10-Secocycloartane (=9,19-cyclo-9,10-secolanostane) triterpenoid saponins: Huangqiyenins M-X from Astragalus membranaceus (Fisch.) Bge.

Phytochemical investigations of the leaves of Astragalus membranaceus (Fisch.) Bge. have led to the isolation of 12 undescribed triterpenoid saponins named huangqiyenins M-X. The structures of the undescribed compounds were determined using NMR and HRESIMS data. The cytotoxicity of these compounds against the RKO and HT-29 colon cancer cell lines was evaluated. Among these compounds, huangqiyenin W exhibited the highest cytotoxic activity against RKO colon cancer cells, whereas huangqiyenin Q and W showed moderate cytotoxic activity against HT-29 colon cancer cells. The network pharmacology results indicated that STAT3, IL-2 and CXCR1 are the correlated targets of huangqiyenin W against colon cancer, with AGE-RAGE and Th17 cell differentiation as the key signaling pathways.

High-Throughput Phytochemical Unscrambling of Flowers Originating from Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) P. K. Hsiao and Astragalus membranaceus (Fisch.) Bug. by Applying the Intagretive Plant Metabolomics Method Using UHPLC-Q-TOF-MS/MS.

Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) P. K. Hsiao (MO) and Astragalus membranaceus (Fisch.) Bug. (ME) are two primary sources of the Astragalus herb, also known as "Huangqi" in China, which is widely applied to treat hypertension, glomerulonephritis, ischemic heart disease, and diabetes mellitus. As two different sources of the Astragalus herb, the chemical profiles of MO and ME may be different. Previous studies showed abundant differences in chemical composition between MO and ME. Therefore, the by-products of MO and ME, such as Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) P. K. Hsiao flower (MOF) and Astragalus membranaceus (Fisch.) Bug. flower (MEF), may have different phytochemical profiles. In this paper, a metabolomics method combined with ultra-high-performance liquid chromatography and electrospray ionization/quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS) was employed to analyze the components of MOF and MEF. Consequently, the results of principal component analysis (PCA) showed that MOF and MEF could be separated clearly. In total, 31 chemical markers differentiating MOF and MEF were successfully identified, including 22 flavonoids, 8 isoflavones and 1 benzopyran. Among them, the contents of 18 components, including Calycosin, Cyanidin-3-O-glucoside, Quercetin, Rutin, Kaempferol, Formononetin, Isomucronulatol and Prim-O-glucosylcimifugin in MEF, were significantly higher than in MOF. In turn, the contents of another 13 components, covering Biochanin A, Tectoridin, Isomucronulatol-7-O-glucoside, Liquiritin, Rhamnetin, etc., were lower in the MEF group than that in the MOF group. It is worth noting that flavonoids, especially flavonoid glycosides, were the primary active chemical ingredients in MOF and MEF. The 18 ingredients in MEF with a higher level carried out diverse activities, like anti-oxidant, anti-inflammatory, anti-bacterial and anti-tumor activities, which led us to speculate that MEF may have greater pharmacological effects and potential development prospects than MOF. The present results displayed that the contents of ingredients in the two different species of plants were radically different, and there was significant uniqueness to the components of MOF and MEF. Our study not only provides helpful chemical information for further quality assessment and active mechanism research of MOF and MEF but also offers scientific support for the resource utilization of MOF and MEF.

Creating a Vaccine-like Supplement against Respiratory Infection Using Recombinant Bacillus subtilis Spores Expressing SARS-CoV-2 Spike Protein with Natural Products.

