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1.
Article in Chinese | WPRIM | ID: wpr-1003774

ABSTRACT

ObjectiveTo analyze the antidepressant quality markers(Q-Marker) of Bupleuri Radix(BP) before and after vinegar-processing by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS), multivariate statistical analysis and network pharmacology. MethodUPLC-Q-TOF-MS was used to analyze the chemical basis of raw and vinegar-processed products of BP, and principal component analysis(PCA) orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to identify the differential components in BP that changed significantly before and after vinegar-processing, which were regarded as candidate quality markers(Q-Marker). Then the disease-drug-component-target network related to antidepressant effect of BP was constructed by network pharmacology, and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined. Rats were randomly divided into blank group, model group, fluoxetine group(2.67 mg·kg-1) and total saponin group(0.72 mg·kg-1), except the blank group, rats in the other groups were subjected to chronic unpredictable mild stress(CUMS). Three weeks after the start of modeling, rats in each administration group were given the corresponding dose of drugs once a day for 4 weeks, and rats in the blank and model groups were given normal saline with dose of 10 mL·kg-1. At 1 day before modeling, 21 days and 28 days after administration, body mass weighing, sucrose preference test and open field test were performed on each group . After 28 days of administration, real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) was used to detect the mRNA expression levels of phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt), mammalian target of rapamycin(mTOR), glycogen synthase kinase-3β(GSK-3β), forkhead box transcription factor O3a(FoxO3a) and β-catenin in hippocampal tissues of rats in each group, while protein expression levels of PI3K, Akt, mTOR and FoxO3a in hippocampal tissues of rats in each group were detected by Western blot. ResultThere were 19 components in BP showed significant changes before and after vinegar-processing, and 9 components such as saikosaponin A, saikosaponin B1, saikosaponin B2, saikosaponin C and saikosaponin D were identified as potential Q-Marker through S-plot differential marker screening. Combined with the disease-drug-component-target network, saikosaponin A, saikosaponin B1, saikosaponin B2 and saikosaponin D were identified as antidepressant Q-Marker of raw and vinegar-processed products of BP. According to the results of pharmacodynamic tests, after 28 d of administration, compared with the blank group, the body mass, sucrose preference index and open field total score of rats in model group, fluoxetine group and total saponin group decreased significantly(P<0.01). Compared with the model group, the body mass, sucrose preference index and open field total score in total saponin group increased significantly(P<0.01). Compared with the blank group, mRNA expression levels of PI3K, Akt, mTOR and β-catenin in hippocampus of rats in the model group decreased significantly(P<0.05), while mRNA expression levels of GSK-3β and FoxO3a increased significantly(P<0.05). Compared with the model group, mRNA expression levels of PI3K, Akt, mTOR and β-catenin in hippocampus of rats in the total saponin group were increased significantly(P<0.05), while mRNA expression levels of GSK-3β and FoxO3a decreased significantly(P<0.05). Compared with the blank group, the protein expression levels of Akt and mTOR in hippocampus of the model group decreased significantly(P<0.01), while the protein expression levels of PI3K and FoxO3a increased significantly(P<0.01). Compared with the model group, the expression level of Akt in hippocampus of the total saponin group increased significantly(P<0.01), the mTOR expression level was increased but not statistically significant, while the protein expression levels of PI3K and FoxO3a decreased significantly(P<0.01). ConclusionThe chemical constituents of BP changed greatly after vinegar-processing, and the antidepressant Q-Marker of raw and vinegar-processed products of BP was determined by chemical basis, pharmacodynamics, network pharmacology and signaling pathway, which provided a reference for further research on quality control, pharmacodynamic substance basis and processing mechanism of BP.

2.
Article in Chinese | WPRIM | ID: wpr-1005266

ABSTRACT

ObjectiveMetabolomics was used to reveal the mechanism of Aconiti Lateralis Radix Praeparata(ALRP) in attenuating toxicity by processing from the aspects of amino acid metabolism, oxidative stress and energy metabolism by analyzing multiple metabolic pathways. MethodTwenty-four rats were randomly divided into control group, raw group and processed group, 8 rats in each group. The raw and processed group were given with 0.64 g·kg-1 of raw ALRP and processed ALRP respectively every day, the control group was given with an equal amount of normal saline once a day. After continuous administration for 7 days, the urine, serum and heart tissue of rats were collected. Pathological examination of the heart was carried out using hematoxylin-eosin(HE) staining, and the activities of lactate dehydrogenase(LDH) and creatine kinase-MB(CK-MB) in serum and cardiac tissues were detected by microplate assay and immunoinhibition assay. The effects of ALRP on rat heart before and after processing were compared and analyzed. Ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to perform urine metabolomics analysis, and multivariate statistical analysis was used to screen for differential metabolites related to ALRP in attenuating toxicity by processing, and pathway enrichment analysis was carried out to explore the processing mechanism. ResultHE staining showed that no obvious pathological changes were observed in the heart tissue of the control group, while obvious infiltration of inflammatory cells such as plasma cells and granulocytes was observed in the heart tissue of the raw group, indicating that the raw ALRP had strong cardiotoxicity. There was no significant difference in HE staining of heart tissue between the processed group and the control group, indicating that the toxicity of ALRP was significantly reduced after processing. Compared with the control group, the activities of LDH and CK-MB were significantly increased in serum and heart tissue of the raw group, and those were significantly decreased in serum and heart tissue of the processed group, suggesting that the myocardial toxicity of processed ALRP was reduced. A total of 108 endogenous differential metabolites associated with the raw ALRP were screened using multivariate statistical analysis in positive and negative modes, of which 51 differential metabolites were back-regulated by the processed ALRP. Biological analysis of the key regulatory pathways and associated network changes showed that the pathways related to toxicity of ALRP mainly included tryptophan metabolism, arginine and proline metabolism, phenylalanine metabolism, aminoacyl-tRNA biosynthesis, alanine, aspartate and glutamate metabolism, etc. The metabolic pathways related to the attenuation of processed ALRP mainly included aminoacyl-tRNA biosynthesis, tryptophan metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism and caffeine metabolism. ConclusionThe processing technology of ALRP in Guilingji can significantly attenuate the cardiotoxicity of raw products, the mechanism mainly involves amino acid metabolism, oxidative stress and energy metabolism, which can provide experimental bases for the research related to the mechanism of toxicity reduction of ALRP by processing and its clinical safety applications.

