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1.
Article in Chinese | WPRIM | ID: wpr-927914

ABSTRACT

With reference to the production process documented in Chinese Pharmacopoeia, this paper prepared the calibrator samples of Xiaochaihu Granules from multiple batches and established a method for fingerprint analysis and content determination that could be used to evaluate Xiaochaihu Granules available in market. Multiple batches of Chinese herbal pieces contained in Xiaochaihu Granules were collected for preparing the calibrator samples according to the process in Chinese Pharmacopoeia. Following the establishment of fingerprints for calibrator samples by UHPLC, the method for determining the contents of saikosaponin B2, saikosaponin B1, baicalin, wogonoside, baicalein, liquiritin, glycyrrhizin G2 and glycyrrhizic acid in Xiaochaihu Granules was established. The experimental results showed that the fingerprints of calibrator samples had 26 common peaks, covering the chemical compounds of main herbs Bupleuri Radix, Scutellariae Radix, Changii Radix, Glycyrrhizae Radix et Rhizoma, and Rhizoma Zingiberis Recens. The similarity of fingerprints for 47 batches of Xiaochaihu Granules from 31 companies with the calibrator sample fingerprint ranged from 0.74 to 0.99, indicating good applicability of the established fingerprint. The contents of main components baicalin, saikosaponin B2, and glycyrrhizic acid in Xiaochaihu Granules were within the ranges of 22.917-49.108 mg per bag(RSD 19%), 0.28-2.19 mg per bag(RSD 62%), and 0.897-6.541 mg per bag(RSD 41%), respectively. The quality difference in saikosaponin B2, and glycyrrhizic acid among different manufacturers was significant. The fingerprint analysis and content determination method for calibrator samples of Xiaochaihu Granules prepared according to the production process in Chinese Pharmacopoeia has been proved suitable for evaluating the quality of Xiaochaihu Granules from different manufacturers. Saikosaponin B2, glycyrrhizic acid, and liquiritin should be added as content control indicators for Xiaochaihu Granules, aiming to further improve the product quality.


Subject(s)
Chromatography, High Pressure Liquid/methods , Drugs, Chinese Herbal/chemistry , Glycyrrhizic Acid/analysis , Rhizome/chemistry , Scutellaria baicalensis
2.
Article in Chinese | WPRIM | ID: wpr-906413

ABSTRACT

The brain and heart has a tight relationship upon physiology and pathology during the development of cardiovascular and cerebrovascular diseases with clinical conditions interacting with each other and complex pathological mechanisms. Clinical studies prove that cerebral diseases such as stroke usually happen with cardiac diseases as complications, and cardiac diseases such as atrial fibrillation can also cause cerebral diseases, which can even aggravate brain atrophy and lead to cognitive impairment. Traditional Chinese medicine (TCM) also believes that “the substance of Shenming is located in the brain and the function in the heart.” Specifically, Yuanshen is in the brain and Shishen is in the heart, which thus makes the heart and brain closely related. If Shenming in any of them is impaired, the other one would also be injured. Therefore, the pathological mechanisms of brain-heart mutual damage have become one of the current research hotspots. This paper, combined with the clinical research status of the brain-heart mutual damage, summarized its pathological mechanisms from the perspectives of inflammatory responses, dysregulation of autonomic nervous system, apoptosis, energy metabolism and oxidative stress. The necessity of “brain-heart concurrent regulation” was proposed and the research progress on the treatment of cerebral and cardiac diseases with TCM represented by Naoxintong capsule was profiled in the light of heart and brain function described by TCM and the holistic concept of TCM treating cerebral and cardiac diseases. This paper reviews the pathological mechanisms of brain-heart mutual damage and the research progress on its treatment with TCM, which can provide reference for the prevention and treatment of cardiovascular and cerebrovascular diseases and further research on them.