Vaccination is the most effective method of combating COVID-19 infection, but people with a psychological fear of needles and side effects are hesitant to receive the current vaccination, and alternative delivery methods may help. Bacillus subtilis, a harmless intestinal commensal, has recently earned a strong reputation as a vaccine production host and delivery vector, with advantages such as low cost, safety for human consumption, and straightforward oral administration. In this study, we have succeeded generating "S spores" by engineering B. subtilis with spore coat proteins resembling the spike (S) protein of the ancestral SARS-CoV-2 coronavirus. With the addition of two immunostimulating natural products as adjuvants, namely Astragalus membranaceus (Fisch.) Bge (AM) and Coriolus versicolor (CV), oral administration of S spores could elicit mild immune responses against COVID-19 infection without toxicity. Mucosal IgA against the S protein was enhanced by co-feeding with AM and CV in an S spores-inoculated mouse model. Faster and stronger IgG responses against the S protein were observed when the mice were fed with S spores prior to vaccination with the commercial COVID-19 vaccine CoronaVac. In vitro studies demonstrated that AM, CV, and B. subtilis spores could dose-dependently activate both macrophages and dendritic cells by secreting innate immunity-related IL-1ß, IL-6, and TNF-α, and some other proinflammatory chemokines and cytokines. In conclusion, the combination of S spores with AM and CV may be helpful in developing a vaccine-like supplement against respiratory infection.

Effect and molecular mechanism research of Astragalus membranaceus on inhibiting intestinal absorption of six alkaloids of Aconitum carmichaelii in spleen deficiency rats.

Objective: To investigate the effect and the mechanism of Astragalus membranaceus (Huangqi in Chinese, HQ) extract on the intestinal absorption of six alkaloids of Aconitum carmichaelii (Fuzi in Chinese, FZ) in rats with spleen deficiency and provide novel insights into the application of HQ on modulating intestinal barrier. Methods: Four-week-old male Sprague-Dawley rats were fed with Xiaochengqi Decoction to induce the spleen deficiency model for 40 d. Single-pass intestinal perfusion model were used to study the effects of HQ extract on the absorption of alkaloids. Protein expression and mRNA levels of MRP2 and BCRP and tight junction proteins (TJ, including Claudin-1, Occludin and ZO-1) were measured using Western blot and real-time PCR, respectively. The location and expression of TJ protein was also investigated by the immunofluorescence method. Results: Compared with the normal group, the protein expression of MRP2, BCRP and TJ proteins in the model group were significantly down-regulated. After oral administration of HQ, the alkaloid absorption in intestinal villi was inhibited, MRP2, BCRP and TJ proteins were up-regulated, the green fluorescence staining of Claudin-1, Occludin, and ZO-1 was enhanced, and a thick layer of mucus was deposited on the surface of the epithelium of the intestinal cavity. Conclusion: HQ as an intestinal barrier modulator improves the physiological changes of the intestinal environment of spleen deficiency to reduce the absorption of toxic components, leading to a decrease in the absorption of drug-like molecules.

Effect and molecular mechanism research of Astragalus membranaceus on inhibiting intestinal absorption of six alkaloids of Aconitum carmichaelii in spleen deficiency rats / 中草药·英文版

Objective: To investigate the effect and the mechanism of Astragalus membranaceus (Huangqi in Chinese, HQ) extract on the intestinal absorption of six alkaloids of Aconitum carmichaelii (Fuzi in Chinese, FZ) in rats with spleen deficiency and provide novel insights into the application of HQ on modulating intestinal barrier. Methods: Four-week-old male Sprague-Dawley rats were fed with Xiaochengqi Decoction to induce the spleen deficiency model for 40 d. Single-pass intestinal perfusion model were used to study the effects of HQ extract on the absorption of alkaloids. Protein expression and mRNA levels of MRP2 and BCRP and tight junction proteins (TJ, including Claudin-1, Occludin and ZO-1) were measured using Western blot and real-time PCR, respectively. The location and expression of TJ protein was also investigated by the immunofluorescence method. Results: Compared with the normal group, the protein expression of MRP2, BCRP and TJ proteins in the model group were significantly down-regulated. After oral administration of HQ, the alkaloid absorption in intestinal villi was inhibited, MRP2, BCRP and TJ proteins were up-regulated, the green fluorescence staining of Claudin-1, Occludin, and ZO-1 was enhanced, and a thick layer of mucus was deposited on the surface of the epithelium of the intestinal cavity. Conclusion: HQ as an intestinal barrier modulator improves the physiological changes of the intestinal environment of spleen deficiency to reduce the absorption of toxic components, leading to a decrease in the absorption of drug-like molecules.

