Study on the Improvement Effect of Ginsenoside Rb 3 Combined with β-asarone on Vascular Dementia Model Mice and Its Mechanism / 中国药房

OBJECTIVE：To study the improvement effects of ginsenoside Rb 3 combined with β-asarone on vascular dementia （VD）model mice and its mechanism. METHODS ：ICR mice were randomly divided into model group ，ginsenoside Rb 3 group（10 mg/kg），β-asarone group （10 mg/kg），drug combination group （ginsenoside Rb 3 10 mg/kg+β-asarone 10 mg/kg），positive control group（donepezil hydrochloride 1 mg/kg）and Akt inhibitor group （LY294002，1 mg/kg），and sham operation group was set up ， with 10 mice in each group. Except for sham operation group ，VD model was induced by four vessel occlusion method in other groups. After modeling ，sham operation group and model group were given constant volume of normal saline ，Akt inhibitor group was given relevant medicine intraperitoneally ，and other groups were given relevant medicine intragastrically ，twice a day ，for consecutive 30 d. After last administration ，the learning and memory ability of mice was detected by avoiding darkness test. The contents of 4-hydroxydecenoic acid （4-HNE），8-hydroxydeoxyguanosine （8-OHdG） and reactive oxygen species （ROS） in hippocampus was detected by ELISA. RT-PCR assay was used ， to detect the mRNA expression of Bcl- 2 and Bax inhippocampus. The protein expression of Bcl- 2 in cortex wadetected by immunofluorescence method. Western blotting deng- assay was used to detect the protein expression of Bcl- 2 and mz1@126.com Bax in hippocampus. RESULTS ： Compared with sham operation group ，the incubation period of avoiding darkness xiaoyinlanlp@126.com test in model group was shortened significantly ； and the number of errors was increased significa ntly；4-HNE，8-OHdG and ROS contents ，mRNA and protein expression of Bax were increased significantly ，and mRNA and protein expression of Bcl- 2 was decreased significantly （P＜0.01）. Compared with model group，the incubation period of avoiding darkness test was prolonged significantly in ginsenoside Rb 3 group，β-asarone group ，drug combination group and positive control group ，the number of errors was decreased significantly ；4-HNE，8-OHdG，ROS contents ， mRNA and protein expression of Bax were decreased significantly ，and mRNA and protein expression of Bcl- 2 were increased significantly，especially in drug combination group （P＜0.05 or P＜0.01）. But the incubation period of avoiding darkness test was shortened significantly in Akt inhibitor group ，and the number of errors was increased significantly ；4-HNE，8-OHdG，ROS contents，mRNA and protein expression of Bax were increased significantly ，and mRNA and protein expression of Bcl- 2 were decreased significantly （P＜0.05）. CONCLUSIONS ：Ginsenoside Rb 3 combined with β-asarone has a protective effect on VD model mice ，and the effect was better than that of each compound alone. The mechanism of which may be associated with anti-oxidative stress and anti-apoptosis of hippocampus.

Determination of ginsenoside Re, pseudo-ginsenoside F11, ginsenoside Rb3, and ginsenoside Rd from flower buds of Panax quinquefolium / 中草药

Objective To establish the determination method of four ginsenosides from flower buds of Panax quinquefolium by Waters Acquity UPLC H-Class with Xevo TQD. Methods The chromatographic separation was performed on a Waters Acquity UPLC BEH C18 column (50 mm × 2.1mm, 1.7 μm). The mobile phase was a mixture of acetonitrile and water containing 0.05% ammonium hydroxide with gradient elution; The flow rate was 0.45 mL/min, and the column temperature was 35 ℃; Multiple reaction monitoring (MRM) acquisition under negative ion scan mode by ESI was also performed. Results The method was established with good precision, stability, repeatability, and accuracy. Ginsenoside Re, pseudo-ginsenoside F11, ginsenoside Rb3, and ginsenoside Rd showed a good linearity in the range of corresponding concentrations. Conclusion The UPLC-MS/MS method is rapid, simple, accurate, reliable, which can be used for the analysis of ginsenosides from flower buds of P. quinquefolium.

