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Int. j. morphol ; 41(1): 308-318, feb. 2023. ilus, tab, graf
Article in English | LILACS | ID: biblio-1430503


SUMMARY: Gastrin plays a vital role in the development and progression of gastric cancer (GC). Its expression is up-regulated in GC tissues and several GC cell lines. Yet, the underlying mechanism remains to be investigated. Here, we aim to investigate the role and mechanism of gastrin in GC proliferation. Gastrin-overexpressing GC cell model was constructed using SGC7901 cells. Then the differentially expressed proteins were identified by iTRAQ analysis. Next, we use flow cytometry and immunofluorescence to study the effect of gastrin on the mitochondrial potential and mitochondria-derived ROS production. Finally, we studied the underlying mechanism of gastrin regulating mitochondrial function using Co-IP, mass spectrometry and immunofluorescence. Overexpression of gastrin promoted GC cell proliferation in vitro and in vivo. A total of 173 proteins were expressed differently between the controls and gastrin- overexpression cells and most of these proteins were involved in tumorigenesis and cell proliferation. Among them, Cox17, Cox5B and ATP5J that were all localized to the mitochondrial respiratory chain were down-regulated in gastrin-overexpression cells. Furthermore, gastrin overexpression led to mitochondrial potential decrease and mitochondria-derived ROS increase. Additionally, gastrin-induced ROS generation resulted in the inhibition of cell apoptosis via activating NF-kB, inhibiting Bax expression and promoting Bcl-2 expression. Finally, we found gastrin interacted with mitochondrial membrane protein Annexin A2 using Co-IP and mass spectrometry. Overexpr ession of gastrin inhibits GC cell apoptosis by inducing mitochondrial dysfunction through interacting with mitochondrial protein Annexin A2, then up-regulating ROS production to activate NF-kB and further leading to Bax/Bcl-2 ratio decrease.

La gastrina juega un papel vital en el desarrollo y progresión del cáncer gástrico (CG). Su expresión está regulada al alza en tejidos de CG y en varias líneas celulares de CG. Sin embargo, el mecanismo subyacente aun no se ha investigado. El objetivo de este estudio fue investigar el papel y el mecanismo de la gastrina en la proliferación de CG. El modelo de células CG que sobre expresan gastrina se construyó usando células SGC7901. Luego, las proteínas expresadas diferencialmente se identificaron mediante análisis iTRAQ. A continuación, utilizamos la citometría de flujo y la inmunofluorescencia para estudiar el efecto de la gastrina en el potencial mitocondrial y la producción de ROS derivada de las mitocondrias. Finalmente, estudiamos el mecanismo subyacente de la gastrina que regula la función mitocondrial utilizando Co-IP, espectrometría de masas e inmunofluorescencia. La sobreexpresión de gastrina promovió la proliferación de células CG in vitro e in vivo. Un total de 173 proteínas se expresaron de manera diferente entre los controles y las células con sobreexpresión de gastrina y la mayoría de estas proteínas estaban implicadas en la tumorigenesis y la proliferación celular. Entre estas, Cox17, Cox5B y ATP5J, todas localizadas en la cadena respiratoria mitocondrial, estaban reguladas a la baja en las células con sobreexpresión de gastrina. Además, la sobreexpresión de gastrina provocó una disminución del potencial mitocondrial y un aumento de las ROS derivadas de las mitocondrias. Por otra parte, la generación de ROS inducida por gastrina resultó en la inhibición de la apoptosis celular mediante la activación de NF-kB, inhibiendo la expresión de Bax y promoviendo la expresión de Bcl-2. Finalmente, encontramos que la gastrina interactuaba con la proteína de membrana mitocondrial Anexina A2 usando Co-IP y espectrometría de masas. La sobreexpresión de gastrina inhibe la apoptosis de las células CG al inducir la disfunción mitocondrial a través de la interacción con la proteína mitocondrial Anexina A2, luego regula el aumento de la producción de ROS para activar NF-kB y conduce aún más a la disminución de la relación Bax/Bcl-2.

Animals , Mice , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Gastrins/metabolism , Annexin A2/metabolism , Mitochondria/pathology , Mass Spectrometry , NF-kappa B , Fluorescent Antibody Technique , Reactive Oxygen Species , Apoptosis , Cell Line, Tumor , Immunoprecipitation , Cell Proliferation , Carcinogenesis , Flow Cytometry
Braz. J. Pharm. Sci. (Online) ; 59: e21283, 2023. tab, graf
Article in English | LILACS | ID: biblio-1439509


Abstract The anecdotal use of Alternanthera sessilis L. as a relief for diabetes has been known in the Philippines for generations, and antidiabetic activity of similar varieties in other countries is likewise documented. However, the compounds responsible for this activity remain unclear. This study aims to isolate the anti-hyperglycemic fraction of local A. sessilis leaves and identify the compounds in this fraction. Methanol extract of A. sessilis leaves and its hexane, ethyl acetate (ASE), and water fractions were administered to alloxan-induced diabetic mice. ASE (250mg/kg) had the highest anti-hyperglycemic activity at 6-h post-treatment (25.81%±12.72%), with almost similar blood glucose reduction rate as metformin (30.13±3.75%, p=0.767). Repeated fractionation employing chromatographic separation techniques followed by in vivo anti-hyperglycemic assay yielded partially purified subfractions. A. sessilis ethyl acetate subfraction 4-2 (100mg/kg) displayed remarkable suppression of blood glucose rise in diabetic mice at 6-h post-treatment (26.45±3.75%, p<0.0001), with comparable activity with metformin (100mg/kg, 27.87±5.65%, p=0.652). Liquid chromatography/mass spectrometry showed eight distinct peaks, with four peaks annotated via the Traditional Chinese Medicine library and custom library for A. sessilis. Among these, luteolin, apigenin, ononin, and sophorabioside were identified as putative compounds responsible for the anti-hyperglycemic activity. This result provided basis for the reported anecdotal claims and potential utility of the local variety of A. sessilis leaves as sources of anti-hyperglycemic agents

