Using Ultra Performance Liquid Chromatography Research Articles

Identification and Evaluation of Diterpenoids from Glandular Trichome Secretions of Air/Sun-Cured Tobacco Germplasm Resources

To explore the multipurpose utilization of tobacco germplasm resources, 80 representative air/sun-cured tobacco germplasms were used as experimental materials to determine the composition and content of the diterpenoids of glandular trichome secretions using ultra-performance liquid chromatography (UPLC). Correlation analysis, cluster analysis, and principal component analysis (PCA) were used to comprehensively evaluate 80 air/sun-cured tobacco germplasms. The results are as follows: (1) 11 chromatographic peaks (Peak1–Peak11) were detected by UPLC, and the coefficient of variation of their contents ranged from 26.3 to 143%. (2) Correlation analysis showed that alpha-cembratriene-diol (α-CBT-diol) and beta-cembratriene-diol (β-CBT-diol) had the highest correlation coefficients (0.97), showing a significantly negative correlation with cis-abienol and a significantly positive correlation with the other diterpenoids. (3) PCA showed that the cumulative contribution rate of the three principal components was 75.70%, and the diterpenoid-rich germplasms were ranked in the following order: X40 (Maiduo) > X48 (Jianpingpiaoba) > X58 (Mianzhu) > X54 (Shifangpipaliu) > X63 (Xuejia5) > X41 (Tangpeng) > X29 (OLOR) > X73 (Criollo) > X44 (Tiebanqing) > X70 (Nicaragua Changxin). The diterpenoid content of X36 was the lowest. These results provide excellent germplasms for plant-based pesticides and fragrance compounds and genetic studies of the diterpenoid metabolism.

Comparative transcriptomic and plastid development analysis sheds light on the differential carotenoid accumulation in kiwifruit flesh.

Carotenoids are colorful lipophilic isoprenoids synthesized in all photosynthetic organisms which play roles in plant growth and development and provide numerous health benefits in the human diet (precursor of Vitamin A). The commercially popular kiwifruits are golden yellow-fleshed (Actinidia chinensis) and green fleshed (A. deliciosa) cultivars which have a high carotenoid concentration. Understanding the molecular mechanisms controlling the synthesis and sequestration of carotenoids in Actinidia species is key to increasing nutritional value of this crop via breeding. In this study we analyzed fruit with varying flesh color from three Actinidia species; orange-fleshed A. valvata (OF), yellow-fleshed A. polygama (YF) and green-fleshed A. arguta (GF). Microscopic analysis revealed that carotenoids accumulated in a crystalline form in YF and OF chromoplasts, with the size of crystals being bigger in OF compared to YF, which also contained globular substructures in the chromoplast. Metabolic profiles were investigated using ultra-performance liquid chromatography (UPLC), which showed that β-carotene was the predominant carotenoid in the OF and YF species, while lutein was the dominant carotenoid in the GF species. Global changes in gene expression were studied between OF and GF (both tetraploid) species using RNA-sequencing which showed higher expression levels of upstream carotenoid biosynthesis-related genes such as DXS, PSY, GGPPS, PDS, ZISO, and ZDS in OF species compared to GF. However, low expression of downstream pathway genes was observed in both species. Pathway regulatory genes (OR and OR-L), plastid morphology related genes (FIBRILLIN), chlorophyll degradation genes (SGR, SGR-L, RCCR, and NYC1) were upregulated in OF species compared to GF. This suggests chlorophyll degradation (primarily in the initial ripening stages) is accompanied by increased carotenoid production and localization in orange flesh tissue, a contrast from green flesh tissue. These results suggest a coordinated change in the carotenoid pathway, as well as changes in plastid type, are responsible for an orange phenotype in certain kiwifruit species.

Monitoring ascorbic acid oxidation with H2O2 and analyzing the food supplement product using parallel factor analysis model of kinetic UV absorption measurements

Ascorbic acid is a functional compound in maintaining the physiological processes of humans who acquire it from dietary sources, and also functions as an antioxidant. Therefore, the quantitation, and degradation of ascorbic acid in the food matrix and its interaction with coexisting components is a very important issue in terms of food safety and human health. Classical analytical methods may not give the expected results in the analysis of more advanced and complex food products obtained with the developing production technology. In this perspective, the parallel factor analysis (PARAFAC) model using kinetic UV absorption measurements was improved for the co-estimation of the kinetics and quantification of ascorbic acid in a food supplement product, which is a commercial effervescent preparation without requiring a prior separation step. The proposed three-way analysis strategy was validated and applied to the analysis of the relevant samples. For example, in the test samples, percent recovery and relative standard deviation were found to be 99.4 % and 1.36 %, respectively. The proposed chemometric strategy would be an alternative approach with a good selectivity for estimating the kinetics and quantification of ascorbic acid in a commercial supplementary product. The assay results of the PARAFAC model was checked by using ultra-performance liquid chromatography (UPLC).

