| Background:Hylomecon japonica（Thunb.）Prantl ＆ Kündig is a perennial herb of the Hylomecon Japonica in Papaveraceae.Mainly distributed in northeast and North China and other areas with an altitude of 300-1800 meters.Hylomecon japonica use rhizome as medicine,can be picked all year round.It has the functions of dispelling rheumatism,eliminating stasis subdue swelling,stopping bleeding and pain.The whole plant contains a variety of biological active ingredients,including alkaloids,phenols,terpenoids,flavonoids,saponins,with antibacterial,anti-inflammatory,anti-cancer and other effects.Objective:1.To establish a method for the content determination of total saponins（THS）and Hylomeconoside A in Hylomecon japonica.2.To optimize THS extraction process and establish HPLC fingerprint of THS.3.To investigate the optimal purification method of THS including purification process and selection of macroporous resin.Methods:1.Use Hylomeconoside A as the standard to configure the reference solution.Analyze the reference substance by colorimetry（Take 0.3ml of the reference solution and volatile completely,add 0.2ml of 8% vanillin-ethanol solution and 5ml of 72% sulfuric acid,shake well,water bath at 90℃ for 15-20 min,measure the absorbance at 510 nm.）and HPLC（A: acetonitrile,B: 0.1% formic acid aqueous solution,peak area was measured at 206nm）.Establish regression equation and validate the methodology.2.The ultrasonic extraction process of total saponins was optimized by four factors:extraction solvent,extraction time,extraction times and solid-liquid ratio.A total of 11 batches of THS were extracted.Establish HPLC fingerprint spectrum and conduct similarity evaluation.3.The optimal type of macroporous resin was determined by static adsorption test,static desorption test and static adsorption curve.Single factor test and Box-Behnken design were used to explore the process parameters of THS purification by macroporous resin.Results:1.The regression equation of Hylomeconoside A measured by colorimetry was y=0.9353X-0.0284,R2=0.9962.The regression equation of Hylomeconoside A measured by HPLC was y=2,782,194.84x+16,522.80,R~2 = 0.999.Both of them have significant regression effect and good linearity.2.The optimal extraction conditions were as follows: 10 g of dried whole grass drenched in 15 times of volume 50% ethanol solution ultrasonic extraction 3 times,1.5h each time.The similarity of fingerprint of total saponins in 11 batches were all greater than0.9.The Contrast fingerprint was obtained by "Similarity evaluation system for chromatographic fingerprint of TCM（2012）".3.Among the seven macroporous resins,NKA-9 resin had the best adsorption and purification effect of total saponins.The optimal condition for the purification of total saponins was obtained,which were: the concentration of sample was 50mg/ m L,the flow rate was 1.9 m L /min,the volume was 1BV,the elution solvent was 50% ethanol,the flow rate was 4.3 m L /min,the volume was 7BV.Conclusion:1.The content determination method of THS and Hylomeconoside A in Hylomecon japonica obtained in the experiment conforms to the standard of Chinese Pharmacopeias and can be used for the quantitative analysis of saponins.2.The HPLC fingerprint of THS showed good similarity,which provided reference for the follow-up study of Hylomecon japonica.3.The purification effect of THS by NKA-9 macroporous resin was good,and the optimized process was stable and feasible. |
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