Absorption and biotransformation of four compounds in the Guizhi decoction in the gastrointestinal tracts of rats

OBJECTIVE:To study the absorption and biotransformation of liquiritin,cinnamic acid,paeoniflorin,and glycyrrhizic acid in the Guizhi decoction(GZD)in the gastrointestinal tracts of rats.METHODS:A simple and reliable high-performance liquid chromatography method was established and validated for the analysis of the four components of GZD simultaneously in the gastrointestinal tracts of rats.Rats were randomly divided into in situ gastrointestinal loop model,in vitro anaerobic culture model,and blank control groups.All rats were fasted for 12 h and anesthetized using 20% urethane.Subsequently,the abdominal cavity of each rat was opened,and the stomach,duodenum,jejunum,ileum,cecum,and colon were ligated.For the in situ gastrointestinal loop model group,2.5 mL of GZD(1.0 g crude drug/mL,37 ℃)were injected into the gastrointestinal tract.The abdominal incision was covered with warm,wet cotton,and animals were maintained at 25 ℃.Then,we collected the gastrointestinal tract content after 1.5 h.For the in vitro anaerobic culture model group,the gastrointestinal tract contents of rats were collected and then cultured in 2.5 mL of GZD in an anaerobic environment at 25 ℃ for 24 h.For the blank control group,rats received the same volume of a normal saline solution instead of GZDHigh performance liquid chromatography was used to detect the liquiritin,cinnamic acid,paeoniflorin,and glycyrrhizic acid concentrations in each group and calculate the absorption and biotransformation rates of each ingredient.RESULTS:Cinnamic acid(low polarity)was more easily absorbed by each gastrointestinal part than the higher-polarity glycosides.However,the absorption rate in the cecum was higher than that in other parts.The four compounds,cinnamic acid,liquiritin,paeoniflorin,and glycyrrhizic acid,were transformed completely within 24 h in the cecum and colon,whereas they were hardly transformed in the stomach,excluding glycyrrhizic acid.In addition,all ingredients had higher biotransformation rates in the distal small intestine than that in the proximal small intestine.CONCLUSION:Although a portion of the glycosides in GZD was directly absorbed as the prototype forms in the gastrointestinal tract,they were primarily metabolized and transformed into their corresponding metabolites by intestinal flora near the distal small intestine before their absorption.     

The Effect of Peony and Licorice Decoction on the Voltage-Gated Sodium Channel Subtype 1.4 Based on Standard Decoction

Objective: The objective of this study is to investigate the inhibitory effect of peony and licorice decoction and its compatibility components on the Nav1.4 voltage?gated sodium channels (VGSCs). Materials and Methods: Writhing test was carried out with ICR mice. Paeonia lactiflora and Glycyrrhiza uralensis group were administrated 0.2 ml of solution of freeze?dried powder dissolved in normal saline with the concentration of 2.94 mg/ml, 1.47 mg/ml, and 0.74 mg/ml using intragastric administration, respectively. Peony and licorice decoction groups were administrated 0.2 ml of solution of freeze?dried powder dissolved in normal saline with the concentration of 5.89 mg/ml, 2.94 mg/ml, and 1.47 mg/ml using intragastric administration, respectively. For electrophysiology studies, each freeze?dried powder was dissolved in DMSO to make 10 mg/ml and 50 mg/ml stock solutions. The electrophysiological recordings were obtained under visual control of a microscope. For UPLC analysis, the freeze?dried powder was dissolved in methanol and then determines the contents of the nine marker compounds. Results: The effect of G. uralensis on incubation period and writhing frequency was significantly better than that of peony and licorice decoction group and P. lactiflora group. The inhibition rate of 50 mg/ml water extracts of the three samples was significantly higher than that of the 10 mg/ml group. Moreover, the water extract of G. uralensis at 50 mg/ml had the strongest inhibitory effect on INav1.4 of the three. Conclusion: The possible mechanism of peony and licorice decoction in relieving spasm and pain is most likely by inhibiting Voltage?Gated Sodium Channel Subtype 1.4.     