Analysis of Racecadotril in Pharmaceutical Formulations Using Ultra Performance Liquid Chromatography (UPLC) Method

Analysis of Racecadotril in Pharmaceutical Formulations Using Ultra Performance Liquid Chromatography (UPLC) Method

#5260 PLASMA AND URINE METABOLOMIC PROFILES REVEAL POTENTIAL BIOMARKERS AND PROVIDE VALUABLE INSIGHTS INTO THE MECHANISMS OF MPO-ANCA-GN

Abstract Background and Aims Currently, the precise mechanism of myeloperoxidase- anti-neutrophil cytoplasmic antibody associated glomerulonephritis (MPO-ANCA-GN) remains elusive. And there is a lack of dependable biomarkers for evaluating disease activity, therapeutic resistance, and risk of relapse. We try to seek valuable biomarkers and explore the mechanism of MPO-ANCA-GN by metabolomics. Method Urine and plasma samples from 63 MPO-ANCA-GN, 31 lupus nephritis (LN), 15 IgA nephropathy (IgAN), and 15 minimal change disease (MCD) patients and 30 HC were collected for metabolomics analysis. The samples were analyzed using Ultra Performance Liquid Chromatography (UPLC) and Tandem mass spectrometry (MS/MS). The different groups of samples were compared using Partial Least Squares-Discriminant Analysis. A Receiver Operator Characteristic (ROC) curve was constructed to assess the performance of the metabolite model in identifying active renal vasculitis. KEGG annotation and enrichment analysis were performed for key pathways. Results All metabolites in urine and plasma samples including hydrophilic compounds and hydrophobic compounds were identified. Compared with the disease control (DC) and HC, we observed 79 significantly upregulated metabolites (VIP>1, FDR<0.05, FC>1.5) and 32 downregulated metabolites (VIP>1, FDR <0.05, FC<0.67) in the plasma samples of MPO-ANCA-GN patients. KEGG enrichment analysis suggested that these upregulated metabolites were mainly associated with phenylalanine metabolism and galactose metabolism and downregulated metabolites were mainly associated with bile secretion, steroid biosynthesis, and ovarian steroidogenesis. Compared with the DC and HC, we identified a total of 122 differential metabolites in the urine samples of MPO-ANCA-GN patients, 40 of which were significantly upregulated (VIP>1, FDR<0.05, FC>1.5) and 68 of which significantly were downregulated (VIP>1, FDR <0.05, FC<0.67). Subsequently, KEGG analysis revealed that these upregulated metabolites were mainly enriched in glycerolipid metabolism, lipid and atherosclerosis, cholesterol metabolism, insulin resistance, regulation of lipolysis in adipocytes, vitamin digestion and absorption, fat digestion and absorption (Corrected P < 0.05). Downregulated metabolites were mainly enriched in steroid hormone biosynthesis (Corrected P < 0.05). Nine MPO-ANCA-GN patients with paired plasma and six MPO-ANCA-GN patients with paired urine during the active and remission phase were included in the study. A total of 504 differential metabolites in plasma samples were identified between the active and remission phase based on VIP value >1, P < 0.05, and FC >1.5 or FC < 0.6. Of these, 425 metabolites were downregulated, while 79 lipid metabolites were upregulated in the active group. MPO-ANCA-GN patients were divided into two groups according to Mayo Clinic/Renal Pathology Society Chronicity Score: Minimal-mild group and Moderate-severe group. In plasma samples, our data showed that 3-Methoxysalicylic Acid, Carnitine C20:5, and isoxanthopterin presented good ability in distinguishing between the minimal-mild group and the moderate-severe group, with an AUC > 0.7. In urine samples, cyclohexylamine and arachidonic acid (AUC > 0.8) can distinguish the two groups well. Conclusion We identified many novel potential metabolic biomarkers in urine and plasma samples for evaluating disease activity, therapeutic resistance, and risk of relapse in patients with MPO-ANCA-GN.