Animals , Male , Female , Mice , Mass Spectrometry/methods , Biological Assay/methods , Plant Leaves/classification , Amaranthaceae/adverse effects , Chromatography, Liquid/methods , Apigenin/agonists
Braz. J. Pharm. Sci. (Online) ; 59: e21726, 2023. tab, graf
Article in English | LILACS | ID: biblio-1439500


Abstract Pterocarpus santalinoides is used in Nigerian ethnomedicine to treat diabetes mellitus. This study aimed to establish the antidiabetic property of the plant, and isolate and characterize its active principle. Dried and pulverized leaves (500 g) of P. santalinoides were extracted with 1.8 L of 80 % hydromethanol by cold maceration. The dried extract (10 g) was partitioned into n-hexane, ethyl acetate (EtOAc), n-butanol, and water. Antidiabetic activitiy-guided isolation by column chromatographic separation of the EtOAc soluble and purification of the sub-fractions by repeated preparative thin layer chromatography (pTLC) yielded a C-glycosyl flavonoid, identified as isovitexin. The chemical structure was elucidated based on high-resolution mass spectroscopy, 1D, and 2D nuclear magnetic resonance spectroscopic analyses. Alloxan-induced diabetic rat model was adopted for antidiabetic screening. The extract of P. santalinoides (100-200 mg/kg), fraction F4 (50 mg/kg), sub-fraction F4.3 (10 mg/kg), and the semi-purified compound F4.3.2 (5 mg/kg) significantly (p<0.05) reduced the fasting blood glucose of alloxan-induced diabetic rats, causing 48.4, 69.4, 57.7 and 64.5 % antidiabetic activity respectively, compared with > 68 % recorded in glibenclamide (2 mg/kg) control. These results reveal that isovitexin is the antidiabetic principle in P. santalinoides

Animals , Male , Rats , Plant Extracts/analysis , Pterocarpus/adverse effects , Hypoglycemic Agents/analysis , Mass Spectrometry/methods , Flavonoids/pharmacology , Magnetic Resonance Spectroscopy/methods , Chromatography, Thin Layer/methods , Diabetes Mellitus/pathology , Acetates/pharmacology
Braz. J. Pharm. Sci. (Online) ; 59: e23017, 2023. tab, graf
Article in English | LILACS | ID: biblio-1505848


Abstract Infusion solutions must be stable from the production stage until the infusion stage. Some infusion fluids contain degradation products, known as advanced glycation end products (AGEs); however, it is unknown whether AGEs exist in parenteral nutrition solutions. We aimed to investigate this question and test the effect of infusion conditions on AGE formation in parenteral nutrition solution. Nine parenteral nutrition solutions were supplied by the pharmacy with which we collaborated. To simulate the infusion conditions, the solutions were held in a patient room with standard lighting and temperature for 24 hours. Samples were taken at the beginning (group A) and the end (24th hour, group B) of the infusion period. The degradation products were 3-deoxyglucosone, pentosidine, N-carboxymethyl lysine, and 4-hydroxynonenal, which we investigated by high-performance liquid chromatography-mass spectrometry (LC-MS) and Q-TOF LC/MS methods. Two of four degradation products, 4-hydroxynonenal and N-carboxymethyl lysine, were detected in all samples, and Group B had higher levels of both compounds compared to Group A, who showed that the quantities of these compounds increased in room conditions over time. The increase was significant for 4-hydroxynonenal (p=0.03), but not for N-carboxymethyl lysine (p=0.23). Moreover, we detected in the parenteral nutrition solutions a compound that could have been 4-hydroxy-2-butynal or furanone

Parenteral Nutrition/adverse effects , Glycation End Products, Advanced/analysis , Parenteral Nutrition Solutions/administration & dosage , Pharmacy/classification , Mass Spectrometry/methods , Patients' Rooms/classification , Lighting/classification , Chromatography, High Pressure Liquid/methods
Braz. J. Pharm. Sci. (Online) ; 59: e21626, 2023. tab, graf
Article in English | LILACS | ID: biblio-1429969


Abstract n our study, we aimed to validate a method based on liquid chromatography-mass spectrometry (LC-MS) to quantify spironolactone (SPI) and its active metabolite canrenone (CAN) simultaneously in plasma samples to support in vivo experiments. Compounds were separated by using a C18 column with the isocratic elution of a mobile phase composed of 0.1% (v/v) formic acid in methanol-water (60:40 v/v) at a flow rate of 0.4 mL min−1. SPI and CAN were detected in na electrospray interface operating in a positive ionization mode and quantified using the selective ion mode monitoring of mass-charge ratios (m/z) of 439.0 for SPI and 363.1 for CAN. After calculating the matrix effect using theoretical equations, we observed the strong interference of plasma in the equipment-generated signal, which required creating analytical curves using the matrix as a solvent. The method was nevertheless linear (r 2 > 0.999) in a concentration range of 0.4-5.0 µg mL−1, as well as precise, with a coefficient of variation less than 5%. SPI's and CAN's recovery rates from the plasma ranged from 87.4% to 112.1%, while their limits of detection (i.e., 0.07 µg mL−1 and 0.03 µg mL−1, respectively) and quantification (i.e., 0.20 µg mL−1 and 0.08 µg mL−1, respectively) in the presence of plasma contaminants were low. Therefore, the bioanalytical method seems to be feasible for quantifying SPI and CAN in plasma

