ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
OBJECTIVE: To study the chemical constituents of roots of Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao. METHODS: The chemical constituents of Astragalus membranaceus (Fisch) Bge. var. mongholicus (Bge) Hsiao were isolated and purified by D101 column chromatography and preparation HPLC. Their structures were identified by physicochemical properties and spectral analysis methods. RESULTS: Seven compounds were obtained and identified as 4, 4′-dimethyl-6′-hydroxychalcone (1), 4-methoxy-4′, 6′-dihydroxychalcone(2), 7, 4′-dihydroxyflavonone(3), 4, 4′, 6′-trihydroxychalcone(4), 4′-hydroxyflavonone-7-O-β-D-glucoside(5), 2′-hydroxy-3′, 4′-dimethylisoflavan-7-O-β-Z)-glucoside(6), and 3, 2′-dihydroxy-3′, 4′-dimethylisoflavan-7-O-β-D-glucoside (7). CONCLUSION: All these compounds were isolated from this plant for the first time.
ABSTRACT
Objective: To determine the contents of astragaloside and flavonoids in four ecotypes of A. membranaceus, which are whip pole type, taproot type, binary type, and chicken feet type, and analyze the correlation between ecotypes and quality. Methods: The contents of astragaloside and flavonoids in A. membranaceus were determined by UPLC, and the measured data were analyzed by principal component analysis (PCA) and cluster analysis (CA). Results: The order of contents of astragaloside and flavonoids in A. membranaceus was whip pole type > taproot type > binary type > chicken feet type; PCA revealed that four ecotypes of ecotypes A. membranaceus could be separated significantly; CA showed that each ecotype of A. membranaceus could be clustered into one class perfectly. Conclusion: The quality of A. membranaceus is closely associated with ecotypes. The quality merits of the order of four ecotypes of A. membranaceus is whip pole type > taproot type > binary type > chicken feet type.
ABSTRACT
Objective: To compare the chemical constituents in traditional planted and two-year-old cultivated Astragali Radix, and to discuss the chemical differences. Methods: The HPLC-ELSD fingerprint of Astragali Radix was collected, and the contents of astragaloside, calycosin-7-O-β-D-glucoside, total polysccharide, and extract were also determined. The similarity analysis, metabolomic multivariate statistical analysis, and quality comparison were performed. Results: The contents of calycosin-7-O-β-D-glucoside and total polysaccharide in traditional planted Astragali Radix were obviously higher than those in two-year-old cultivated Astragali Radix. But the contents of astragaloside and extract showed no difference. The two kinds of Astragali Radix could be distinguished by HPLC-ELSD metabolomic analysis. The differential metabolites were calycosin-7-O-β-D-glucoside, ononin, peaks 4, 5, 13, astragalosides I and II. Conclusion: HPLC-ELSD metabolomic approach combined with the content determination of index components could be used for the quality evaluation of traditional planted and two-year-old cultivated Astragali Radix.
ABSTRACT
To develop a method for the quality control of Astrgalus membranaceus (Fisch.) Bge. powder for injection (AMPI). Methods TLC-spectrophotometry was used to determine the content of astragaloside A in AMPI. The mobile phase was chloroform-methanol-water (65∶30∶10) and detected at the wave length of 418 nm. Results The regression equation within the spotted range of 80～240 μg was A=1.538 C+ 0.189, with good linearity, r=0.999 8. The average recovery rate was 98.68% and RSD=2.52%(n=5). Conclusion The method used simple and easily available instruments and the rusults obtained were reliable and specific, suitable for the quality control of AMPI.
ABSTRACT
Object An attempt to seek after an intrinsic inhibitor present in the seed of Astragalus membranaceus (Fisch.) Bge.. Methods Crude ethereal extract of the seed was prepared and treated on paper chromatography. Inhibitory effects of different fractions with different Rf value were tested on Brassica chinensis L. and wheat germination. Effect of steeping the seed in warm water at 41 ℃ or 45 ℃ for different periods of time was also studied.Results Seed of A. membranaceus does contain strong intrinsic inhibitor. The portion of its ethereal extract with Rf 0.9 showed the most strong inhibition for the germination of Brassica, and the fraction with Rf 1.0 can inhibit the growth of the tender Brassica root, steeping with warm water can remove most of the intrinsic inhibitor, which also inhibits the growth of both aerial and underground parts of wheat sprouts, but without effect on its seed germination. It also showed strong inhibition of seed germination and growth of tender root of A. membranaceus. Conclusion Besides the low water permeability of the seed peel, the intrinsic inhibitor present in A. membranaceus is another essential factor that retard its germination.
ABSTRACT
Objective: To establish the method of purifying astragaloside. Methods: Astrageloside was determined by HPLC fingerprinting to compare macroporous resin absorbing method with extraction refine by n butyl alcohol. Results: The HPLC fingerprints of each method were difference. Conclusion: AB 8 macroporous resin is better than the others for purifying astrageloside.