The Neuroprotective Effects of Ginsenoside Rd Pretreatment in a Rat Model of Spinal Cord Ischemia-Reperfusion Injury

ABSTRACT Introduction: Paraplegia may develop as a result of spinal cord ischemia-reperfusion injury in patients who underwent thoracoabdominal aortic surgery. The objective of this research is to determine the neuroprotective effects of ginsenoside Rd pretreatment in a rat model of spinal cord ischemia-reperfusion injury. Methods: Sprague-Dawley rats (n=36) were randomly assigned to three groups. The sham (n=12) and control (n=12) groups received normal saline orally. The Rd group (n=12) received ginsenoside Rd (100 mg/kg) orally 48 hours before the induction of spinal cord ischemia. Spinal cord ischemia was induced by aortic occlusion using a Fogarty balloon catheter in the Rd and control groups. A neurological assessment according to the motor deficit index and a histological evaluation of the spinal cord were performed. To evaluate the antioxidant activity of ginsenoside Rd, malondialdehyde levels and superoxide dismutase activity were determined. Further, the tissue levels of tumor necrosis factor-alpha and interleukin-1 beta were measured. Results: The Rd group showed significantly lower motor deficit index scores than did the control group throughout the entire experimental period (P<0.001). The Rd group demonstrated significantly greater numbers of normal motor neurons than did the control group (P=0.039). The Rd group exhibited decreased malondialdehyde levels (P<0.001) and increased superoxide dismutase activity (P=0.029) compared to the control group. Tumor necrosis factor-alpha and interleukin-1 beta tissue levels were significantly decreased in the Rd group (P<0.001). Conclusion: Ginsenoside Rd pretreatment may be a promising treatment to prevent ischemia-reperfusion injury in patients who undergo thoracoabdominal aortic surgery.

Effects of total ginsenosides from Panax ginseng stems and leaves on gut microbiota and short-chain fatty acids metabolism in acute lung injury mice / 中国中药杂志

This study aimed to investigate the biological effects and underlying mechanisms of the total ginsenosides from Panax ginseng stems and leaves on lipopolysaccharide(LPS)-induced acute lung injury(ALI) in mice. Sixty male C57BL/6J mice were randomly divided into a control group, a model group, the total ginsenosides from P. ginseng stems and leaves normal administration group(61.65 mg·kg~(-1)), and low-, medium-, and high-dose total ginsenosides from P. ginseng stems and leaves groups(15.412 5, 30.825, and 61.65 mg·kg~(-1)). Mice were administered for seven continuous days before modeling. Twenty-four hours after modeling, mice were sacrificed to obtain lung tissues and calculate lung wet/dry ratio. The number of inflammatory cells in bronchoalveolar lavage fluid(BALF) was detected. The levels of interleukin-1β(IL-1β), interleukin-6(IL-6), and tumor necrosis factor-α(TNF-α) in BALF were detected. The mRNA expression levels of IL-1β, IL-6, and TNF-α, and the levels of myeloperoxidase(MPO), glutathione peroxidase(GSH-Px), superoxide dismutase(SOD), and malondialdehyde(MDA) in lung tissues were determined. Hematoxylin-eosin(HE) staining was used to observe the pathological changes in lung tissues. The gut microbiota was detected by 16S rRNA sequencing, and gas chromatography-mass spectrometry(GC-MS) was applied to detect the content of short-chain fatty acids(SCFAs) in se-rum. The results showed that the total ginsenosides from P. ginseng stems and leaves could reduce lung index, lung wet/dry ratio, and lung damage in LPS-induced ALI mice, decrease the number of inflammatory cells and levels of inflammatory factors in BALF, inhibit the mRNA expression levels of inflammatory factors and levels of MPO and MDA in lung tissues, and potentiate the activity of GSH-Px and SOD in lung tissues. Furthermore, they could also reverse the gut microbiota disorder, restore the diversity of gut microbiota, increase the relative abundance of Lachnospiraceae and Muribaculaceae, decrease the relative abundance of Prevotellaceae, and enhance the content of SCFAs(acetic acid, propionic acid, and butyric acid) in serum. This study suggested that the total ginsenosides from P. ginseng stems and leaves could improve lung edema, inflammatory response, and oxidative stress in ALI mice by regulating gut microbiota and SCFAs metabolism.

Excellent appearance of Dao-di Ginseng Radix et Rhizoma and interaction mechanism between genetic and environmental factors: a review / 中国中药杂志

