Study on chemical constituents of Notoginseng Radix et Rhizoma and network pharmacology of its anti-inflammatory mechanism / 中草药

Objective: To analyze the main chemical constituents of Notoginseng Radix et Rhizoma (NRR) by ultra performance liquid chromatography quadrupole-time-of-flight hybrid mass spectrometry (UPLC-Q-TOF-MS), and to study on the mechanism of NRR with multi-components, multi-targets, and multi-pathways for the treatment of inflammatory based on network pharmacology. Methods: The main chemical components in NRR were analyzed by UPLC-Q-TOF-MS. DAVID database was used to analyze gene ontology (GO) enrichment analysis and Kyoto gene and genome encyclopedia (KEGG) pathway analysis. In addition, Cytoscape 3.6.1 software was used to draw network interaction diagrams, and Image GP tool was used to draw GO bubble diagrams. Results: A total of 22 active components (ginsenoside Rh1, ginsenoside Rg1, and monolaurin) of NRR and 31 related targets (EGFR, STAT3, MAPK14) were screened. GO and KEGG pathway enrichment analysis revealed that active components of NRR acted on EGFR, STAT3, MAPK14, IL2 targets, and may regulate pathways in cancer, Cytokine-cytokine receptor Interaction, CAMs and so on. Conclusion: This study reflects the characteristics of multi-components, multi-targets, and multi-pathways of NRR in the aspect of anti-inflammatory, which may provides new ideas and methodology for further research on NRR.

HPLC fingerprint of steamed Panax Notoginseng and content determination of multi-components / 中草药

Objective To establish the HPLC fingerprint and simultaneous method for the determination of 11 saponins in steamed Panax notoginseng from different origins. Methods Agilent Zorbax SB-C18 (250 mm × 4.6 mm, 5 μm) column was adopted, the mobile phase consisted of acetonitrile-water with gradient elution at the flow rate of 1.0 mL/min (elution program: 0-30 min, 20% acetonitrile; 30-60 min, 20%-45% acetonitrile; 60-88 min, 45%-75% acetonitrile; 88-98 min, 75% acetonitrile; 98-100 min, 75%-20% acetonitrile), and the detection wavelength was 203 nm. The establishment of steamed P. notoginseng HPLC fingerprint, and determination method of notoginsenoside R1, ginsenoside Rg1, Re, Rb1, 20S-Rh1, 20R-Rh1, Rd, Rk3, Rh4, 20S-Rg3, and 20R-Rg3 index components were studied methodologically. The content of 11 saponins in 10 batches was determined. Results The HPLC fingerprint was establish, and thirty common peaks were selected as characteristic peaks of steamed P. notoginseng. The similarities of different samples from 10 areas were 0.941, 0.938, 0.945, 0.951, 0.913, 0.909, 0.920, 0.928, 0.917, and 0.919. In quantitative analysis, eleven saponins were separated well and the average content was 5.274, 20.515, 2.838, 23.651, 3.476, 1.407, 5.239, 1.784, 1.580,0.904, and 0.294 mg/g, respectively. Additionally, all calibration curves showed good linear regression relationship, with correlation indexes of 0.999 7, 0.999 5, 0.999 5, 0.999 7, 0.999 7, 0.999 6, 0.999 7, 0.999 6, 0.999 7, 0.999 7, and 0.999 6; The average recoveries were 101.23%, 98.52%, 97.67%, 99.62%, 98.17%, 98.92%, 99.44%, 99.14%, 100.25%, 98.23%, and 96.89%, with RSDs of 1.35%, 1.58%, 2.44%, 1.05%,1.48%, 1.56%, 0.85%, 2.34%, 2.85%, 1.25%, and 1.08%. Conclusion This method is sensitive, accurate and reproducible. It can be used to provide a reference for the standard and evaluation of quality of steamed P. notoginseng.

