the assays. Furthermore, the sPLS-DA models correctly classified wine samples according to both withering time and yeast strains, providing a much broader characterization of wine metabolome with respect to what was obtained from the individual techniques. Significant variations were notably observed in the accumulation of amino acids, monosaccharides, and polyphenolic compounds throughout the withering process, with a lower error rate in sample classification (7.52%). In conclusion, this strategy demonstrated a high capability to integrate large omics datasets and identify key metabolites able to discriminate wine samples based on their characteristics.

Proton Nuclear Magnetic Resonance (1H NMR) Metabolomics Study in Serum, Urine, and Cystic Fluid for Differentiating Fertility and Staging of Intra-abdominal Hydatid Cyst in Adults.

Background: Cystic echinococcosis (CE) is a parasitic zoonosis caused by the tapeworm Echinococcus granulosus. Over the past few years, a lot of research has been done on liver illnesses using metabolomics techniques to identify biomarkers which could identify the diseases in its early stages. The present study was done to explore biomarkers in serum, urine, and cystic fluid which would help in differentiating, staging, and assessing fertility of intra-abdominal hydatid cyst by using proton nuclear magnetic resonance (1H NMR) metabolomics. Materials and methods: In the study, 28 subjects (16 cases and 12 controls) were enrolled. Staging of hydatid cysts was performed using ultrasonography. In patients complying with case and control definition, blood, urine, and cystic fluid were collected for complete blood count, urine culture, Echinococcus IgG enzyme-linked immunosorbent assay (ELISA), and metabolomic analysis. The 17, 15, and 11 metabolites in serum, urine, and cystic fluid samples were quantified, respectively, to differentiate between case and control group. Results: In this study, we observed that there was a significant downregulation of succinate metabolite in urine samples of cases, down-regulation of five metabolites (isoleucine, valine, histidine, tyrosine and formate) and upregulation of alanine in cystic fluid of cases. Conclusion: Current study demonstrates that metabolomics can be used non-invasively for rapid diagnosis of CE. This is one of the very few studies, which used 1H NMR spectroscopy, to analyze the profile of metabolites in serum, urine, and cystic fluid in cases of CE and controls. How to cite this article: Raj N, Pandey A, Roy R, et al. Proton Nuclear Magnetic Resonance (1H NMR) Metabolomics Study in Serum, Urine, and Cystic Fluid for Differentiating Fertility and Staging of Intra-abdominal Hydatid Cyst in Adults. Euroasian J Hepato-Gastroenterol 2024;14(1):30-34.

Exploring Salivary Metabolic Alterations in Type 2 Diabetes: Implications for Dental Caries and Potential Influences of HbA1c and Vitamin D Levels.

Diabetes mellitus is considered to be the most common health issue affecting almost 1 in 11 adults globally. Oral health complications including xerostomia, periodontal disease, dental caries, and soft tissue lesions are prevalent among individuals with diabetes, and therefore an understanding of the potential association between salivary metabolites and dental caries progression would enable the early detection and prevention of this non-communicable disease. Therefore, the aim of this study was to compare salivary biomarkers between individuals with type 2 diabetes (T2DM) with those without this disorder (ND) using 1H NMR-based metabolomics strategies. The objectives were to identify T2DM-associated biomarker signatures and their potential impact on dental caries. In addition, HbA1c and vitamin D levels were also analysed for this purpose. METHODS: Stimulated whole-mouth saliva (SWS) samples were collected from T2DM and ND (n = 30 in each case) participants randomly selected from a group of 128 participants recruited for this case-control study. All participants were advised to refrain from eating, drinking, and smoking for at least 1-2 h prior to sample collection. Following preparation, SWS supernatants underwent 1H NMR analysis at an operating frequency of 800 MHz, and the dataset acquired was analysed using a range of multivariate metabolomics techniques. RESULTS: Metabolomics analysis of data acquired demonstrated that, together with up- and downregulated blood HbA1c and vitamin D levels, key salivary discriminators between these two classifications included lactate, taurine, creatinine, α-glucose, and formate to a lesser extent. The bacterial catabolites lactate and formate were both significantly upregulated in the T2DM group, and these have previously been implicated in the pathogenesis of dental caries. Significance analysis of metabolites (SAM)-facilitated AUROC analysis yielded an 83% accuracy for this distinction. CONCLUSION: In conclusion, this study highlights the significant differences in salivary metabolites between individuals with T2DM and healthy controls. Such differences appear to be related to the development and progression of dental caries in T2DM patients.