Anti-oxidative Activities of Ethanol Extracts from Both Wild Plant and Suspension Cell Cultures of Rheum franzenbachii

Objective To evaluate the content of rhaponticin and anti-oxidative activities of theethanol extracts from both the wild plants and suspension cell cultures of Rheum franzenbachii.Methods Quantitative analysis of rhaponticin was performed by HPLC.The anti-oxidative activities of the ethanol extracts were evaluated using 2,2-diphenyl-1-picrylhydrazyl(DPPH)scavenging assays.Results The content of rhaponticin in the roots of the wild plant was 4.36 mg/g,while the content was only 1.59 mg/g in the leaves.The content of rhaponticin in suspension cells cultured on Murashige and Skoog(MS)medium supplemented with 1.0 mg/L 6-benzylaminopurine(6-BAP)and 2.0 mg/L 2,4-dicholorophenoxy acetic acid(2,4-D)was 17.64 mg/g,which increased by 4.05 times compared with the content in the roots of the wild plants.The roots of wild plants displayed the strongest anti-oxidative activity,followed by thesuspension cells 5 and 6,and the scavenging percent was 91.96%,91.23%,and 89.27%,respectively,at the concentration of 100μg/mL.The IC50 values were 2.477,15.644,and 31.415μg/mL,respectively.In particular,the DPPH scavenging activity of the ethanol extracts from the roots of the wild plant was generally comparable to the control of ascorbic acid(VC),and the IC50 value of the extracts was lower than that of VC(2.502μg/mL).Conclusion Rhaponticin production in the cell culture can be modulated and the accumulation can be increased.The roots of the wild plant display the strongest anti-oxidative activity.These results suggest that R.franzenbachii could hold a good potential source for human health.     

Discovery and identification of quality markers of Sparganii Rhizoma based on zebrafish thrombosis model

Objective: The aim of the present study was to determine the Q-markers of Sparganii Rhizoma against thrombus through an integration of investigations on its antithrombotic effect, content determination and spectrum-effect correlation analysis. Methods: Based on the concept of Q-Marker, Sparganii Rhizoma was investigated for the identification of chemical component. The pharmacological effects on arachidonic acid-induced thrombosis in zebrafish were also investigated. The material basis in ethanol extract was determined by HPLC-UV. Furthermore, the potential Q-markers were analyzed and predicted according to the effect-chemical correlation analysis. Finally, the anti-thrombotic Q-markers were verified through the anti-thrombotic test of monomer components. Results: The model of thrombosis zebrafish was established with larvae exposed to 100 μmol/L arachidonic acid for 1 h. Nine ingredients in Sparganii Rhizoma were identified as 5-hydroxymethylfurfural, vanillic acid, ferulic acid, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, vanillin, protocatechuic acid, p-coumaric acid and isoferulic acid. According to the determination effect of zebrafish thrombosis model and HPLC content analysis results, all the other contents present positive correlation except 5-hydroxymethylfurfural, and the P values of three representative potential Q-markers (ferulic acid, protocatechuic acid and p-coumaric acid) were 0.002, 0.001 and 0.026, respectively. Conclusion: Sparganii Rhizoma showed a dose-dependent effect on the recovery of reducing cardiac red blood cell on zebrafish model. Three phenolic acids (ferulic acid, protocatechuic acid and p-coumaric acid) were proved to possess the anti-thrombotic effects which could be regarded as the potential Q-markers for quality assessment of Sparganii Rhizoma.     

Cloning and Expression Analysis on 3-Hydroxy-3-methyl-glutaryl-coenzyme A Reductase from Aquilaria sinensis