Development of UPLC-UV-ELSD Method for Fatty Acid Profiling in Polysorbate 80 and Confirmation of the Presence of Conjugated Fatty Acids by Mass Spectrometry, UV Absorbance and Proton Nuclear Magnetic Resonance Spectroscopy

Polysorbate 80 (PS80), a chemical substance composed of sorbitol, ethylene glycol, and fatty acids, is commonly used in pharmaceutical drug products to stabilize formulations. However, recent studies have demonstrated that PS80 may hydrolyze over time and the released free fatty acids (FFAs) may lead to particle formation. Naming conventions of fatty acids in current pharmacopeia and in products’ certificates of analysis (CoA) of PS80 do not typically distinguish between isomeric species of fatty acids in PS80. Thus, methods to fully characterize the fatty acid species present in PS80 raw materials are needed to enhance quality control strategies of pharmaceuticals using PS80. Here, extended effort is taken to characterize fatty acids in hydrolyzed PS80 raw materials and elucidate the identities of isomeric fatty acid species. In this work, a method was developed and optimized for separation and detection of fatty acids in alkaline hydrolyzed PS80 raw materials using ultra performance liquid chromatography (UPLC) with ultra-violet (UV) detection and evaporative light scattering detection (ELSD). Fatty acids not specified in the current pharmacopeias were detected in PS80 raw material by the developed LC-UV-ELSD method including conjugated forms of linoleic and linolenic fatty acid species. Their identities were orthogonally confirmed by retention time agreement with analytical standards, accurate mass by high resolution mass spectrometry, UV absorbance, and proton nuclear magnetic resonance spectroscopy. The detected conjugated fatty acids are theoretically more hydrophobic and less soluble than their unconjugated counterparts and may increase the propensity of PS80 to form particles upon hydrolysis. This work highlights the need for better quality control of PS80 raw material, as it may eventually play a critical role in product quality of therapeutic proteins.

Determination of four aflatoxins in feeds by high throughput automated immunoaffinity magnetic beads purification-ultra performance liquid chromatography

Aflatoxin (AFT) is an extremely toxic and highly toxic carcinogenic substance. This is particularly problematic due to the risk of aflatoxin contamination in raw feed materials and products during production, transportation, and storage. In this study, immunoaffinity magnetic beads (IMBs) were prepared for the purification of four aflatoxins (aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2)). The aflatoxin contents were then determined rapidly and accurately using ultra performance liquid chromatography (UPLC). More specifically, the coupling ratio of magnetic beads (MBs) to the aflatoxin monoclonal antibody was initially optimized, wherein an MB volume of 1 mL and an antibody content of 2.0 mg was found to meet the purification requirements of this method. The magnetic properties of the MBs and the IMBs were then investigated using a vibrating sample magnetometer (VSM) at room temperature. As a result, the maximum saturation super magnetizations of the MBs and the IMBs were determined to be 28.61 and 23.22 emu/g, respectively, indicating that the saturation magnetization intensity of the IMBs was reduced by coupling with a non-magnetic antibody. However, the saturation magnetization intensity remained sufficiently high to permit magnetic separation from the solution. In addition, the appearance of the IMBs was examined using a biomicroscope, and it was clear that the magnetic cores were wrapped in agarose gel. Furthermore, the reaction time between the IMBs and the aflatoxins was investigated, and the optimal reaction time for meeting the purification requirements was determined to be 2 min. The stability of the IMBs was then evaluated under refrigerated storage conditions at 4 ℃. It was found that the prepared IMBs maintained a high aflatoxin enrichment capacity for at least eight months. Through the examination of three different extraction solutions, a mixture of acetonitrile and water (70∶30, v/v) was found to be optimal for the extraction of aflatoxins from the feed samples. Moreover, five sample dilutions and purification effects were also examined, and phosphate-buffered saline (containing 0.5% Tween-20) was selected as the preferred sample dilutant. With the optimized conditions, the effectiveness of using IMB for the purification of different feed samples was investigated. The resulting UPLC chromatogram showed no spurious peaks close to the target peaks, demonstrating a good purification performance. Following matrix spiking (5, 20, and 40 μg/kg, calculated based on AFB1) of the four feed samples (i. e., soybean meal, distillers dried grains with solubles, pig feed, and chicken feed), the spiked recoveries of the four aflatoxins ranged from 91.1% to 119.4% with a relative standard deviation (RSD) of <6.9%. In addition, the inter-day precision was 4.5% to 7.5%, and the method exhibited a good reproducibility. Subsequently, the developed method was used to detect AFB1 using reference materials. The test value was 18.6 μg/kg with an accuracy of 110.3%, thereby constituting satisfactory results. Upon testing 21 randomly purchased feed samples using this method, four of these samples contained AFB1, and the test results obtained using the developed method and stable isotope dilution LC-MS/MS were comparable. It was therefore apparent that the IMB purification method combined with UPLC analysis exhibited a good accuracy for aflatoxin determination. Thus, an automatic purification system was established to facilitate the operation and use of IMBs. This system was able to purify 24 samples simultaneously in 30 min. An IMB purification kit for was also designed and produced for aflatoxin detection in feed samples. The kit contained the sample dilutant, IMBs, the washing solution, and the eluent. After extraction of the feed sample, the extraction solution was added to the sample wells provided in the kit, and the purification system automatically completed the steps of aflatoxin enrichment, impurity washing, and elution of the target toxin. It should be noted that the purification process does not require the operator to manually add the solution, thereby simplifying operation. Overall, the purification method established in this study achieved the high-throughput and automatic purification of the four aflatoxins in feed samples.