3.
Article in Chinese | WPRIM | ID: wpr-906187

ABSTRACT

Objective:To investigate the effective constituents and mechanism of Tingli Dazao Xiefeitang for the treatment of heart failure through network pharmacology and high resolution mass spectrometry technique as well as molecular docking technique. Method:Chemical components and potential targets in Descurainiae Semen Lepidii Semen and Jujubae Fructus were searched by referring to literature and BATMAN-TCM database. The disease targets were searched in GeneCards database with ''heart failure'' as the key word. STRING platform was used to construct protein-protein interaction(PPI) network based on common targets of drugs and disease. The network topology was analyzed by using Cytoscape 3.7.2 software to obtain the core targets eventually. Kyoto encyclopedia of genes and genomes(KEGG) enrichment analysis of the core targets was conducted through DAVID database to draw a network of “herb-compound-target-pathway”. Based on the results of the network pharmacology research above,high resolution mass spectrometry was used to analyze the decoction and confirm the selected active components. AutoDock 4.2.6 software was used for molecular docking verification of key active components and related targets. Result:A total of 85 components were obtained in Descurainiae Semen Lepidii Semen,and 49 components were obtained in Fructus Ziziphi Jujubae. A total of 1 078 drug targets and 1 549 disease targets were identified. After PPI analysis and network topology analysis,23 core targets and 33 active components were obtained,involving 19 signaling pathways (<italic>P</italic><0.05). Mass spectrometry analysis results indicated that 18 components such as isovanillic acid,descuraininA and kaempferol-7-<italic>O-β-D</italic>-glucopyranoside were confirmed in decoction. Molecular docking analysis results indicated that 6 core components (degree value top 6),such as isovanillic acid,descurainin A and kaempferol-7-<italic>O-β-D</italic>-glucopyranoside,had good binding activity with silent information regulatory factor 1(SIRT1),interleukin(IL)1B,protein kinase B<italic>α</italic>(AKT1) and tumor necrosis factor(TNF). The compound recipe for heart failure mainly involved mitogen-activated protein kinase(MAPK),hypoxia inducible factor-1(HIF-1),Mammalian target of rapamycin(mTOR) and other signaling pathways. Conclusion:This study preliminarily investigated the effective constituents and mechanism of Tingli Dazao Xiefeitang in the treatment of heart failure. It can provide references for the precise clinical medication and screening of quality control markers,as well as the discovery of active components in the treatment of heart failure.

4.
Article in Chinese | WPRIM | ID: wpr-887939

ABSTRACT

Astragali Radix is one of the most commonly used medicinal materials. In recent years, its cultivated varieties and a variety of adulterants have flooded the market, which makes its quality uneven, and the development of quality control methods has become a research hotspot. Therefore, figuring out the quality markers of Astragali Radix is of great significance for its comprehensive evaluation. In this study, the fingerprints of 15 batches of Astragali Radix were established by HPLC, and the main components causing intergroup differences were screened out by PLS-DA. On the basis of literature review and network pharmacology analysis, the targets and pathways of active ingredients were obtained from SwissTargetPrediction, PubChem Compound and other databases, and then the "component-target-pathway" network was constructed with Cytoscape 3.7.1 for the prediction of potential quality markers. Twenty-eight common peaks were identified in the established fingerprint, and three differential components were selected as potential quality markers for Astragali Radix, which were astragaloside Ⅳ, calycosin-7-O-β-D-glucoside and ononin. The proposed method based on HPLC fingerprint of Astragali Radix is convenient and feasible, facilitating the improvement in its quality control.


Subject(s)
Astragalus Plant , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Plant Roots , Quality Control
5.
Article in Chinese | WPRIM | ID: wpr-921748