Objective To clone the full-length cDNA of 3-hydroxy-3-methylglutaryl-coenzyme A reductase(HMGR)from Aquilaria sinensis(AsHMGR1)and to analyze its expression profile in different tissues and in response to different treatments.HMGR is the first rate-limiting enzyme for sesquiterpene synthesis in the mevalonate pathway.Methods RT-PCR and RACE were used to clone the full-length cDNA of HMGR from A.sinensis based on the conserved HMGR gene fragments.The bioinformatic analysis was performed on its nucleic acid and protein sequence.The expression profile of AsHMGR1 in different tissues and in response to different treatments was analyzed by quantitative RT-PCR.Results The full-length AsHMGR1 cDNA was 2026 bp,containing a 1719 bp open reading frame which encoded a protein of 572 amino acids.Amino acid sequence homology alignment and phylogenetic analysis demonstrated that AsHMGR1 belonged to the HMGR gene family.The detection of tissue expression patterns showed that AsHMGR1 was mainly expressed in the stem,followed by roots and branches.AsHMGR1 could be stimulated by methyl jasmonate and H2O2to varying degrees in a time-dependent manner.Conclusion These data will provide a foundation for further investigation on AsHMGR1 functions and regulatory mechanisms in sesquiterpene synthesis in A.sinensis.     

HPLC Fingerprint and LC-TOF-MS Analysis on Extract from Roots of Gentiana macrophylla

Objective Establishing a fingerprint method to identify the characteristic chemicals in the roots of Gentiana macrophylla and evaluate their quality. Methods RP-HPLC was developed for fingerprint analysis and determination of four ingredients in G. macrophylla roots from different sources. LC-ESI-TOF-MS was employed to identify the chromatographic peaks of the fingerprint. Results Five common peaks were identified by comparing their retention time with reference secoiridoid glucosides. Eight major peaks in chromatographic fingerprint were analyzed by on-line LC-ESI-TOF-MS. Four secoiridoid glucosides were identified based on their MS data. Conclusion The method is specific and could be served for the quality identification and comprehensive evaluation of G. macrophylla.     

Reduning Injection prevents carrageenan-induced inflammation in rats by serum and urine metabolomics analysis

Objective: To elucidate the anti-inflammatory mechanism of Reduning Injection (RDN) by analyzing the potential biomarkers and metabolic pathways of the carrageenan-induced inflammatory model from the overall metabolic level. Methods: Rat inflammatory model was established by carrageenan. UPLC-Q-TOF/MS was used to detect and analyze changes of endogenous metabolites in the serum and urine of carrageenan-induced inflammatory rats. Combined with multivariate analysis and databases analysis, inflammatory-related potential biomarkers were screened and identified to analyze possible metabolic pathways. The reliability and biological significance of these biomarkers was verified by metabolic network analysis and correlation analysis with pharmacodynamic indicators. Results: A total of 16 potential biomarkers were screened and identified by multivariate analysis and metabolite databases, among which 13 species could be adjusted by RDN. The metabolism pathway analysis revealed that histidine metabolism, sphingolipid metabolism, and tyrosine metabolism were greatly disturbed. Their biomarkers involved urocanic acid, sphingosine, and norepinephrine, all of which showed a callback trend after RDN treatment. The three biomarkers had a certain correlation with some known inflammatory-related small molecules (histamine, arachidonic acid, Leukotriene B4, and PGE2) and pharmacodynamic indicators (IL-6, IL-1β, PGE2 and TNF-α), which indicated that the selected biomarkers had certain reliability and biological significance. Conclusion: RDN has a good regulation of the metabolic disorder of endogenous components in carrageenan-induced inflammatory rats. And its anti-inflammatory mechanism is mainly related to the regulation of amino acid and lipid metabolism. This research method is conducive to the interpretation of the overall pharmacological mechanism of Chinese medicine.     

Pyrolysisi Characteristics of Radix Rhizoma Rhei,Cortex Moudan Radicis,and Radix San-guisorbac and correlations with the carbonizing process of Chinese herbs简