Active ingredients screening by cell membrane chromatography and simultaneous quantitation of ginsenosides in bulk drug of secondary ginsenoside H dripping pills / 中草药

To establish a HPLC-MS/MS method for simultaneous determination and active ingredients screening of pseudoginsenoside RT5, 20(S)-ginsenoside Rh1 and 20(S)-ginsenoside Rh2 by cell membrane chromatography (CMC) in secondary ginsenoside H dripping pills (SGHDP). Methods The samples were separated on Century SIL BDS C18 column (250 mm × 4.6 mm, 5 μm) eluted with 0.2% formic acid aqueous solution-acetonitrile in a gradient mode, and the target compounds were analyzed by positive ion multiple reaction monitoring (MRM) mode, and active ingredients of SGHDP obtained in solid-phase of biomembrane by CMC technology were determined at the same time. Results The linear ranges of pseudoginsenoside RT5, 20(S)-ginsenoside Rh1, and 20(S)-ginsenoside Rh2 were 0.095-0.235, 0.042-0.168, and 0.105-0.419 mg/mL; the extraction recoveries were 99.95%, 100.12%, and 100.06%; and RSD were 1.06%, 0.96%, and 0.91%, respectively. The contents of pseudoginsenoside RT5, 20(S)-ginsenoside Rh1, and 20(S)-ginsenoside Rh2 in SGHDP were 21.24%, 21.42%, and 29.70%, respectively. 20(S)-Ginsenoside Rh2 was the active ingredient obtained by biomembrane using as a new quality control maker for SGHDP. Conclusion The developed method is accurate and reliable for the determination of three ginsenosides in SGHDP, and provides a new reference for quality control of SGHDP. 20(S)-Ginsenoside Rh2 is a immobilization component of red cell membrane, speculated to be the active ingredient of SGHDP, which is in consistent with previous studies on antitumor and antidepression.

Determination of content changes of ginsenoside Re, Rg1, Rb1, Rg3, Rh1, and Rh2 before and after pulping of mountain cultivated ginseng by HPLC / 中草药

Objective To analyze the content changes of six kinds of ginsenosides Re, Rg1, Rb1, Rg3, Rh1, and Rh2 after pulping of mountain cultivated ginseng. Methods The HPLC-UV method was performed on an Innoval ODS-2 chromatographic column (250 mm × 4.6 mm, 5 μm), gradient elution of acetonitrile and water with column temperature 30 ℃, at a flow rate of 1.0 mL/min, and detected at 203 nm with injection volume as 20.0 μL. Results The content of six kinds of ginsenosides Re, Rg1, Rb1, Rg3, Rh1 and Rh2 were changed from 0.651, 0.506, 0.363, 0.014, 0.023, 0.031 mg/g to 0.517, 0.413, 0.105, 0.122, 0.214, 0.098 mg/g after pulping of mountain cultivated ginseng. The calibration curve was liner within 2.5-100 mg/L for ginsenoside Re, Rg1, Rb1, Rg3, Rh1, and Rh2, respectively, with the correlation r2 > 0.999 5 and perfect precision, stability, and repeatability. The average recoveries ranged from 95% to 105%, and RSD values varied from 1.25% to 3.5%. Conclusion The content of six kinds of ginsenosides Re, Rg1, Rb1, Rg3, Rh1, and Rh2 in mountain cultivated ginseng were changed after the pulping. The content of ginsenoside Re, Rg1, and Rb1 was reduced and rare ginsenoside Rg3, Rh1, and Rh2 was increased by 8.7 times, 9.3 times, and 3.2 times respectively after the pulping. The HPLC method for simultaneous determination of six kinds of ginsenosides has good accuracy and reliability and can provide scientific basis for the quality evaluation of mountain cultivated ginseng pulp.