ABSTRACT

This Radix study cum aims Melle to explore(HRPM)the on efficacy spleen differences deficiency between syndrome.modeling Astragali A Radix of Praeparata110cum rats Melle were(ARPM)randomized fatigue),and into rats Hedysari a Praeparata(n qi total irregular HRPM male diet,SD diarrhea,control were(n Yiqi=10)the=100).Pill group fied and model a modeling group,group Buzhong After(BYP)(through ARPM and the HRPM-H),classimedium-dose into(ARPM-M raised group,and high-dose(ARPM-H each and Rats BYP and under HRPM-M),normal and low-dose and(ARPM-L in and group HRPM-L)were groups,continuously10rats induced.were in group.the in group the were18.9,control given group were g·kg~(-1)conditions while those the the model Rats respectively in18.912.6,BYP kg~(-1)water extract,decoction those in ARPM/HRPM-H,the-M,dosage lasted and of-L groups treated the with control and model6.3group g·rewere motilin determined m L·kg~(-1)·day~(-1).days.of dose Spleen ARPM/HRPM of in water.morning,The at the10Rats spleen in index group thymus and index ceived equal calculated.(MTL),distilled tissue administration to15observe Then the and Routine of each group D-xylose,were was(IL-2),the subjected HE stainingγ(IFN-γ),lower to the pathological changes.(IgA),blood gastric indexes,mucosa index,interleukin-2group.interferon group immunoglobulin of A and spleen pepsin index,of in Ig A,IL-2spleen IFN-γ,control each MTL,levels Rats pepsin the in model(P<0.01),had higher levels routine(P<0.01),blood and indexes,more thymus lesions D-xylose,the and in index,level decreased HRPM-L of IL-2severe compared spleen with than the those model in group.thymus group.that(P<0.05group,P<0.01)index administration thymus groups Ig A or spleen as that and in spleen routine Except index,spleen the Ig A,index,group and were in in ARPM-M model group,group,index,indexes,P<0.01)and thymus MTL index,those in ARPM-L insignificantly Ig A,different pepsin from other those in the the blood index,compared IFN-γ,group,(P<0.05The D-xylose,model MTL,spleen and lesions high-dose in each administration administration groups group increased relieved.blood or comparison as of with HRPM in as the folARPM and the effect in and were white and result than ARPM and is of lows:(P stronger<0.05),of medium-dose high-dose HRPM HRPM on IL-2cell high-dose of(WBC)and count medium-dose the HRPM and corresponding doses than IFN-γmore ARPM the obvious effect(P<0.05of on evident(P<0.05of impact P<0.01),on low-dose between the on corresponD-xylose P<0.01),doses ding MTL doses than Meanwhile,in of or more high-dose,and medium-dose,difference HRPM the and indexes.corresponding there of ARPM in or IL-2no levels in the HRPM-L effect and two groups,on but conclusion,other the both functions IFN-γwas group no was difference more the than recovery that of the and ARPM-H between(IL-2,P<0.01;ARPM-L recovery HRPM the IFN-γ,P<0.05).HRPM-H and obvious therapeutic in rats group qi In ARPM dose have are certain equivalent,effects on with spleen function deficiency.the Specifically,is the better difference immunomodulatory of two at g·low kg~(-1).and but the promote immunomodulatory the of former rats significantly ARPM.than that between of the later two at in the dose>18.9HRPM promotion can of better digestion digestion absorption and may absorption due of than The immunoregulation and be to the difference in clinical medication.


Subject(s)
Animals , Astragalus Plant , Drugs, Chinese Herbal , Plant Roots , Rats , Spleen
6.
Article in Chinese | WPRIM | ID: wpr-921718

ABSTRACT

An UPLC-MS/MS method was developed to simultaneously determine complanatoside A and complanatoside B in rat plasma with rutin as the internal standard and applied to examine the effect of salt-processing on pharmacokinetics of these two flavonoid glycosides. The pharmacokinetic parameters were estimated using DAS 3.2.6 and subjected to independent sample t-test with SPSS 23.0. No significant difference in T_(max) of complanatoside B was observed between the raw and processed groups; however, in the processed group, C_(max) and AUC_(0-12 h) of complanatoside B increased obviously(P<0.05), while MRT_(0-12 h) decreased from(3.34±0.44) h to(1.81±0.36) h(P<0.05). C_(max) [(14.72±11.13) μg·L~(-1)] and MRT_(0-24) [(3.93±0.26) h] of complanatoside A in the raw group were statistically different from those [(35.64±21.99) μg·L~(-1),(1.43±0.24) h] in the processed group(P<0.05). As a result, salt-processing can facilitate the in vivo adsorption and accelerate the excretion of complanatoside A and complanatoside B.