3.
Article in Chinese | WPRIM | ID: wpr-1039637

ABSTRACT

ObjectiveTo establish a qualitative analysis method for the chemical constituents of the reference sample of Xiao Xumingtang, and to establish the fingerprint of 15 batches of Xiao Xumingtang, so as to evaluate the quality consistency among batches. MethodAccording to the key information of Xiao Xumingtang in the Key Information Table of Ancient Famous Classical Formulas(25 Formulas), the reference sample of this formula was prepared, and it was detected by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS). The chemical components were identified by self-constructed database, consulting relevant literature, and comparing with the reference substances, and the components were assigned by comparing with single drug samples and negative samples lacking single drug. The fingerprint of the reference sample of Xiao Xumingtang was established using high performance liquid chromatography(HPLC), and the common peaks were assigned and identified through single drug samples and negative samples lacking single drug. ResultBased on the information of MS fragments, relevant literature, and database retrieval, a total of 64 compounds were identified and inferred from the reference sample of Xiao Xumingtang, including 31 flavonoids, 8 terpenoids, 12 triterpenoid saponins, 2 phthalides, 3 phenylpropanoids, 2 gingerols, 5 alkaloids, and 1 cyanoside. Among them, 21 were derived from Scutellariae Radix, 10 from stir-fried Glycyrrhizae Radix et Rhizoma, 9 from Ginseng Radix et Rhizoma, 8 from Paeoniae Radix Alba, 4 from Saposhnikoviae Radix, 3 from Stephaniae Tetrandrae Radix, 3 from Chuanxiong Rhizoma, 2 from Aconiti Lateralis Radix Praeparata, 2 from Zingiberis Rhizoma Recens, 1 from Ephedrae Herba, and 1 from Armeniacae Semen Amarum. The established HPLC fingerprint of the reference sample of Xiao Xumingtang had 23 common peaks, among which, peaks 1 and 2 were derived from Paeoniae Radix Alba, peaks 3 and 7 from Saposhnikoviae Radix, peaks 4, 8 and 9 from Stephaniae Tetrandrae Radix, peaks 10, 17, 18, 20 and 21 from stir-fried Glycyrrhizae Radix et Rhizoma, peaks 11-16, 19 and 22 from Scutellariae Radix, peak 5 from Chuanxiong Rhizoma, peak 23 from Zingiberis Rhizoma Recens, peak 6 was the common component of stir-fried Glycyrrhizae Radix et Rhizoma and Scutellariae Radix. A total of 10 compounds including albiflorin(peak 1), paeoniflorin(peak 2), cimicifugoside(peak 3), 5-O-methylvisammioside(peak 7), baicalin(peak 11), sec-O-glucosylhamaudol(peak 13), oroxylin A-7-O-β-D-glucuronide(peak 15), wogonoside(peak 16), glycyrrhizic acid(peak 21) and 6-gingerol(peak 23) were identified. The similarities of 15 batches of reference samples were>0.999, indicating that the reference samples had good consistency. ConclusionThrough the identification of the chemical constituents in the reference sample of Xiao Xumingtang, it is clear that the composition of the samples is mainly composed of flavonoids and triterpenoid saponins. The established fingerprint can basically reflect the overall chemical characteristics of the reference sample of Xiao Xumingtang, which can provide a basis for the quality research of its compound preparations.

4.
Article in Chinese | WPRIM | ID: wpr-1006568

ABSTRACT

ObjectiveTo establish a qualitative and quantitative analysis method for chemical constituents in Liu Junzitang(LJZT), and to clarify its material basis. MethodThe chemical constituents in LJZT were analyzed by ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS), and the resulting compounds were identified by using databases, such as MassBank, PubChem, ChemSpider, Traditional Chinese Medicine Systems Pharmacology Database and Analytical Platform(TCMSP), and by combining with relevant literature. UPLC was used to establish a quantitative method for analysis of 9 compounds in LJZT, including liquiritin, hesperidin, lobetyolin, liquiritigenin, glycyrrhizic acid, nobiletin, tangeretin, atractylenolide Ⅱ and Ⅰ. ResultBy combining the relevant literature, database and MS information, a total of 79 compounds were identified from LJZT, including 31 flavonoids, 15 terpenoids, 14 nitrogen-containing compounds, 6 phenylpropanoids, 6 organic acids and 7 other compounds. The established quantitative analytical method for the nine representative components showed good linearity within their respective linear ranges, and the precision, stability, reproducibility and recovery were in accordance with the requirements. The quantitative results showed that the contents of liquiritin, hesperidin, lobetyolin, liquiritigenin, glycyrrhizic acid, nobiletin, tangeretin, atractylenolide Ⅱ and Ⅰ in LJZT were 0.376 5, 2.602 1, 0.082 6, 0.128 1, 1.778 6, 0.015 7, 0.006 7, 0.030 4, 0.003 2 mg·g-1, respectively. ConclusionThe established method can quickly, sensitively and accurately analyze the chemical constituents in LJZT, clarify that the material basis of LJZT is mainly flavonoids, terpenoids and nitrogen-containing compounds, and simultaneously determine the contents of the 9 components, which can lay a foundation for the research on quality control, mechanism and clinical application of LJZT.