Plasma , Mass Spectrometry/methods , Spironolactone/analysis , Canrenone/analysis , Chromatography, Liquid/methods , Pharmacokinetics , Androgen Antagonists/adverse effects
São Paulo; s.n; s.n; 2023. 81 p. graf, tab.
Thesis in Portuguese | LILACS | ID: biblio-1437408


Com base nas perturbações fosfoproteômicas de moléculas associadas ao ciclo celular em células infectadas pelo coronavírus causador da síndrome respiratória aguda grave (SARSCoV)-2, a hipótese de inibidores do ciclo celular como uma terapia potencial para a doença de coronavírus 2019 (COVID-19) foi proposta. No entanto, o cenário das alterações do ciclo celular em COVID-19 permanece inexplorado. Aqui, realizamos uma análise integrativa de sistemas imunológicos de proteoma publicamente disponível (espectrometria de massa) e dados de transcriptoma (sequenciamento de RNA em massa e de célula única [scRNAseq]), com o objetivo de caracterizar mudanças globais na assinatura do ciclo celular de pacientes com COVID-19. Além de módulos de co-expressão de genes significativos enriquecidos associados ao ciclo celular, encontramos uma rede interconectada de proteínas diferencialmente expressas associadas ao ciclo celular (DEPs) e genes (DEGs) integrando dados moleculares de 1.480 indivíduos (974 pacientes infectados por SARS-CoV-2 e 506 controles [controles saudáveis ou indivíduos com outras doenças respiratórias]). Entre esses DEPs e DEGs estão várias ciclinas (CCNs), ciclo de divisão celular (CDCs), quinases dependentes de ciclinas (CDKs) e proteínas de manutenção de minicromossomos (MCMs). Embora os pacientes com COVID-19 compartilhem parcialmente o padrão de expressão de algumas moléculas associadas ao ciclo celular com outras doenças respiratórias, eles exibiram uma expressão significativamente maior de moléculas associadas ao ciclo celular relacionadas à gravidade da doença. Notavelmente, a assinatura do ciclo celular predominou nos leucócitos do sangue dos pacientes, mas não nas vias aéreas superiores. Os dados de scRNAseq de 229 indivíduos (159 pacientes com COVID- 19 e 70 controles) revelaram que as alterações das assinaturas do ciclo celular predominam nas células B, T e NK. Esses resultados fornecem uma compreensão global única das alterações nas moléculas associadas ao ciclo celular em pacientes com COVID-19, sugerindo novas vias putativas para intervenção terapêutica

Based on phosphoproteomics perturbations of cell cycle-associated molecules in severe acute respiratory syndrome coronavirus (SARS-CoV)-2-infected cells, the hypothesis of cell cycle inhibitors as a potential therapy for Coronavirus disease 2019 (COVID-19) has been proposed. However, the landscape of cell cycle alterations in COVID-19 remains mostly unexplored. Here, we performed an integrative systems immunology analysis of publicly available proteome (mass spectrometry) and transcriptome data (bulk and single-cell RNA sequencing [scRNAseq]), aiming to characterize global changes in the cell cycle signature of COVID-19 patients. Beyond significant enriched cell cycle-associated gene co-expression modules, we found an interconnected network of cell cycle-associated differentially expressed proteins (DEPs) and genes (DEGs) by integrating molecular data of 1,480 individuals (974 SARS-CoV- 2 infected patients and 506 controls [either healthy controls or individuals with other respiratory illness]). Among these DEPs and DEGs are several cyclins (CCNs), cell division cycle (CDCs), cyclin-dependent kinases (CDKs), and mini-chromosome maintenance proteins (MCMs). Although COVID-19 patients partially shared the expression pattern of some cell cycleassociated molecules with other respiratory illnesses, they exhibited a significantly higher expression of cell cycle-associated molecules associated with disease severity. Notably, the cell cycle signature predominated in the patients blood leukocytes but not in the upper airways. The scRNAseq data from 229 individuals (159 COVID-19 patients and 70 controls) revealed that the alterations of cell cycle signatures predominate in B, T, and NK cells. These results provide a unique global comprehension of the alterations in cell cycle-associated molecules in COVID-19 patients, suggesting new putative pathways for therapeutic intervention