Exploring the Anti-Inflammatory and Antioxidant Potential, Metabolite Composition and Inorganic Profile of Cistus monspeliensis L. Aerial Parts and Roots.

This work focuses on Cistus monspeliensis L. aerial parts (AP) and roots (R) extracts, investigating the anti-inflammatory and antioxidant potential of the two organs in comparison. At dosages between 1.56 and 6.25 µg/mL, both extracts showed a protective effect against LPS inflammatory stimulus on a macrophage cell line (RAW 264.7). Interestingly, only R was able to significantly reduce both IL-1ß and IL-6 mRNA gene expression in the presence of LPS. Moreover, the treatment of a neuroblastoma cell line (SH-SY5Y) with AP and R at 6.25 µg/mL increased the cell survival rate by nearly 20% after H2O2 insult. However, only R promoted mitochondria survival, exhibited a significantly higher production of ATP and a higher activity of the enzyme catalase than the control. Both AP and R had similar primary metabolites; in particular, they both contained 1-O-methyl-epi-inositol. Labdane and methoxylated flavonoids were the most characteristic compounds of AP, while R contained mainly catechins, gallic acid, and pyrogallol derivatives. Considering the importance of elemental composition in plants, the inorganic profile of AP and R was also investigated and compared. No potentially toxic elements, such as Pb, were detected in any sample.

Usefulness of the 1H NMR Multisuppression Approach for the Global Characterization of Monovarietal Extra-Virgin Olive Oils.

Extra-virgin olive oil (EVOO) is one of the most appreciated vegetable oils worldwide, but its high price makes it prone to suffer adulteration with lower quality oils. Therefore, it is important to have methodologies able to study EVOO composition as a whole in a simple and fast way, in order to guarantee its quality and safety. For this purpose, in this study, commercial samples of five Spanish olive cultivars (Arbequina, Arroniz, Cornicabra, Hojiblanca, Picual) were studied by Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy, using standard and multisuppression pulses. The aim was to explore the possibility of 1H NMR use to characterize in a single run and in a global way the composition of these monocultivar oils, regarding not only their main components (fatty acids supported on triglycerides) but also minor ones (squalene, sterols, diterpenic wax esters of phytol and geranylgeraniol, phenolic and secoiridoid derivatives, like tyrosol, hydroxytyrosol, oleacein, oleocanthal, and lignans, among others, and aldehydes). The use of univariate and multivariate statistical analyses confirmed the presence of compositional features that were specific to some olive varieties. The Arbequina and Arroniz oils showed the most characteristic features that allowed for clearly differentiating them from the others. In contrast, the discrimination between the Cornicabra, Hojiblanca and Picual oils was not so easily achieved.

NMR-based analysis of impact of siRNA mixing conditions on internal structure of siRNA-loaded LNP.