5.
Article in Chinese | WPRIM | ID: wpr-1016846

ABSTRACT

ObjectiveUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS) was used to identify the metabolites of harmine in rats, in order to explore the differences in distribution of metabolites in rats after single dose(40 mg·kg-1) intragastric administration of harmine, as well to speculate the metabolic pathways. MethodSD rats were given a single dose of harmine by intragastric administration. Plasma, bile, urine and feces samples were collected after administration, and the samples were processed for determination by UPLC-Q-TOF-MS. The separation was performed on an ACQUITY UPLC™ HSS T3 columu(2.1 mm×100 mm, 1.8 μm) with acetonitrile(A)-0.1% formic acid aqueous solution(B) as mobile phase for gradient elution(0-2 min, 5%A; 2-9 min, 5%-35%A; 9-9.5 min, 35%-100%A; 9.5-12 min, 100%A; 12-12.5 min, 100%-5%A; 12.5-14 min, 5%A), the mass spectra were obtained in positive ion mode with electrospray ionization(ESI), the scanning range was m/z 50-1 200. The metabolites of harmine were identified based on the information of the obtained compounds and the literature data, and the metabolic pathways were hypothesized. ResultA total of 42 compounds(harmine and its metabolites) were identified in rats, including 27 in plasma, 17 in bile, 26 in urine and 13 in feces. The metabolic pathways involved in these 42 metabolites included monohydroxylation, dihydroxylation, demethylation, glucuronidation and sulfation. ConclusionHarmine can undergo phase Ⅰ and phase Ⅱ metabolic reactions in rats, and the prototype drug is metabolized rapidly in vivo, and the metabolites are mainly excreted by the kidneys, which can provide a reference basis for the pharmacodynamics and material basis of harmine.

6.
Article in Chinese | WPRIM | ID: wpr-1021967

ABSTRACT

BACKGROUND:Traumatic spinal cord injury primarily relies on scale assessment and imaging examinations in clinical practice.However,there are limitations in predicting the prognosis of the injury.Therefore,the use of metabolomics technology for biomarker screening is significant for estimating the extent of damage,injury and recovery,as well as developing new therapies. OBJECTIVE:To characterize the metabolic features of patients with traumatic spinal cord injury using metabolomics technology and explore potential biomarkers and disrupted metabolic pathways. METHODS:Serum and urine samples were collected from 20 patients with traumatic spinal cord injury(observation group)and 10 healthy subjects(control group).Metabolites were analyzed and multivariate statistical analysis was then performed for data processing to screen differential metabolites.Metabolic pathway enrichment was performed using MetaboAnalyst software.Logistic regression was applied to construct a biomarker combination model,and its relationship with the American Spinal Injury Association grading was analyzed. RESULTS AND CONCLUSION:Significant differences in 160 and 73 metabolites were detected in the serum and urine samples of the two groups,respectively.Pathway enrichment analysis showed evident disturbances in lipid metabolism after traumatic spinal cord injury,including sphingolipid,arachidonic acid,α-linolenic acid,and arachidonic acid metabolism,as well as glycerophospholipid and inositol phosphate biosynthesis.The combination of two identified biomarkers,telmisartan and quercetin glycoside,showed a correlation with the American Spinal Injury Association grading in both serum and urine levels.Thus,metabolomics technology provides assistance in further understanding the pathological mechanisms of traumatic spinal cord injury and screening therapeutic targets.The identified metabolic biomarker combination may serve as a reference for assessing the severity of traumatic spinal cord injury.

7.
Article in Chinese | WPRIM | ID: wpr-973752

ABSTRACT

ObjectiveBased on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MSE) technique, we identified qualitatively the metabolites of aristolochic acid(AAs) in rat in order to analyze the metabolic differences between water extract of Aristolochiae fructus(AFE) and Aristolochic acid Ⅰ(AAⅠ). MethodSD rats were selected and administered AFE(110 g·kg-1·d-1) or AAⅠ(5 mg·kg-1·d-1) by oral for 5 days, respectively. Serum, urine and feces were collected after administration. Through sample pretreatment, ACQUITY UPLC BEH C18 column(2.1 mm×100 mm, 1.7 μm) was used with the mobile phase of 0.01% formic acid methanol(A)-0.01% formic acid water(B, containing 5 mmol·L-1 ammonium acetate) for gradient elution(0-1 min, 10%B; 1-7 min, 10%-75%B; 7-7.2 min, 75%-95%B; 7.2-10.2 min, 95%B; 10.2-10.3 min, 95%-10%B; 10.3-12 min, 10%B) at a flow rate of 0.3 mL·min-1. Positive ion mode of electrospray ionization(ESI+) was performed in the scanning range of m/z 100-1 200. In combination with UNIFI 1.9.4.053 system, the Pathway-MSE was used to qualitatively analyze and identify the AAs prototype and related metabolites in biological samples(serum, urine and feces), and to compare the similarities and differences of metabolites in rats in the subacute toxicity test between AFE group and AAⅠ group. ResultCompared with AAⅠ group, 6, 10, 13 common metabolites and 14, 20, 30 unique metabolites were identified in biological samples(serum, urine and feces) of AFE group, respectively. Moreover, the main AAs components always followed the metabolic processes of demethylation, nitrate reduction and conjugation. Compared with common metabolites in AAⅠ group, prototype components of AAⅠ in serum and most metabolic derivatives of AAⅠ[AAⅠa, aristolochic lactam Ⅰ(ALⅠ)a, 7-OHALⅠ and its conjugated derivatives] in biological samples were significantly increased in AFE group(P<0.05, P<0.01), except that the metabolic amount of ALⅠ in feces of AFE group was remarkably lowed than that of AAⅠ group(P<0.01). In addition, a variety of special ALⅠ efflux derivatives were also identified in the urine and feces of the AFE group. ConclusionAlthough major AAs components in AFE all show similar metabolic rules as AAⅠ components in vivo, the coexistence of multiple AAs components in Aristolochiae Fructus may affect the metabolism of AAⅠ, and achieve the attenuating effect by increasing the metabolic effection of AAⅠ and ALⅠ.