Humans , Male , Female , Patients/classification , Cell Cycle/immunology , COVID-19/pathology , Respiratory Tract Diseases/pathology , Mass Spectrometry/methods , Killer Cells, Natural/classification , Chromosomes/metabolism , Sequence Analysis, RNA/instrumentation , Coronavirus/pathogenicity , Proteome/analysis , Transcriptome/immunology
São Paulo; s.n; s.n; 2023. 131 p. tab, graf, ilus.
Thesis in Portuguese | LILACS | ID: biblio-1437606


myrsine coriacea (Sw.) R. Br. ex Roem. & Schult. (Primulaceae) conhecida popularmente como capororoquinha ou capororoca, é amplamente distribuída nas regiões sul e sudeste do Brasil. As espécies desse gênero apresentam um potencial antioxidante e anti-inflamatório, que pode ser acessado na busca de novos ativos para o tratamento de desordens pigmentares da pele. Desta forma, este trabalho teve como objetivos avaliar o potencial antitirosinase e antioxidante de extratos e frações de M. coriacea e identificar os possíveis compostos responsáveis por essas atividades. Foram realizados ensaios para avaliar o potencial antioxidante das amostras através do método do DPPH, enquanto a capacidade hipopigmentante das amostras foi avaliado pela inibição da enzima tirosinase. Como complemento, foram determinados os teores de compostos fenólicos totais e flavonoides através dos métodos colorimétricos empregando o reagente Folin-Ciocalteau e AlCl3. Adicionalmente, os extratos de M. coriacea tiveram avaliados seus potenciais citotóxicos utilizando diferentes linhagens tumorais humanas. O perfil fitoquímico de M. coriacea foi analisado por cromatografia a gás acoplada com espectrometria de massas (CG-EM) e cromatografia em camada delgada (CCD) com padrões. Nessas análises foram identificados 34 compostos, sendo o ácido palmítico e o palmitato de etila os compostos majoritários nas amostras de M. coriacea. O extrato bruto das folhas apresentou o maior teor de fenólicos totais, enquanto a fração de acetato de etila das folhas teve o maior teor de flavonoides. Contudo, o extrato bruto dos frutos apresentou a melhor atividade antioxidante de todas as amostras analisadas, apresentando também a melhor atividade antitirosinase. Dentre os compostos anotados, mandenol, ácido -linoleico e o linolenato de etila foram os compostos considerados como possíveis inibidores da tirosinase, com boa interação molecular com a enzima nas análises de ancoragem molecular in silico. Das amostras analisadas com relação a inibição de crescimento frente as células tumorais, a amostra da fração de clorofórmio das folhas foi a que apresentou potencial antitumoral frente as células de adenocarcinoma de cólon (HCT116)

myrsine coriacea (Sw.) R. Br. ex Roem. & Schult. (Primulaceae) popularly known as capororoquinha or capororoca, is widely distributed in southern and southeastern Brazil. Myrsine species have an antioxidant and anti-inflammatory potential, which can be accessed in the search for new actives for the treatment of skin pigmentation disorders. Thus, this work aimed to evaluate the antityrosinase and antioxidant potential from extracts and fractions of M. coriacea and to identify the probable compounds responsible for these activities. Assays were performed to evaluate the antioxidant potential of the samples using the DPPH method, while the hypopigmentation capacity of the samples was evaluated by the tyrosinase inhibition. As a complement, the amounts of total phenolic compounds and flavonoids were determined through colorimetric methods using the Folin-Ciocalteau reagent and AlCl3. Additionally, M. coriacea extracts had their cytotoxic potential evaluated using different human tumor cell lines. M. coriacea phytochemical profile was obtained by gas chromatography coupled with mass spectrometry (GC-MS) and thin layer chromatography (TLC) with standards. In these analyses, 34 compounds were identified, with palmitic acid and ethyl palmitate as the major compounds in M. coriacea samples. The leaf crude extract presented the highest total phenolics contents, while the leaf ethyl acetate fraction had the highest flavonoid amounts. However, the fruit crude extract showed the best antioxidant and antityrosinase activities of all analyzed samples. Among the annotated compounds, mandenol, -linoleic acid and ethyl linolenate were the compounds considered as putative tyrosinase inhibitors, presenting good molecular interaction with the enzyme active site in the in silico molecular docking analysis. The leaf chloroform fraction was the only sample that showed an antitumor potential against colon adenocarcinoma cells (HCT116)

Monophenol Monooxygenase/analysis , Primulaceae/metabolism , Myrsine/classification , Fruit/classification , Antioxidants/analysis , Mass Spectrometry/methods , Skin Pigmentation/immunology , Chromatography, Thin Layer/methods , Hypopigmentation/pathology
Braz. J. Pharm. Sci. (Online) ; 59: e21088, 2023. tab, graf
Article in English | LILACS | ID: biblio-1439546


Abstract The present study was aimed at conducting phytochemical analysis and evaluating the in vitro antifungal and antioxidant activities of the essential oil obtained from the fruits of J. oxycedrus L. Hydro-distillation was used to extract the essential oil from the fruits of Juniper oxycedrus. The essential oil was analyzed using gas chromatography with a flame ionization detector (GC-FID) and gas chromatography coupled with mass spectrometry (GC/MS). The antioxidant activity of the essential oil against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was determined in vitro using varying concentrations of the essential oil and vitamin C as a standard antioxidant compound. A disc diffusion test was employed to evaluate the antifungal activity of the essential oil against two test fungal strains, Penicillium citrinum, and Aspergillus niger. The results revealed that 49 constituents were identified in fruit oil, representing 91.56% of the total oil and the yield was 1.58%. Juniper fruit oil was characterized by having high contents of ß-pinene (42.04%), followed by limonene (15.45%), sabinene (9.52%), α-pinene (5.21%), (E)-caryophyllene (3.77%), ρ-cymene (1.56%), caryophyllene oxide (2.02%), and myrcene (1.02%). The radical scavenging activity (% inhibition) of the essential oil was highest (81.87± 2.83%) at a concentration of 200 µg/mL. The essential oil of J. oxycedrus exhibited antifungal activity against A. niger and P. citrinum with minimum inhibitory concentration values (MIC) ranging from 2.89 to 85.01 µl/mL. The findings of the study reveal that the antioxidant and antifungal properties of J. oxycedrus essential oil and their chemical composition are significantly correlated