Effects of ginsenoside Rh1 on expression of inflammatory factors in mouse asthma model / 中国病理生理杂志

AIM:To observe the effects of ginsenoside Rh 1 on the levels of inflammatory factors in serum and bronchoalveolar lavage fluid(BALF),and the pathological changes of the lung tissues in an experimentally induced mouse asthma model.METHODS:Male BALB/c mice(n=40)were divided into 4 groups:normal control group,asthma mo-del group,and low-dose(40 mg· kg-1 · d-1 )and high-dose(80 mg· kg-1 · d-1 )ginsenoside Rh1 groups.The bron-chial asthma mouse model was established by the method of ovalbumin induction and excitation ,and during the excitation period,the mice were daily treated with ginsenoside Rh 1 for 2 weeks.At 24 h after the final dose of ginsenoside Rh 1,the mice were sacrificed.The number of eosinophils(EOS)and the concentrations of interleukin(IL)-4,IL-5 and interferon(IFN)-γin BALF were determined.The levels of IgG and IgE in serum were measured ,and the expression of transforming growth factor(TGF)-β1 and the pathological changes in lung tissues were evaluated.RESULTS:Ginsenoside Rh1 inhibi-ted the increases in the number of EOS and the concentrations of IL-4,IL-5,IFN-γand IgE,reversed the increased ex-pression of TGF-β1,and improved the pathological changes of the lung tissues in asthmatic mice.CONCLUSION:Gin-senoside Rh1 improves the immuno-inflammatory profile and pathological changes in the experimentally induced mouse asth -ma model,implying its potential therapeutic effect on asthma.

Discovery of ginsenosides from Ligusticum chuanxiong and its significance / 中草药

Objective To investigate the chemical constituents of the rhizomes of Ligusticum chuanxiong and discuss the significance of first discovery of ginsenosides from the plant. Methods The compounds were isolated and repeatedly purified by column chromatographies such as macroperous resin, Sephadex LH-20, silica gel, and preparative TLC as well as semi-preparative RP-HPLC. Their structures were elucidated by physicochemical properties, NMR, and MS spectral analyses. Results Three ginsenoside compounds were isolated from the n-butanol extracts of rhizomes of L. chuanxiong, and their structures were identified as (20S)-ginsenoside Rh1 (1), (20R)-ginsenoside Rh1 (2), and (20R)-ginsenoside Rg3 (3). Conclusion Ginsenosides are isolated from the genus Ligusticum (Umbelliferae) for the first time, it is of great significance for clarifying pharmacodynamic material basis of the rhizomes of L. chuanxiong. These results also provide the reference data for further verifying the relevance of plant evolution and traditional efficacy between L. chuanxiong and Panax ginseng.

Advances in biotransformation of protopanaxatriol rare ginsenoside / 中草药

Ginseng is a traditional medicine in Asian countries. Ginsenoside has the main active ingredient, exhibit cardiovascular, tumor, and central nervous system activities. In particular, protopanaxatriol-type ginsenosides Rh1, exhibits anti-inflammatory, anti-allergic, and memory improvement activities. Ginsenoside Rh1 is only found in trace amounts in Panax ginseng, Panax pseudoginseng var. notoginseng, and Panax quinquefolius. Biotransformation of rare ginsenosides has become an effective way. In this paper, the research progress of transformation of ginsenoside saponins by biotransformation to produce rare ginsenoside Rh1 is reviewed, which provides a useful reference for the further development and preparation of ginsenoside Rh1.

Explore of synthesis method of different configuration pseudo-ginsenoside Rg2, pseudo-ginsenoside Rh1, and pseudo-PPT / 中草药