A new diphenyl butanedione from Astragalus membranaceus of northern Shanxi / 药学学报

Seven compounds were isolated from Astragalus membranaceus of northern shaanxi by silica gel and Sephadex LH-20 column chromatographies. Their chemical structures were identified on the basis of their physical and chemical properties. These compounds were elucidated as astragaloside IV (1), formononetin (2), calycosin (3), 1-(4-hydroxyphenyl)-4-(2,4-hydroxyphenyl)-2-hydroxy-1,4-but anedione (4), (E)-4-methylcinnamic acid (5), quercetin (6), and uridine (7). Compound 4 is a new compound and compound 5 was isolated from the plants of Astragalus Linn. for the first time. The results of in vitro antitumor activity assay showed that compound 4 could inhibit the proliferation of A549 with IC50 values of 11.41 μmol·L-1.

Integration of network pharmacology and molecular docking technology reveals the mechanism of the herbal pairing of Codonopsis Pilosula (Franch.) Nannf and Astragalus Membranaceus (Fisch.) Bge on chronic heart failure.

BACKGROUND: The herbal pairing of Dangshen (DS) [Codonopsis pilosula (Franch.) Nannf.] and Huangqi (HQ) [Astragalus membranaceus (Fisch.) Bge.] (DHP) is a traditional Chinese herbal medicine that is frequently used to treat chronic heart failure (CHF) in China. However, the pharmacological mechanism of DHP has not been fully elucidated. This is the first study aimed to reveal the active mechanism of DHP in the treatment of CHF by using network pharmacology methods. METHODS: The active ingredients of DHP were obtained from the TCMSP database, and the potential targets of DHP were predicted using the SwissTargetPrediction database. CHF-related targets were searched by the DisGeNET and GeneCards databases. The common targets between the disease and herbs were obtained using a Venn diagram. The STRING database was utilized to obtain the protein-protein interaction data. Next, we used Cytoscape 3.7.2 software to construct and analyze the herb-ingredient-potential targets-disease network. Topology analysis was used to identify the key ingredients and hub genes. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the Metascape database to reveal the mechanism. Furthermore, molecular docking simulation was performed using AutoDock Vina software to assess the affinity of the key ingredients and hub genes. RESULTS: Five key ingredients and six hub genes were screened. The six hub genes were closely related to PI3K /AKT or ERK1/2 pathways. The KEGG pathways mainly involved the TNF signaling pathway, calcium signaling pathway, and cancer-related pathways. The GO enrichment analysis results showed that DHP might act on biological processes including positive regulation of kinase activity and cellular response to nitrogen compound via the three above-mentioned pathways in the treatment of CHF. Finally, the molecular docking results showed that the five key ingredients exhibited strong affinities to the six hub genes. CONCLUSIONS: This study revealed the molecular mechanism that the flavonoids in DHP may alleviate endothelial dysfunction and cardiac hypertrophy via regulation of the TNF pathway and its downstream PI3K/Akt or ERK1/2 signaling pathways, or improve excitation-contraction coupling by regulating calcium signaling pathway, thereby improving CHF. These results provide insights for further experimentation on its pharmacological effects.

Cloning and expression of three PAL genes in Astragalus membranaceus / 中草药

Objective To disclose the molecular mechanism of calycosin-7-O-β-D-glucoside (CG) accumulation in Astragalus membranaceus, we cloned PAL genes and analyzed the expression patterns of them and changes of CG contents in different tissues of A. membranaceus. Methods PAL genes were cloned with the methods of homology cloning and RACE technique using the total RNA as template and the analysis of bioinformatics on the cloned genes was carried out, gene expressions in root, stem, and leaf were determined with real-time PCR method, and CG content in root, stem, and leaf were analyzed by HPLC methods. Results Three PAL genes were cloned from A. membranaceus. The genbank accession number was KY086279 (AmPAL1), KY086280 (AmPAL2), and KY086281 (AmPAL3), respectively; The full-length cDNA of them was 2 508 bp, 2 401 bp, and 2 498 bp, respectively; And they all consisted of 2 157 bp open reading frame encoding 718 amino acids. Deduced AmPAL proteins had typical active sequences of PAL proteins, they were homology with other PAL proteins, and they shared the highest identities with PAL proteins of leguminous plants. Phylogenetic tree analysis showed AmPAL1 belonged to the different sub-class with the sub-class of AmPAL2 and AmPAL3. Real-time PCR analysis indicated that expression levels of AmPALs were different from each other, the expression level of AmPAL1 was the highest, the expression level of AmPAL2 was the next, and that of AmPAL3 was lowest in all detected tissues, and only the expression levels of AmPAL2 was similar to the changes of CG contents in different tissues (root > stem > leaf). Conclusion The cloned AmPAL1, AmPAL2, and AmPAL3 from A. membranaceus were typical genes of PAL, each might have different function in developing of different tissues, and AmPAL2 might involve in CG accumulation in different tissues.