Subject(s)
Animals , Astragalus Plant , Chromatography, High Pressure Liquid , Chromatography, Liquid , Glycosides , Rats , Semen , Tandem Mass Spectrometry
7.
Article in Chinese | WPRIM | ID: wpr-773120

ABSTRACT

Chemical constituents of the Fufang Huangbai Ye( FFHB) were analyzed and identified by UPLC-ESI-LTQ-OrbitrapMS. The analysis was performed on an Waters HSS T3 reverse phase column( 2. 1 mm×100 mm,1. 8 μm). The mobile phase consisting of 0. 1% aqueous formic acid( A) and acetonitrile( B) was used with gradient elution,and the flow rate was 0. 3 mL·min~(-1).Based on the information of the accurate mass,the multistage fragment ions,the mass spectrometric data of the standard substance and the relative reference literature,the structure of the chemical constituents in FFHB were identified. Based on the identified compounds,network pharmacology study,including target prediction,functional enrichment,and molecular docking was applied to screen out the main active substances for treatment of diabetes foot and explore the potential mechanism. The results showed that a total of 138 compounds were identified,including 28 alkaloids,16 flavonoids,11 phenylethanoid glycosides,9 cycloolefins,11 cyclohexylethanol derivatives,28 phenolic acids and derivatives,3 lignans,4 terpenes,28 volatile oils and the others. Further,36 active substances for diabetes foot were screened out,and the functional enrichment showed the potential mechanism of FFHB were mainly seven functional items including inflammatory response,growth factor activity. This study combining the UPLC-LTQ-Orbitrap-MS technology and the network pharmacology provide a useful reference and basis for active compounds,quality control markers and the pharmacological mechanism of FFHB for diabetic foot treatment.


Subject(s)
Chromatography, High Pressure Liquid , Diabetic Foot , Drug Therapy , Drugs, Chinese Herbal , Pharmacology , Humans , Mass Spectrometry , Molecular Docking Simulation , Phytochemicals , Pharmacology
8.
Article in Chinese | WPRIM | ID: wpr-802182

ABSTRACT

Objective:In this paper,the network pharmacology method was used to explore the material basis and the mechanism of Magnoliae Officinalis Cortex(Houpo) on depressive disorder. Method:Firstly,the main chemical components of Houpo were gathered from CNKI,SciFinder,traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP) and other databases.Next,the potential targets of the chemical ingredients in Houpo were searched and selected by BATMAN-TCM database.The targets of depressive disorder were collected from HPO database.Then all the targets were entered into the search tool(String database) for the retrieval of protein-protein interactions so as to confirm antidepressant chemistries and their related targets.Furthermore,the functional enrichment analysis was carried out through the David database.Based on these above results,the networks of "drug targets-disease targets" and "compounds-targets-pathways" of Houpo on depressive disorder were built by Cytoscape v3.5.1 software,respectively.Network topology analysis was used to screen the key targets and the corresponding components.Then molecular docking verification of "component-target proteins" was further conducted. Result:A total of 16 active compounds involving in 74 key targets for depressive disorder were selected and confirmed from 138 chemical components of Houpo.Molecular docking analysis showed that compared with other components,ten volatile components in the 16 active compounds had good binding activities with the top 5 key targets[the top 5 of degree value,including insulin receptor(INS),mitogen-activated protein kinase 1(MAPK1),guanine nucleotide binding protein alpha inhibition 3(GNAI3),phosphatidylinositol 3-kinase receptor 1(PIK3R1) and selective receptor B1(GNB1)] and the 3 direct acting targets of popular drugs for depression[muscarinic acetylcholine receptor M2(CHRM2),5-hydroxytryptamine receptor(HTR)2B and HTR2C].The functional enrichment analysis showed the antidepressant mechanism of Houpo mainly involved neurotrophin signaling pathway,MAPK signaling pathway,calcium signaling pathway and neuroactive ligand-receptor interaction,etc. Conclusion:This study reveals the active ingredients and the mechanism of anti-depression of Houpo based on network pharmacology,a total of 16 key active ingredients related to anti-depression are selected.This paper can provide references for development of antidepressants and the discovery of quality markers of Houpo for anti-depression.