This study aimed to assess the applicability of solution-state 1H NMR for molecular-level characterization of siRNA-loaded lipid nanoparticles (LNP). Dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, MC3) was used as an ionizable lipid, and siRNA-loaded LNPs were prepared by pre-mixing and post-mixing methods. The pre-mixing method involved mixing an acidic solution containing siRNA with an ethanolic lipid solution using a microfluidic mixer. The pre-mixed LNP was prepared by dialyzing the mixed solution into the phosphate buffered saline (PBS, pH 7.4). The post-mixed LNP was prepared by mixing the siRNA solution with empty LNP in an acidic condition with and without ethanol, resulting in post-mixed LNP (A) and (B), respectively. Both pre-mixed and post-mixed LNPs formed LNP particles with an average diameter of approximately 50 nm. Moreover, the ratio of encapsulated siRNA to lipid content in each LNP particle remained constant regardless of the preparation method. However, small-angle X-ray scattering measurements indicated structural variations in the siRNA-MC3 stacked bilayer structure formed in the LNPs, depending on the preparation method. Solution-state 1H NMR analysis suggested that the siRNA was incorporated uniformly into the LNP core for pre-mixed LNP compared to post-mixed LNPs. In contrast, the post-mixed LNPs contained siRNA-empty regions with local enrichment of siRNA in the LNP core. This heterogeneity was more pronounced in post-mixed LNP (B) than in post-mixed LNP (A), suggesting that ethanol facilitated the homogeneous mixing of siRNA with LNP lipids. The silencing effect of each siRNA-loaded LNP was reduced in the order of pre-mixed LNP, post-mixed LNP (A), and post-mixed LNP (B). This suggested that the heterogeneity of the siRNA-loaded LNP could cause a reduction in the silencing effect of the incorporated siRNA inside LNPs. The present study highlighted that NMR-based characterization of siRNA-loaded LNP can reveal the molecular-level heterogeneity of siRNA-loaded LNP, which helps to optimize the preparation conditions of siRNA-loaded LNP formulations.

Effect of water content on gelatinization functionality of flour from sprouted sorghum.

Sorghum starch granules are encapsulated in a rigid protein matrix that prevents the granules from fully swelling and gelatinizing. Sprouting and subsequent drying treatment can affect the gelatinization properties of sorghum starch. This study aimed to evaluate the gelatinization properties of flours from unsprouted (US) and sprouted (S50, S40) sorghum dried at 50 °C (6h) and 40 °C (12h), respectively. Swelling power (Sp), thermal properties (DSC) and 1H molecular mobility and dynamics were evaluated at different water contents (38-91%). Sp increased with increasing water content, with S40 showing the lowest values, probably due to prolonged amylase activity and thus starch breakdown. Sprouting increased gelatinization temperatures; however, these differences disappeared for high water contents (82 and 91%). From a molecular point of view, sprouted samples showed a decrease in protons associated to the rigid protein matrix and starch structures. 1H CPMG results showed the presence of 4 populations at 38% water content. The evolution of the more mobile population with increasing water content supported the assignment of more mobile water fraction to this population. Sprouting decreased the mobility of populations in unheated samples, suggesting an increase in molecular bonds between flour biopolymers and water. After heating, however, increased molecular mobility in S40 indicated the formation of a weaker network between starch, protein, and water at the molecular level. These results suggest that post-sprouting drying treatment influences sorghum gelatinization, with potential modulation by water content. This study contributes to understanding the application of sprouted sorghum in foods with different moisture content.

Acute toxicity profiling of medicinal herb Ardisia elliptica leaf extract by conventional evaluations and proton nuclear magnetic resonance (NMR) metabolomics.

Background and aim: Interest in the safety of herbal medicine is growing rapidly regarding knowledge and challenges in natural products. Hence, this study aimed to reveal the toxicological profile of Ardisia elliptica, a traditional medicinal plant used in the treatment of various illnesses. Experimental procedure: Acute toxicity study was performed on female and male Sprague Dawley rats with a single oral administration of 2000 mg/kg BW of 70% ethanolic A. elliptica leaf extract, using a combination of conventional investigations and 1H-NMR-based metabolomics approaches. Results: Physical, hematological, biochemical, and histopathological assessments demonstrated the usual rat profile, with no mortality and delayed toxicity 14 days after administration. 1H NMR serum metabolomics depicted similar metabolites between normal and treated groups. Nevertheless, 1H NMR of urinary metabolomics revealed perturbation in carbohydrate, amino acid, and energy metabolism within 24h after extract administration, while no accumulation of toxic biomarkers in the collected biological fluids on Day 14. A minor gender-based difference revealed the influence of sex hormones and different energy expenditure on response to extract treatment. Conclusion: This study suggested that 2000 mg/kg BW of 70% ethanolic A. elliptica leaf extract is considered as safe for consumption and offered a comprehensive overview of the response of physiological and metabolic aspects applicable to food and herbal product development.