Therapeutic effect of Panax notoginseng saponins combined with cyclophosphamide in mice bearing hepatocellular carcinoma H22 cell xenograft / 南方医科大学学报

OBJECTIVE@#To investigate the therapeutic effects of total saponins from Panax notognseng (PNS) combined with cyclophosphamide (CTX) in mice bearing hepatocellular carcinoma H22 cell xenograft.@*METHODS@#We examined the effects of treatment with different concentrations of PNS on H22 cell proliferation for 24 to 72 h in vitro using CCK8 colorimetric assay. Annexin V/PI double fluorescence staining was used to detect the effect of PNS on apoptosis of H22 cells. Mouse models bearing H22 cell xenograft were established and treated with CTX (25 mg/kg), PNS (120, 240 or 480 mg/kg), alone or in combinations. After treatments for consecutive 10 days, the mice were euthanized for examinations of carbon clearance ability of the monocytes and macrophages, splenic lymphocyte proliferation, tumor necrosis factor (TNF-α), interleukin-2 (IL-2), serum hemolysin antibody level, blood indicators, and the tumor inhibition rate.@*RESULTS@#Treatment with PNS concentration-dependently inhibited the proliferation and significantly promoted apoptosis of cultured H22 cells (P < 0.01). In the tumor-bearing mouse models, PNS alone and its combination with CTX both resulted in obvious enhancement of phagocytosis of the monocyte-macrophages, stimulated the proliferation of splenic lymphocytes, promoted the release of TNF-α and IL-2 and the production of serum hemolysin antibody, and increased the number of white blood cells, red blood cells and lymphocytes in the peripheral blood. Treatment with 480 mg/kg PNS combined with CTX resulted in a tumor inhibition rate of 83.28% (P < 0.01) and a life prolonging rate of 131.25% in the mouse models (P < 0.05).@*CONCLUSION@#PNS alone or in combination with CTX can improve the immunity and tumor inhibition rate and prolong the survival time of H22 tumor-bearing mice.

Effect of Notoginseng Total Saponins on Changing Multidrug Resistance in HepG2/ Adriamycin Cells Through ERK/Akt Signaling / 中国实验方剂学杂志

Objective:To investigate the effect of notoginseng total saponins （TNS） on adriamycin （Adr） resistance in HepG2/Adr cells and the expression and activity of the mechanisms as the modulators of multi-drug resistance， so as to explore the possible mechanism of extracellular signal-regulated kinase （ERK） and protein kinase B （Akt） signaling pathways in reversing the resistance of HepG2/Adr cells mechanism. Method:Effect of TNS on HepG2/Adr cell proliferation was detected by thiazole blue （MTT） method. HepG2/Adr cells were treated with different concentrations （100， 50， 25， 0 mg·L^-1） of TNS and （20 μmol·L^-1） Adr respectively， and a blank group was set. The high-content screening platform was used to detect the accumulation of Adr in HepG2/Adr cells after 40 minutes， 3 hours and 6 hours. Western blot was used to detect the expression of P-glycoprotein /multidrug resistance/ATP binding cassette subfamily B member 1（P-gp/MDR1/ABCB1） and other drug resistance-related proteins and the main protein expression of ERK/Akt signaling pathway. The change of MDR1 on cell membranes was observed by laser confocal microscopy. Result:Compared with HepG2 cells， the expression of MDR1 in HepG2/Adr cells was significantly increased （<italic>P</italic><0.01）. Compared with the Adr group， the half-inhibitory concentration （IC₅₀） of TNS （25， 50， 100 mg·L^-1） and Adr （20 μmol·L^-1） co-administration group on HepG2/Adr cells <italic>in vitro</italic> significantly reduced （<italic>P</italic><0.01）， and the highest reversal multiple was 10 times. Compared with the Adr group， the co-administration group could significantly increase the accumulation of Adr in the cells （<italic>P</italic><0.05） in a dose-dependent manner. Compared with the blank group， the co-administration group could significantly reduce MDR1， ABC semitransporter （ABCG2）， multidrug resistance associated protein （MRP1）， ERK， phosphorylated extracellular regulatory protein kinase （p-ERK）， Akt， phosphorylated protein kinase B （p-Akt）， mammals， rapamycin target protein （mTOR） and phosphorylated mammalian rapamycin target protein （p-mTOR） （<italic>P</italic><0.05）， with the same results in the doxorubicin group. Compared with the blank group， there was no significant difference in the distribution and fluorescence intensity of MDR1 on the cell membrane between the Adr group and the notoginseng total saponins （25 mg·L^-1） group. Compared with the blank group and the doxorubicin group， TNS could significantly reduce the distribution of MDR1 on the cell membrane （<italic>P</italic><0.05）. Conclusion:TNS can inhibit the ERK/Akt pathway， reduce the expression of MDR1， and significantly increase the accumulation of doxorubicin in HepG2/Adr cells， which may be one of the mechanisms of notoginseng total saponins in reversing resistance.