9.
Article in Chinese | WPRIM | ID: wpr-802023

ABSTRACT

Quality marker(Q-marker) is a new concept and pattern for quality control of traditional Chinese medicine(TCM),which will lead the development direction for quality control of TCM.Among them,how to characterize the overall quality attribute of TCM and its biological effect,is a critical scientific problem in the study of Q-marker.In this paper,integrated pharmacology is utilized to screen out and confirm the Q-marker from the complex system of TCM,so as to solve the critical scientific problem.System biology in vivo is firstly applied to establish the correlation of chemical fingerprints of TCM,their metabolic fingerprints,network targets,biological effects and efficacy of TCM,which is used to preliminary screen out Q-marker of TCM.Following that,a pharmacological method in vitro,including intestinal absorption in vitro coupled with bioactivity assessment,is employed to simultaneously determine the absorbed doses of TCM and evaluate their biological activity.Furthermore,data mining is utilized to establish the exact quantitative mathematic model between Q-marker of TCM and bioactivity.Meanwhile,two representative examples,including Yuanhu Zhitong tablets,Xinsuning capsules,are introduced to identify Q-marker of TCM and establish their quality standards related with bioactivity,which will be beneficial to improve the level of quality control of TCM and ensure the effectiveness and safety of clinical applications.

10.
Article in Chinese | WPRIM | ID: wpr-801717

ABSTRACT

Objective: To analysis and identify the chemical components in Trichosanthis Fructus by UPLC-LTQ-Orbitrap-MS. Method: Samples of Trichosanthis Fructus were extracted by ultrasonic with 70% methanol after smashing and sifting by 40 mesh sieve. Thermo ScientificTM DionexTM UltiMateTM 3000 Rapid Separation LC system performed UPLC separations with Waters HSS T3-C18(2.1 mm×100 mm,1.8 μm) column. The mobile phase was 0.1% formic acid water(A)-methanol(B) with a gradient elution. The volume flow was 0.3 mL ·min-1. A Thermo ScientificTM LTQ-Orbitrap mass spectrometer equipped with a ESI probe was employed. The samples were respectively scanned in MS1 and MS2 mode of positive and negative ions. According to the chromatographic peak separation,mass signal intensity,and the number of molecular ions in MS1 model,the extraction condition,chromatogram and mass spectrum parameters were optimized. The chemical compounds were identified by the accurate mass measurement of molecular ions and fragment ion and comparation with reference substance. Result: 91 chemical compositions in Trichosanthis Fructus were totally identified,including 14 amino acids,5 monoterpenoids,5 tetracyclic triterpenoids,1 pentacyclic triterpene,14 flavonoids, 17 organic acids,3 polysaccharides,7 nucleotides,7 alkaloids and nitrogen compounds,2 volatile components,1 phytosterol,5 other compositions. Conclusion: The established UPLC-LTQ-Orbitrap-MS method can be used to quickly analyze and identify the main chemical constituents of Trichosanthis Fructus. The chemical information concerning the constituents in Trichosanthis Fructus could be helpful to the quality control and further studies of Trichosanthis Fructus.