AIM： The aim of the work is to study the pyrolysis characteristics of Radix Rhizoma Rhei, Cortex Moudan Radicis, and Radix Sanguisorbae in an inert atmosphere of argon （Ar）, and to investigate the mechanism of the carbonizing process of the three traditional Chinese herbs. METHODS： The pyrolysis characteristics of the crude materials and their extracts were studied by thermogravimetry-mass spectrometry （TG-MS） in a carrier gas of argon, coupled with Fourier transform infrared spectrometry （FTIR） and scanning electron microscopy （SEM） methods. Correlation of the pyrolysis behaviors with the carbonizing process by stir-frying of traditional Chinese medicines was made. RESULTS： Within the temperature range of 200-300 ℃, which is the testing range for the study of the carbonizing process of Chinese herbs, the temperatures indicated by the maximum weight loss rate peak of the above three extracts were taken as the upper-limit temperatures of the carbonizing process of the herbs, and which were 200, 240 and 247 ℃ for Radix Rhizoma Rhei, Cortex Moudan Radicis, and Radix Sanguisorbae, respectively. The ion monitoring signal peaks detected by the TG-MS method corresponded with reports that the level of chemical components of traditional Chinese medicinal materials would decrease after the carbonizing process. It was confirmed by Fourier transform infrared spectrometry （FTIR） and scanning electron microscopy （SEM） methods that better results of ＂medicinal property preservation＂ could be obtained by heating at 200 ℃ for Radix Rhizoma Rhei, at about 250 ℃ for Cortex Moudan Radicis, and Radix Sanguisorbae, as the relative intensity values of the common peaks were among the middle of their three carbonized samples by programmed heating. CONCLUSION： The upper-limit temperatures of the carbonizing process for Radix Rhizoma Rhei, Cortex Moudan Radicis and Radix Sanguisorbae were 200, 240 and 247 ℃ respectively. It is feasible to research the mechanism and technology of the carbonizing process of traditional Chinese medicinal materials using thermogravimetry, Fourier transform infrared spectrometry, and scanning electron microscopy methods.     

Comparison of anti-bacterial activity of three types of di-O-caffeoylquinic acids in Lonicera japonica flowers based on microcalorimetry简

The anti-bacterial activities of three types of di-O-caffeoylquinic acids （diCQAs） in Lonicerajaponica flowers, a traditional Chinese medicine （TCM）, on Bacillus shigae growth were investigated and compared by microcalorimetry. The three types of diCQAs were 3, 4-di-O-caffeoylquinic acid （3, 4-diCQA）, 3, 5-di-O-caffeoylquinic acid （3, 5-diCQA）, and 4, 5-di-O-caffeoylquinic acid （4, 5-diCQA）. Some qualitative and quantitative information of the effects of the three diCQAs on metabolic power-time curves, growth rate constant k, maximum heat-output power Pro, and the generation time tG, total heat output Qt, and growth inhibitory ratio I of B. shigae were calculated. In accordance with a thermo-kinetic model, the corresponding quantitative relationships of k, Pm, Qt, I and c were established. Also, the half-inhibitory concentrations of the drugs （IC50） were obtained by quantitative analysis. Based on the quantity-activity relationships and the IC50 values, the sequence of inhibitory activity was 3, 5-diCQA 〉 4, 5-diCQA 〉 3, 4-diCQA. The results illustrate the possibility that the caffeoyl ester group at C-5 is the principal group that has a higher affinity for the bacterial cell, and that the intramolecular distance of the two caffeoyl ester groups also has an important influence on the anti-bacterial activities of the diCQAs.     

Chemical Characteristics Combined with Bioactivity for Comprehensive Evaluation of Tumuxiang Based on HPLC-DAD and Multivariate Statistical Methods

Background: The dried roots of Inula helenium L.(IH) and Inula racemosa Hook f.(IR) are used commonly as folk medicine under the name of "tumuxiang(TMX)". Phenolic acid compounds and their derivatives, as main active constituents in IH and IR, exhibit prominent anti-inflammation effect.Objective: To develop a holistic method based on chemical characteristic and anti-inflammation effect for systematically evaluating the quality of twenty-seven TMX samples(including 18 IH samples and 9 IR samples) from different origins.Methods: HPLC fingerprints data of AL(Aucklandia lappa Decne.) whose dried root was similar with HR was added for classification analysis. The HPLC fingerprints of twenty-seven TMX samples and four AL samples were evaluated using hierarchical clustering analysis(HCA) and principle component analysis(PCA). The spectrum-efficacy model between HPLC fingerprints and anti-inflammatory activities was investigated by principal component regression(PCR) and partial least squares(PLS).Results: All samples were successfully divided into three main clusters and peaks 7, 9, 11, 22, 24 and 26 had a primary contribution to classify these medicinal herbs. The results were in accord with the appraisal results of herbs. The spectrum-efficacy relationship results indicated that citric acid, quinic acid, caffeic acid-β-D-glucopyranoside, chlorogenic acid, caffeic acid, 1,3-O-dicaffeoyl quinic acid, tianshic acid and 3β-Hydroxypterondontic acid had main contribution to anti-inflammatory activities.Conclusion: This comprehensive strategy was successfully used for identification of IH, IR and AL, which provided a reliable and adequate theoretical basis for the bioactivity relevant quality standards and studying the material basis of anti-inflammatory effect of TMX.     