Mechanism of simplified formula of Tuoli Xiaodu Powder in promoting of diabetic wound healing based on molecular docking and network pharmacology / 中草药

Objective: To investigate the active ingredients and molecular mechanism of Astragalus membranaceus var. mongholicus, Angelica sinensis, Angelicae dahurica, and Gleditsia sinensis in Tuoli Xiaodu Powder in promoting of diabetic wound healing. Methods: UPLC-Q-TOF/MS in positive and negative ion modes was applied to analyze the components in the ethanol extract from Tuoli Xiaodu Powder. Molecular docking technology was used to predict the targets proteins of these components. The function and pathway annotations of target proteins were performed through relevant databases such as Uniprot and KEGG. The drug components-target-function diagram was constructed using Cytoscape software. Results: Twenty-eight compounds containing flavonoids, saponins, coumarins, alkaloids, and triterpenoids were identified in positive and negative ion modes. Among these compounds, 17 compounds could interact with 17 target proteins, and there were 210 pairs of component-target relationships by analyzing the results of molecular docking. Among them, five targets were related to immune regulation, six targets were related to antibacterial and anti-inflammatory effects, six targets were related to cell differentiation, 10 targets were related to cell migration, six targets were related to angiogenesis, two targets were related to stimulation of epithelial growth factor, six targets were related to vasodilation, and two targets were related to estrogen. Conclusion: The flavonoids, saponins, coumarins, steroids, and triterpenoids contained in the simplified formula possess many biological effects such as antibacterial, anti-inflammatory, immune regulation, and angiogenesis. These functions may be related to its modulation of NF-κB, PI3K/Akt/eNOS, and MAPK pathway through regulating NF-κB, MAPK, PI3K, and ERK2 targets.

Nutritional component analysis and resource value evaluation of flowers of Astragalus membranaceus var. mongholicus / 中草药

Objective To analyze and evaluate the chemical components from flowers of Astragalus membranaceus var. mongholicus, and to investigate the potential value of the medicinal plant resources. Methods UV-Vis spectrophotometry was used to determine the total contents of polysaccharides and water-soluble protein. HPLC-PDA/ELSD method was used to determine monosaccharides and oligosaccharides and GC-MS method was used to determine volatile components and the fatty acids in the flowers of A. membranaceus var. mongholicus. UPLC-TQ-MS method was used to analyze the nucleosides and amino acids. Results The flowers of A. membranaceus var. mongholicus contain abundant polysaccharides (47.02 mg/g), water-soluble protein (470.66 mg/g), fructose (45.46 mg/g), glucose (8.71 mg/g), and sucrose (1.05 mg/g). There were 32 kinds of volatile components detected in the flowers of A. membranaceus var. mongholicus, in which oxy-derivatives were the main components. In addition, six nucleosides and 15 amino acids were detected in the flowers of A. membranaceus var. mongholicus, and their total contents were 2.77 mg/g and 6.52 mg/g, respectively. Eight fatty acids in the flowers of A. membranaceus var. mongholicus were also detected, in which myristic acid, palmitic acid, and oleic acid were the main components. Conclusion This study investigated the composition and content of various nutritional components of the flower of A. membranaceus var. mongholicus, which provides a scientific basis for its utilization and development.