11.
Article in Chinese | WPRIM | ID: wpr-801715

ABSTRACT

Objective: Taking electronic-eye (visual analyzer) technique,based on the powder color of Andrographis Herba,to investigate the applicability of electronic-eye technique and evaluate the quality of Andrographis Herba with different commercial specifications. Method: HPLC was employed to determine contents of andrographolide,dehydroandrographolide,14-deoxyandrographolide,neoandrographolide in 50 batches of Andrographis Herba with different commercial specifications(stems,leaves and aerial parts).Color of these samples were measured by electronic-eye technique.The data were analyzed by principal component analysis(PCA) and Pearson correlation analysis.The ability of electronic-eye to distinguish the different commercial specifications of Andrographis Herba was investigated and the correlation of chroma space system parameters (L*,a*,b*) with active components was investigated. Result: There was remarkable difference in contents of 4 diterpenoids in Andrographis Herba from different parts,their contents in leaves was the highest,followed by the aerial parts(mixture of stems and leaves),and their contents in stems was the lowest.The results of PCA was divided into two classes,namely the stem part,leaf and aerial parts,indicating that electronic-eye could be used to distinguish the quality of Andrographis Herba.The correlation results showed that there were significant negative correlation(PL*(lightness value) and the contents of andrographolide,dehydroandrographolide,14-deoxyandrographolide,neoandrographolide and the total content of these 4 components.In addition,L* of samples that did not conform to the lower limit of determination in the 2015 edition of Chinese Pharmacopeia was ≥ 69.5,and the L* of more than 90% of the samples in accordance with the requirements was Conclusion: Electronic-eye technique provides a new method and idea for the quality evaluation of Andrographis Herba.

12.
Article in Chinese | WPRIM | ID: wpr-771539

ABSTRACT

Intestinal absorption liquid was prepared by using everted intestinal sac method; meanwhile, its recipes were decomposed or restructured. Platelet aggregation activity was examined by biochemical tests and a microplate reader. One or more kinds of Chinese medicines which displayed inhibiting activity in Naoxintong Capsules were screened through separation and combination of prescription. The results showed that Naoxintong Capsules could inhibit ADP-induced platelet aggregation. Recipe decomposition and restructuring results showed that Salviae Miltiorrhizae Radix et Rhizoma, Paeoniae Radix Rubra, Cinnamomi Ramulus and Hirudo were the main effective medicines in inhibiting platelet aggregation. Furthermore, Cinnamomi Ramulus played a vital role in inhibiting activity among those four kinds of Chinese medicines. Coumarin derived from intestinal absorption liquid of Cinnamomi Ramulus had inhibiting activity in the range of 50-200 μmol·L⁻¹, and other ingredients such as cinnamyl alcohol and cinnamaldehyde also had inhibiting activities. In conclusion, Salviae Miltiorrhizae Radix et Rhizoma, Paeoniae Radix Rubra, Cinnamomi Ramulus and Hirudo are the main components for inhibiting ADP-induced platelet aggregation, and Cinnamomi Ramulus has the most strongest inhibiting activity in Naoxintong Capsules.


Subject(s)
Adenosine Diphosphate , Capsules , Drugs, Chinese Herbal , Intestinal Absorption , Platelet Aggregation
13.
Article in Chinese | WPRIM | ID: wpr-687266

ABSTRACT

Cardiovascular and cerebrovascular diseases (CCDs) are the primary causes of death in Chinese adults. With the increase in morbidity and mortality rates and the decrease in the age of onset, CCD becomes a very natural target for traditional Chinese medicine. Schisandrae Chinensis Fructus (SCF) is the dry ripe fruit of Schisandra chinensis (Turcz.) Baill., which features a sweet and sour taste and the effects of calming the heart and tranquilizing the mind. It is mainly used for treatment of dysphoria and palpitation, insomnia and dreamful sleep due to the lack of spirit preservation. The main components of SC include lignans, volatile oils and polysaccharides. This review summarized the pharmacological effects of SC and its active components in the treatment of CCDs. The results showed that SCF and its active components protect against cardiovascular diseases mainly through the antioxidant, apoptosis inhibition and anti-inflammatory mechanisms. In addition, they protect against cerebrovascular diseases mainly by increasing energy metabolism, regulating autophagy and inhibiting apoptosis, antioxidant, and regulating nerve neurotransmitters and circadian genes. In conclusion, lignans are the most active components in SCF. This study provides a reference for the clinical research and utilization of SCF, as well as the application basis for co-treatment of cardiovascular and cerebrovascular diseases.