Bifidobacterium longum CCFM1077 Attenuates Hyperlipidemia by Modulating the Gut Microbiota Composition and Fecal Metabolites: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

An increasing number of studies have indicated that gut microbiota and its metabolites are crucial in the development of hyperlipidemia. Bifidobacterium longum (B. longum) CCFM1077 has been shown to have lipid-lowering effects in animals. This study aimed to evaluate the potential of B. longum CCFM1077 in lowering the lipid levels in patients with hyperlipidemia and investigate the effect of this bacterium on serum lipid abnormalities, gut microbiota, and fecal metabolites in these patients. This study was a six-week, randomized, double-blind, and placebo-controlled pilot clinical trial. Subjects with hyperlipidemia (N = 62) were randomly assigned to receive placebo (N = 31) or B. longum CCFM1077 (1 × 1010 colony-forming units (CFUs) per day; N = 31). Serum lipid levels including total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), total triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) were examined at the baseline and interventional endpoints. Changes in the gut microbiota composition and diversity were measured based on 16S ribosomal RNA (rRNA) sequencing of the V3–V4 region at the end of the intervention period. Non-targeted metabolomics of the feces was performed using ultra-performance liquid chromatography (UPLC)-Q-Exactive Orbitrap/mass spectrometer. Oral administration of B. longum CCFM1077 for six weeks significantly decreased the serum levels of TC (p < 0.01) and LDL-C (p < 0.01) in patients with hyperlipidemia. B. longum CCFM1077 treatment markedly increased gut microbiota diversity and the relative abundance of anti-obesity-related genera, including Lactobacillus, Butyricicoccus, Bifidobacterium, and Blautia, whereas it decreased the relative abundance of obesity-related genera, including Alistipes, Megamonas, and Catenibacterium. Additionally, some key metabolites (bile acids (BAs), biotin, and caffeine) and their corresponding metabolic pathways (primary BA biosynthesis, and taurine and hypotaurine, biotin, purine, and caffeine metabolisms) were enriched by B. longum CCFM1077, and thus it may lower lipid levels. B. longum CCFM1077 is a probiotic strain with the potential to lower serum TC and LDL-C levels patients with hyperlipidemia. The underlying mechanism may be related to the increased abundance of anti-obesity-related genera and fecal metabolites. These findings provide a foundation for future clinical applications of lipid-lowering probiotics in managing individuals with hyperlipidemia.

Assessment of Purity, Functionality, Stability, and Lipid Composition of Cyclofos-nAChR-Detergent Complexes from Torpedo californica Using Lipid Matrix and Macroscopic Electrophysiology

The main objective of the present study was to find detergents that can maintain the functionality and stability of the Torpedo californica nicotinic acetylcholine receptor (Tc-nAChR). We examined the functionality, stability, and purity analysis of affinity-purified Tc-nAChR solubilized in detergents from the Cyclofos (CF) family [cyclofoscholine 4 (CF-4), cyclofoscholine 6 (CF-6), and cyclofloscholine 7 (CF-7)]. The functionality of the CF-Tc-nAChR-detergent complex (DC) was evaluated using the Two Electrode Voltage Clamp (TEVC) method. To assess stability, we used the florescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) methodology. We also performed a lipidomic analysis using Ultra-Performance Liquid Chromatography (UPLC) coupled to electrospray ionization mass spectrometry (ESI–MS/MS) to evaluate the lipid composition of the CF-Tc-nAChR-DCs. The CF-4-Tc-nAChR-DC displayed a robust macroscopic current (− 200 ± 60 nA); however, the CF-6-Tc-nAChR-DC and CF-7-Tc-nAChR-DC displayed significant reductions in the macroscopic currents. The CF-6-Tc-nAChR and CF-4-Tc-nAChR displayed higher fractional florescence recovery. Addition of cholesterol produced a mild enhancement of the mobile fraction on the CF-6-Tc-nAChR. The lipidomic analysis revealed that the CF-7-Tc-nAChR-DC displayed substantial delipidation, consistent with the lack of stability and functional response of this complex. Although the CF-6-nAChR-DC complex retained the largest amount of lipids, it showed a loss of six lipid species [SM(d16:1/18:0); PC(18:2/14:1); PC(14:0/18:1); PC(16:0/18:1); PC(20:5/20:4), and PC(20:4/20:5)] that are present in the CF-4-nAChR-DC. Overall, the CF-4-nAChR displayed robust functionality, significant stability, and the best purity among the three CF detergents; therefore, CF-4 is a suitable candidate to prepare Tc-nAChR crystals for structural studies.Graphical abstract