Effects of ecological factors on ginsenosides synthesis and its key enzyme genes expression / 中草药

Objective To explore the effect of ecological factors and the expression of key enzyme genes on the synthesis and accumulation of ginsenosides. Methods Cultivated four-year-old ginseng were used as test materials, the expression of key enzyme genes (HMGR, FPS, SS, SE, DS, β-AS, CYP82D47, CYP716A47) in the biosynthesis of ginsenosides of roots in different growth periods was determined by real-time quantitative PCR, determination of the content of eight ginsenosides (ginsenoside Rg1, Re, Rf, Rb1, Rb2, Rb3, Rc, Rd) in roots by HPLC, the meteorological data were collected by a small weather station, correlation analysis was performed with SPSS. Results The expression of key enzyme genes in the period of flowering to fruit ripening was higher than the root growing after fruit period and the withering period, the expression of key enzyme genes involved in the synthesis of ginsenosides was influenced by each other, and the expression of key enzyme genes in ginseng roots showed a positive correlation with the accumulation of ginsenosides; The contents of ginsenoside Rg1, Rb1, Re, and Rc were higher in the roots of ginseng, eight kinds of monomer ginsenosides content dynamic changes trend is different; Temperature, photosynthetically active radiation, soil water potential are important ecological factors for ginsenosides synthesis in roots, temperature was significantly negatively correlated with ginsenoside Rb1 and Rd (P < 0.05), PAR can significantly promote the formation of ginsenoside Rg1 (P < 0.05), soil water potential was significantly negatively correlated with ginsenoside Rb1 (P < 0.05); Grey correlation analysis results showed that the major ecological factors that influenced ginsenosides content in ginseng roots were temperature, PAR and relative humidity, the grey correlation between the expression of the key enzyme genes with the content of ginsenosides is less than ecological factors with the content of ginsenosides, under the guidance of ecological factors, the expression of the key enzyme genes regulate the synthesis and accumulation of ginsenosides. Conclusion The dynamic changes of the expression of key enzyme genes and the content of ginsenosides in ginseng were determined, it provides a theoretical basis for elucidating the physiological and ecological mechanism of ginsenoside synthesis and the quality control of Radix Ginseng.

Simultaneous determination of 15 ginsenosides from Panacis Majoris Rhizoma by HPLC-ESI-MS/MS method / 中草药

Objective: A high-performance liquid chromatography-tandem mass spectrometric method for the simultaneous determination of 15 ginsenoside compounds from Panacis Majoris Rhizoma (PMR) was developed. Methods: A Waters Sunfire ™ C18 column (150 mm × 4.6 mm, 5 μm) was used for the separation. The mobile phase consisted of A (H2O + 0.05% HCOOH) and B (CH3CN + 0.05% HCOOH) using a gradient elution. For the quantification of ginsenosides, the multiple reaction monitoring (MRM) mode of the mass spectrometer was applied and the declustering potential (DP), collision energy (CE), and collision cell exit potential (CXP) were optimized to perform automatic on-line MS/MS experiments during the chromatographic separation. Results: By using the optimized method, the linearity range of 15 analytes was 0.000 9 to 2 952.592 3 μg/mL with more than 0.999 determination coefficient (r) of linear regressions, the detection limits of the 15 ginsenosides ranged from 0.003 to 626.554 ng/mL, the limits of quantitation ranged from 0.075 to 1 762.150 ng/mL, the recoveries of 15 ginsenosides in the samples were 98.15%-101.12% with relative standard deviation (RSD) that ranged from 0.82% to 2.15%. Conclusion: The proposed LC-MS/MS method is accurate and reproducible in accordance with TCM guidelines, showing high sensitivity, rapidness, and recovery. This method allows the assessment of various ginsenosides in a single analytical run providing an innovative tool to control Panacis Majoris Rhizoma materials quantification.

Effect of Ginsenoside Re and Rb3 on mice with viral myocarditis / 中国小儿急救医学

Objective To study the role of Ginsenoside Re and Rb3 on mice with viral myocarditis (VMC) and explore the mechanisms. Methods BALB/C mice were infected by coxsackievirus B3 ( CVB3) to establish VMC model. The mice were divided into control group,virus group and Ginsenoside Re and Rb3 treatment group(treatment group). On day 5,day 10,day 20 after infection,the level of serum crea-tine phosphokinase-MB( CK-MB) was detected. Then myocardial sections stained with Masson′s trichrome were used to observe the distribution of mice myocardial collagen fibers, quantify collagen volume fraction (CVF),and detect the levels of superoxide dismutase(SOD). Results (1)The level of CK-MB peaked on day 5,and decreased afterwards[day 5:(463. 68 ± 47. 62) U/L; day 10:(588. 81 ± 56. 09) U/L; day 20:(340. 48 ± 58. 22) U/L,respectively]. While the levels of CK-MB in treatment group were lower than those in virus group[day 5:(378. 69 ± 56. 02) U/L;day 10:(452. 56 ± 67. 78) U/L; day 20:(327. 13 ± 47. 20) U/L,respectively] in the same point. There were significant differences between groups on day 5 and day 10 (P<0. 01). (2) In viral group,the blue staining degree gradually increased with time in myocardial sections stained with Masson′s trichrome,especially in myocardial necrosis area. Compared with treatment group,CVF increased significantly in virus group on day 10 and day 20 ( day 10:6. 52% ± 2. 34% vs. 8. 94% ± 1. 67%;day 20:7. 00% ± 1. 53% vs. 10. 46% ± 1. 74%,P<0. 01). The levels of SOD in myocardial sections in virus group were lower than those of control group[day 5:(48. 83 ± 17. 74) U/L;day 10:(61. 41 ± 14. 58) U/L;day 20:(66. 26 ± 18. 97) U/L,respectively,P<0. 05],but in treatment group,the level of SOD could beimprovedsignificantly[day5:(72. 07 ±24. 85)U/L;day10:(83. 22 ±19. 52)U/L;day20:(92. 00 ± 20. 46) U/L, respectively, P < 0. 05]. Conclusion Because of the inhibition of oxygen radicals and oxidative stress, Ginsenoside Re and Rb3 can protect myocardial tissue. Ginsenoside Re and Rb3 can effectively reduce the extent of myocardial fibrosis. The mechanism may be related with the reduced peroxide level in vivo.