Oils, Volatile/analysis , Juniperus/adverse effects , Phytochemicals/analysis , Fruit/classification , Morocco/ethnology , Antioxidants/pharmacology , Mass Spectrometry/methods , In Vitro Techniques/methods , Microbial Sensitivity Tests/methods , Chromatography, Gas/methods , Antifungal Agents/pharmacology
Article in English | WPRIM | ID: wpr-971678


Angelicae Sinensis Radix (AS) is reproted to exert anti-depression effect (ADE) and nourishing blood effect (NBE) in a rat model of depression. The correlation between the two therapeutic effects and its underlying mechanisms deserves further study. The current study is designed to explore the underlying mechanisms of correlation between the ADE and NBE of AS based on hepatic metabonomics, network pharmacology and molecular docking. According to metabolomics analysis, 30 metabolites involved in 11 metabolic pathways were identified as the potential metabolites for depression. Furthermore, principal component analysis and correlation analysis showed that glutathione, sphinganine, and ornithine were related to pharmacodynamics indicators including behavioral indicators and hematological indicators, indicating that metabolic pathways such as sphingolipid metabolism were involved in the ADE and NBE of AS. Then, a target-pathway network of depression and blood deficiency syndrome was constructed by network pharmacology analysis, where a total of 107 pathways were collected. Moreover, 37 active components obtained from Ultra Performance Liquid Chromatography-Triple-Time of Flight Mass Spectrometer (UPLC-Triple-TOF/MS) in AS extract that passed the filtering criteria were used for network pharmacology, where 46 targets were associated with the ADE and NBE of AS. Pathway enrichment analysis further indicated the involvement of sphingolipid metabolism in the ADE and NBE of AS. Molecular docking analysis indciated that E-ligustilide in AS extract exhibited strong binding activity with target proteins (PIK3CA and PIK3CD) in sphingolipid metabolism. Further analysis by Western blot verified that AS regulated the expression of PIK3CA and PIK3CD on sphingolipid metabolism. Our results demonstrated that sphingolipid metabolic pathway was the core mechanism of the correlation between the ADE and NBE of AS.

Rats , Animals , Rats, Sprague-Dawley , Molecular Docking Simulation , Network Pharmacology , Drugs, Chinese Herbal/chemistry , Metabolomics/methods , Mass Spectrometry
Article in Chinese | WPRIM | ID: wpr-970572


The ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to conduct the qualitative analysis of the monoterpene chemical components from Paeoniae Radix Rubra. Gradient elution was performed on C_(18) HD(2.1 mm×100 mm, 2.5 μm) column with a mobile phase of 0.1% formic acid(A) and acetonitrile(B). The flow rate was 0.4 mL·min~(-1) and the column temperature was 30 ℃. MS analysis was conducted in both positive and negative ionization modes using electrospray ionization(ESI) source. Qualitative Analysis 10.0 was used for data processing. The identification of chemical components was realized by the combination of standard compounds, fragmentation patterns, and mass spectra data reported in the literature. Forty-one monoterpenoids in Paeoniae Radix Rubra extract were identified. Among them, 8 compounds were reported in Paeoniae Radix Rubra for the first time and 1 was presumed to be the new compound 5″-O-methyl-galloylpaeoniflorin or its positional isomer. The method in this study realizes the rapid identification of monoterpenoids from Paeoniae Radix Rubra and provides a material and scientific basis for quality control and further study on the pharmaceutical effect of Paeoniae Radix Rubra.

Chromatography, Liquid , Drugs, Chinese Herbal , Mass Spectrometry , Monoterpenes
Article in Chinese | WPRIM | ID: wpr-970480


The chemical constituents in stem leaf, root, and flower of Ixeris sonchifolia were identified by the ultra performance li-quid chromatography coupled with linear ion trap quadrupole-orbitrap mass spectrometry(UPLC-LTQ-Orbitrap-MS~n). The separation was performed on an Acquity UPLC BEH C_(18) column(2.1 mm×100 mm, 1.7 μm) with a mobile phase of water(containing 0.1% formic acid, A)-acetonitrile(B) with gradient elution. With electrospray ionization source, the data of 70% methanol extract from stem leaf, root and flower of I. sonchifolia were collected by high-resolution full-scan Fourier transform spectroscopy, data dependent acquisition, precursor ion scan, and selected ion monitoring in the negative and positive ion modes. The compounds were identified based on accurate molecular weight, retention time, fragment ions, comparison with reference standard, Clog P and references. A total of 131 compounds were identified from the 70% methanol extract of I. sonchifolia, including nucleosides, flavonoids, organic acids, terpenoids, and phenylpropanoids, and 119, 110, and 126 compounds were identified from the stem leaf, root and flower of I. sonchifolia, respectively. In addition, isorhamnetin, isorhamnetin-7-O-sambubioside and caffeylshikimic acid were discovered from I. sonchifolia for the first time. This study comprehensively analyzed and compared the chemical constituents in different parts of I. sonchifolia, which facilitated the discovery of effective substances and the development and application of medicinal material resources of I. sonchifolia.