Study on the Composition and Mechanism of Santiao Decoction in Treating Insomnia Based on UPLC and Network Pharmacology and Molecular Docking Technology.

To identify the chemical components of Santiao Decoction (STD) using Ultra Performance Liquid Chromatography (UPLC) and to conduct a network pharmacological study of STD for the treatment of insomnia based on this technique. An ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm) was used to identify the chemical components of STD by relative molecular weight, mass spectrometry information, and comparison with the control. The active ingredients of the formula and their corresponding gene targets and targets for insomnia were retrieved from several databases, and a visual network diagram of "drug-active ingredient-target-disease" was constructed using Cytoscape 3.8.2 software, and GO functional annotation and KEGG pathway enrichment analysis were performed using various databases such as DAVID. Five active ingredients were identified from STD by UPLC technique, 268 active ingredients of STD were screened from the TCMSP database, and 109 genes related to STD for insomnia were screened by network pharmacology, among which IL6, MMP9, VEGFA, IL10, CCL2 may be the key targets of STD for insomnia. KEGG pathway analysis showed that STD acts on membrane rafts, plasma membrane micro-regions, and other related pathways, such as Toll-like receptor signaling pathway, prolactin signaling pathway, dopaminergic synapse, relaxin signaling pathway, ErbB signaling pathway, steroid hormone biosynthesis and NF-kappa B signaling pathway for regulation. The active ingredients in STD, such as (+)-catechin, Swertisin, quercetin, baicalein, and wogonin, may act on IL6, CCL2, VEGFA, MMP9, and other targets to regulate Toll-like receptor signaling pathway, ErbB signaling pathway, NF-kappa B and other signaling pathways, and exert certain therapeutic effects on insomnia, which provide a reference and basis for further research on the mechanism of action of STD.

Modulation of the biological network of lumbar spinal stenosis by Tongdu Huoxue Decoction based on clinical metabolomics.

Objective: To explore the clinical efficacy and metabolic mechanism of Tongdu Huoxue Decoction (THD) in treating lumbar spinal stenosis (LSS). Methods: A total of 40 LSS patients and 20 healthy participants were recruited from January 2022 to June 2022. The patients' pre- and post-treatment visual analogue scale (VAS) and Japanese Orthopaedic Association (JOA) scores were recorded. ELISA kits were used to assess pre- and post-treatment levels of serum Interleukin-1beta (IL-1β), Alpha tumour necrosis factor (TNF-α) and prostaglandin E2 (PGE2). Finally, the patients' pre- and post-treatment and healthy human sera were subjected to extensively targeted metabolomics using Ultra Performance Liquid Chromatography (UPLC) to identify potential differential metabolites and metabolic pathways using multivariate statistical analysis. Results: Compared to the pre-treatment (group A), the patients' VAS scores decreased significantly (p < 0.05), while JOA scores increased significantly (p < 0.05) post-treatment (group B), indicating that THD could effectively improve the pain and lumbar spine function of LSS patients. Moreover, THD could effectively inhibit the expression of IL-1β, TNF-α and PGE2-associated inflammatory factors in serum. Regarding metabolomics, the levels of 41 differential metabolites were significantly different in the normal group (group NC) compared to group A, and those were significantly restored after treatment with THD, including chenodeoxycholic acid 3-sulfate, taurohyodeoxycholic acid, 3,5-Dihydroxy-4-methoxybenzoic acid, pinocembrin. These biomarkers are mainly involved in purine metabolism, steroid hormone biosynthesis and amino acid metabolism. Conclusion: This clinical trial demonstrated that THD is effective in improving pain, lumbar spine function and serum levels of inflammation in patients with LSS. Moreover, its mechanism of action is related to the regulation of purine metabolism, steroid hormone biosynthesis and the expression of key biomarkers in the metabolic pathway of amino acid metabolism.