Chemical constituents in roots of Astragalus membranaceus / 中草药

Objective: To study the chemical constituents in the roots of Astragalus membranaceus. Methods: The constituents were isolated and purified by silica gel, ODS, polyamide and Sephadex LH-20 column chromatography. Their structures were elucidated by NMR spectra. Results: Twenty-three compounds were isolated from the roots of A. membranaceus, covering 14 flavonoids, eight triterpenoid saponins and one phenylpropanoid. They were identified as 2'-methoxyisoliquiritigenin (1), echinatin (2), licochalcone B (3), 3', 4', 7-trihydroxyflavone (4), 4', 7-dihydroxyflavone (5), 3', 7, 8- trihydroxy-4'-methoxyisoflavone (6), 4-hydroxycinnamic acid (7), calycosin (8), formononetin (9), 2', 4, 4'-trihydroxychalcone (10), pendulone (11), liquiritigenin (12), pratensein (13), calycosin-7-O-β-D-glucopyranoside (14), ononin (15), astragaloside IV (16), cyclocanthoside E (17), isoastragaloside II (18), astragaloside II (19), astragaloside III (20), isoastragaloside I (21), brachyoside B (22), and cycloaraloside A (23). Conclusion: Compounds 1-7 are isolated from the plants of Astragalus Linn. for the first time.

Research on purification and activity characterization of AmPR-10 ribonuclease from Astragalus membranaceus / 中草药

Objective To establish a stable and rapid separation and purification method for Astragalus membranaceus (Am) pathogenesis-related protein-10 (AmPR-10) using an automatic intelligent protein purification system AKTA Avant 25, and analyze its physiochemical and biological activity. Methods Am was extracted by Tris-HCl buffer. The crude extract was captured by anion exchange chromatography, and finely separated by hydrophobic chromatography and gel filtration chromatography. The relative molecular weight of AmPR-10 was measured by MALDI-TOF/TOF mass spectrometry, the protein identification was determined by mass spectrometry and MS/MS Ion Search, the glycoprotein identification was estimated by periodic acid-Schiff method, and the ribonuclease activity and effect factors were analyzed by agarose gel electrophoresis. Results The electrophoretically pure AmPR-10 was obtained by three-step purification of Q Sepharose Fast Flow, Butyl Sepharose High Performance and SuperdexTM 75 10/300 GL from the crude extraction. The relative molecular weight of AmPR-10 was 16 801. AmPR-10 was highly homologous to PR-10 and has no carbohydrate chains. Incubated at 56 ℃ for 30 min, AmPR-10 exhibited significant ribonuclease activity to total RNA of mammalian cells. The activity was insensitive to NaCl, pH value and mental ions, and weekly inhibited by 0.5 mol/L NaCl, pH 9.0, Mg2+ and Co2+. The activity was the same at EDTA as high as 20 mmol/L. Conclusion The three-step method of exchange chromatography-hydrophobic chromatography-gel filtration chromatography, a stable and rapid separation and purification method of AmPR-10, can be applied for other Chinese herbs. AmPR-10 might play an important role in resistance against virus.

Aromatic Constituents from the Stems of Astragalus membranaceus (Fisch.) Bge. var. Mongholicus (Bge.) Hsiao.

Four new aromatic constituents, astraflavonoids A (1), B (2), C (3), and astramemoside A (4), along with sixteen known ones 5-20 were obtained from the stems of A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. Their structures were elucidated by chemical and spectroscopic methods. Among the known isolates, 14 was obtained from the Astragalus genus for the first time, while 7-12, 18-20 were isolated from the species for the first time. The effects of the compounds obtained from the plant on glucose consumption were analyzed in differentiated L6 myotubes in vitro, whereby compounds 1, 2, 3, 7, 8, 10, 11, 14, 15 and 18 displayed significant promoting effects on glucose consumption in L6 myotubes. Among them, the activities of 1, 2 and 7 were comparable to that of insulin, which suggested that these compounds may be involved in glucose metabolism and transport.

Bioactive cyclolanstane-type saponins from the stems of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao.

