| Objective:①To compare the HPLC-ELSD information of flavonoids, saponin,calycosin and formononetin content and total flavonoid content in different growth years Astragalus membranaceus,as its cultivation and harvesting reference.②To purify APS in annual Astragalus membranaceus,discuss it's effect on the release of NO in mice of RAW264.7.③To observe the effects of micro-milling of the annual Astragalu (MMAA) on the immunosuppression and nephrotoxicity induced by cyclophosphamide(CTX)in mice.Methods:①The saponin in Astragalus membranaceus and the content of calycosin and formononetin compared by HPLC-ELSD. Column is Agilent Zorbax SB-C18 column [(250×4.6) mm,5um, Thermo],mobile phase is acetonitrile-water, gradient elution 90 minutes,flow rate is 0.8 mL-min,-1the elution time is 90 min,Column temperature is 30℃,Detector parameters:Drift tube temperature is 105℃.Carrier gas pressure is 2.70 bar;Saponins in one, two and three yearth Astragalus membranaceus is compared by TLC. Use chloroform-methanol-water (13:6:2) as the agent,10% sulfuric acid in ethanol as color agent;the flavonoids in Astragalus membranaceus and the content of calycosin and formononetin is compared by HPLC-DAD. The column is BDS Hypersil C18 column [(250×4.6) mm,5um, Thermo], diode array detector (DAD), the mobile phase is acetonitrile-water, gradient elution for 50 minutes, flow rate is 1.0 mL-min-1, the detection wavelength is 254 nm. Flavonoids in Astragalus membranaceus is compared by TLC. use toluene-ethyl acetate-formic acid (8:5:1) solution as the agent,the second-benzaldehyde 10%sulfuric acid in ethanol as color agent. Colorimetric technique is used to determine the total flavonoid content. Phenol-sulfuric acid method was used to determine and compare the diferent polysaccharides in diferent-year-old Astragalus;Water extraction,alcohol precipitation,dialysis and Sephadex column method is used to purify polysaccharide,the molecular weight is determined by gel chromatography②Griess method is used to compare the effect of diferente concentrations of APS on the release of NO mouse macrophage RAW264.7.③The Kunming mice were randomly divided into four groups, they are control group,model group,large dose and small dose group of MMAA.The mice in small and large dose groups of MMAA were intragastrically administrated of 2,4 g-kg-1 of MMAA for 11 days,and in normal control and model groups were administrated with the same volume water. On the 5 th and 10 th day,the mice in all groups except control group were injected with 80 mg-kg-1 of CTX,the same volume of physiological saline is injected in control group.On the 11th day,the weights of immune organs,white blood cell counts (WBC) and the blood urea nitrogen (BUN) were detected.Kunming mice were processed as the same methods above.On the. 3rd,6th and 9th day,the mice in all groups except control group were intraperitoneally injected wiht CTX 80 mg-kg-1,same voume physiological saline in control group.On the 10 th day,the MDA levels in renal tissue was detected.Results:①There are twelve main common peaks in the fingerprint of saponin in different growth years Astragalus membranaceus,the RSD of the relative retention time of peaks are between 0.112-0.394%(less than 3%), while the RSD the total relative peak area of peak are between 27.23-143.23%(all greater than 3%). TLC profiles are basically the same map, the position where Rf are same face the same spot, but the spot size is different.There are seven main common peaks in the finger print of flavonoids in different growth years Astragalus membranaceus,the RSD of the relative retention time of peaks are between 0.008-0.097%(less than 3%),while the RSD the total relative peak area of peak are between 9.61-72.95%(all greater than 3%).TLC profiles are basically the same map, the position where Rf are same face the same spot, but the spot size is different;The total flavonoids are 1.02,1.23 and 1.79 mg.g-1, respectively. Calycosins are 0.337,0.121 and 0.119 mg.g-1,formononetins are 0.101,0.031 and 0.032μg.g-1.Polysaccharides:42.31,23.96 and 32.52 g-kg-1;3 different components are isolated, the weight-average molecular weights of APS40%Ⅰ,ASP60%IⅠand ASP 60%Ⅱwere 3.0×105,1.5×105And 0.45×105.②The results showed that Astragalus Polysaccharides of ASP40%Ⅰ, ASP60%Ⅰand ASP60%Ⅱcould stimulated RAW 264.7 cells secrete NO among 6.25-200,3.125～200 and 3.125～200μg.mL-1.③Compared with normal control group,in immunosuppressive mice,the weight of thymus and spleen,the WBC were decreased significantly.Compared with model group,the thymus weights and the WBC of mice in small and large dosage groups of MMAA were increased and the renal tissue MDA content markedly decreased,and the blood urea nitrogen content were significantly decreased in large dosage group of M MAA.Conclusion:①saponin types In the annual is consistent to the two and three year-old Astragalus membranaceus, but there is an obvious difference in the quality of single saponins. The content of flavonoid in Astragalus membranaceus is increased with the increase of growth period, the types of flavonoids remain in coincidence. In the TLC,saponins and flavonoids of Annual Astragalus membranaceus are basically the same in color and shape as two or three-year Astragalus membranaceus,but the spots vary in size.②APS ASP40%Ⅰ, ASP60%Ⅰand ASP60%Ⅱcould stimulate RAW 264.7 cells to release NO,the stronger the smaller the molecular weight; in small doses, there is a positive correlation brtween the role of APS and the concentration.③MMAA has the effects of improving immune and antagonizing renal toxicity caused by cyclophosphamide in mice. |
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