Method validation for quantitative analyzing aflatoxin productivity in Aspergillus sp. isolated from soybean paste

Non-aflatoxigenic Aspergillus oryzae and aflatoxigenic A. flavus cannot be clearly identified by partial sequencing of the internal transcribed spacer (ITS) and 18S ribosomal ribonucleic acid (18S rRNA) regions. This study aimed to compare the accuracy among three aflatoxin detection methods using ultra-performance liquid chromatography (UPLC), high-performance liquid chromatography (HPLC), and an enzyme-linked immunosorbent assay (ELISA) kit and to select the non-aflatoxigenic Aspergillus sp. isolated from soybean paste. All analytical methods were suitable according to the international standards of Codex Alimentarius FAO-WHO (CODEX) or the Ministry of Food and Drug Safety (MFDS). UPLC exhibited the best of limit of detection (LOD) and limit of quantification (LOQ). Based on UPLC, HPLC, and the ELISA kit assay, the P5 and P7 strains isolated from soybean paste had 1,663.49, 1,468.12, and ⟩20 μg/kg and 1,470.08, 1,056.73, and ⟩20 μg/kg, respectively, detected and re-identified as A. flavus. In contrast, the P3 and P4 strains (A. oryzae), which were detected below the MFDS standards in all assays, were confirmed as non-aflatoxigenic fungi. Among the methods evaluated for quantitative analysis of aflatoxin, UPLC and HPLC are superior in terms of accuracy, and the ELISA kit rapidly detects low concentrations of aflatoxin. Furthermore, this study demonstrates that any Aspergillus sp. isolated for use as a fermentation starter should be analyzed for potential aflatoxin production using UPLC and HPLC for accurate quantitative analysis or ELISA for the rapid detection of low-level concentrations of aflatoxin.

Molecular mechanisms of the Guizhi decoction on osteoarthritis based on an integrated network pharmacology and RNA sequencing approach with experimental validation.

Background: Our aim was to determine the potential pharmacological mechanisms of the Guizhi decoction (GZD) in the treatment of osteoarthritis (OA) through an integrated approach of network pharmacological analyses, RNA sequencing (RNA-seq), and experimental validation. Methods: The quality control and identification of bioactive compounds of the GZD were carried out by using ultra-performance liquid chromatography (UPLC), and their OA-related genes were identified through overlapping traditional Chinese medicine systems pharmacology database (TCMSP), DrugBank and SEA Search Server databases, and GeneCards. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were implemented after constructing the component-target network. RNA-seq was used to screen differentially expressed genes (DEGs) under intervention conditions with and without the GZD in vitro. The crossover signaling pathways between RNA-seq and network pharmacology were then analyzed. Accordingly, protein-protein interaction (PPI) networks, GO, and KEGG analysis were performed using the Cytoscape, STRING, or DAVID database. The OA rat model was established to further verify the pharmacological effects in vivo. Hematoxylin-eosin (H&E) and safranin O/fast green (S-O) staining were used to grade the histopathological features of the cartilage. We verified the mRNA and protein expressions of the key targets related to the TNF signaling pathways in vivo and in vitro by qPCR, Western blotting (WB), and immunofluorescence assay. In addition, we also detected inflammatory cytokines in the rat serum by Luminex liquid suspension chip, which included tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β). Results: Eighteen compounds and 373 targets of the GZD were identified. A total of 2,356 OA-related genes were obtained from the GeneCards database. A total of three hub active ingredients of quercetin, kaempferol, and beta-sitosterol were determined, while 166 target genes associated with OA were finally overlapped. The RNA-seq analysis revealed 1,426 DEGs. In the KEGG intersection between network pharmacology and RNA-seq analysis, the closest screening relevant to GZD treatment was the TNF signaling pathway, of which TNF, IL-6, and IL-1β were classified as hub genes. In consistent, H&E and S-O staining of the rat model showed that GZD could attenuate cartilage degradation. When compared with the OA group in vivo and in vitro, the mRNA levels of TNF-α, IL-1β, IL-6, matrix metalloproteinase 3 (MMP3), and matrix metalloproteinase 9 (MMP9) were all downregulated in the GZD group (all p < 0.05). The expression levels of anabolic proteins (Col2α1 and SOX9) were all higher in the GZD group than in the OA group (p < 0.05), while the expression levels of the catabolic proteins (MMP9 and COX-2) and TNF-α in the GZD group were significantly lower than those in the OA group (p < 0.05). In addition, the expression levels of TNF, IL-6, and IL-1β were upregulated in the OA group, while the GZD group prevented such aberrations (p < 0.01). Conclusion: The present study reveals that the mechanism of the GZD against OA may be related to the regulation of the TNF signaling pathway and inhibition of inflammatory response.

