| Aconitum carmichaeli Debx is belong to the buttercup family, monkshood genera, It has the effect of warming the spleen and kidney, it also can dispel the coldness, In clinical, it is used in treating the various pains, coronary heart disease and the shock.In this study, the subject of the research is the fibrous roots of Aconitum carmichaeli Debx, seven chemical constituents have been isolated and identified, and then the contents of the two alkaloids were determined.1. The extraction, separation, purification, structural identification of the chemical constituentsIn this sduty, seven compounds have been separated from the fibrous roots of Aconitum carmichaeli Debx, They were identified by various spectral approaches such as IR,MS,NMR as songoramine (A), songorine (B), neoline (C), aconitine (D), hypaconitine (E), sitosterol (F), daucosterol (G). They were all isolated from the fibrous roots of Aconitum carmichaeli Debx for the first time.1.1 Extraction50 kg dry fibrous roots of Aconitum carmichaeli Debx, mixed with 95% ethanol were extracted by hot reflux for three times,3 kg extract was obtained. The extract was concentrated, acidated, ethyl acetate extracted, alkalized, ethyl acetate extracted, as a result, two part extracts were obtained.1.2 Isolation and purificationThe two part extraction were column chromatographied by silica gel and recrystallizated repeatedly, Compound A-G were obtained.1.3 Structural identificationAccording to their physical and chemical properties and their data of spectrum, and comparing to the literatures, the chemical structures of all compounds were identified as songoramine (A), songorine (B), neoline (C), aconitine (D), hypaconitine (E), sitosterol (F), daucosterol (G).2 The determination of Songoramine (A) and Songorine (B)2.1 Instrument, reagents and reference substancesChromatograph:Agilent 1200 HPLCColumn:Extend-C18 (ODS,4.6×250mm 5μm)Detector:V WD Ultraviolet detectorBalance:Sartorius YDPOZ-ODReagents:methanol (HPLCgrade), acetonitrile (HPLCgrade), chloroform, ethyl acetate, petroleum ether, triethylamine, distilled water twice.Reference substance:compound A (97.5%), compound B (98.2%)2.2 Methodology investigation1) Chromatographic conditionsColumn:Extend-C18 (4.6×250mm ODS,5μm)Temperature:25℃Mobile phase:methanol-0.1% triethylamine water (60:40)Flow rate:1.0mL.min-1Wavelength:220 nm2) The Linear testIn the range of 0.1～10.0μg, the linearity of compound A and compound B were good. The regression equations of A and B were S=385.73 m+7.5019 (r=0.9999) and S= 477.8 m+45.813 (r=0.9999) respectively.3) The LOD and LOQThe LOD of compound A was 0.002μg, and the LOQ of compound A was 0.05μg;The LOD of compound B was 0.008μg, and the LOQ of compound B was 0.04μg.4) Precision testThe same sample was tested 6 times in parallel. The RSD of peak area was 0.86% and 0.65% respectively.5) Repeatability testAccording to the determination results 6 times, the RSD of the content of compound A was 1.48%; The RSD of the content of compound B was 1.15%.6) Stability testThe same sample was determinated once every 2 hours within 10 hours. The RSD of retention time and the peak area were 1.67% and 0.40% respectively for compound A, and were 1.02% and 0.46% respectively for compound B. The results showed that compound A and compound B have good stability within 10 hours.7) Recoveries testThe results showed the average recoveries of compound A and compound B were 99.75% (RSD1.49%)and 99.27% (RSD 1.46%) respectively.2.3 The contents determinationThe compound A and compound B of the fibrous roots of Aconitum carmichaeli Debx was 0.057 mg.g-1 and 0.11 mg.g-1 respectively.The results of the paper can provide a science basis for the further research to the fibrous roots of Aconitum carmichaeli Debx. |
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