The effects of water regulation on the biosynthesis of calycosin-7-O-β-D-glucoside in 2-year-old Astragalus membranaceus var. mongholicus were studied,and the mechanism was explained from the aspects of key enzyme gene expression and antioxidant enzyme system. The content of calycosin-7-O-β-D-glucoside was determined by HPLC,and the expression levels of six key enzyme genes( PAL,4 CL,CHS,CHI,IFS,13'H) in the synthesis pathway were analyzed by q RT-PCR. The activities of protective enzymes and contents of osmoregulation substances and malondialdehyde were also determined. In the water deficit group,the maximum concentration of calycosin-7-O-β-D-glucoside was 0. 49 mg·g-1 on the 24 th day of treatment. In the whole water regulation,the water deficit group outweighed the water adequate group in osmoregulation substance and MDA contents. The activities of A. membranaceus var.mongholicus antioxidant enzymes SOD,POD,and CAT increased during the initial period of water regulation,but decreased with time.The expression of PAL,CHS,and 13'H in the water deficit group was at a low level,and the 4 CL had active expression,slightly lower than that in the water adequate group. The expression of CHI and IFS elevated rapidly when water deficit occurred. Correlation analysis showed that the content of calycosin-7-O-β-D-glucoside was positively correlated with CHI expression( P<0. 01) and IFS expression( P<0. 05). Therefore,water regulation can change the accumulation pattern of calycosin-7-O-β-D-glucoside,and water deficit may be an effective way to increase its content. CHI and IFS are the key genes in response to water deficit.
Astragalus propinquus/genetics* ; Biosynthetic Pathways ; Glucosides ; Isoflavones ; Water

To identify and verify the active ingredients from Astragalus membranaceus on hypertensive cardiac remodeling based on network pharmacology and heart RNA-sequencing data. The monomers of A. membranaceus and their intervention target database were established by using network pharmacology. The genes associated to cardiac remodeling were then screened by analyzing cardiac RNA-sequencing data. An overlap between genes related to cardiac remodeling and targets of ingredients form A. membranaceus was collected to obtain monomers with protective effect on hypertensive cardiac remodeling. Angiotensin Ⅱ(AngⅡ)-induced mouse cardiac remodeling model was used to validate the protective effect of active ingredients from A. membranaceus on hypertensive cardiac remodeling. Finally, a total of 81 monomers and 1 197 targets were enrolled in our database. Mouse RNA-sequencing data showed that 983 genes were significantly up-regulated and 465 genes were down-regulation in myocardial tissues of the cardiac remodeling mice as compared with blank group mice, respectively. Ninety-two genes were found via overlapping between genes related to cardiac remodeling and targets, involving 59 monomers from A. membranaceus. Further research found that vanillic acid(VA) could intervene 27 genes associated with hypertensive cardiac remodeling, ranking top 1. Meanwhile, VA could significantly inhibit AngⅡ-induced increase in ratio of heart weight to body weight and heart weight to tibial length, ANP and BNP mRNA levels in myocardial tissues, myocardial tissue damage, cardiac fibrosis level and cardiac hypertrophy level in vivo. Those results showed that network pharmacology screen-based VA has protective effect on AngⅡ-induced cardiac remodeling.
Angiotensin II ; Animals ; Astragalus propinquus/chemistry* ; Heart ; Hypertension/genetics* ; Mice ; Protective Agents/pharmacology* ; Vanillic Acid/pharmacology* ; Ventricular Remodeling/genetics*

Astragalus membranaceus (Radix Astragali, RA) and Atractylodes macrocephala (Rhizoma Atractylodis Macrocephalae, RAM) are often used to treat gastrointestinal diseases. In the present study, we determined the effects of polysaccharides extracts from these two herbs on IEC-6 cell migration and explored the potential underlying mechanisms. A migration model with IEC-6 cells was induced using a single-edged razor blade along the diameter of cell layers in six-well polystyrene plates. The cells were grown in control media or media containing spermidine (5 μmol·L, SPD), alpha-difluoromethylornithine (2.5 mmol·L, DFMO), 4-Aminopyridine (40 μmol·L, 4-AP), the polysaccharide extracts of RA or RAM (50, 100, or 200 mg·L), DFMO plus SPD, or DFMO plus polysaccharide extracts of RA or RAM for 12 or 24 h. Next, cytosolic free Ca ([Ca]) was measured using laser confocal microscopy, and cellular polyamine content was quantified with HPLC. Kv1.1 mRNA expression was assessed using RT-qPCR and Kv1.1 and RhoA protein expressions were measured with Western blotting analysis. A cell migration assay was carried out using Image-Pro Plus software. In addition, GC-MS was introduced to analyze the monosaccharide composition of both polysaccharide extracts. The resutls showed that treatment with polysaccharide extracts of RA or RAM significantly increased cellular polyamine content, elevated [Ca] and accelerated migration of IEC-6 cells, compared with the controls (P < 0.01). Polysaccharide extracts not only reversed the inhibitory effects of DFMO on cellular polyamine content and [Ca], but also restored IEC-6 cell migration to control level (P < 0.01 or < 0.05). Kv1.1 mRNA and protein expressions were increased (P < 0.05) after polysaccharide extract treatment in polyamine-deficient IEC-6 cells and RhoA protein expression was increased. Molar ratios of D-ribose, D-arabinose, L-rhamnose, D-mannose, D-glucose, and D-galactose was 1.0 : 14.1 : 0.3 : 19.9 : 181.3 : 6.3 in RA and 1.0 : 4.3 : 0.1 : 5.7 : 2.8 : 2.2 in RAM. In conclusion, treatment with RA and RAM polysaccharide extracts stimulated migration of intestinal epithelial cells via a polyamine-Kv1.1 channel activated signaling pathway, which facilitated intestinal injury healing.
Animals ; Astragalus propinquus ; chemistry ; Atractylodes ; chemistry ; Cell Line ; Cell Movement ; drug effects ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Intestines ; cytology ; drug effects ; Kv1.1 Potassium Channel ; genetics ; metabolism ; Polyamines ; metabolism ; Polysaccharides ; chemistry ; isolation & purification ; pharmacology ; Rats ; Rhizome ; chemistry ; Signal Transduction ; drug effects ; rhoA GTP-Binding Protein ; metabolism