14.
Chinese Herbal Medicines ; (4): 282-289, 2014.
Article in Chinese | WPRIM | ID: wpr-842351

ABSTRACT

Objective: To assess the quality of Astragali Radix from different areas based on the biological evaluation and chemical analysis. Methods: The bioassay method of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity for Astragali Radix was established. The parameters of DPPH assay including sample extraction time, reaction time, repeatability, and stability were detected. Furthermore, a method of HPLC-MS was developed to simultaneously determine calycosin. -7-O-glucoside, ononin, formononetin, and astragaloside IV in Astragali Radix samples. And the total flavonoids and total saponins were detected by spectrophotometry. The relationship between DPPH evaluation and chemical analysis was studied by Pearson correlation analysis. Results: Twelve batches of Astragali Radix from different origins showed a wide range of DPPH radical scavenging activities (IC50 = 1.395-9.894 μg/mL). Based on DPPH assay, Sample 10 derived from Inner Mongolia Autonomous Region (IC50 = 1.395 μg/mL) showed the best quality of all samples. Chemical analysis showed that different compounds selected as indices would cause different results for quality evaluation. Pearson correlation analysis revealed that the contents of total flavonoids (P = 0.032), calycosin-7-glucoside (P = 0.035), and astragaloside IV (P = 0.010) were positively correlated with DPPH radical scavenging activity. Conclusion: Except for chemical analysis, DPPH radical scavenging activity can be used as a good alternative to assess and control the quality of Astragali Radix.

15.
Article in Chinese | WPRIM | ID: wpr-336719

ABSTRACT

<p><b>OBJECTIVE</b>To construct and identify lentiviral vector containing human ILK-shRNA and mda7 gene.</p><p><b>METHODS</b>Based on the human ILK gene sequences, RNAi target sequences were designed and cloned into the lentiviral vector pSicoR-eGFP by restriction endonuclease HpaI and XhoI double digestion and T4 DNA ligase ligation. Based on the human mda7 gene sequences, PCR primers were designed to clone the full-length mda7, and were cloned into the lentiviral vector pLVX-Puro. After the candidate clones were identified by DNA sequencing, the recombinant plasmid and the three packaging plasmids were co-transfected into the human embryonic kidney 293T cells by lipofectamine 2000 to produce the lentiviral particles. Human prostate cancer PC-3 cells were infected with the constructed lentiviral vector. The ILK and mda7 expression levels in PC-3 cells were quantified by qPCR and Western blot, respectively. The effect of ILK and mda7 on proliferation and migration of PC-3 cells were assessed by MTT method and Transwell assay, respectively.</p><p><b>RESULTS</b>ILK-pSicoR-eGFP and mda7-pLVX-Puro lentiviral vectors were successfully constructed. Strong green fluorescence was observed in the 293T cells under the fluorescent microscope after co-transfection of 293T cells with 4 plasmids of lentiviral vector. The transfection efficiency of the collected virus exceeded 90% in the 293T cells and the PC-3 cells were infected with the lentiviral particles with high efficiency. The A and B lentiviral vector inhibited the expression of ILK at both the mRNA and protein levels in PC-3 cells significantly. The mda7-pLVX-Puro lentiviral vector increased the expression of mda7 in PC-3 cells, and the ability was maintained for one month. Within 96 h, ILK and mad7 significantly inhibited the proliferation and migration of PC-3 cells (Ps<0.05).</p><p><b>CONCLUSION</b>The lentiviral vectors of ILK knockdown and mda7 over-expression have been successfully constructed and identified. The recombinant lentivirus can efficiently infect human prostate cancer PC-3 cells, in which ILK expression is inhibited and mda7 is over-expressed.</p>


Subject(s)
Cell Line , Genetic Vectors , Humans , Interleukins , Genetics , Lentivirus , Genetics , Plasmids , Genetics , Protein Serine-Threonine Kinases , Genetics , RNA, Small Interfering , Genetics , Transfection
16.
Article in Chinese | WPRIM | ID: wpr-350693