Effect of Astragaloside Ⅳ Combined with Notoginseng Total Saponins on Angiogenesis After Transplantation of BMSCs in Rats with Cerebral Ischemia / 中国实验方剂学杂志

Objective:To investigate the effect of astragaloside Ⅳ（AST Ⅳ）and Notoginseng total saponins （NTS） combined with bone marrow mesenchymal stem cell （BMSC） transplantation on neural repair and angiogenesis in rats with cerebral ischemia. Method:The rats were randomly divided into a sham operation group， a model group， low- and high-dose AST Ⅳ + NTS groups， a BMSC infusion group， and low- and high-dose BMSC infusion+AST Ⅳ （10 and 20 mg·kg^-1） + NTS group （25， 50 mg·kg^-1）. BMSCs were isolated and purified by whole bone marrow adherent culture. The positive expression of surface markers of BMSCs （CD29， CD90， CD34， and CD45） was detected by flow cytometry. The focal cerebral ischemia model was established by middle cerebral artery occlusion （MCAO）. The PKH26-labeled BMSCs were injected into the tail vein of rats in the BMSC infusion group， once a day. The rats in the combination groups received BMSC injection once a day and intragastric administration of drugs twice a day. Other groups were administered twice a day by gavage. The sham operation group and the model group received the same amount of normal saline. Symptoms and signs of neurological deficits were assessed by the Longa method and the cerebral infarction rate was determined by TTC staining. The survival and vascularization ［double positive expression of PKH26/vascular endothelial growth factor （VEGF）］ after transplantation of BMSCs were observed by the immunofluorescence method. The protein expression of Ang1 and TGF-<italic>β</italic>₁ was measured by Western blot. Result:BMSCs were properly isolated and cultured. The identification of surface markers CD29， CD90， CD34， and CD45 was consistent with the characteristics of BMSCs. The neurological deficit score and cerebral infarction rate of the model group were significantly increased （<italic>P</italic><0.01）. All drugs and cell transplantation could alleviate the above pathological changes in varying degrees. The strongest effect was observed in high-dose BMSC infusion+AST Ⅳ+NTS group （<italic>P</italic><0.01）， which was superior to those in the AST Ⅳ+NTS groups or the BMSC infusion group. BMSC injection helped cells survive in the ischemic brain tissues and promoted angiogenesis， and this effect could be enhanced by the combination with drugs. After cerebral ischemia， the expression of Ang1 and TGF-<italic>β</italic>₁ was increased， and the effect in the BMSC infusion+AST Ⅳ+NTS groups was the strongest （<italic>P</italic><0.01）. Conclusion:AST Ⅳ combined with NTS can promote the survival of transplanted BMSCs and facilitate angiogenesis after target repair of damaged blood vessels after cerebral ischemia. The mechanism may be related to the improvement of the local microenvironment in the brain after cerebral ischemia and the promotion of the survival and differentiation of transplanted stem cells.

Composition of Ophiopogon Polysaccharide, Notoginseng Total Saponins and Rhizoma Coptidis Alkaloids Inhibits the Myocardial Apoptosis on Diabetic Atherosclerosis Rabbit / 中国结合医学杂志