Preparation and Quality Evaluation of Total Leaves Saponins of Panax Notoginseng Liposomal Gels / 中国药师

Objective:To study the preparation of total leaves saponins of panax notoginseng liposomal gels and evaluate the quali-ty. Methods:The total leaves saponins of panax notoginseng liposomes were prepared by the film-dispersion method, and the formula was optimized by the orthogonal experimental design. A high-speed centrifugation method was applied to determine the encapsulation efficiency used as the evaluation index for the optimization of the preparation process. Then the gels were prepared and an HPLC meth-od was used to determine the content of ginsenoside Rb3 . The stability was preliminarily studied as well. Results:The preparation was semi-solid, and viscous gels with low skin irritation and good stability. The quality was accordance with the relevant provisions in Chi-nese Pharmacopoeia (2010 edition). Conclusion: The gels are with the properties of simple preparation process, reliable detection methods and stable and controllable quality.

Effects of GSRb3 on GABA in brain tissue of rats with injury of hypoxia / 临床神经病学杂志

Objective To observe the influence of ginsenoside Rb3(GSRb3) on ?-aminobutyric acid (GABA) in brain tissue of rats with injury of hypoxia.Methods 20 rats were randomly divided into A, B, C, D and E group. The models of cerebral anoxia in group A and C were made by hypopiesia and hypoxia, and the models of hypoxia-reoxgenation were made in group B and D. 24 h and 1 h before establishment of models, ginsenoside Rb3 was injected peritoneally in rats of group C and D, respectively. The number and form of GABA-like immunoreactive neurons in hippocampus CA1 area of rats after brain hypoxia-reoxgenation were investigated with immunohistochemistry.Results (1)Compared with group E, GABA-like immunoreactive neuron density in CA1 area of group A and B decreased, presented with light staining and absence of prominency. GABA-like immunoreactive neuron density in CA1 area in group C and D was significantly higher than that in group A and B, but the shape was similar to group E. (2) The number of GABA-like immunoreactive neuron in CA1 area was 7.7?2.83 (group A), 10.1?2.08 (group B), 30.9?2.02 (group C), 33.1?4.2 (group D) and 16.9?1.05 (group E), respectively. The numbers were lower in group A and B than in group E, however higher in group C and D than in group E(all P

Absorptive Characteristics of Ginsenoside Rb_3 in Caco-2 Cell Monolayer Model / 中国药房

OBJECTIVE:To study the absorptive characteristics of ginsenoside Rb3 in Caco-2 cell monolayer model. METHO-DS:The ginsenoside Rb3 cell samples underwent high speed centrifugation, then the supernatant was collected and determined by LC-ESI-MS/MS method in which the mobile phase consisted of acetonitrile-1 mmol?L-1 ammonium formate water solution (34 ∶ 66) with ginsenoside Rg2 as internal standard, and the tandem mass spectrometry was operated in negative electrospray ionization in a multiple reaction monitoring (MRM) mode, with detection ions m/z1077.7→m/z 783.4 for ginsenoside Rb3 and m/z 783.6→m/z 475.1 for ginsenoside Rg2.The concentration of ginsenoside Rb3 across the Caco-2 cell monolayer model was determined and the apparent permeability coefficient(Papp) of ginsenoside Rb3 was calculated. RESULTS: The calibration curve for ginsenoside Rb3 was linear in the range of 50～2 000 ng?mL-1,with intra-day precision and inter-day precision at less than 15%. P(AP-BL) from apical side (AP) to basolateral side (BL) was 3.22?10-6 cm?s-1, whereas P(BL-AP) from BL to AP was 6.0?10-6 cm?s-1,and the ratio of P(BL-AP)/P(AP-BL) was 1.86.CONCLUSION:The LC-ESI/MS/MS method is simple and sensitive, and it is applicable for the study of the absorptive characteristics of ginsenoside Rb3 in Caco-2 cell monolayer model.