8.
Article in Chinese | WPRIM | ID: wpr-973755

ABSTRACT

ObjectiveTo study the potential quality marker (Q-marker) of Tinosporae Radix associated with efficacy of "relieving sore throat" based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), multivariate statistical analysis (MSA), and network pharmacology. MethodUPLC-Q-TOF-MS was used to identify the main chemical components in 18 batches of Tinosporae Radix. On this basis, principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were employed to screen out the main marker components that caused differences between groups. Moreover, network pharmacology technology was applied to predict the potential "sore throat-relieving" components, and the molecular docking between the common components resulting from MSA and network pharmacology and the core targets was carried out to verify the marker components. ResultA total of 17 compounds, including alkaloids, diterpenoid lactones, and sterols, were identified by UPLC-Q-TOF-MS. Five main differential components were found by MSA: Columbamine, jatrorrhizine, palmatine, menisperine, and columbin. Network pharmacology analysis yielded six compounds: tetrahydropalmatine, palmatine, menisperine, fibleucin, neoechinulin A, and columbin which were selected as potential "sore throat-relieving" components of Tinosporae Radix. They may relieve sore throat by acting on interleukin-6, epidermal growth factor receptor, prostaglandin G/H synthase 2, matrix metalloproteinase-9, proto-oncogene tyrosine-protein kinase Src and other targets, and regulating Hepatitis B, influenza A, human T-cell virus infection, human cytomegalovirus infection, coronavirus disease-2019, and other signaling pathways. The common active components in Tinosporae Radix resulting from MSA and network pharmacology analysis were palmatine, menisperine, and columbin, which had high binding affinity with six core targets and can be used as the Q-marker components of Tinosporae Radix in "relieving sore throat". ConclusionThis study predicts the "sore throat-relieving" Q-marker of Tinosporae Radix, which lays a basis for developing the quality standard of Tinosporae Radix based on the efficacy and improving the quality evaluation system of the medicinal.

9.
Article in Chinese | WPRIM | ID: wpr-975167

ABSTRACT

ObjectiveTo analyze the migrating components absorbed into blood of the aqueous extract of Euphorbia helioscopia, and to explore the pharmacodynamic material basis of the aqueous extract of E. helioscopia against chronic obstructive pulmonary disease(COPD). MethodUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) was used to detecte the migrating components absorbed into blood of rats after intragastric administration of aqueous extract of E. helioscopia. An Agilent RRHD SB-C18 column(3 mm×100 mm, 1.8 μm) was used with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as the mobile phase for gradient elution(0-15 min, 5%-30%B; 15-20 min, 30%-50%B; 20-30 min, 50%-95%B; 30-35 min, 95%-5%B), and the detection wavelength of 190-800 nm, column temperature of 40 ℃, flow rate of 0.3 mL∙min-1 and injection volume of 4 μL. The electrospray ionization(ESI) was used in positive and negative ion modes, and the detection range was m/z 50-1 250. Network pharmacology was used to screen out the key components and the key targets of COPD through the interaction analysis. Metascape database was used to predict the molecular function, biological process, cellular composition and signal pathways mainly involved in the anti-COPD effect of E. helioscopia. Molecular docking technique was used to determine the affinity of key targets with key components. ResultA total of 29 migrating components absorbed into blood of rats were identified after intragastric administration of aqueous extract of E. helioscopia, 9 of which were prototype components and 20 were metabolites. Network pharmacological analysis showed that luteolin, quercetin, apigenin, naringenin and helioscopinolide C were the key components of E. helioscopia against COPD, and vascular endothelial growth factor A(VEGFA), albumin(ALB), protein kinase B1(Akt1), tumor necrosis factor(TNF) and interleukin-6(IL-6) were the key targets. Molecular docking results showed that one diterpene lactone(helioscopinolide C) and three flavonoids(naringenin, luteolin, apigenin) in the migrating components absorbed into blood all had strong binding activity to the key targets of E. helioscopia against COPD. ConclusionNaringenin, helioscopinolide C, luteolin and apigenin may be the main anti-COPD active substances of E. helioscopia.

10.
Article in Chinese | WPRIM | ID: wpr-965664

ABSTRACT

ObjectiveTo develop a quality control method for the simultaneous determination of multiple active components in Nymphaeae Flos aiming at the problems of the single index for quality control and the relatively low overall quality control level. MethodUltra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS)was used to identify and select the index components for quality control with the mobile phase of 0.1% formic acid aqueous solution(A)-acetonitrile(B)for gradient elution (0-2 min, 3%-8%B; 2-4 min, 8%-10%B; 4-13 min, 10%-15%B; 13-19 min, 15%-20%B; 19-26 min, 20%-45%B) at a flow rate of 0.4 mL·min-1, detection wavelength of 350 nm, electrospray ionization(ESI), negative ion scanning mode, ion source temperature of 120 ℃, scanning range of m/z 100-1 200, transmit collision energy of 6 eV for low-energy scanning and 25-50 eV for high-energy scanning. High performance liquid chromatography(HPLC)was used to establish the quality control method for the simultaneous determination of multi-index components with the mobile phase of 0.2% phosphoric acid aqueous solution(A)-acetonitrile(B) for gradient elution(0-30 min, 12%-15%B; 30-60 min, 15%-22%B; 60-90 min, 22%-40%B)and detection wavelength of 350 nm. The preparation method of the test solution for content determination was refluxing extraction for 60 min with 80 times the amount of 70% methanol. ResultBy comparing the retention time, ultraviolet absorption characteristics, MS and MS/MS spectrometric signals in the samples with the reference substances, 8 active components with high contents, including brevifolincarboxylic acid, ellagic acid, rutin, nicotiflorin, astragalin, quercetin, quercetin-3-methylether and kaempferol, were identified qualitatively from Nymphaeae Flos, which were selected as the index components for quality control. Under the established HPLC conditions, the above 8 components could be well separated(resolution>1.5), and showed good linearity(r=0.999 9)between the concentration ranges of 1.99-99.6, 1.76-176, 1.52-75.8, 3.60-180, 0.964-96.4, 1.18-118, 1.94-96.8, 1.04-104 mg·L-1 and the peak areas, respectively. The detection limits of them were 10-49 μg·L-1, and the limits of quantitation were 34-164 μg·L-1. The average recoveries were 97.12%-103.1% with the relative standard deviations (RSDs) were 1.1%-2.2%. ConclusionA quality control method for simultaneous determination of the multiple active components in Nymphaeae Flos have been developed, which is simple, accurate and reproducible, and it can provide a scientific basis for the formulation of quality standard of this herb and lay a research foundation for the transformation of Uygur hospital preparations containing Nymphaeae Flos into new drugs.