Dao-di medicinal materials produced in a specific environment always present excellent appearance and high quality. Because of the unique appearance, Ginseng Radix et Rhizoma is regarded as a paradigm in the research on excellent appearance. This paper systematically summarized the research progress in the genetic and environmental factors influencing the formation of the excellent appearance of Ginseng Radix et Rhizoma, aiming to provide reference for the quality improvement of Ginseng Radix et Rhizoma and the scientific connotation of Dao-di Chinese medicinal materials. The Ginseng Radix et Rhizoma with high quality generally has a robust and long rhizome, a large angle between branch roots, and the simultaneous presence of a robust basal part of rhizome, adventitious roots, rhizome bark with circular wrinkles, and fibrous roots with pearl points. The cultivated and wild Ginseng Radix et Rhizoma have significant differences in the appearance and no significant difference in the population genetic diversity. The differences in the appearance are associated with cell wall modification, transcriptional regulation of genes involved in plant hormone transduction, DNA methylation, and miRNA regulation. The rhizosphere soil microorganisms including Fusarium and Alternaria, as well as the endophytes Trichoderma hamatum and Nectria haematococca, may be the key microorganisms affecting the growth and development of Panax ginseng. Cultivation mode, variety, and root exudates may be the main factors influencing the stability of rhizosphere microbial community. Ginsenosides may be involved in the formation of the excellent appearance. However, most of the available studies focus on the partial or single factors in the formation of Dao-di medicinal materials, ignoring the relationship within the complex ecosystems, which limits the research on the formation mechanism of Dao-di medicinal materials. In the future, the experimental models for the research involving genetic and environmental factors should be established and mutant materials should be developed to clarify the internal relationship between factors and provide scientific support for the research on Dao-di medicinal materials.

Protective effect of Guanxin Danshen formula on myocardial ischemiareperfusion injury in rats

Purpose: Myocardial ischemia/reperfusion injury (MIRI) leads to myocardial tissue necrosis, which will increase the size of myocardial infarction. The study examined the protective effect and mechanism of the Guanxin Danshen formula (GXDSF) on MIRI in rats. Methods: MIRI model was performed in rats; rat H9C2 cardiomyocytes were hypoxia-reoxygenated to establish a cell injury model. Results: The GXDSF significantly reduced myocardial ischemia area, reduced myocardial structural injury, decreased the levels of interleukin (IL-1ß, IL-6) in serum, decreased the activity of myocardial enzymes, increased the activity of superoxide dismutase (SOD), and reduced glutathione in rats with MIRI. The GXDSF can reduce the expression of nucleotide- binding oligomerization domain, leucine-rich repeat and pyrin domain containing nod-like receptor family protein 3 (NLRP3), IL-1ß, caspase-1, and gasdermin D (GSDMD) in myocardial tissue cells. Salvianolic acid B and notoginsenoside R1 protected H9C2 cardiomyocytes from hypoxia and reoxygenation injury and reduced the levels of tumor necrosis factor α (TNF-α) and IL-6 in the cell supernatant, decreasing the NLRP3, IL-18, IL-1ß, caspase-1, and GSDMD expression in H9C2 cardiomyocytes. GXDSF can reduce the myocardial infarction area and alleviate the damage to myocardial structure in rats with MIRI, which may be related to the regulation of the NLRP3. Conclusion: GXDSF reduces MIRI in rat myocardial infarction injury, improves structural damage in myocardial ischemia injury, and reduces myocardial tissue inflammation and oxidative stress by lowering inflammatory factors and controlling focal cell death signaling pathways.

Protective effects of ginsenosides on macrophages subjected to simulated weightlessness / 数字中医药（英文）

@#[Objective] To investigate the evolution of inflammation under conditions and the effects of ginsenosides on macrophages subjected to the simulated weightlessness, with the aim of mitigating the inflammation. [Methods] Initially, genes related to weightlessness, inflammation, and immunity were identified in the GeneCards database. Then, Search Tool for the Retrieval of Interaction Gene/Proteins (STRING) protein network analysis was conducted to determine the core targets involved in the weightlessness-induced inflammation. Subsequently, Label-Free Quantitative (LFQ) proteomics was carried out to discern the distinctive genes within ginsenoside-treated Tohoku Hospital Pediatrics-1 (THP-1) cells. Next, utilizing the outcomes of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, the biological processes and signaling pathways in which ginsenosides predominately engaged were scrutinized, and the primary targets of ginsenosides in combating weightlessness-induced inflammation were examined. Finally, enzyme-linked immunosorbent assay (ELISA) was performed to detect the secretion levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor (TNF)-α from lipopolysaccharide (LPS)-induced THP-1 cells under simulated weightlessness conditions, as well as during the weightlessness recovery period following treatment with ginsenosides. [Results] A total of 2 933 genes associated with inflammation, 425 genes linked to weightlessness, and 4 564 genes connected to immunity were retrieved from the GeneCards database. Protein-protein interaction (PPI) networks were generated to identify pivotal targets associated with weightlessness-induced inflammation such as IL-1β, IL-6, TNF, and albumin (ALB). It was found that ginsenosides primarily participated in the regulation of various inflammationrelated signaling pathways and pathways related to pathogenic microorganism infections. Moreover, it has a significant impact on the expression of proteins such as cluster of differentiation 40 (CD40), IL-1β, and poly ADP-ribose polymerase 1 (PARP1). As revealed in the simulated weightlessness cell test, ginsenosides exhibited a remarkable capacity to attenuate the secretion of inflammatory factors, specifically IL-6 and TNF-α (P < 0.000 1), in THP-1 macrophages following induction by LPS under simulated weightlessness conditions. In addition, it reduced the secretion of IL-1β, IL-6, IL-8, and TNF-α (P < 0.000 1) during the weightlessness recovery phase [Conclusion] Weightlessness can disrupt several inflammation-related signaling pathways, but ginsenosides were shown to mitigate the release of various inflammatory factors in macrophages subjected to simulated weightlessness, thereby exerting a protective role against inflammation. This study has laid a theoretical groundwork for further exploring the potential application of ginsenosides in safeguarding against LPS induced inflammation in a weightlessness environment.

