Pharmaceutical Research Of Huannao Yicong Fang And Preparative Isolation And Purification Of Effective Components From Rhizoma Chuanxiong

In this article, the extraction techniques of Huannao Yicong Fang, Chinese medicine compound prescription containing five herbs which are Panax ginseng, Polygonum multiflorum, Grass-leaved sweetflag, Coptis root and Rhizoma Chuanxiong were investigated, including determination, the optimum solution in extraction, separation and purification. Additional, the method to separate three active compounds senkyunolide-Ⅰ, senkyunolide-H and ferulic acid from Rhizoma Chuanxiong by high speed counter-current chromatography (HSCCC) was also studied.By the results of single and orthogonal experiments, the optimum extraction condition of Huannao Yicong Fang is as follows:80% ethanol, solid to liquid ratio is 1:20, the reflux time is 90min,3 times. To study the optimal technology for the purification of total flavonoids from Huannao Yicong Fang, AB-8 resin was chosen for the separation of total flavonoids because of its higher adsorption capacity and desorption ratio than that of other resins. The highest adsorption capacity of AB-8 was achieved when initial concentration, feed flow rate, feed pH and feed volume were 5.13 mg·mL-1,3.35 mL·min-1, pH 3.3 and 10 bed volumes respectively. The saturated resin was first washed with 5 BV of water to remove impurities, and then a purified product was obtained by eluting the resin with 6 BV of 70%(v/v) aqueous ethanol. The optimal diameter length ratio was 1:5 and AB-8 resin could be reused for 5 times.High-speed countercurrent chromatography (HSCCC), being a support-free liquid-liquid partition method, eliminates irreversible adsorption of sample onto the solid support, and has been widely used in preparative separation of natural products. Three active compounds senkyunolide-Ⅰ, senkyunolide-H and ferulic acid were successfully isolated and purified from the extracts of Rhizoma Chuanxiong by high-speed counter-current chromatography (HSCCC). Based on the principle of the partition coefficient values (k) for target compounds, an optimized volume ratio of 3:7:4:6 (v/v) was selected for the HSCCC separation. In a single run,400mg of the crude extract yielded pure senkyunolide-I (6.4 mg), senkyunolide-H (1.7 mg), and ferulic acid (4.4 mg) with the purities of 98%,93% and 99%, respectively. The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three active compounds were identified by MS and 1H NMR.