OBJECTIVE@#To investigate the effects of Composition of Ophiopogon polysaccharide, Notoginseng total saponins and Rhizoma Coptidis alkaloids (CONR) on myocardial apoptosis of diabetic atherosclerosis (DA) rabbits METHODS: Sixty male New Zealand white rabbits were randomly divided into 6 groups [control group, model group, CONR high-dose group (450 mg/kg), CONR medium-dose group (150 mg/kg), CONR low-dose group (50 mg/kg), and simvastatin group] by using a completely random method, 10 in each group. DA model was established by intravenously injected alloxan combined with high-fat diet and abdominal aortic balloon injury. After mediation for 10 weeks, fasting blood glucose (FBG), glycosylated hemoglobin (GHB), glycosylated serum protein (GSP), fructoseamine (FRA), aldose reductase (AR), advanced glycation end products (AGEs) in serum were measured by enzyme linked immunosorbent assay (ELISA) method; the expression of receptor of AGEs (RAGE) in myocardial tissue were observed by immunohistochemical method; and p-Jun N-terminal kinase (p-JNK), caspase-3, B-cell lymphoma-2 (bcl-2) protein expression in myocardial tissue were measured by Western blotting. The myocardial apoptosis was detected by TdT-mediated dUTPnick-end labeling (TUNEL) method, and apoptosis index (AI) was calculated.@*RESULTS@#Compared with the control group, serum FBG, GHB, GSP, FRA, AR, AGEs and the expression of myocardium RAGE, p-JNK, caspase-3 proteins, as well as apoptosis index (AI) were significantly increased and bcl-2 protein was significantly decreased in the model group (P<0.01). Compared with the model group, the levels of serum FBG, GHB, GSP, FRA and AR showed a significant decline in CONR high- and medium-dose groups (P<0.01). FBG and GHB showed a significant decline in CONR low-dose group (P<0.01). Compared with the model group, the expression of serum AGEs and myocardium RAGE, p-JNK and caspase-3 protein as well as AI were significantly decreased and bcl-2 protein was significantly up-regulated in all treatment groups (P<0.01); high-dose CONR had the most significant effect on abovementioned indices compared with other treatment groups (P<0.01). Middle-dose CONR had better effect on serum AGEs compared with the low-dose group (P<0.01); middle-dose CONR and simvastatin groups had better effect on the expression of caspase-3, bcl-2 protein, myocardium apoptosis compared with the CONR low-dose group (P<0.01).@*CONCLUSION@#CONR may effectively inhibit myocardial apoptosis on DA rabbits by intervening AGEs-RAGE and JNK, caspase-3, and bcl-2 protein expressions.

Anti-fatigue Analysis of Common Mechanisms of Interaction of Ginseng Radix et Rhizoma "Tonifying Qi" and Notoginseng Radix et Rhizoma "Enriching Blood" / 中国实验方剂学杂志

Objective:To analyze the common active ingredients， potential target genes and pathways of Ginseng Radix et Rhizoma "Tonifying Qi" and Notoginseng Radix et Rhizoma "Enriching blood" in alleviating fatigue based on the network pharmacology technology. And the compound ingredients of total Ginsenoside Ginseng Root and Notoginseng total Saponins were selected to verify the core target genes in vitro. Method:The main active ingredients and related targets of Ginseng Radix et Rhizoma and Notoginseng Radix et Rhizoma were screened by traditional Chinese medicine systems pharmacology （TCMSP）. The data of fatigue genes were established by GeneCards comprehensive database and Human Mendelian Genetic Integrated Database（OMIM）. Depending， The data sets of fatigue-related genes are established based on the data bank of GeneCards and OMIM. The intersecting genes of drugs and disease were obtained by R software. Cytoscape software was used to establish the regulatory network among the active ingredients， drug targets and fatigue-related genes. PPI network of intersecting genes was constructed by STRING 11.0 software， and the core genes were screened by CytoHubba software and Matthews correlation coefficient （MCC） algorithm. Based on the results of network analysis， 24 male SPF ACR mice were randomly divided into control group， total Ginsenoside Ginseng Root group （0.08 g·kg-1） and Notoginseng total Saponins group （0.08 g·kg-1）. The corresponding drugs were given for 3 weeks. The expressions of core genes in muscle tissue were detected by real-time fluorescence quantitative PCR. Result:The 20 active components and 181 drug targets were screened from TCMSP. 33 intersecting genes of diseases and drugs were obtained when compared with GeneCards and OMIM comprehensive database using R software. 10 core genes including aryl hydrocarbon receptor （AHR）， androgen receptor （AR）， glutathione S-transferase P1 （GSTP1）， cysteine proteinase-3（Caspase-3）， cytochrome p450 enzyme 3A4 （CYP3A4）， intercellular adhesion molecule 1 （ICAM1） and nuclear factor kappa B inhibitor alpha （NFKBIA） were screened out by the algorithm of MCC. Total Ginsenoside Ginseng Root and Notoginseng total Saponins had no significant effect on GSTP1 and ICAM1 genes， but they could significantly inhibit the expressions of AHR， CYP3A4， Caspase-3， NFKBIA and AR （P<0.05，P<0.01）， and there were no significant difference in anti-fatigue effect between total Ginsenoside Ginseng Root and Notoginseng total Saponins groups. Conclusion:The mechanism of anti-fatigue of Ginseng Radix et Rhizoma and Notoginseng Radix et Rhizoma may be related to the regulation of AHR， CYP3A4 and Caspase-3 genes， and there is no significant difference in their anti-fatigue effects， through the analysis of network and experimental verification.