11.
Article in Chinese | WPRIM | ID: wpr-984596

ABSTRACT

ObjectiveA rapid method for identification of chemical constituents in Puerariae Lobatae Radix dispensing granules was established in order to clarify the material basis. MethodThe chemical constituents of Puerariae Lobatae Radix dispensing granules was qualitatively analyzed by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS) under positive and negative ion modes, and the chromatographic conditions were on an ACQUITY UPLC HSS T3 column(2.1 mm×100 mm, 1.8 μm) with 0.1% formic acid aqueous solution(A)-0.1% formic acid acetonitrile solution(B) as mobile phase for gradient elution(0-4 min, 5%-10%B; 4-10 min, 10%-15%B; 10-20 min, 15%-16%B; 20-27 min, 16%-31%B; 27-33 min, 31%-59%B; 33-42 min, 59%-95%B; 42-42.1 min, 95%-5%B; 42.1-45 min, 5%B), the flow rate was 0.35 mL·min-1, the column temperature was 40 ℃, the injection volume was 5 μL, and electrospray ionization(ESI) was selected. Then these chemical constituents were comprehensively identified based on PeakView 1.2, PubChem, ChemicalBook, ChemSpider, comparative control profiles and literature information. ResultA total of 128 chemical constituents were identified from the dispensing granules, including 60 flavonoids, 26 organic acids, 7 glycosides, 6 coumarins, 3 nucleosides and 26 other compounds. By focusing on the cleavage patterns of flavonoids, organic acids, glycosides, coumarins, nucleosides and other compounds, 12 compounds that have not been reported in Puerariae Lobatae Radix species were identified from the dispensing granules. ConclusionThe established method can systematically and rapidly identify the chemical constituents in Puerariae Lobatae Radix dispensing granules, and cleared it composition is mainly flavonoids and organic acids. Laying a foundation for the study of the material basis, mechanism of action and clinical application of the dispensing granules.

12.
Article in Chinese | WPRIM | ID: wpr-997667

ABSTRACT

ObjectiveTo explore the material basis of bile-processed Coptidis Rhizoma clearing excessive fire of liver-gallbladder based on ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry(UPLC-Q-TOF/MS) metabolomics and molecular docking. MethodUPLC-Q-TOF/MS metabolomics was used to analyze the chemical constituents of Coptidis Rhizoma, water-processed Coptidis Rhizoma and bile-processed Coptidis Rhizoma. Chromatographic separation was achieved with 0.1% formic acid aqueous solution(A)-acetonitrile(B) as the mobile phase in gradient elution(0-2 min, 5%B; 2-20 min, 5%-65%B; 20-40 min, 65%-10%B; 40-45 min, 10%B; 45-46 min, 10%-95%B; 46-49 min, 95%B), and electrospray ionization(ESI) was applied and operated in positive and negative ion modes, the acquisition range was m/z 80-1 200. Based on this, partial least squares-discriminant analysis(PLS-DA) and variance analysis were used to screen the differential compounds among the three products of Coptidis Rhizoma. Network pharmacology and molecular docking were used to verify the degree of association between differential compounds and excessive fire of liver-gallbladder syndrome. ResultA total of 33 chemical constituents were identified, including 2 phenolic acids, 5 binding bile acids and 26 alkaloids. And 16 differential compounds were identified by multivariate statistical analysis, including 11 alkaloids and 5 binding bile acids. Pathway enrichment analysis in the Kyoto Encyclopedia of Genes and Genomes(KEGG) database yielded 8 pathways related to excessive fire of liver-gallbladder, and the key protein phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform(PIK3CA) was obtained according to the "component-target-pathway" network analysis. Molecular docking results showed that 11 alkaloids had good binding ability with PIK3CA. ConclusionPorcine bile is unique in the processing of bile-processed Coptidis Rhizoma, which can promote the production and dissolution of 11 alkaloids, including berberine and dihydrochelerythrine. Based on the results of molecular docking and reported pharmacological experiments, it can be concluded that 16 different compounds such as berberine, dihydrochelerythrine and taurohyodeoxycholic acid are the material basis of bile-processed Coptidis Rhizoma.

13.
Article in Chinese | WPRIM | ID: wpr-930100

ABSTRACT

Objective:This study aimed to determinate ten phenols (polydatin, resveratrol, rhein, emodin, chrysophanol, physcion, oxyresveratrol, 2,3,5,4'-tetera-hydroxystilbene-2-O-β-D-glucoside, (+)-catechin and (-)-epicatechin) in Polygoni Cuspidati Rhizoma simultaneously based on the high-resolution multiple reaction monitoring (MRMHR) mode of ultra- performance liquid chromatography- quadrupole/time-of-flight mass spectrometry. Methods:The assay was performed on Waters ACQUITY UPLC BEH C18 (2.1 mm× 100 mm, 1.7 μm) column using acetonitrile-0.1% formic acid as the mobile phase. The flow rate was 0.2 ml/min. The MRMHR mode was adopted for quantification.Results:The analyzed compounds showed good linearity relationships ( r2>0.999). The intra- and inter-batch relative standard deviations were all <5% and the recovery rate was between 96.28%-103.23%. The content of polydatin was the highest, followed by resveratrol and emodin. However, the contents of chrysophanol and oxyresveratrol were relatively low and some batches were unqualified. The contents of analyzed compounds varied significantly among the ten batches. Conclusion:The proposed UPLC-Q/TOF-MS method was successfully established to determinate ten phenols in Polygoni Cuspidati Rhizoma simultaneously, which could provide technical support for the quality evaluation of Polygonum cuspidatum.