Blood and synovial tissue metabolomics of rheumatoid arthritis rats treated with less polar ginsenosides / 中华风湿病学杂志

Objective:The therapeutic effect of less polar ginsenosides on rats with rheumatoid arthritis was studied, and the metabolic pathway that produce anti-inflammatory effect of less polar ginsenosides was identified.Methods:Rats were randomly divided into the control group, the model group, methotrexate treatment group, and high dose, medium dose, and low dose less polar ginsenosides groups. After 30 days of oral administration, less polar ginsenosides reduced the disease activity significantly in rats with rheumatoid arthritis. Blood and ankle synovial tissue metabolisms were measured by ultra performance liquid chromatography (UPLC) tandem mass spectrometry (MS) to explore the mechanism of less polar ginsenosides.The resulting data were subjected to principal component analysis and orthogonal partial least squares discriminant analysis(OPLS-DA).Results:Compared with the model group, erythrocyte sedimentation rate and RF decreased significantly in the high dose of less polar ginsenosides ( P<0.01). Metabolomics showed that R2X and R2Y of serum OPLS-DA were 0.626 and 0.904 respectively. The R2X and R2Y of synovial OPLS-DA were 0.429 and 0.689 respectively. Major differential metabolites were identified in the model group of rats, including arachidonic acid, valine, linoleic acid, and guanine nucleoside, etc. The main differential metabolites were identified in rats in the high dose group of less polar ginsenosides, including linoleic acid, betaine, eicosapentaenoic acid, alanine, methionine sulfoxide, isoleucine, etc. Conclusion:The metabolic spectrum has shown that inflammation is associated with linoleic acid metabolism, valine, leucine and isoleucine degradation, arachidonic acid metabolism. Less polar ginsenosides regulatethe linolenic acid metabolism, methionine metabolism and glucose alanine cycle.

Functional annotation map of natural compounds in traditional Chinese medicines library: TCMs with myocardial protection as a case

The chemical complexity of traditional Chinese medicines (TCMs) makes the active and functional annotation of natural compounds challenging. Herein, we developed the TCMs-Compounds Functional Annotation platform (TCMs-CFA) for large-scale predicting active compounds with potential mechanisms from TCM complex system, without isolating and activity testing every single compound one by one. The platform was established based on the integration of TCMs knowledge base, chemome profiling, and high-content imaging. It mainly included: (1) selection of herbal drugs of target based on TCMs knowledge base; (2) chemome profiling of TCMs extract library by LC‒MS; (3) cytological profiling of TCMs extract library by high-content cell-based imaging; (4) active compounds discovery by combining each mass signal and multi-parametric cell phenotypes; (5) construction of functional annotation map for predicting the potential mechanisms of lead compounds. In this stud TCMs with myocardial protection were applied as a case study, and validated for the feasibility and utility of the platform. Seven frequently used herbal drugs (Ginseng, etc.) were screened from 100,000 TCMs formulas for myocardial protection and subsequently prepared as a library of 700 extracts. By using TCMs-CFA platform, 81 lead compounds, including 10 novel bioactive ones, were quickly identified by correlating 8089 mass signals with 170,100 cytological parameters from an extract library. The TCMs-CFA platform described a new evidence-led tool for the rapid discovery process by data mining strategies, which is valuable for novel lead compounds from TCMs. All computations are done through Python and are publicly available on GitHub.

Discovery and confirmation of protein action site AK1 of ginsenosides in brain based on DARTS technology / 中国中药杂志

This study aims to explore the targets of ginsenosides in brain based on drug affinity responsive target stability(DARTS) technology. Specifically, DARTS technology was combined with label-free liquid chromatography tandem mass spectrometry(LC-MS) to screen out the proteins in the brain that might interact with ginsenosides. Based on the screening results, adenylate kinase 1(AK1) was selected for further confirmation. First, the His-AK1 fusion protein was yielded successively through the construction of recombinant prokaryotic expression vector, expression of target protein, and purification of the fusion protein. Biolayer interferometry(BLI) was employed to detect the direct interaction of Rg_1, Re, Rb_1, Rd, Rh_2, F1, Rh_1, compound K(CK), 25-OH-PPD, protopanaxa-diol(PPD), and protopanaxatriol(PPT) with AK1, thereby screening the ginsenoside monomer or sapogenin that had strong direct interaction with the suspected target protein AK1. Then, the BLI was used to further determine the kinetic parameters for the binding of PPD(strongest interaction with AK1) to His-AK1 fusion protein. Finally, molecular docking technology was applied to analyze the binding properties between the two. With DARTS and LC-MS, multiple differential proteins were screened out, and AK1 was selected based on previous research for target verification. Fusion protein His-AK1 was obtained by prokaryotic expression, and the response(nm) of Re, Rg_1, Rd, Rb_1, Rh_1, Rh_2, F1, PPT, PPD, 25-OH-PPD, and CK with His-AK1 was respectively 0.003 1, 0.001 9, 0.042 8, 0.022 2, 0.013 4, 0.037 3, 0.013 9, 0.030 7, 0.140 2, 0.016 0, and 0.040 8. The K_(on), K_(off), and K_D values of PPD and His-AK1 were determined by the BLI as 1.22×10~2 mol~(-1)·L·s~(-1), 1.04×10~(-2) s~(-1), 8.52×10~(-5) mol·L~(-1). According to the molecular docking result, PPD bound to AK1 with the absolute value of the docking score of 3.438, and hydrogen bonds mainly formed between the two. Thus, AK1 is one of the protein action sites of ginsenosides in the brain. The direct interaction between ginsenoside metabolite PPD and AK1 is the strongest.