Changes in Lipidomics, Metabolomics, and the Gut Microbiota in CDAA-Induced NAFLD Mice after Polyene Phosphatidylcholine Treatment.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in most parts of the world. Although there is no first-line drug approved for the treatment of NAFLD, polyene phosphatidylcholine (PPC) is used by clinicians to treat NAFLD patients. This study aimed to evaluate the efficacy of PPC on a mice model of NAFLD, and to study the PPC's mechanism of action. The mice were fed a choline-deficient, L-amino acid-defined (CDAA) diet to induce NAFLD and were subsequently treated with PPC. The treatment effects were evaluated by the liver index, histopathological examination, and routine blood chemistry analyses. Lipidomics and metabolomics analyses of 54 samples were carried out using ultraperformance liquid chromatography (UPLC) coupled to a mass spectrometer to select for changes in metabolites associated with CDAA diet-induced NAFLD and the effects of PPC treatment. The intestinal flora of mice were extracted for gene sequencing to find differences before and after the induction of NAFLD and PPC treatment. PPC significantly improved the CDAA diet-induced NAFLD condition in mice. A total of 19 metabolites including 5 polar metabolites and 14 lipids showed marked changes. In addition, significant differences in the abundance of Lactobacillus were associated with NAFLD. We inferred that the protective therapeutic effect of PPC on the liver was related to the supplement of phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin (PC, LPC, and SM, resectively) and acylcarnitine metabolism. This study developed a methodology for exploring the pathogenesis of NAFLD and can be extended to other therapeutic agents for treating NAFLD.

Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1)

Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (kcat/Km) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b5 produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that –as in human CYP17A1–these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b5 protein in the lyase reactions.

The growth characteristics and lignans contents of Schisandra chinensis fruits from different cultivation regions

We developed and validated an optimized method for quantifying lignans using ultra-performance liquid chromatography (UPLC) and performs correlation analysis of growth characteristics and contents of lignans. The methods for determining lignans were validated by measuring the linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision using UPLC. All calibration curves showed good linearity (r2 ≥ 0.9998) within the tested ranges. The limit of detection (LOD) and limit of quantification (LOQ) were 0.003–0.02 µg/mL and 0.01–0.07 µg/mL, respectively. The precision of analysis was less than 3%. The recoveries of quantified compounds ranged from 98.4 to 101.68%. Growth characteristics of fruits were negatively correlated with content of total marker compounds. The results of this study can be used to quality-control of S. chinensis fruits used as medicinal raw materials.

Effect of Grinding Process Parameters and Storage Time on Extraction of Antioxidants from Ginger and Nutmeg.

The aim of this study was to optimize the grinding process parameters (mesh size of grinder sieve (X1), the peripheral velocity of the grinding wheels (X2)), and the storage time (X3) of ground ginger rhizome and nutmeg to obtain ethanol and ethanol-water extracts with improved antioxidant properties. The optimal conditions were estimated using response surface methodology (RSM) based on a three-variable Box-Behnken design (BBD) in order to maximize the antioxidant capacity (AC) determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) methods, and the total phenolic content (TPC) was determined by the Folin-Ciocalteu (F-C) method in spice extracts. Additionally, the phenolic acid profiles in extracts from optimized conditions were analyzed using ultra-performance liquid chromatography (UPLC). It was found that the optimal preparation conditions for antioxidant extraction were dependent on the spice source and solvent type. The best antioxidant properties in nutmeg extracts were achieved for X1 = 1.0 mm, X2 = 40-41 Hz and X3 = 7 days, whereas the optimized parameters for ginger extracts were more varied (1.0-2.0 mm, 43-50 Hz and 1-9 days, respectively). The ginger extracts contained 1.5-1.8 times more phenolic acids, and vanillic, ferulic, gallic, and p-OH-benzoic acids were dominant. In contrast, the nutmeg extracts were rich in protocatechuic, vanillic, and ferulic acids.

Bacillus thuringiensis CHGP12 uses a multifaceted approach for the suppression of Fusarium oxysporum f. sp. ciceris and to enhance the biomass of chickpea plants.