Effect of solution environment on ultrafiltration separation of Panax notoginseng total saponins based on molecular state / 中草药

Objective: To clarify the effect of solution environment on ultrafiltration separation of Panax notoginseng total saponins (PNS) based on the molecular state. Methods: In the experiment, the transmittance and surface tension were selected as indexes for analyzing the effect of ethanol, inorganic salts, surfactants, and pH on the molecular state of PNS. And then, ethanol, NaCl, and pH were selected as influencing factors to analyze the separation rule of notoginsenoside R1 (R1) and ginsenoside Rb1 (Rb1). Results: The intermolecular interaction force of saponins was weakened by increasing the ethanol concentration; The pH value promoted saponin ionization, increased critical micelle concentration, and increased PNS ultrafiltration transmittance; The salting out effect of inorganic salt reduced the critical micelle concentration and PNS transmittance; The surfactant type was related to the ultrafiltration separation behavior of PNS. Rb1 was more sensitive to the factors than R1 by response surface methodology. Conclusion: The effect of solution environmental factors on the ultrafiltration separation of PNS was clarified by the combination of single factor analysis and response surface methodology. And the saponins can be separated purposefully by dynamically adjusting the molecular state.

Simultaneous Quantitative Determination of Five Saponins in Notoginseng Total Saponins by HPLC with a Monolithic Column / 中国药师

Objective:To establish a rapid HPLC method for the quantitative determination of 5 saponins ( notoginsenoside R1 , ginsenoside Rg1 ,ginsenoside Re,ginsenoside Rb1 and ginsenoside Rd) in Notoginseng total saponins using a monolithic column. Meth-ods:The analysis was performed on a Merck Chromolith Performance RP-18e column (100 mm × 4. 6 mm,2μm) with gradient elution of acetonitrile and water. The detection wavelength was set at 203 nm. Results:Satisfactory separation of all analytes was obtained in 20 min. All calibration curves showed good linearity within the testing ranges (r≥0.9998). The average recoveries were between 98. 6% and 100. 4%. The RSDs were less than 2. 1% (n=6). Conclusion:The method is efficient and accurate for the quality con-trol of Notoginseng total saponins.

Simultaneous Quantitative Determination of Five Saponins in Notoginseng Total Saponins by HPLC with a Monolithic Column / 中国药师

Objective:To establish a rapid HPLC method for the quantitative determination of 5 saponins ( notoginsenoside R1 , ginsenoside Rg1 ,ginsenoside Re,ginsenoside Rb1 and ginsenoside Rd) in Notoginseng total saponins using a monolithic column. Meth-ods:The analysis was performed on a Merck Chromolith Performance RP-18e column (100 mm × 4. 6 mm,2μm) with gradient elution of acetonitrile and water. The detection wavelength was set at 203 nm. Results:Satisfactory separation of all analytes was obtained in 20 min. All calibration curves showed good linearity within the testing ranges (r≥0.9998). The average recoveries were between 98. 6% and 100. 4%. The RSDs were less than 2. 1% (n=6). Conclusion:The method is efficient and accurate for the quality con-trol of Notoginseng total saponins.

Preparation of notoginseng total saponins-tanshinone composite particles by solvent deposition method and their characterization / 中草药

Objective: To synchronously inhale Chinese materia medica compound using particle as inhalation drug delivery system, notoginseng total saponins-tanshinone composite particle was prepared. Methods: The composite particle of notoginseng total saponins-tanshinone was prepared by solvent deposition method and was characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), differential thermal analysis (DTA), particle size analysis, and high-performance liquid chromatography (HPLC). Results: The notoginseng total saponins-tanshinone composite particle was successfully prepared by solvent deposition method, the results of characterization proved that tanshinone was coated on the notoginseng total saponins core particle. Conclusion: The preparation of composite particle provides an effective way for synchronous inhalation of Chinese materia medica compound prescription and technical support for the preparation of compound dry powder inhalations.