Drugs, Chinese Herbal/chemistry , Methanol , Chromatography, High Pressure Liquid/methods , Mass Spectrometry , Asteraceae
Article in English | WPRIM | ID: wpr-982702


Viscum coloratum (Kom.) Nakai is a well-known medicinal plant. However, the optimal harvest time for V. coloratum is unknown. Few studies were performed to analyze compound variation during storage and to improve post-harvest quality control. Our study aimed to comprehensively evaluate the quality of V. coloratum in different growth stages, and determine the dynamic variation of metabolites. Ultra-performance liquid chromatography tandem mass spectrometry was used to quantify 29 compounds in V. coloratum harvested in six growth periods, and the associated biosynthetic pathways were explored. The accumulation of different types of compounds were analyzed based on their synthesis pathways. Grey relational analysis was used to evaluate the quality of V. coloratum across different months. The compound variation during storage was analyzed by a high-temperature high-humidity accelerated test. The results showed that the quality of V. coloratum was the hightest in March, followed by November, and became the lowest in July. During storage, compounds in downstream steps of the biosynthesis pathway were first degraded to produce the upstream compounds and some low-molecular-weight organic acids, leading to an increase followed by a decrease in the content of some compounds, and resulted in a large gap during the degradation time course among different compounds. Due to the rapid rate and large degree of degradation, five compounds were tentatively designated as "early warning components" for quality control. This report provides reference for better understanding the biosynthesis and degradation of metabolites in V. coloratum and lays a theoretical foundation for rational application of V. coloratum and better quality control of V. coloratum during storage.

Viscum/chemistry , Plants, Medicinal/chemistry , Chromatography, Liquid , Mass Spectrometry , Metabolomics
Article in Chinese | WPRIM | ID: wpr-986016


Objective: To establish a inductively coupled plasma mass spectrometry method for the determination of trace cobalt and tungsten in human urine. Methods: The authors used 1% nitric acid solution as diluent in October-December 2021, the sample dilution factor and internal standard element were optimized by single factor rotation experiment, and the difference between the working curve and the standard curve was compared. Results: The method uses working curve to determine cobalt and tungsten in urine, the linear range of this method was 0.0~10.0 μg/L, the correlation coefficient was 0.999 9, the detection limits respectively were 0.005 μg/L (cobalt) and 0.09 μg/L (tungsten), the recoveries of samples respectively were 87.0%~100.2% (cobalt) and 89.4%~104.8% (tungsten), the relative standard deviations respectively were 0.4%~4.4% (cobalt) and 0.6%~3.8% (tungsten) . Conclusion: A simple and rapid method for determination of cobalt and tungsten in urine has been established. This method has the advantages of simple operation, high sensitivity, low detection limit and good stability. It is suitable for determination of cobalt and tungsten in urine of all kinds of people.

Humans , Cobalt/analysis , Tungsten/analysis , Spectrum Analysis , Nitric Acid , Mass Spectrometry
Article in Chinese | WPRIM | ID: wpr-981409


To study the quality control of three traditional Chinese medicines derived from Gleditsia sinensis [Gleditsiae Sinensis Fructus(GSF), Gleditsiae Fructus Abnormalis(GFA), and Gleditsiae Spina(GS)], this paper established a multiple reaction monitoring(MRM) approach based on ultra-high performance liquid chromatography-triple quadrupole-linear ion-trap mass spectrometry(UHPLC-Q-Trap-MS). Using an ACQUITY UPLC BEH C_(18) column(2.1 mm × 100 mm, 1.7 μm), gradient elution was performed at 40 ℃ with water containing 0.1% formic acid-acetonitrile as the mobile phase running at 0.3 mL·min~(-1), and the separation and content determination of ten chemical constituents(e.g., saikachinoside A, locustoside A, orientin, taxifolin, vitexin, isoquercitrin, luteolin, quercitrin, quercetin, and apigenin) in GSF, GFA, and GS were enabled within 31 min. The established method could quickly and efficiently determine the content of ten chemical constituents in GSF, GFA, and GS. All constituents showed good linearity(r>0.995), and the average recovery rate was 94.09%-110.9%. The results showed that, the content of two alkaloids in GSF(2.03-834.75 μg·g~(-1)) was higher than that in GFA(0.03-10.41 μg·g~(-1)) and GS(0.04-13.66 μg·g~(-1)), while the content of eight flavonoids in GS(0.54-2.38 mg·g~(-1)) was higher than that in GSF(0.08-0.29 mg·g~(-1)) and GFA(0.15-0.32 mg·g~(-1)). These results provide references for the quality control of G. sinensis-derived TCMs.

Flavonoids/analysis , Alkaloids , Chromatography, High Pressure Liquid/methods , Mass Spectrometry , Drugs, Chinese Herbal
Article in Chinese | WPRIM | ID: wpr-981322


This paper explored the chemical constituents of Boswellia carterii by column chromatography on silica gel, Sephadex LH-20, ODS column chromatography, and semi-preparative HPLC. The structures of the compounds were identified by physicochemical properties and spectroscopic data such as infrared radiation(IR), ultra violet(UV), mass spectrometry(MS), and nuclear magnetic resonance(NMR). Seven diterpenoids were isolated and purified from n-hexane of B. carterii. The isolates were identified as(1S,3E,7E,11R,12R)-11-hydroxy-1-isopropyl-4,8,12-trimethyl-15-oxabicyclo[10.2.1]pentadeca-3,7-dien-5-one(1),(1R,3S,4R,7E,11E)-4,8,12,15,15-pentamethyl-14-oxabicyclo[11.2.1]hexadeca-7,11-dien-4-ol(2), incensole(3),(-)-(R)-nephthenol(4), euphraticanoid F(5), dilospirane B(6), and dictyotin C(7). Among them, compounds 1 and 2 were new and their absolute configurations were determined by comparison of the calculated and experimental electronic circular dichroisms(ECDs). Compounds 6 and 7 were obtained from B. carterii for the first time.