Co-cultured adventitious roots of Echinacea pallida and Echinacea purpurea inhibit lipopolysaccharide-induced inflammation via MAPK pathway in mouse peritoneal macrophages

Objective: In order to elucidate the biological activity of the co-cultured adventitious roots (ARs) of Echinacea pallida and Echinacea purpurea and provide theoretical basis for its application, and the anti-inflammatory activities and potential mechanisms of co-cultured ARs were studied. Methods: The experimental materials were obtained by bioreactor co-culture technology and used in the activity research. In this study, mouse macrophages induced by lipopolysaccharide (LPS) were used as in vitro model. Different concentrations of AR extract (50?400 g/mL) were used to treat cells. The expression of pro-inflammatory cytokines was determined using enzyme linked immunosorbent assay. The inducible nitric oxide synthase and cyclooxygenase-2 expression, mitogen-activated protein kinase (MAPK) phosphorylation, and the inhibitor of nuclear factor-kappa B-α levels were determined by the Western blot analysis. Results: In the co-cultured ARs, total flavonoids and total caffeic acid were determined, and the contents of both bioactive compounds were significantly higher than those ARs from the single-species culture. Compared with the control group, the large amount of pro-inflammatory mediators was released after LPS stimulation. However, in the extract groups with different concentrations (25, 50, and 100 g/mL), the production of these pro-inflammatory mediators was inhibited in a dose-dependent manner. Furthermore, the levels of phosphorylation of MAPK proteins, including p-p38, p-c-Jun N-terminal kinase, and p-extracellular regulated protein kinases were significantly (P < 0.05) decreased in the extract groups, revealing that the AR extract probably involved in regulating the MAPK signaling pathway. Conclusion: Collectively, our findings suggested that the co-cultured ARs of E. pallida and E. purpurea can inhibit production of pro-inflammatory mediators in mouse peritoneal macrophages and possess the anti-inflammatory effect by regulating MAPK signaling pathways.     

Common mechanism of Citrus Grandis Exocarpium in treatment of chronic obstructive pulmonary disease and lung cancer

Objective: “Same treatment for different diseases” is a unique treatment strategy in traditional Chinese medicine. Two kinds of malignant respiratory diseases endanger human health-chronic obstructive pulmonary disease (COPD) and lung cancer. Citrus Grandis Exocarpium (Huajuhong in Chinese, HJH), a famous herbal, is always applied by Chinese medicine practitioners to dispersion the lung toresolve phlegm based on “syndrome differentiation and treatment” theory. However, the common mechanism for HJH’s treatment of COPD and lung cancer is not clear. Methods: In this study, based on network pharmacology and molecular docking technology, the common mechanism of HJH in the treatment of COPD and lung cancer was studied. The active ingredients and related targets of HJH were integrated from TCMSP, BATMAN-TAM, STP, and Pubchem databases. The standard names of these targets were united by UniProt database. Targets of COPD and lung cancer were enriched through GeneCards, NCBI (Gene), Therapeutic Target Database, and DisGeNET (v7.0) databases. Then the intersection targets of HJH and diseases were obtained. The STRING network and the Cytoscape 3.7.2 were used to construct PPI network, the DAVID database was used to perform GO and KEGG analysis. Then Cytoscape 3.6.1 was used to build “ingredient-target-signal pathway” network. Finally, AutoDock 1.5.6 software was used to perform molecular docking of key proteins and molecules. Results: Eleven active ingredients in HJH were obtained by searching the database, corresponding to 184 HJH-COPD-lung cancer targets intersection. The results of biological network analysis showed that naringenin, the active component in HJH, could mainly act on target proteins such as AKT1, EGFR. Then through positive regulation of vasoconstriction and other biological processes, naringenin could regulate estrogen signaling pathway, VEGF signaling pathway, HIF-1 signaling pathway, ErbB signaling pathway, PI3K-Akt signaling pathway to play an important role in the treatment of both COPD and lung cancer. Conclusion: Network pharmacology was employed to systematically investigate the active ingredients and targets of HJH in treatment of COPD and lung cancer. And then, the common pharmacodynamic network of HJH for the two malignant respiratory diseases was firstly described. Furthermore, naringenin was proved to strongly bind with AKT1 and EGFR. It may provide the scientific basis for understanding the “Same treatment for different diseases” strategy in traditional Chinese medicine and inspirit subsequent drug discovery for COPD, lung cancer and other malignant lung diseases.     