~1H-qNMR quantification of total ginsenosides in Shenmai Injection / 中国中药杂志

A content determination method based on ~1H-qNMR was developed for the determination of total ginsenosides in Shenmai Injection. The parameters were optimized with CD_3OD as the solvent, dimethyl terephthalate as the internal standard, the peak at δ 8.11 as the internal standard peak, and the peaks at δ 1.68 and δ 0.79 as quantitative peaks of total ginsenosides. The developed ~1H-qNMR-based method was validated methodologically. The results showed that the method could achieve accurate measurement of total ginsenosides in Shenmai Injection in the range of 0.167 6-3.091 1 mmol·L~(-1). The developed ~1H-qNMR-based method for total ginsenosides is simple in operation, short in analysis time, strong in specificity, independent of accompanying standard curve, and small in sample volume, which can serve as a reliable mean for the quality control of Shenmai Injection. This study is expected to provide new ideas for the development of quantification methods of total ginsenosides.

Effects of ginsenoside Rg1 on the expression of neuronal autophagosome-related proteins in brain slices of a rat model of Alzheimer's disease / 中华老年医学杂志

Objective:To explore the effects and molecular mechanisms of ginsenoside Rg1 on the expression of neuronal autophagosome-related proteins in a rat model of Alzheimer's disease(AD).Methods:Six-week-old SD rats were decapitated to prepare hippocampal brain slices.The slices were randomly divided into the blank control group, the model group, the low-concentration, medium-concentration and high-concentration Rg1 groups, with 10 in each group.In the model group, Aβ 1-42(final concentration: 5 μmol/L)was added into an artificial cerebrospinal fluid(CSF)for 2 h treatment.The low-concentration, medium-concentration and high-concentration Rg1 groups were treated with Aβ 1-42(final concentration: 5 μmol/L)for 2 h, and then treated with Rg1(final concentrations: 60 μmol/L, 120 μmol/L, 240 μmol/L, respectively)for 3 h. The blank control group was not given any intervention drugs.At the end of intervention, histological changes of hippocampal brain slices in each group were examined via hematoxylin-eosin(HE)staining.Autophagosomes in hippocampal brain slices of each group were detected using transmission electron microscopy.The expression levels of autophagy-related proteins(P62, LC3-Ⅱ/LC3-Ⅰ), Aβ 1-42and shank protein in hippocampal brain slices of each group were detected with Western blot. Results:The results of HE staining showed that the arrangement of hippocampal neurons were disordered in the model group, with death and depletion of neurons.The arrangement and depletion of hippocampal neurons in each Rg1 group were less severe compared with the model group, with most significant improvement seen in the high-concentration Rg1 group.The results of transmission electron microscopy showed that the number of autophagosomes in brain slices in the model group was significantly higher than that in the blank control group, while each Rg1 group had fewer autophagosomes than the model group.The results of Western blot showed that, compared with the blank control group, levels of Shank1, P62 and LC3-Ⅰ proteins in brain slices were decreased(all P<0.05), while levels of Aβ 1-42and LC3-Ⅱ protein were significantly increased(all P<0.05)in the model group.Compared with the model group, levels of Shank1, P62 and LC3-Ⅰ proteins in brain slices were increased(all P<0.05), while levels of Aβ 1-42and LC3-Ⅱ protein were decreased( P<0.05)in each Rg1 group.These changes were the most significant in the high-concentration Rg1 group. Conclusions:Ginsenoside Rg1 may inhibit autophagy by up-regulating the expression of Shank1, P62 and LC3-Ⅰ proteins in hippocampal brain slices of rats in the AD model, thus playing protective roles in brain neurons.