Molecular Structure , Boswellia/chemistry , Diterpenes/chemistry , Mass Spectrometry
Rev. Inst. Adolfo Lutz ; 81: e37179, mar.1, 2022. tab
Article in Portuguese | LILACS, CONASS, ColecionaSUS, SES-SP, SESSP-IALPROD, SES-SP, SESSP-IALACERVO | ID: biblio-1414590


Os cereais são uma importante fonte alimentícia e econômica, dentre eles se destaca o trigo e sua farinha. Durante o cultivo, visando o controle de pragas, frequentemente são utilizados os agrotóxicos, porém seu uso abusivo pode acarretar danos à saúde humana e ao meio ambiente. O trabalho teve como objetivo avaliar a presença de resíduos de agrotóxicos em amostras de farinha de trigo comercializadas no estado de São Paulo, utilizando o método QuEChERS (Rápido, Fácil, Barato, Efetivo, Robusto e Seguro) modificado, seguido de análise por CG-EM/EM e CLUE-EMAR para identificação e quantificação. No total, 124 ingredientes ativos (i.a.) estavam dentro dos critérios de aceitação para linearidade, limites de detecção e quantificação, exatidão e precisão (intervalo de confiança de 95%; k = 2). Os resultados de 25 amostras indicaram a presença de bifentrina, fenitrotiona, clorpirifós, deltametrina, lambda-cialotrina e pirimifós-metílico em farinha de trigo comum e os quatro últimos i.a. também foram detectados em farinha de trigo orgânica. O pirimifósmetílico foi detectado em 92% das amostras. Os i.a. encontrados nas amostras estavam abaixo do Limite Máximo de Resíduos (LMR) estabelecidos pela Agência Nacional de Vigilância Sanitária (Anvisa) para farinha de trigo e as amostras foram consideradas próprias para o consumo (AU).

Cereals are an important economic factor and food source, which wheat and its flour stand out. Pesticides are often employed to control harmful organisms, but their abusive use can cause damage to human health and the environment. This study aimed to evaluate the presence of pesticide residues in wheat flour samples commercialized in the state of São Paulo ­ Brazil, using the modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method, followed by GC-MS/MS and UHPLC-HRMS analysis for identification and quantification. Thus, 124 active ingredients (a.i.) were within acceptable criteria for linearity, limits of detection and quantification, accuracy (trueness), and precision (95% confidence level; k = 2). The results for 25 wheat flour samples showed the presence of bifenthrin, fenitrothion, chlorpyrifos, deltamethrin, lambda-cyhalothrin, and pirimiphos-methyl, and the last four a.i. were also detected in organic wheat flour. Pirimiphos-methyl was detected in 92% of all samples. All compounds were found below the Maximum Residue Levels (MRL) established by Brazilian Health Regulatory Agency (Anvisa) for wheat flour and the samples were considered suitable for consumption (AU).

Mass Spectrometry , Agrochemicals , Flour
Acta bioquím. clín. latinoam ; 56(1): 17-31, ene. 2022. graf
Article in Spanish | LILACS, BINACIS | ID: biblio-1402943


Resumen La espectrometría de masas (MALDI-TOF MS) permite la identificación de microorganismos directamente de las colonias en pocos minutos. En este estudio se ha desarrollado y evaluado un protocolo reducido para identificar microorganismos directamente de las botellas de hemocultivos positivos en 30 minutos con una alta sensibilidad y especificidad, utilizando MALDITOF. Un total de 2535 hemocultivos positivos fueron estudiados por el método directo de MALDI-TOF MS, a partir de una alícuota de sangre de las botellas y el método de colonia, utilizando los cultivos desarrollados en medios sólidos. Del total de hemocultivos positivos incluidos en este estudio, 2381 (93,9%) fueron monomicrobianos y 146 (5,8%) polimicrobianos. Mil trescientos treinta (55,9%) de los aislamientos correspondieron a cocos gram positivos, 922 (38,7%) a bacilos gram negativos, 60 (2,5%) a anaerobios, 36 (1,5%) a bacilos gram positivos y 13 a levaduras. La concordancia global entre ambos métodos fue del 81,7% a nivel de especie (90,0% para bacilos gram negativos, 76,7% para cocos gram positivos y 33,3% para bacilos gram positivos). Se identificó al menos un germen en el 88% de las botellas positivas con desarrollo polimicrobiano. Los resultados del presente estudio demostraron que el protocolo basado en MALDI-TOF MS permite la identificación microbiana directamente de hemocultivos positivos en un tiempo corto, con una alta precisión, con excepción de los bacilos gram positivos.