14.
Article in Chinese | WPRIM | ID: wpr-942325

ABSTRACT

ObjectiveTo establish the quality standard of Liangditang benchmark samples. MethodUltra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) was used to qualitatively analyze the chemical composition of Liangditang on the basis of molecular and fragment ion peak information with cracking law. The mobile phase was methanol (A)-0.05% phosphate aqueous solution (B) for gradient elution (0-10 min, 5%-23.5%A; 10-20 min, 23.5%A; 20-58 min, 23.5%-63%A; 58-60 min, 63%-90%A), the flow rate was 0.8 mL·min-1, and the detection wavelength was 254 nm. Electrospray ionization was employed under positive ion mode, the detection range was m/z 100-1 700. Key quality attributes and sources were determined by comparing with single medicine and reference substances. Through mass transfer analysis of multiple batches from decoction pieces to benchmark samples, high performance liquid chromatography (HPLC) for determining the contents of index components and HPLC detection of characteristic maps were established. Through the determination of 15 batches of benchmark samples, the content range of the index components and the common peaks of the characteristic map were determined. Thin layer chromatography (TLC) was applied to the identification of 5 medicines in the formula. Moisture and dry extract yield of the benchmark samples were determined by drying method. ResultA total of 27 compounds were inferred from the benchmark samples of Liangditang, among which 9 compounds were confirmed by comparison with the control, including catalpol, harpagide, gallic acid, albiflorin, paeoniflorin, verbascoside, angoroside C, cinnamic acid and harpagoside. A method for determining the characteristic maps of the benchmark samples were established and 13 peaks were assigned, and the characteristic peaks were mainly derived from wine-processed products of Rehmanniae Radix, Scrophulariae Radix and wine-processed products of Paeoniae Radix Alba. The similarity between the characteristic map of 15 batches of benchmark samples and the control characteristic map was >0.9. Methods for the determination of paeoniflorin, harpagoside, L-hydroxyproline and glycine were established, and the contents of these four components in 15 batches of benchmark samples were within ±30% of the corresponding mean value, and the transfer rate of decoction pieces to the benchmark samples was stable and controllable. TLC was established to identify 5 prescription drugs (except Ejiao) with two kinds of test solutions, and the results showed that the method had good specificity. The average dry extract yield was 48.06%, and the average moisture was 5.58%, which were within the range of ±10% and ±30% of their mean values, respectively. ConclusionThe quality standard of Liangditang benchmark samples was as follows:the similarity between the benchmark samples and the control characteristic map is >0.9, the contents of paeoniflorin, harpagoside, L-hydroxyproline and glycine are 217-403, 24-46, 634-1 178, 1 253-2 328 mg per dose, the dry extract yield is 43.0%-53.0%, the moisture is 4.0%-7.0%, under the set detection conditions, the benchmark samples have corresponding characteristic spots by comparing with the control herbs of 5 medicines. This quality standard is stable and reliable, which fills the gap in the quality control of Liangditang, and can provide a reference for the establishment of the quality standard of Liangditang granules.

15.
Article in Chinese | WPRIM | ID: wpr-943095

ABSTRACT

ObjectiveTo analyze changes of the chemical composition in Euodiae Fructus before and after processing with Coptidis Rhizoma decoction, so as to provide scientific basis for elucidating the processing mechanism of this decoction pieces. MethodUltra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed on a Titank C18 column (2.1 mm×100 mm, 1.8 μm), the mobile phase was 0.1% formic acid aqueous solution-acetonitrile for gradient elution, the column temperature was set at 40 ℃, the flow rate was 0.25 mL·min-1. Electrospray ionization (ESI) was used to scan in positive and negative ion modes, and the scanning range was m/z 50-1 250. The chemical constituents in Euodiae Fructus were identified before and after processing by reference substance comparison, database matching and literature reference, and MarkerView™ 1.2.1 software was used to normalize the obtained data, SIMCA-P 14.1 software was employed to perform principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) on MS data of raw and processed products to screen the differential components before and after processing. ResultA total of 50 compounds were identified, including 48 kinds of stir-fried products with Coptidis Rhizoma decoction and 44 kinds of raw products. After processing, six compounds were added, including danshensu, noroxyhydrastinine, oxyberberine, 13-methylberberrubine, protopine and canadine. However, two kinds of compounds, including (S)-7-hydroxysecorutaecarpine and wuchuyuamide Ⅱ, were not detected after processing. In general, after processing, the overall contents of phenolic acids and flavonoids decreased significantly, the overall content of limonoids increased, and the overall content of alkaloids did not decrease insignificantly. The results of PCA and OPLS-DA showed that there were significant differences in the composition and content of the chemical components of Euodiae Fructus before and after processing, and a total of 12 variables such as quercetin, dihydrorutaecarpine and dehydroevodiamine were obtained by screening. ConclusionEuodiae Fructus stir-fried with Coptidis Rhizoma decoction mainly contains phenolic acids, flavonoids, limonoids and alkaloids. The composition and content of the chemical components have some changes before and after processing. The addition of processing excipients and hot water immersion are the main reasons for the difference, which can provide experimental basis for interpretation of the processing mechanism of this characteristic processed products of Euodiae Fructus.