Bacillus species synthesize antifungal lipopeptides (LPs) making them a sustainable and eco-friendly management option to combat Fusarium wilt of chickpea. In this study, 18 endophytic Bacillus strains were assessed for their antifungal activity against Fusarium oxysporum f. sp. ciceris (FOC) associated with Fusarium wilt of chickpea. Among them, 13 strains produced significant inhibition zones in a direct antifungal assay while five strains failed to produce the inhibition of FOC. Bacillus thuringiensis CHGP12 exhibited the highest inhibition 3.45 cm of FOC. The LPs extracted from CHGP12 showed significant inhibition of the pathogen. Liquid chromatography-mass spectrometry (LC-MS) analysis confirmed that CHGP12 possessed the ability to produce fengycin, surfactin, iturin, bacillaene, bacillibactin, plantazolicin, and bacilysin. In an in vitro qualitative assay CHGP12 exhibited the ability to produce lipase, amylase, cellulase, protease, siderophores, and indole 3-acetic acid (IAA). IAA and gibberellic acid (GA) were quantified using ultra-performance liquid chromatography (UPLC) with 370 and 770 ng mL-1 concentrations of IAA and GA respectively. Furthermore, the disease severity showed a 40% decrease over control in CHGP12 treated plants compared to the control in a glasshouse experiment. Moreover, CHGP12 also exhibited a significant increase in total biomass of the plants namely, root and shoot growth parameters, stomatal conductance, and photosynthesis rate. In conclusion, our findings suggest that B. thuringiensis CHGP12 is a promising strain with high antagonistic and growth-promoting potential against Fusarium wilt of chickpea. © 2022 Society of Chemical Industry.

Comparative Studies on a Standardized Subfraction of Red Onion Peel Ethanolic Extract (Plant Substance), Quercetin (Pure Compound), and Their Cell Mechanism and Metabolism on MDA-MB-231.

This study indicates the presence of quercetin in subfraction F1 and the standardized value of F1 derived from research using ultra-performance liquid chromatography (UPLC) and AlCl3 colorimetric assays, which further proved that both F1 and quercetin are potential growth inhibitors in MDA-MB-231 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In the process, staining of F1-treated cells with annexin/propidium iodide (PI) reduced cell proliferation and induced only S and G2 phases of cell cycle arrest in the treated cells by flow cytometry. Quercetin reduced cell proliferation by inducing apoptosis and S phase arrest. The 5′-bromo-2′-deoxyuridine (BrdU) incorporation of DNA synthesis in MDA-MB-231 cells was also inhibited after F1 and quercetin treatments. F1 and quercetin induced CYP1A1 and CYP1B1 gene expression, but only F1 induced CYP2S1 gene expression in the treated cells. Both F1 and quercetin inhibited the proliferation of MDA-MB-231 cells in different ways, but F1 is likely a better potential anticancer agent derived from the green approach towards breast cancer treatment.

Dominant changes in the breast muscle lipid profiles of broiler chickens with wooden breast syndrome revealed by lipidomics analyses

BackgroundChicken is the most consumed meat worldwide and the industry has been facing challenging myopathies. Wooden breast (WB), which is often accompanied by white striping (WS), is a serious myopathy adversely affecting meat quality of breast muscles. The underlying lipid metabolic mechanism of WB affected broilers is not fully understood.ResultsA total of 150 chickens of a white-feathered, fast-growing pure line were raised and used for the selection of WB, WB + WS and control chickens. The lipids of the breast muscle, liver, and serum from different chickens were extracted and measured using ultra performance liquid chromatography (UPLC) plus Q-Exactive Orbitrap tandem mass spectrometry. In the breast, 560 lipid molecules were identified. Compared to controls, 225/225 of 560 lipid molecules (40.2%) were identified with differential abundance (DA), including 92/100 significantly increased neutral lipids and 107/98 decreased phospholipids in the WB/WB + WS groups, respectively. The content of monounsaturated fatty acids (MUFA) was significantly higher, and the polyunsaturated fatty acids (PUFA) and saturated fatty acids (SFA) were significantly lower in the affected breasts. In the liver, 434 lipid molecules were identified, and 39/61 DA lipid molecules (6.7%/14.1%) were detected in the WB and WB + WS groups, respectively. In the serum, a total of 529 lipid molecules were identified and 4/44 DA lipid molecules (0.8%/8.3%) were detected in WB and WB + WS group, respectively. Compared to controls, the content of MUFAs in the serum and breast of the WB + WS group were both significantly increased, and the content of SFAs in two tissues were both significantly decreased. Only five lipid molecules were consistently increased in both liver and serum in WB + WS group.ConclusionsWe have found for the first time that the dominant lipid profile alterations occurred in the affected breast muscle. The relative abundance of 40.2% of lipid molecules were changed and is characteristic of increased neutral lipids and decreased phospholipids in the affected breasts. Minor changes of lipid profiles in the liver and serum of the affected groups were founded. Comprehensive analysis of body lipid metabolism indicated that the abnormal lipid profile of WB breast may be independent of the liver metabolism.