Objective To explore an efficient preparation method of pseudo-ginsenoside Rg2, pseudo-ginsenoside Rh1, and pseudo-PPT, as to provide theoretical basis for the preparation of pseudo-ginsenosides and pseudo-PPT. Methods Ginsenosides Re, Rh1, and PPT as raw material, via a simple three-step called acetylation, elimination-addition and saponification achieve the preparation of 20 (E/Z)-pseudo-ginsenoside Rg2, 20 (E/Z)-pseudo-ginsenoside Rh1, and 20 (E/Z)-pseudo-PPT. The detailed structure elucidation of the compounds were obtained by NMR, HR-ESI-MS, and IR. Results The production rates of 20 (E/Z)-pseudo-ginsenoside Rg2, 20 (E/Z)-pseudo-ginsenoside Rh1, and 20 (E/Z)-pseudo-PPT were 41%/13%, 43%/11%, and 56%/15%, respectively. Among them, 20 (Z)-pseudo-PPT was identified as new triterpenoid. Conclusion The method through the price relatively cheap and easy gain reactants ginsenoside Re, ginsenoside Rh1, and PPT prepared active better pseudo-ginsenoside Rg2, pseudo-ginsenoside Rh1, and pseudo-PPT, the method for the preparation of other types of pseudo-ginsenoside provides a new train of thought. At the same time, the method is simple and the yield is high.

Simultaneous quantification of ginsenoside Rg1 and its metabolites by HPLC-MS/MS: Rg1 excretion in rat bile, urine and feces

Ginsenoside Rg1 (Rg1), the major effective component of ginseng, has been shown to have multiple bioactivities, but low oral bioavailability. The aim of this study was to develop a simple, sensitive and rapid high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which could be used to validate and quantify the concentrations of Rg1 and its metabolites in Sprague-Dawley rat bile, urine, and feces after oral administration (25 mg/kg). Calibration curves offered satisfactory linearity (>0.995) within the determined ranges. Both intra-day and inter-day variances were less than 15%, and the accuracy was within 80-120%. The excretion recoveries of Rg1, ginsenoside Rh1 (Rh1), and protopanaxatriol (Ppt) in bile, urine, and feces combined were all greater than 70%. The fecal excretion recoveries of Rg1, Rh1, and Ppt were 40.11%, 22.19%, and 22.88%, respectively, whereas 6.88% of Rg1 and 0.09% of Rh1 were excreted in bile. Urinary excretion accounted for only 0.04% of Rg1. In conclusion, the observed excretion profiles for Rg1 and its metabolites after oral administration are helpful for understanding the poor oral bioavailability of Rg1 and will aid further investigations of Rg1 as a pharmacologically active component.

Protopanaxatriol Ginsenoside Rh1 Upregulates Phase II Antioxidant Enzyme Gene Expression in Rat Primary Astrocytes: Involvement of MAP Kinases and Nrf2/ARE Signaling

Oxidative stress activates several intracellular signaling cascades that may have deleterious effects on neuronal cell survival. Thus, controlling oxidative stress has been suggested as an important strategy for prevention and/or treatment of neurodegenerative diseases. In this study, we found that ginsenoside Rh1 inhibited hydrogen peroxide-induced reactive oxygen species generation and subsequent cell death in rat primary astrocytes. Rh1 increased the expression of phase II antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1, superoxide dismutase-2, and catalase, that are under the control of Nrf2/ARE signaling pathways. Further mechanistic studies showed that Rh1 increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to the antioxidant response element (ARE), and increased the ARE-mediated transcription activities in rat primary astrocytes. Analysis of signaling pathways revealed that MAP kinases are important in HO-1 expression, and act by modulating ARE-mediated transcriptional activity. Therefore, the upregulation of antioxidant enzymes by Rh1 may provide preventive therapeutic potential for various neurodegenerative diseases that are associated with oxidative stress.

The Change of Ginsenoside Composition in the Ginseng (Panax ginseng) Flower Buds by the Ultrasonication and Vinegar Process

The purpose of this study was to develop a new ginseng (Panax ginseng) flower buds extract with the high concentration of ginsenoside Rg3, Rg5, Rk1, Rh1 and F4, the Red ginseng special component. Chemical transformation from the ginseng saponin glycosides to the prosapogenin was analyzed by the HPLC. The ginseng flower buds were processed at the several treatment conditions of the ultrasonication (Oscillator 600W, Vibrator 600W) and vinegar (about 14% acidity). The result of UVGFB-480 was the butanol fraction of ginseng flower buds that had been processed with ultrasonication and vinegar for 480 minutes gained the highest amount of ginsenoside Rg5 (3.548%), Rh1 (2.037%), Rk1 (1.821%), Rg3 (1.580%) and F4 (1.535%). The ginsenoside Rg5 of UVGFB-480 was found to contain 14.3 times as high as ginseng flower buds extracts (GFB, 0.249%).