Investigation of Adsorption and Desorption Properties of Total Ginsenosides on Anion-exchange Resins and Its LC-MS Analysis / 中国实验方剂学杂志

Objective:To compare the adsorption and desorption properties of different anion exchange resins for total ginsenosides， clarify their adsorption/desorption mechanism， and establish a simple protocol for the purification of total ginsenosides. Method:The adsorption and desorption properties of five different resins （D301， D315， D312， D330， D201） on total ginsenosides were evaluated with specific adsorption capacity， specific desorption capacity， desorption rate and recovery rate as indices. The adsorption kinetics and thermodynamics of the selected resin and D101 macroporous resin were investigated by pseudo-first-order and pseudo-second-order kinetic models， as well as Langmuir and Freundlich isothermal adsorption models， and the differences of adsorption mechanism between anion exchange resin and conventional macroporous resin were elucidated. The dynamic adsorption and desorption experiments were used to determine the optimum chromatographic parameters for anion exchange resin. After verifying the purification process of total ginsenosides， nine individual ginsenosides were qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry （LC-MS）. Result:D301 anion exchange resin was obviously superior to the other four kinds of anion exchange resin， the optimum parameters were set as follows：pH 8 of loading solution， loading volume of 2 BV， loading speed of 4 BV·h^-1， eluted with 3 BV of water and 20% ethanol for the impurities， eluted with 8 BV of 80% ethanol with elution speed of 4 BV·h^-1. After purified by D301 resin， the enrichment coefficients of 9 monomer ginsenosides were simultaneously increased to different degrees， the overall enrichment coefficient was up to 5.3， the recovery rate for the total amount of these ginsenosides was calculated to be 80.9%， and the purity of total ginsenosides in Ginseng Radix et Rhizoma extract increased from 17.07% to 91.19%. Conclusion:D301 anion exchange resin is suitable for rapid and practical purification of total ginsenosides， hence allowing for the enrichment of high-purity total ginsenosides from Ginseng Radix et Rhizoma via one-dimensional column chromatography.

Establishment of UPLC-PDA Fingerprint of Baoyuantang Substance Benchmark and Identification of Its Chemical Constituents / 中国实验方剂学杂志

Objective:To establish the fingerprint of Baoyuantang substance benchmark， and to analyze and identify the common peaks. Method:A total of 15 batches of Baoyuantang substance benchmark were prepared， ultra performance liquid chromatography-diode array detector method （UPLC-PDA） was used to establish the fingerprint of the substance benchmark， and the methodology was developed. The chromatographic conditions were as follows：ACQUITY UPLC BEH Shield C₁₈ column （2.1 mm×100 mm， 1.7 μm）， mobile phase of 0.05% formic acid solution （A） and 0.05% formic acid acetonitrile solution （ B） for gradient elution （0-0.5 min， 5%-19%B； 0.5-6 min， 19%B； 6-10 min， 19%-27%B； 10-20 min， 27%-45%B； 20-20.1 min， 45%-95%B； 20.1-23 min， 95%B）， the flow rate of 0.4 mL·min^-1， the column temperature of 30 ℃， the detection wavelength at 203 nm and 260 nm， and the injection volume of 2 μL. Similarity evaluation system of traditional Chinese medicine fingerprint （2012 edition） was used to establish the fingerprint and generate the control fingerprint. The chemical constituents of Baoyuantang substance benchmark were identified by comparison of standard substances and UPLC-electrospray ionization tandem mass spectrometry （UPLC-ESI-MS/MS） with full information tandem mass spectrometry （MS^E） and scanning range of <italic>m</italic>/<italic>z</italic> 50-1 200. Result:The similarities of 15 batches of Baoyuantang substance benchmark were above 0.90 by comparing with the control fingerprint. There were 37 common peaks， 22 of which were identified through UPLC-ESI-MS/MS， including liquiritin， violanthin， ginsenoside Rg₁， ginsenoside Rb₁， ginsenoside Re and so on. These components were all from Astragali Radix， Ginseng Radix et Rhizoma， Zingiberis Rhizoma Recens and Glycyrrhizae Radix et Rhizoma. Conclusion:This method is accurate， stable and reliable， which will basically reflect the overall chemical composition characteristics of Baoyuantang， and it provides experimental basis for development of the granules of this famous classical formulas.

Research progress on naturally-occurring and semi-synthetic ocotillol-type ginsenosides in the genus Panax L. (Araliaceae) / 中国天然药物

Ocotillol (OT)-type ginsenosides, one subtype of ginsenosides, consist of a dammarane skeleton and a tetrahydrofuran ring. Most naturally-occurring OT-type ginsenosides exist in Panax species, particularly in Panax quinquefolius, which may be attributed to the warm and humid climate of its native areas. Till now, merely 28 types of naturally-occurring OT-type ginsenosides have been isolated. In contrast, semi-synthesized OT-type ginsenosides are attracted considerable attentions. These ginsenosides can be obtained through oxidation and cyclization of side chains of dammarane-type ginsenosides, and other methods, which may change their physical and chemical properties and further improve their bioavailabilities. It is also notable that the pharmacological activities of ginsenosides are closely related to the stereoisomers caused by the configuration at C-20. Semi-synthesis of OT-type ginsenosides can facilitate our understanding of the biosynthesis, transformation and metabolism of OT-type ginsenosides in the body. This review will systematically summarize the research progress on naturally-occurring and semi-synthetic OT-type ginsenosides, which provides a theoretical basis for their bioactivity-guided research.