Metabolomic characteristics of spontaneously hypertensive rats under chronic stress and the treatment effect of Danzhi Xiaoyao Powder,a traditional Chinese medicine formula

Objective: Numerous studies have demonstrated the close relationship between chronic stress and blood pressure(BP). Hypertensive subjects exhibit exaggerated reactions to stress, especially higher BP. The mechanisms by which stress affects pre-existing hypertension still need to be explored. Danzhi Xiaoyao Powder(DP), a historical traditional Chinese medicine formula, is a promising treatment for BP control in hypertensive patients under stress. The present study investigated the metabolomic disruption caused by chronic stress and the treatment effect and mechanism of DP.Methods: Spontaneously hypertensive rats(SHRs) were subjected to chronic restraint stress(CRS) for 4 weeks. BP was measured via the tail-cuff method, and anxiety-like behavior was quantified using the elevated-plus-maze test. Meanwhile, DP was administered intragastrically, and its effects were observed. Global metabolomic analysis was performed using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, followed by multivariate statistical analysis to detect differential metabolites and pathways.Results: DP alleviated the CRS-induced increase in BP and anxiety-like behavior. Systematic metabolic differences were found among the three study groups. A total of 29 differential plasma metabolites were identified in both positive-and negative-ion modes. These metabolites were involved in triglyceride metabolism, amino acid(phenylalanine, tryptophan, and glycine) metabolism, and steroid hormone pathways.Conclusion: These findings expose the metabolomic disturbances induced by chronic stress in SHRs and suggest an innovative treatment for this disorder.     

Mechanism Research of Chonglou as a Pain Killer by Network Pharmacology

Objective: The objective of this study is to screen the therapeutic targets of pain of traditional Chinese medicine Chonglou and explore the relevant mechanism by network pharmacology techniques and methods. Materials and Methods: The chemical components of Chonglou were collected according to chemistry database and related literature. SwissADME was used to collect the potential active ingredients from all the chemical components of Chonglou and SwissTarget Prediction was utilized to predict their targets. The genes related to pain were collected from GeneCards and Online Mendelian Inheritance in Man databases. Joint genes were uploaded to the online string database for the analysis and the PPI network was constructed. The “Chonglou-active component-target-pain” network was constructed by Cytoscape 3.7.1 software, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed for key target proteins. The top three active components with most targets in the network were docked with the target proteins by the molecular docking technique. Results: A total of nine potential active compounds of Chonglou, 264 potential target genes, 2385 targets of pain disorder, and 128 common targets for drug and disease were screened. One hundred and thirty?one GO items were identified by the GO enrichment analysis, and 23 related signaling pathways were identified by the KEGG pathway enrichment analysis. Molecular?docking results show that pennogenin is the optimal butt ligand of PIK3CA, STAT3, mitogen?activated protein kinase 14, and ADORA1. Conclusion: It is preliminarily revealed that Chonglou might treat pain through multiple targets, multiple biology processes and multiple pain-related signaling pathways, providing reference for the subsequent experimental research.     