Abstract Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) enables the identification of microorganisms directly from colonies within minutes. In this study this technology was adapted and tested for use with blood culture bottles, thus allowing identification in 30 minutes once the blood culture is detected as positive by the automate. A total of 2535 blood culture bottles reported as positive were tested by MALDI-TOF MS directly from positive blood culture bottles and colonies. A total of 2381 (93.9%) and 146 (5.8%) of the positive blood cultures were monomicrobial and polymicrobial, respectively. And 1330 (55.9%), 922 (38.7%), 60 (2.5%), 36 (1.5%) and 13 of the isolates were gram-positive cocci (GPC), gram-negative bacilli (GNB), anaerobic bacteria, gram-positive bacilli (GPB) and yeast respectively. Concordance between both methods was 81.7% (76.7% of GPC, 90% of GNB, 74.2% of anaerobic bacteria and 33.3% of GPB) in monomicrobial cultures. Eighty eight per cent of the polymicrobial cultures were identified correctly in at least one of the two bacteria. The results of the present study show that this fast, MALDI-TOF MS based method allows microbial identification directly from positive blood culture in a short time, with a high accuracy, with the exception of gram-positive bacilli.

Resumo A espectrometria de massa (MALDI-TOF MS) permite a identificação de microorganismos diretamente das colônias em minutos. Nesse estudo, foi desenvolvido um protocolo reduzido para identificar microrganismos diretamente das garrafas de hemoculturas positivas em 30 minutos com alta sensibilidade e especificidade, utilizando MALDI-TOF. Um total de 2535 hemoculturas positivas foram relatadas -o método direto de MALDI-TOF MS, a partir de uma alíquota de sangue dos vidros e o método de colônia, a partir das culturas desenvolvidas em meios sólidos. Do total de hemoculturas positivas incluídas neste estudo, 2.381 (93,9%) eram monomicrobianas e 146 (5,8%) eram polimicrobianas. Mil trezentos e trinta (55,9%) dos isolados corresponderam a cocos gram-positivos, 922 (38,7%) bacilos gram-negativos, 60 (2,5%) anaeróbios, 36 (1,5%) bacilos gram-positivos e 13 leveduras. A concordância geral entre os dois métodos foi de 81,7% em nivel de especie (90,0% para bacilos gram-negativos, 76,7% para cocos gram-positivos e 33,3% para bacilos gram-positivos). Pelo menos um germe foi identificado em 88% dos vidros positivos com desenvolvimento polimicrobiano. Os resultados do presente estudo demonstraram que o protocolo baseado em MALDI-TOF MS permite a identificação microbiana diretamente de hemoculturas positivas em um curto espaço de tempo, com alta precisão, com exceção de bacilos gram-positivos.

Mass Spectrometry , Gram-Positive Rods , Microbiology , Technology , Time , Bacteria , Yeasts , Glass Industry , Sensitivity and Specificity , Gram-Positive Cocci , Guidelines as Topic , Cocos , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Culture , Growth and Development , Blood Culture , Lasers , Methods
Ciênc. rural (Online) ; 52(2): e20200894, 2022. ilus, tab, graf
Article in English | VETINDEX, LILACS | ID: biblio-1339655


Seed germination is a complex process controlled by many factors, in which physical and biochemical mechanisms are involved and the mobilization of reserves is crucial for this process to occur. Although, seed reserve mobilization is usually thought to be a post-germination process, seed reserve proteins mobilization occurs during germination. This study quantified seed proteins of bean genotypes during different hydration times, in order to understand the process of protein mobilization and whether there is relationship of this biochemical component with seed vigor. This study was conducted using seeds with different levels of vigor, genotypes with highest (13, 42, 55 and 81) and lowest (07, 23, 44, 50, IPR-88-Uirapurú and Iapar 81) physiological quality. High vigor genotypes showed greater efficiency in hydrolysis and mobilization of protein component, because they presented low globulins content in cotyledons at radicle protrusion in relation to low vigor genotypes (07, 23 and 50). The protein alpha-amylase inhibitor, observed in all genotypes, is involved with the longer time needed for radicle protrusion, according to the band intensity difference in genotypes 07, 44 and Iapar 81.

A germinação de sementes é um processo complexo controlado por muitos fatores, nos quais mecanismos físicos e bioquímicos estão envolvidos e a mobilização de reservas é decisiva para que esse processo ocorra. Embora a mobilização de reservas de sementes seja considerada um processo pós-germinativo, a mobilização das proteínas de reserva de sementes ocorre durante a germinação. Este estudo teve como objetivo quantificar as proteínas de sementes de genótipos de feijão durante os diferentes tempos de hidratação, a fim de compreender o processo de mobilização proteica e se há relação desse componente bioquímico com o vigor das sementes. Este estudo foi realizado utilizando sementes com diferentes níveis de vigor, genótipos com maior (13, 42, 55 e 81) e menor (07, 23, 44, 50, IPR-88-Uirapurú e Iapar 81) qualidade fisiológica. Os genótipos de alto vigor apresentaram maior eficiência na hidrólise e mobilização do componente proteico, pois apresentaram baixo teor de globulinas nos cotilédones na protrusão radicular em relação aos genótipos de baixo vigor (07, 23 e 50). A proteína inibidora da alfa-amilase, observada em todos os genótipos, está envolvida com o maior tempo necessário para a protrusão da radícula, de acordo com a diferença de intensidade da banda nos genótipos 07, 44 e Iapar 81.

Seeds/chemistry , Genetic Variation/genetics , Proteins/analysis , Phaseolus/embryology , Mass Spectrometry , Electrophoresis, Polyacrylamide Gel
J. venom. anim. toxins incl. trop. dis ; 28: e20210042, 2022. graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1360568


Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)

Animals , Mass Spectrometry/instrumentation , Spider Venoms/analysis , Spiders , Protein Isoforms/biosynthesis , Hyaluronoglucosaminidase , Pharmaceutical Preparations