Analysis of chemical constituents in Shenmai Injection by LC-Q-TOF-MS and LC-IT-MS / 中国中药杂志

The chemical constituents in Shenmai Injection(SMI) were qualitatively analyzed by using liquid chromatography/quadrupole time-of-flight mass spectrometry(LC-Q-TOF-MS) and liquid chromatography-ion trap-mass spectrometry(LC-IT-MS). The analysis was performed on an Agilent Zorbax SB-C_(18)(4.6 mm×250 mm, 5 μm) and gradient elution was carried out with 0.05% formic acid solution-acetonitrile as mobile phase at a flow rate of 0.6 mL·min~(-1) and a column temperature of 30 ℃. Mass spectrometry data of the components in SMI were collected in negative ion mode. The structures of components were speculated and identified by analyzing mass spectrometry data, comparing with standards, and referring to related literature. A total of 64 components in SMI were estimated, and the structures were confirmed in 16 of them by comparison with standards. Fifty-six compounds derived from Ginseng Radix et Rhizoma Rubra included 34 protopanaxadiol ginsenosides, 19 protopanaxatriol ginsenosides, 1 oleanane ginsenosides and 2 other glycosides. Eight compounds derived from Ophiopogonis Radix included 7 steroidal saponins, and 1 monoterpene glycoside. The results of this study would provide an important theoretical basis for the improvement of the quality control standards and the discovery of effective constituents in SMI.

Effect of ginsenosides on serous metabonomic profiles in cerebral ischemia-reperfusion rats based on ~1H-NMR / 中国中药杂志

Serum metabonomic profiles of the model of focal cerebral ischemia reperfusion is established with the suture-occluded method by Longa to study the effect of ginsenosides. In this study, 48 rats were randomly divided into six groups: sham-operated group, pathological model group, positive drug group(6 mg·kg~(-1)·d~(-1)) and high, medium, low-dose ginsenosides groups(200, 100, 50 mg·kg~(-1)·d~(-1)). They are given intragastric administration respectively with same amount of 0.5% CMC-Na,nimodipine and ginsenoside for 5 days. At 2 h after the final administration, the model was established with the suture-occluded method, and free radical-scavenging activity changes of ginsenoside were observed by maillard reaction, and Longa was possible used as a renoprotective agent-occluded method. At the end of 24 h after the reperfusion, the hemolymph of rats in each group was collected, and the ~1H-NMR spectrum was collected after being treated by certain methods, and analyzed by principal component analysis(PCA). Compared with sham-operated group, pathological model group showed significant increases in the levels of lactate, glutamate, taurine, choline, glucose and methionine, but decreases in the levels of 3-hydroxybutyrate and phosphocreatine/creatine in serum. After treatment with ginsenosides, lipid, 3-hydroxybutyrate and phosphocreatine/creatine were increased in the serum of ginsenosides group rats, but with decreases in lactate and glutamate. The results showed that ginsenosides could regulate metabolic disorders in rats with focal cerebral ischemia reperfusion, and promote a recovery in the process of metabolism. It's helpful to promote the metabolic changes in rats with focal cerebral ischemia reperfusion via ~1H-NMR, and lay a foundation to develop ginsenosides as a new drug to treat ischemic cerebral paralysis.

Effect of Steaming Time and Pressure on Contents of Six Ginsenosides in Ginseng Radix et Rhizoma by UPLC-QqQ-MS/MS / 中国实验方剂学杂志

Objective::The processing method of red ginseng was determined by comparing the effects of different steaming time and pressure on the total content of six ginsenosides. Method::The contents of ginsenoside Rg1, Re, Rf, Rb1, Rc and Rb2 were determined by ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS). The Waters ACQUITY UPLC BEH C8 column (2.1 mm×100 mm, 1.7 μm) was used. The mobile phase was 0.1% formic acid aqueous solution (A) and 0.1% formic acid acetonitrile solution (B) for gradient elution (0-4 min, 81%-79%A; 4-6.3 min, 79%-75%A; 6.3-6.5 min, 75%-71%A; 6.5-9.5 min, 71%A; 9.5-16.5 min, 71%-68.5%A; 16.5-16.6 min, 68.5%-60%A; 16.6-19 min, 60%-100%A). The flow rate was set at 0.4 mL·min-1 and the column temperature was set at 35 ℃. The mass spectrographic analysis employed electrospray ionization (ESI) and negative ion collection mode with capillary ionization voltage of 2.5 kV, desolvation temperature of 350 ℃, desolvation gas flow of 700 L·h-1 and cone gas flow of 50 L·h-1. Multiple reaction monitoring (MRM) mode was used to collect information, the collection range was m/z 100-1 500, detection was performed by MRM mode at m/z 799.59-637.49 for ginsenoside Rg1, m/z 945.54-475.79 for ginsenoside Re, m/z 799.59-475.49 for ginsenoside Rf, m/z 1 107.59-783.97 for ginsenoside Rb1, m/z 1 077.58-783.96 for ginsenoside Rc, m/z 1 077.75-191.19 for ginsenoside Rb2. Result::When the steaming time was 3 hours, the total mass fraction of six ginsenosides in each sample group was 7.099 8-16.768 5 mg·g-1, and the total amount of the six ginsenosides in atmospheric steaming was 2.5-12.6 times of that in pressurized steaming, which was obviously better than that in pressurized steaming. Conclusion::Under the conditions of this experiment, the best processing method of red ginseng is atmospheric steaming for 3 hours with fresh ginseng.

