Extraction, Separation And Purification Of Catalpol And Stachyose From Rehmannia Glutinosa Libosch

Rehmannia glutinosa Libosch(R.glutinosa),a member of the Scrophulariaceae family,is widely used in Traditional Chinese Medicines to treat metabolic disorders. Some large-scale GAP bases for planting resource species of R.glutinosa have been established in China,which is beneficial in exploiting bioactive substances from R. glutinosa.Catalpol and stachyose are two of major bioactive compounds rich in the root of R.glutinosa,however,much concern was just focused on separation of catalpol or stachyose from the root,respectively..Therefore,it will play a vital role in improving economic benefits to obtain stachyose from the remainders of R.glutinosa extracts free of catalpol.In this study catalpol and stachyose were sequentially separated and purified from the extracts of R.glutinosa.The extraction of catalpol by using extraction at room temperature(ERT),heat reflux extraction(HRE) and ultrasound-assisted extraction(UAE)from R.glutinosa was investigated.Results showed that the extraction yield of catalpol by UAE was 83 %higher than that by ERT and 42%higher than that by HRE.Then,the optimum conditions for UAE were established:68%(v/v) ethanol aqueous solution as extraction solvent,the ratio of solvent to solid of 5:1 mL/g,the ultrasonic treatment time of 36 min.The optimal yield of catalpol was 1.95%.Then,the preparative separation of crude catalpol solution with macroporous resins was studied.The results showed that H103 resin offered the best adsorption and desorption capacity for catalpol among ten macroporous resins tested in this study, and its adsorption isotherm data were well fitted to Langmuir and Freundlich model. Dynamic adsorption and desorption experiments have been carried out with the column packed by H103 resin to optimize the separation process of catalpol from R. glutinosa extracts.The optimum parameters for adsorption and desorption were sample solution catalpol concentration 6.15 mg/mL,flow rate 1.0 mL/min, ethanol-water(70:30,v/v),elution flow rate 0.5 mL/min and elution volume 3.33 BV. Then,a purified product(Ⅰ) containing 62.39%catalpol was obtained after treatment with H103 resin.The product(Ⅰ) was further refined by silica gel and Sephadex LH-20 column chromatography,respectively.The purity of catalpol from the product(Ⅱ) after refinement by Sephadex LH-20 column exceeded 94.1%with recovery yield of 82.3%;however,the purity of catalpol reached up to 81.3%with recovery yield of 45.6%after refinement by silica gel column.The extraction of stachyose from the remainders of R.glutinosa extracts free of catalpol by a microwave-assisted process and a heat reflux process was studied.A uniform design was used to obtain the optimal conditions of microwave-assisted extraction(MAE).The optimum parametes for MAE were microwave power 528 W, microwave disposal time 60 s,ratio of solvent to solid of 20:1 mL/g and times of radiation twice.Based on the MAE conditions optimized above,the extraction yield of stachyose reached 23.24%,which was as 1.45 times as that by HRE.Then,the crude stachyose solution was pre-treated by microfiltration and separated by ultrafiltration membrane of MWCO 10 K.Da.The optimum separation by ultrafiltration was achieved under the conditions of ultrafiltration pressure 0.10 MPa, temperature 50℃,VCR 8.And the content of stachyose in retentate was increased from 28.3%to 40.5%with the decoloration ratio of 72.4%.The retentate was further decolored by active carbon,aluminium oxide and five kinds of ion exchange resins, respectively.The results showed that active carbon possessed the highest decoloration capacity among the seven adsorbents.And the optimum conditions for decoloration by active carbon were as follows:temperature 50℃,concentration of active carbon 0.3 g/L,decoloration time 2.5h and pH4.0.Finally,the decoloration ratio of retentate reached 99.1%.The stachyose solution after decoloration was concentrated,and then precipitated.The content of stachyose in precipitate reached up to 60%with recovery yield of 36.4%.The obtained precipitate was refined by active carbon column chromatography,and the purity of stachyose in final product was increased to 92% with recovery yield of 76.7%.This study will provide a potential approach for large-scale separation and purification of catalpol and stachyose for their wide pharmaceutical use.