20(S)-Ginsenoside-Rf2, a novel triterpenoid saponin from stems and leaves of Panax ginseng / 中草药

Objective: To study the chemical constituents of saponins in the stems and leaves of Panax ginseng. Methods: The chemical constituents were isolated and purified by various chromatographic methods, and the chemical structures were identified by NMR and MS data analyses. Results: A total of 39 compounds were isolated and identified. Among them, 17 compounds were determined as ginsenoside Re (1), 20(S)-ginsenoside Rh1 (2), 20(R)-ginsenoside Rh1 (3), ginsenoside Rh5 (4), 20(E)-ginsenoside F4 (5), ginsenoside F2 (6), 20(S)-ginsenoside Rg3 (7), 20(R)-ginsenoside Rg3 (8), 20(S)-ginsenoside Rf2 (9), 20(R)-ginsenoside Rf2 (10), 20(S)- protopanaxadiol (11), 20(R)-protopanaxadiol (12), 20(S)-ginsenoside Rh2 (13), 20(R)-ginsenoside Rh2 (14), 20(S)-protopanaxatriol (15), 20(R)-protopanaxatriol (16), and ginsenoside Rd (17). Conclusion: Compound 9 is a new saponin. Compounds 2-10, 13, and 14 are rare ginsenosides.

Promotion on in vitro percutaneous absorption of trace ginsenoside Rh1 using imidazole type-ionic liquids / 中草药

Objective: To evaluate the in vitro percutaneous absorption profiles of ginsenoside Rh1 using ionic liquids [BMIM][Cl] as a novel permeation enhancer and to investigate the mechanism. Methods: Mice skin and porcine skin were used as skin model. Oil acid, isopropyl myristate, and menthol were used as comparing skin enhancers. The solubility of Rh1 (with or without enhancer) in water was measured by shake-flask method. Skin permeation experiment was performed using Franz diffusion cells. Skin structure change after treatment of [BMIM][Cl] was measured by FTIR. Results: By comparing with commonly used enhancers, 5% [BMIM][Cl] significantly increased the solubility of Rh1 and gave an excellent improvement on the skin penetrability of Rh1. The FTIR results suggested that [BMIM][Cl] accelerated the drug skin permeation by disrupting the lipid bilayer of skin stratum corneum. Conclusion: [BMIM][Cl] can serve as a novel skin permeation enhancer, and show a broad application prospect in transdermal drug delivery system.

Insulin-like growth factor- II induces mTOR pathway change in Rh1 sarcoma cells / 第二军医大学学报

Objective To observe the effect of insuiin-iike growth factor-II (IGF-2) on the growth and the mTOR pathway of Rh1 sarcoma cells. Methods Rh1 cells were cultured routinely, and were treated with IGF-2 at a final concentration of 10 ng/ml after starving with pure RPMI 1640 medium. The growth of cells was analyzed by flow cytometry 72 h after IGF-2 treatment. The phosphorylation of S6 and Akt (s473) proteins were examined by Western blotting analysis at 5, 10, 20, 30, and 60 min after IGF-2 treatment. Results IGF-2 treatment promoted the survival and inhibited the apoptosis of Rh1 cells compared with the control group. IGF-2 also increased the phosphorylation of S6 in a time-dependent manner. However, the phosphorylation of Akt(s473) was relatively stable in Rh1 cells. Conclusion IGF-2 can gradually increase the function of S6 in the mTOR pathway, and the function of Akt (s473) is kept relatively stable.