Establishment of Fingerprints and Comparative Study on Ginsenoside Content of Panax notoginseng Flower with Different Growing Years / 中国药房

OBJECTIVE：To compare chemical composition types and ginsenoside content of Panax notoginseng flowers with different growing years ，and to explore the effect of growing year on the quality of P. notoginseng flowers. METHODS ：Each 10 batches of biennial，triennial and quadrennial P. notoginseng flower were collected and determined by HPLC. The determination was performed on Shim-pack GIST C 18 column with mobile phase consisted of acetonitrile- 0.05％ phosphoric acid solution （gradient elution ）at the flow rate of 0.5 mL/min. The column temperature was set at 30 ℃，and the detection wavelength was set at 203 nm. The sample size was 20 μL. Similarity Evaluation System of TCM Chromatogram Fingerprint was used to establish the fingerprint of 30 batches of samples ，identify the diagnostic components and analyze the similarity. Cluster analysis was conducted by using SPSS 22.0 software. The contents of ginsenoside Rb 1，Rb2，Rb3 and Rc in 30 batches of P. notoginseng flower with different growing years were determined by above HPLC . The quality control analysis was conducted by using SPSS 22.0 software. RESULTS：Established fingerprint showed good precision ，stability and reproducibility. There were good linear relationship （R2＞ 0.999），quantitative limit ，precision，stability，repeatability and accuracy of the content determination method . Six common components as ginsenoside Rb 1， Rb2， Rb3 and Rc were Δ 基金项目：云南省地方高校联合专项（No.KX182504Y） identified in P. notoginseng flower with different growing ＊助教，硕士。研究方向：中药资源开发 。电话：0876-2684947。 E-mail：wshuangzaiqiang@163.com years by fingerprint ；ginsenoside Rd was identified in triennial # 通信作者 ：研究员，硕士。研究方向 ：中药资源开发 。电话： P. notoginseng flower. The similarities of the fingerprints 0876-8883731。E-mail：gaomingju@163.com among 10 batches of biennial ，triennial and quadrennial P. 中国药房 2020年第31卷第8期 China Pharmacy 2020Vol. 31 No. 8 ·969· notoginseng flower were 0.881，0.952 and 0.945，respectively. The similarity among samples with different growing ye ars was more than 0.817. Thirty batches of P. notoginseng flower could be grouped into 4 categories，the category Ⅱ was quadrennial samples，the category Ⅲ was triennial samples ，while the categories Ⅰ and Ⅳ were mostly biennial samples and a small number of triennial and quadrennial samples. RSDs of 4 ginsenosides contents and their total contents in biennial samples were 8.90％-21.43％ and total saponin contents were 11.65％-17.76％，respectively. RSDs of 4 ginsenosides contents and their total contents in triennial samples were 6.45％-14.23％，and total saponin contents were 15.74％-19.30％. RSDs of 4 ginsenosides contents and their total contents in quadrennial samples were 7.50％-18.86％，and total saponin contents were 15.92％-20.16％. The results of quality control analysis showed that biennial samples mainly distributed in the areas of Ⅱ and Ⅲ ；triennial and quadrennial samples mainly distributed in the areas of Ⅰ and Ⅱ ；the order of ginsenosides content was Ⅰ ＞Ⅱ ＞Ⅲ. CONCLUSIONS：Chemical components of P. notoginseng flower with different growing years are generally close in types but there still a re some differences ，among which the content of ginsenosides in biennial samples is lower ，fluctuates more ，and the overall quality is slightly poor ；the content of ginsenosides in triennial and quadrennial samples is higher ，fluctuates less ，and the overall quality is higher and tends to be stable.

Study on Improvement Effects of Total Ginsenosides on D-galactose Induced PC 12 Cell Senescence and Its Me- chanism / 中国药房