Twelve cyclolanstane-type saponins including six new ones, astrolanosaponins A1 (1), A2 (2), B (3), C (4), D (5), and E (6) were obtained from the stem of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, and their structures were elucidated by chemical and spectroscopic methods. Of the known ones, cycloastragenol-3-O-ß-D-glucopyranoside (7), astraverrucin II (8), cycloaraloside E (9), huangqiyenin A (10), and huangqiyenin B (11) were isolated from the species first. Meanwhile, compounds 1-3, 5-9, and aleksandroside I (12) showed inhibitory effects on triglyceride accumulation in HepG2 cells.

Oleanane type saponins from the stems of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao.

Astragalus membranaceus is a Traditional Chinese Medicine (TCM) belonging to the Leguminosae family. It has been used as antidiabetic, cardioprotective, and hepatoprotective in the TCM clinic. From the stems of A. membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, 14 oleanane type saponins (1-14) including eight new ones, astroolesaponins A (1), B (2), C1 (3), C2 (4), D (5), E1 (6), E2 (7), and F (8) were obtained, and their structures were elucidated by chemical and spectroscopic methods. Compounds 1-5, 7, 8, 11, and 13 showed decreased effects on triglyceride levels in sodium oleate induced HepG2 cells.

Quality differences of medical material, raw decoction pieces, and processed products of Astragalus membranaceus var. mongholicus / 中草药

Objective: To study the quality differences of medical material, raw decoction pieces, and processed products of Astragalus membranaceus var. mongholicus. Methods: The raw decoction pieces and processed products were obtained from genuine medicinal materials of A. membranaceus var. mongholicus. The reasons for the quality differences were analyzed by comparing the contents of astragaloside IV, calycosin-7-O-β-D-glucopyranoside, formononetin, total polysaccharides, saponins, and flavones. Results: With content analysis, the sequence was found as follows: astragaloside IV (medical material > raw decoction piece > honey-fried piece > alcohol-fried piece > salt-fried piece > fried piece); calycosin-7-O-β-D-glucopyranoside, formononetin, and total polysaccharides (medical material > raw decoction pieces > alcohol-fried piece > salt-fried piece > honey-fried piece > fried piece), total flavones (medical material > alcohol-fried piece > raw decoction pieces > salt-fried piece > honey-fried piece > fried piece), total saponins (medical material > honey-fried piece > raw decoction pieces > alcohol-fried piece > salt-fried piece > fried piece). Conclusion: The temperature and supplementary material may play the main roles for quality differences of A. membranaceus var. mongholicus.

Optimization of extraction technology of two immune active proteins from Astragalus membranaceus var. mongholicus / 中草药

Objective: To optimize the extraction technology of immune active glycoproteins AmPR10-16kD and HQGP-2 from Astragalus membranaceus var. mongholicus (AMM). Methods: The optimized extraction temperature conditions were investigated by circular dichroism of water-soluble protein involving in AmPR10-16kD and HQGP-2 with secondary structure from AMM. The optimized extraction technology was investigated using single factor test and orthogonal test with gray value of water-soluble protein AmPR10-16kD and HQGP-2 as the index which was determined by Image of gel graphical analysis software. In this study, the effects of temperature, solid-liquid ratio, time, solvent, granularity, and times on gray value were investigated, for which the inhibitory effect of water-soluble protein was determined as an evidence by CCK-8 method, and the content of water-soluble protein is determined as an evidence by BCA method. Results: The optimized extraction technique for proteins AmPR10-16kD and HQGP-2 in AMM was established, that was 5.0 g powder of AMM over the No.4 sieve, olvent Tris-HCl, solid-liquid ratio 1:16 and 60 min for extraction at the temperature of 40 ℃ and being mixed under 100 r/min. The water-soluble protein extract rate in the orthogonal test analysis was 65 mg/g, of which inhibitory effect was 90.90% at a concentration of 90 μg/mL. Conclusion: The optimal extraction conditions could accurately reflect the relative amounts of AmPR10-16kD and HQGP-2 maximum extraction rate, providing a stable, reasonable, and feasible extraction process for further study of the bioactive substance of AMM.