ABSTRACT

<p><b>OBJECTIVE</b>To establish a method to determine underivatized endogenous amino acids in brain tissues after cerebral ischemia based on RRLC-QQQ.</p><p><b>METHOD</b>Diamonsil chromatographic column C18 (4.6 mm x 250 mm, 5 microm) was adopted to determine 12 amino acids in 15 min, with acetonitrile-0.1% formic acid for gradient elution. The flow rate was set at 0.5 mL x min(-1). With ESI as the ion source, positive ion scanning mode was adopted for multi-reaction monitoring.</p><p><b>RESULT</b>Each amino acid standard curve (AAs) showed good linear relationship within the detection range (r > 0.996), with the limit of detection of less than 11%, the limit of quantitation of less than 3.09 microg x L(-1). The RSD of intra- and inter-day precisions at high, middle and low concentrations were less than 11%.</p><p><b>CONCLUSION</b>The determination results of actual samples showed that compared with the levels of AAs of the sham operation group, all of the remaining amino acids apart from N-acetyl-aspartate increased in brain tissues. Some amino acids showed significant changes in a time-dependent manner after the operation. The method is so simple, rapid and sensitive that it can be used for finding biological metabolite markers of cerebral ischemia, and exploring cerebral ischemia molecular mechanisms and synergistic mechanism of combined administration of multi-component traditional Chinese medicines.</p>


Subject(s)
Amino Acids , Metabolism , Animals , Brain , Metabolism , Brain Ischemia , Metabolism , Chromatography, High Pressure Liquid , Methods , Male , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Tandem Mass Spectrometry , Methods
17.
Article in Chinese | WPRIM | ID: wpr-332420

ABSTRACT

<p><b>OBJECTIVE</b>Conducting the gene characterization of the E6 and E7 gene of human papillomavirus 16 (HPV16) isolated from 15 cases of cervical cancer at Beijing.</p><p><b>METHODS</b>Overlapping primers were designed according to the full-length genomes of E6 and E7 from the GenBank and PCR was used to amplify the E6 and E7 fragments. TA clone was used to select a purified clone in order to have better and valuable sequencing results. Nucleotide and amino acid sequence were analyzed by the Sequencer, Bioedit, Mega et al.</p><p><b>RESULTS</b>8 of 15 (8/15) cervical samples contained HPV E6 and E7 gene, and 4 had Asian type like and 4 had Europe prototype like. There were two nucleotide mutation at E6 position 178 (T-->G,D25E) and at E7 position 647 (A-->G, N29S) in 4 Asian type like viruses. There were one nucleotide mutation at E6 position 335 (C-->T, H78Y) in 1 of 4 Europe prototype like virus. In the cervical cancer samples, 8 of 15 contained the HPV16E 6 and E7 gene. HPV16 E6 and E7 can not be detected in denosquamous carcinoma and adenocarcinoma.</p><p><b>CONCLUSION</b>HPV16 is the main etiology of the cervical carcinoma. The HPV16 infectious ratio of squamous carcinoma is more than the ratio of adenocarcinoma. 178th nucleotide in E6 gene is the very important site to distinguish the Asia and the Europe prototype strain like. 178 nucleotide in E6 and 647 nucleotide in E7 are the frequent mutation site in cervical carcinoma. Analysis based on the E6 and E7 gene sequence of HPV 16 isolates suggests that naturally occurring sequence variants of E6 and E7 gene may have identify the oncogenic properties.</p>


Subject(s)
Adenocarcinoma , Virology , Carcinoma, Squamous Cell , Virology , China , Female , Human papillomavirus 16 , Chemistry , Genetics , Humans , Mutation , Oncogene Proteins, Viral , Chemistry , Genetics , Papillomavirus E7 Proteins , Papillomavirus Infections , Virology , Repressor Proteins , Chemistry , Genetics , Sequence Homology, Amino Acid , Uterine Cervical Neoplasms , Virology
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