16.
Article in Chinese | WPRIM | ID: wpr-940402

ABSTRACT

ObjectiveTo systematically analyze the chemical components of QiLing Wenshen (QLWS) formula and explore the key active components and mechanism of the formula in the treatment of polycystic ovary syndrome (PCOS). MethodThe chemical components of QLWS formula were systematically identified by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MSE) combined with comparison with reference substances, literature data, and databases. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and SwissADME were employed to screen the active components for network pharmacological analysis. SwissTargetPrediction, GeneCards, DisGeNET, and DrugBank were used to obtain the potential components and targets of the formula for the treatment of PCOS. The protein-protein interaction (PPI) network was constructed via STRING database for further screening of the core targets. Gene ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of core targets were carried out with DAVID database. Molecular docking was performed in MOE 2019. ResultA total of 90 components of QLWS formula were identified, and 32 active components and 45 core targets for treating PCOS were obtained. GO annotation obtained 429 terms and KEGG pathway enrichment screened out 110 signaling pathways, mainly involving phosphatidylin-ositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, estrogen signaling pathway, and hypoxia inducible factor-1 (HIF-1) signaling pathway. The molecular docking revealed that key active components in QLWS formula were icariin, salvianolic acid A\B\C, wogonin, magnoflorine, etc., which may play a role in treating PCOS through regulating mitogen-activated protein kinase 1 (MAPK1), epidermal growth factor receptor (EGFR), mitogen-activated protein kinase 3 (MAPK3), etc. ConclusionThis study preliminarily predicted that several key active components of QLWS formula could treat PCOS via multiple targets and multiple pathways based on UPLC-Q-TOF/MSE and network pharmacology, which could provide ideas and references for the study of pharmacodynamic material basis and mechanism of action of the formula.

17.
Article in Chinese | WPRIM | ID: wpr-940803

ABSTRACT

ObjectiveBased on ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), the changes of endogenous markers in rat plasma at the different stage, namely modeling and administration of Shenling Baizhusan (SLBZS), and the mechanism of SLBZS in the treatment of ulcerative colitis (UC) were studied. MethodIn the modeling stage, rats were randomly divided into normal group, spleen deficiency with dampness retention-UC (SDDR-UC) and pure-UC (P-UC) model group. In the administration stage, SLBZS was given to the above two different model groups. After modeling and administration, rat plasma was collected and determined by UPLC-Q-TOF/MS. The mobile phase was 0.1% formic acid aqueous solution (A)-acetonitrile (B) for gradient elution (in positive ion mode:0-2 min, 99%A; 2-9 min, 99%-73%A; 9-10 min, 73%-44%A; 10-13 min, 44%-38%A; 13-19 min, 38%-28%A; 19-21 min, 28%-2%A; 21-23 min, 2%A; 23-25 min, 2%-10%A; 25-27 min, 10%-99%A; in negative ion mode:0-2 min, 85%A; 2-3 min, 85%-65%A; 3-5.5 min, 65%-44%A; 5.5-8 min, 44%-25%A; 8-10 min, 25%-2%A; 10-16 min, 2%-85%A). The electrospray ionization (ESI) temperature was 120 ℃ under the positive and negative ion modes, and the acquisition range was 50-1 000. Partial least squares-discriminant analysis (PLS-DA) was used to analyze the changes of endogenous metabolites in the above two different model rats from the different stage. MetaboAnalyst 5.0 was used to analyze the metabolic pathways of these identified metabolites. ResultSixteen potential biomarkers were screened and identified in the modeling stage, among which 11 potential biomarkers were common in the two model rats, which mainly affected the primary bile acid biosynthesis pathway. Twenty-three potential biomarkers were screened and identified during the administration stage, among which 3 potential biomarkers were shared by the two model rats, and SDDR-UC and P-UC model rats had 11 and 9 potential biomarkers, respectively. It mainly affected 6 pathways such as purine metabolism, pentose phosphate pathway, pyrimidine metabolism, retinol metabolism, primary bile acid biosynthesis and steroid hormone synthesis. ConclusionThe primary bile acid biosynthesis pathway appears in the different stage of modeling and administration of UC, showing a dynamic change process. The therapeutic effect of SLBZS on SDDR-UC rats may be related to inhibiting the expression of nuclear transcription factor -κB (NF-κB) signaling pathway, activating farnesoid X receptor (FXR) and enhancing the expression of cytochrome P450.

18.
Article in Chinese | WPRIM | ID: wpr-905921

ABSTRACT

Objective:To control the quality of the reference sample of Wenjingtang by establishing the specific chromatograms. Method:On the basis of analyzing 15 batches of Wenjingtang freeze-dried powder samples, a high performance liquid chromatography (HPLC) specific chromatogram analysis method of Wenjingtang was established. The system adaptability was investigated and the retention time, relative retention value and deviation caused by different chromatographic columns and instruments were calculated by using the same brand of chromatographic columns, four different brands of chromatographic columns and instruments from three different manufacturers. The precision, repeatability and stability of this method was further completed. The possible chemical components of the freeze-dried powders were speculated and identified by ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS<italic><sup>n</sup></italic>). Chromatographic separation was performed on ACQUITY UPLC BEH C<sub>18</sub> column (2.1 mm×100 mm, 1.7 μm) with acetonitrile (A)-0.1% formic acid aqueous solution (B) as mobile phase for gradient elution (0-2.8 min, 10%A; 2.8-8.0 min, 10%-18%A; 8.0-12.2 min, 18%-25%A; 12.2-15.3 min, 25%-40%A; 15.3-17.4 min, 40%A; 17.4-20.5 min, 40%-90%A), and column temperature was set at 30 ℃ with flow rate of 0.4 mL·min<sup>-1</sup>. Mass spectrometry was performed on electrospray ionization, data were collected under positive and negative ion modes, and the detection range was <italic>m</italic>/<italic>z</italic> 50-1 600. Result:Ten characteristic peaks were selected as the distinguishing features in this specific chromatograms, and eight of them were identified by comparing with the reference standards, including paeoniflorin (peak 1), liquiritin apioside (peak 2), liquiritin (peak 3), ferulic acid (peak 4), iquiritigenin (peak 6), cinnamaldehyde (peak 8), paeonol (peak 9)and glycyrrhizic acid (peak 10). By mass spectrometry analysis, 30 compounds were identified, and the source of medicinal materials were assigned. It mainly contained triterpenoid saponins and flavonoids from Glycyrrhizae Radix et Rhizoma, ginsenosides from Ginseng Radix et Rhizoma, monoterpenoid glycosides and tannins from Paeoniae Radix Alba, steroids in Achyranthis Bidentatae Radix, phenolic acids in Angelicae Sinensis Radix. Conclusion:The established characteristic chromatographic analysis method of Wenjingtang is simple, stable and repeatable. The chemical composition of the freeze-dried powder of Wenjingtang is basically defined by mass spectrometry identification and source attribution, which can provide reference for the development and quality control of Wenjingtang in the future.