OBJECTIVE：To study the improvement effects of total ginsenosides on the senescence of PC 12 cells induced by D-galactose and its mechanism. METHODS ：Rat pheochromocytoma （PC12）cells were treated with D-galactose to establish cell senescence model. CCK- 8 method was used to screen the D-galactose modeling concentration and total ginsenosides concentration. Normal control group ，model group ，total ginsenosides low and high concentration groups were set up. Cell senescence ，cell apoptosis rate ，apoptotic cycle and mitochondrial membrane potential （MMP），cell adenosine triphosphate （ATP）and reactive oxygen species （ROS）levels in each group were detected. The expression of apoptosis related proteins [B lymphoma 2（Bcl-2）and its related egg X protein （Bax），cytochrome C （Cyt-C）] and oxidative damage related proteins [nuclear factor 2 related factor 2 （Nrf2），heme oxygenase 1（HO-1）] were detected. In addition ，positive drug group [ 5 mmol/L N-acetyl-L-cysteine（NAC）] and positive control group [ D-galactose+5 mmol/L NAC] were set up to compare the levels of oxidative damage related proteins. RESULTS：D-galactose could significantly inhibit the survival rate of PC 12 cells，with a critical concentration of 20 mg/mL. The total ginsenosides could significantly increase the survival rate of D-galactose induced senescent cells with a median effective concentration（EC50）of 65 μg/mL，and then the low and high concentrations of total ginsenosides were set at 55 and 65 μg/mL. Compared with normal control group ，the number of aging cells increased ，the apoptotic rate and percentage of G 1 phase were significantly increased i n model group. the percentage of S phase ，MMP and ATP contents ，the protein expression of Bcl- 2 and Cyt-C in mitochondria were decreased significantly ，whileROS content ，the protein expression of Bax ，Nrf2 and Cyt-C protein in endochylema were increased significantly （P＜0.05 or P＜0.01）. Compared with model group ，the number of E-mail：sunqiao150509@163.com aging cells reduced ，the apoptosis rates and percentage of G 1 phase were significantly decreased in total ginsenosides low and high concentration groups ，the percentage of S phase ，the contents of MMP and ATP （except for low concentration group ），protein expression of Bcl- 2，Nrf2 and HO- 1 as well as protein expression of Cyt-C in mitochondria were increased significantly ；ROS level （except for low concentration group ）and Bax protein as well as protein expression of Cyt-C were decreased significantly. The protein expression of Nrf 2 and HO- 1 were increased significantly in positive control group （P＜0.05 or P＜0.01）， but it was lower than that of total ginsenosides groups . CONCLUSIONS：Total ginsenosides can improve D-galactose induced senescence of P 12 cells，the mechanism of which may be related to activating Nrf 2 antioxidant signal pathway to antagonize D-galactose induced oxidative stress and alleviating mitochondrial dysfunction.

20()-Protopanaxatriol promotes the binding of P53 and DNA to regulate the antitumor network multiomic analysis

Although the tumor suppressor P53 is known to regulate a broad network of signaling pathways, it is still unclear how certain drugs influence these P53 signaling networks. Here, we used a comprehensive single-cell multiomics view of the effects of ginsenosides on cancer cells. Transcriptome and proteome profiling revealed that the antitumor activity of ginsenosides is closely associated with P53 protein. A miRNA-proteome interaction network revealed that P53 controlled the transcription of at least 38 proteins, and proteome-metabolome profiling analysis revealed that P53 regulated proteins involved in nucleotide metabolism, amino acid metabolism and "Warburg effect". The results of integrative multiomics analysis revealed P53 protein as a potential key target that influences the anti-tumor activity of ginsenosides. Furthermore, by applying affinity mass spectrometry (MS) screening and surface plasmon resonance fragment library screening, we confirmed that 20()-protopanaxatriol directly targeted adjacent regions of the P53 DNA-binding pocket and promoted the stability of P53-DNA interactions, which further induced a series of omics changes.

Penicillium sp. YJM-2013 induces ginsenosides biosynthesis in Panax ginseng adventitious roots by inducing plant resistance responses / 中草药·英文版

Objective: Fusarium oxysporum is a common pathogenic fungus in ginseng cultivation. Both pathogens and antagonistic fungi have been reported to induce plant resistance responses, thereby promoting the accumulation of secondary metabolites. The purpose of this experiment is to compare the advantages of one of the two fungi, in order to screen out more effective elicitors. The mechanism of fungal elicitor-induced plant resistance response is supplemented. Methods: A gradient dilution and the dural culture were carried out to screen strains. The test strain was identified by morphology and 18 s rDNA. The effect of different concentrations (0, 50, 100, 200, 400 mg/L) of Penicillium sp. YJM-2013 and F. oxysporum on fresh weight and ginsenosides accumulation were tested. Signal molecules transduction, expression of transcription factors and functional genes were investigated to study the induction mechanism of fungal elicitors. Results: Antagonistic fungi of F. oxysporum was identified as Penicillium sp. YJM-2013, which reduced root biomass. The total ginsenosides content of Panax ginseng adventitious roots reached the maximum (48.95 ± 0.97 mg/g) treated with Penicillium sp. YJM-2013 at 200 mg/L, higher than control by 2.59-fold, in which protopanoxadiol-type ginsenosides (PPD) were increased by 4.57 times. Moreover, Penicillium sp. YJM-2013 activated defense signaling molecules, up-regulated the expression of PgWRKY 1, 2, 3, 5, 7, 9 and functional genes in ginsenosides synthesis. Conclusion: Compared with the pathogenic fungi F. oxysporum, antagonistic fungi Penicillium sp. YJM-2013 was more conducive to the accumulation of ginsenosides in P. ginseng adventitious roots. Penicillium sp. YJM-2013 promoted the accumulation of ginsenosides by intensifying the generation of signal molecules, activating the expression of transcription factors and functional genes.