Simultaneous Quantitative Analysis of Five Components in Angelica sinensis and Angelica acutiloba Acclimatized Growing in Vietnam by High-Performance Liquid Chromatography with Photodiode Array Detector

Background: Chinese Danggui(Angelica sinensis) and Japanese Danggui(Angelica acutiloba) are evaluated as the same in using and critical of quality control in Vietnamese Pharmacopoeia. In Vietnam, Japanese Danggui were acclimatized and cultivated in several regions in recent years. Despite the huge climatic difference between Vietnam and Japan, Japanese Danggui grows very well in Vietnam. However, there are no studies to assess the overall quality of this medicinal herbs. Aims and Objectives: The aim of this study is to develop a method to identify and assay simultaneously 5 component compounds of these crude herbs: chlorogenic acid, ferulic acid, scopoletin, xanthotoxin and ligustilide by high-performance liquid chromatography with photodiode array detector(HPLC-PDA). Materials and Methods: The procedure was optimized by using column Phenomenex Gemini 5 μm PR C18, 15 x 4,6 mm with the detection wavelength set at 321 nm for detector PDA and mobile phase was composed of(A) ACN and(B) aqueous solution 1% of acid acetic using a gradient elution. Analytes were performed at 30°C with a flow rate of 1.0 mL/min. Results: The developped method were fully validated for linearity, accuracy, recovery and met the validation requirements, proved practical applicability for the quality control of these herbs and related products. Conclusion: The method was successfully applied to the quantification of five markers simultaneously from collected samples.     

Microbial inoculants and garbage fermentation liquid reduced root-knot nematode disease and As uptake in Panax quinquefolium cultivation by modulating rhizosphere microbiota community

Objective: To find a suitable ecological cultivation measure to solve the problem of root-knot nematode disease of Panax quinquefolium (Panacis Quinquefolii Radix) and the heavy metals accumulating in its roots. Methods: Three-year-old P. quinquefolium was treated with four different combinations of microbial inoculant (MI) and garbage fermentation liquid (GFL) [the joint application of ‘TuXiu’ MI and Fifty potassium MI (TF), the combination use of ‘No. 1’ MI and Fifty potassium MI (NF), ‘Gulefeng’ poly-γ-glutamic acid MI (PGA), GFL], and the untreated control (CK). Here, high-throughput sequencing, ICP-MS and UPLC were employed to systematically characterize changes of microbial diversity and structure composition, heavy metals (As, Cd and Pb) content and ginsenoside content among different treatments. Results: The results revealed that different MIs and GFL could increase the root dry weight of P. quinquefolium, PGA enhanced it by 83.24%, followed by GFL (49.93%), meanwhile, PGA and GFL were able to lessen root-knot nematode disease incidence by 57.25% and 64.35%. The treatment of PGA and GFL can also effectively reduce heavy metals in roots. The As content in GFL and PGA was decreased by 52.17% and 43.48% respectively, while the Cd and Pb contents of GFL and PGA was decreased somewhat. Additionally, the content of total ginsenosides was increased by 42.14% and 42.07%, in response to TF and NF, respectively. Our metagenomic analysis showed that the relative abundance of particular soil microbial community members related to the biocontrol of root-knot nematode disease and plant pathogen (i.e., Chaetomium in NF, Xylari in GFL, and Microascus in PGA), heavy metal bioremediation (Hyphomacrobium in PGA and Xylaria in GFL), and nitrogen fixation (Nordella and Nitrospira in TF) was significantly increased; notably, potential harmful microflora, such as Plectosaphaerella and Rhizobacter, were more abundant in the control group. Conclusion: MI and GFL could improve the quality of P. quinquefolium by modifying its rhizosphere microbial community structure and composition, both of them are beneficial to the development of ecological cultivation of P. quinquefolium.     