19.
Article in Chinese | WPRIM | ID: wpr-906062

ABSTRACT

Objective:To compare the chemical constituents of Puerariae Flos from three different varieties of <italic>Pueraria montana</italic> var. <italic>lobata</italic>, <italic>P. montana</italic> var. <italic>thomsonii</italic> and <italic>P</italic>. <italic>montana</italic> var<italic>. montana</italic>. Method:Ultra-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was used with the mobile phase of 0.1% formic acid aqueous solution (A)-acetonitrile (B) for gradient elution (0-20 min, 10%-30%B; 20-30 min, 30%-55%B; 30-35 min, 55%-95%B; 35-37 min, 95%B; 37-40 min, 95%-10%B), the flow rate was 0.25 mL·min<sup>-1</sup>. Electrospray ionization (ESI) was used to scan and collect MS data in positive and negative ion modes with scanning range of <italic>m</italic>/<italic>z</italic> 50-1 500. The chemical components from different sources of Puerariae Flos were identified in combination with the chemical composition database and literature information. After the obtained data were normalized by MarkerView<sup>TM</sup> 1.2.1, they were imported into SICMA-P 14.1 software for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to select the main differentiated components among the three different varieties. Result:A total of 35 compounds were identified from three different varieties of Puerariae Flos, including 22 isoflavones, 6 flavonoids and 7 saponins. The flowers of <italic>P</italic>. <italic>lobata</italic>, <italic>P. montana</italic> var. <italic>thomsonii</italic> and <italic>P</italic>. <italic>montana</italic> var<italic>. montana</italic> contained 32, 35, 33 compounds, respectively. And 18 differential compounds were screened under the positive and negative ion modes, including kakkalide, tectoridin, 6″-<italic>O</italic>-xylosyl-tectoridin, 4'-methyltectorigenin-7-glucoside, glycitin, 6″-<italic>O</italic>-xylosyl-glycitin, irisolidone, kaikasaponin Ⅲ, 6″-<italic>O</italic>-malonylglycitin, kakkalidone, tectorigenin, rutin, soyasaponin BB, vitexin, biochanin A, genistin, kakkatin, azukisaponin Ⅱ. Conclusion:This research is the first to systematically study the chemical constituents of the flower of <italic>P</italic>. <italic>montana</italic> var<italic>. montana</italic>, although the flower of <italic>P</italic>. <italic>montana</italic> var<italic>. montana</italic> is used as adulterants, it has high contents of tectoridin and 6″-<italic>O</italic>-xylosyl-tectoridin, which has great potential for development. The efficacy components such as kakkalide and tectoridin in Puerariae Flos from the three sources of varieties are obviously different, and it is necessary to carefully consider the application of these three varieties as Puerariae Flos.

20.
Article in Chinese | WPRIM | ID: wpr-906091

ABSTRACT

Objective:To identify the anti-acetylcholinesterase active ingredients in <italic>Aconitum tanguticum</italic>, so as to lay the foundation for finding new anti-Alzheimer's disease (AD) drugs. Method:The anti-acetylcholinesterase active fractions of <italic>A. tanguticum</italic> were screened by the modified Ellman's method, and the chemical composition of the active fraction was analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). The chromatographic separation was performed on an ACQUITY UPLC BEH C<sub>18</sub> column (2.1 mm×50 mm, 1.7 μm) with acetonitrile (A)-0.4% ammonia aqueous solution (B) as mobile phase for gradient elution, and the column temperature was set at 30 ℃ with the flow rate of 0.4 mL·min<sup>-1</sup>. Phase A of the dichloromethane fraction changed with time as follows:0-3 min, 5%A; 3-7 min, 5%-20%A; 7-11.5 min, 20%-33%A; 11.5-15.5 min, 33%-50%A; 15.5-20.5 min, 50%-80%A; 20.5-23 min, 80%-85%A; 23-25 min, 85%-95%A. Phase A of the <italic>n</italic>-butanol fraction changed with time as follows:0-2 min, 5%A; 2-8 min, 5%-20%A; 8-11 min, 20%-33%A; 11-15 min, 33%-95%A. Mass spectrometry was performed on electrospray ionization, data were collected in positive ion mode, and the detection range was <italic>m</italic>/<italic>z</italic> 100-1 500. Result:Both the dichloromethane and <italic>n</italic>-butanol fractions had a certain inhibitory effect on acetylcholinesterase, their half inhibitory concentration (IC<sub>50</sub>) values were (64±4.4) mg·L<sup>-1</sup> and (85.7±3.8) mg·L<sup>-1</sup>, respectively. By UPLC-Q-TOF-MS/MS analysis, a total of 21 alkaloids were identified from the dichloromethane fraction, and 11 alkaloids were identified from <italic>n</italic>-butanol fraction. Guan-fu base Ⅰ, found in both fractions, was first discovered in <italic>A. tanguticum</italic>. Conclusion:Diterpene alkaloids are the main anti-acetylcholinesterase substances of <italic>A. tanguticum</italic>, which is worth further exploration.

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