Genetic diversity analysis of germplasm of authentic Astragalus membranaceus in Inner Mongolia / 中草药

Objective: To establish an optimum reaction system suitable for ISSR analysis of Astragalus membranaceus and to analyze the genetic diversity of wild populations in Inner Mongolia. Methods: A stable and reliable ISSR reaction system was set up combining the concentration gradient of the single factor test and orthogonal test. The genetic diversity of 30 A. membranaceus populations in nine zones of Inner Mongolia was analyzed using NTSYS2.1 software. Results: The optimal ISSR reaction system (20 μL) contained 10 × PCR buffer 2.0 μL, 1.5 mmol/L MgCl2, 0.4 mmol/L deoxyribonucleotide triphosphate (dNTP), 1.5 U Taq DNA polymerase, 0.5 μmol/L primer, and 40 ng template DNA. A total of 169 amplified loci were detected by 15 ISSR primers, in which 157 loci were polymorphic loci with the percentage of 75%~100%. The genetic distance amplitude ranged between 0.242 7-0.730 8. The clustering analysis showed that 30 A. membranaceus populations could be divided into two categories, and most of them corresponded to the geographical distribution. Conclusion: ISSR-PCR reaction system for A. membranaceus is stable and reliable. Wild resources of A. membranaceus in Inner Mongolia have higher genetic diversity. The genetic relationship of the populations is correlated with its geographic location.

miRNA library and thermodynamic stability of Astragali Radix decoction pieces / 中草药

Objective: To establish a small RNA library of Astragalus membranaceus, in order to understand the intake and absorption of miRNAs in human body, and to lay the foundation for pharmacological research of Astragali Radix decoction pieces (ARP). Methods: The decoction of A. membranaceus after desiccation, high temperature and microwave treatment was used as sample, and using high-throughput sequencing technology, the unknown miRNA sequence was obtained. Results: Totally 9931049 small RNA sequences were identified from ARP by the high-throughput sequencing technology. After compared with all hairpin-forming precursors in miRBase 21 database, the miRNA library of ARP decoction was established, and one conserved miRNA and 9 novel miRNA sequences were confirmed. Twelve class I small interfering RNAs (siRNAs) were obtained by further bioinformatic analysis and we found that the 5' terminal was unstable while the 3' terminal was stable thermodynamically. Conclusion: The miRNAs expression profiles of ARP is first revealed by thermodynamic treatment. Our study suggests that class I siRNA can be used as a potential biochemistry components of phytomedicine, which might contribute to the epigenetic studies on Chinese materia medica.

Comparative Study on Determination of Astragaloside, AstragalosideⅠ, AstragalosideⅡ and AstragalosideⅢ in Mongolia Radix Astragali from Various Habitats / 世界科学技术-中医药现代化

The HPLC-ELSD method was used in the content determination of astragaloside, astragalosideⅠ, astragalosideⅡand astragalosideⅢ in Mongolia Radix Astragali (Astragalus membranaceus(Fisch.) Bge. var. mongholicus(Bge.) Hsiao) among 16 batches from various habitats. The DIKMA Diamonsil C18 (150 mm× 4.6 mm, 5μm) was adopted with acetonitrile and water as the mobile phase at a gradient mode program. The flow rate was 1.0 mL·min-1. And the column temperature was 30℃. The ELSD detector parameters were the drift tube temperature at 90℃, and the air flow rate of 2.8 L·min-1. The SPSS 16.0 software was used in the cluster analysis of content determination. The results showed that when the injection volume was within the range of 0.093 2-1.02μg (r = 0.999 5), 0.789-8.78μg (r = 0.999 7), 0.506-3.13μg (r = 0.999 6), and 0.016 1-1.38μg (r = 0.999 2) for astragaloside, astragalosideⅠ, astragalosideⅡ and astragalosideⅢ, respectively, the average recoveries were 97.55%, 98.61%, 99.68%, 98.58%with RSD of 1.2%, 1.3%, 1.3%, 1.2%, respectively. The results of cluster analysis showed that the single using of astragaloside as index was unable to differentiate Mongolia Radix Astragali from various habitats. However, the simultaneous determination of 4 types of astragalosides as indexes can differentiate Mongolia Radix Astragali from various habitats. It was concluded that the method was simple, quick and accurate, which can directly reflect the quality status of Mongolia Radix Astragali from different origins. It also provided new ideas for the quality control of Mongolia Radix Astragali.