Study On Separation And Purification Technology And Microwave Green Extraction Method Of Active Components Of Coumarin From Angelicae Pubescentis Radix

Angelicae Pubescentis Radix,as a commonly used Traditional Chinese Medicine,was first recorded in Shennong Materia Medica,and was often used to treat arthritis in clinic.Coumarins are the active components of the Angelicae Pubescentis Radix,which have a variety of pharmacological activities.Therefore,it is very important to extract and separate the active components of coumarin.Macroporous resin is a kind of adsorption materials with high adsorption performance,reusable and environment-friendly,which has been widely used in the separation and purification of pharmacologically active natural products.At the same time,microwave-assisted extraction has been used to extract natural active products in recent years because of its advantages of high efficiency,energy saving and environmental protection.In order to better develop and make use of Angelicae Pubescentis Radix,develop an efficient method to obtain the active ingredient coumarin from Angelicae Pubescentis Radix.In this paper,the extraction and separation of coumarin were studied by macroporous resin and microwave-assisted extraction.This paper was mainly carried out from the following four aspects.The HPLC-FLD fingerprint of the extract of Angelicae Pubescentis Radix was established,and the fingerprint of the extract was studied by high performance liquid chromatography?HPLC?.The gradient elution was carried out with methanol-water,and the Excitation wavelength was 325 nm,Emission wavelength was 400 nm.The similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine was used.As a result,a HPLC fingerprint detection method with good precision,stability and reproducibility was established.The similarity evaluation was carried out using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System,the result showed that the fingerprint of 10 batches of water extract showed 16 common peaks.The similarity was more than 0.86,and four common peaks were identified including columbianetin-?-D-glucopyranoside?columbianetin?angelol A and columbianetin acetate.In addition,the contents of four coumarins in the water extract were determined,methodology verification is accurate and reliable.This indicated that the established fingerprint detection method can be used for the quality control of Angelicae Pubescentis Radix water extract.A useful and economical method for the purification of columbianetin-?-D-Glucopyranoside from Angelicae Pubescentis Radix extract.Among the five different tested macroporous resins,GDX-201 resin possess best adsorption and desorption capacities for columbianetin-?-D-glucopyranoside.Moreover,the adsorption kinetic curve was well fitted to the pseudo second order equation?R²>0.99?.The liquid film diffusion model and the intra-particle diffusion model show that the rate-controlling step of the adsorption process is intra-particle diffusion.The Langmuir isotherms models?R²>0.99?could describe the whole adsorption process which were exothermic and spontaneous according to the result of thermodynamics tests.After one pretreatment,the content of columbianetin-?-D-glucopyranoside in the product was increased 29.61-fold from 0.45%to 13.32%with yield of 88.03%by an experiment of lab-scale enlargement.Then,columbianeti-?-D-glucopyranoside was further purified by PHPLC and its purity was more than 98%.Additionally,the analgesic activity of the columbianetin-?-D-glucopyranoside was assessed by hot plate test.The result indicated that the columbianetin-?-D-glucopyranoside was obviously increased the latency to pain response in hot plate test.This study demonstrated that Columbianetin-?-D-glucopyranoside could be used as potentially natural analgesic ingredients in pharmaceutical products.An efficient method was established by combining macroporous resins?MARs?with preparative high performance liquid chromatography?PHPLC?to enrich and separate three coumarins?columbianetin acetate,osthole and columbianadin?from Angelicae Pubescentis Radix?APR?.Five different macroporous resins?D101,AB-8,DA-201,HP-20 and GDX-201?were used to assess the adsorption and desorption characteristics of three coumarins.The result demonstrated that HP-20 resin possessed the best adsorption and desorption capacities for these three coumarins.Moreover,the adsorption dynamics profiles of three coumarins were well fitted to the pseudo second order equation?R²>0.99?for the HP-20 resin.The adsorption process was described by the Freundlich model?R²>0.99,0.2748?1/n?0.3103?.The contents of columbianetin acetate,osthole and columbianadin in the product were increased 10.81-fold,19.98-fold and 19.89-fold after enrichment,respectively.Three coumarins were further purified by PHPLC and the purities of them reached above 98%.It was concluded that the new method combining MARs with PHPLC was efficient and economical for enlarging scale separation and enrichment of columbianetin acetate,osthole and columbianadin with anti-inflammatory effect from the APR extract.A green and quick ionic liquid coupled with microwave assisted extraction method?IL-MAE?was established to simultaneously extract six coumarins from Angelicae pubescentis Radix.Among the eight tested ionic liquid,The 100 m M of[C₁₂MIM]Br showed the best extraction efficiency.Based on the single factor experiment results,the effect of three parameters,extraction time?2-4 min?,microwave power?200-400 W?,and amount of drug?50-150 mg?on Total coumarins content were investigated and optimised using a Box Behnken design?BBD?.The optimal extraction conditions as follows:300 W microwave radiation power,3.3 min microwave radiation time,100 mg amount of drug.Besides,the extraction kinetic curves of total coumarins showed good linear correlation?R²>0.90?based on the first-order diffusion kinetic model.The thermodynamic parameters of Gibbs free energy change??G<0?and enthalpy free energy??H>0?revealed that the IL-MAE process was endothermic and spontaneous.In this study,the technological parameters of IL-MAE extraction of coumarins from Angelicae Pubescentis Radix were optimized and the possible extraction mechanism of the extraction process was discussed for the first time.