Saponin Accumulation in Flower Buds of Panax notoginseng

Objective Panax notoginseng is an important Chinese medicinal plant. Saponin accumulation is higher in the flower buds than in the other parts of P. notoginseng. However, the flower bud compositions have not yet been quantified. The aim of this study is to investigate the formation and accumulation of saponins in the flower buds of P. notoginseng from different populations and at different growth years. Methods Fourteen types of P. notoginseng with different growing durations and from different areas of Wenshan County, Yunnan Province were collected. We separated P. notoginseng individually into the flower buds, stems, leaves, and roots at the places where it has the highest saponin content. An efficient high-performance liquid chromatography(HPLC) method was developed for simultaneously quantifying two active saponins, ginsenoside Rb1 and ginsenoside Rb3, in the flower buds of P. notoginseng. The total saponin content was determined by using a quantitative vanillin-sulfuric acid colorimetric method. HPLC method was used to establish the fingerprints of 13 saponins and then quantify the composition in the whole plant of P. notoginseng. Results The saponin contents of different parts differ significantly, and the total saponin content and those of Rb1 and Rb3 do not entirely correlated. The flower buds of P. notoginseng contain 27.79% of total saponins, which is the highest saponin content in the whole plant. Fingerprint result showed that different saponins were appeared in different parts of the plant, i.e. flower buds, stems, leaves, and roots.Conclusion The saponin contents from the flower buds of P. notoginseng vary depending on the growth area and duration. The fingerprints show that the saponin contents and compositions vary depending on the part of P. notoginseng. These results are useful for the pharmacological evaluation and quality control of P. notoginseng.     

Triterpenes from Euphorbia hirta and their cytotoxicity

AIM： To investigate the chemical constituents of the stems, leaves and roots of Euphorbia hirta, and to test for the cytotoxic and antimicrobial potentials of the major constituents of the plant. METHODS： The compounds were isolated by silica gel chromatography and their structures were elucidated by NMR spectroscopy. The cytotoxicity tests were conducted using the 3-（4, 5-dimethylthiazol-2-yl）-2, 5-diphenyltetrazolium bromide （MTT） assay, while the antimicrobial tests employed the agar well method. RESULTS： The air-dried stems of E. hirta afforded taraxerone 1, a mixture of 25-hydroperoxycycloart-23-en-3β-ol （2a） and 24-hydroperoxycycloart-25-en-3β-ol （2b） （sample 2） in a 2 ： 1 ratio, and another mixture of cycloartenol （3a）, lupeol （3b）, α-amyrin （3e） and fl-amyrin （3d） （sample 3） in a 0.5 ： 4 ： 1 ： 1 ratio. The air-dried leaves of E. hirta yielded sample 2 in a 3 ： 2 ratio, sample 3 in a 2 ： 3 ： 1 ： 1 ratio, phytol and phytyl fatty acid ester, while the roots afforded sample 2 in a 2 ： 1 ratio, sample 3 in a 2 ： 1 ： 1 ： 1 ratio, a mixture of cycloartenyl fatty acid ester 4a, lupeol fatty acid ester 4b, α-amyrin fatty acid ester 4c and β-amydn fatty acid ester 4d （sample 4） in a 3 ： 2 ： 1 ： 1 ratio, linoleic acid, β-sitosterol and squalene. Compound 1 from the stems, sample 2 from the leaves, and sample 3 from the stems were assessed for cytotoxicity against a human cancer cell line, colon carcinoma （HCT 116）. Sample 2 showed good activity with an ICs0 value of 4.8 μg.mL-1, while 1 and sample 3 were inactive against HCT 116. Sample 2 was further tested for cytotoxicity against non-small cell lung adenocarcinoma （A549）. It showed good activity against this cell line with an ICs0 value of 4.5 μg.mL-1. Antimicrobial assays were conducted on 1 and sample 2. Results of the study indicated that I was active against the bacteria： Pseudomonas aeruginosa and Staphylococcus aureus, but was inactive against Escherichia coli and Bacillus subtilis. Sample 2 was active against the bacteria： Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli and fungi： Candida albicans and Trichophyton mentagrophytes. It was inactive against Bacillus subtilis and Aspergillus niger. CONCLUSIONS： The triterpenes： 2a, 2b, 3a, 3b, 3c and 3d were obtained from the stems, roots and leaves of E. hirta. Taraxerol （1） was only isolated from the stems, the leaves yielded phytol and phytyl fatty acid esters, while the roots afforded 4a-4d, linoleic acid, β-sitosterol, and squalene. Triterpene 1 and sample 2 were found to exhibit antimicrobial activities. Thus, these compounds are some of the active principles of E. hirta which is used in wound healing and the treatment of boils. The cytotoxic properties of sample 2 imply that triterpenes 2a and 2b contribute to the anticancer activity of E. hirta.