Study On The Endogenous Enzymatic-assisted Extraction,Separation And Purification Of Baicalein And Wogonin From Radix Scutellariae

In the present study, the endogenous-baicalinase catalyzed-hydrolysis (EBCH) of baicalin combined with negative pressure cavitation (NPC) system has been researched to evaluate the effect of cavitation on the hydrolysis and extraction efficiency by comparing their reaction kinetic and mass transfer kinetic models, respectively. The effects of the main influential factors, i.e. vacuum degree, liquid/solid ratio, the value of pH, ethanol concentration, extraction cycles on the extraction yields were evaluated. Preparative separation and purification of the hydrlysis procucts baicalein and wogonin from Scutellariae Radix extracts was carried out by liquid liquid extraction, low-pressure preparative silica gel column chromatography, low temperature crystallization and recrystallization. The optimum process of hydrlysis, extraction, separation and purification of baicaliein and wogonin were established and the HPLC method for determination and quantification of the target compounds was developed, which provides the scientific basis for the rational and efficient use of Scutellariae radix resources.1. Determination of baicalin, wogonoside, baicalein and wogonin by HPLC-UVChromatographic conditions:Agilent1100series HPLC system; column:Agilent Eclipse HiQ Sil C18column (250mm×4.6mm i.d.,5μm) and a MercuryMS Guard cartridges (10×4.0mm i.d.,5μm); column temperature:30℃; detection wavelength:276nm; mobile phase:0.05%formic acid aqueous solution (A)-acetonitrile (B); gradient elution:0-3min20%(B),3-15min20-45%(B),15-20min45-60%(B),3min60%(B); injection volume:10μL; flow rate:1mL/min.2. The endogenous-baicalinase catalyzed-hydrolysis (EBCH) of baicalin and wogonoside in Radix ScutellariaeThe effects of cavitation systems on the hydrolysis efficiency was studied by using reaction kinetic models, and the optimum conditions of EBCH of baicalin and wogonoside combined with NPC were as follows:Vacuum degree:-0.08MPaLiquid/solid ratio:20:1mL/gTemperature:50℃Time:60minpH:5The results of reaction kinetic showed that the NPC improve the hydrolysis reaction rate as three times fast as that in the conventional procedure. Under the optimum conditions, baicalin and wogonoside were transformed into baicalein and wogonin, and the transformation ratio reached98.21%and96.60%at50℃for60min, respectively. Hence, baicalein and wogonin increased from17.974±0.304and6.047±0.752mg/g to98.255±2.052and23.286 ±0.945mg/g, which were about4.47-fold and2.86-fold higher than those of the raw sample without enzymatic hydrolysis, respectively3. Negative pressure cavitation extraction of the hydrolysate baicalein and wogoninThe optimum parameters of NPC extraction of baicalein and wogonin were as follows:Extraction solvent:80%(v/v) aqueous ethanolVacuum degree:-0.08MPaLiquid/solid ratio:20:1mL/gExtraction time:20minExtraction cycle:3The yields of baicalein and wogonin were reached to98.255and23.286mg/g at the optimum conditions described above, proving that NPC was an efficient method to improve the extraction efficiency by accelerating the mass transfer.4. Separation and purification of baicalein(B) and wogonin(W)Preparative separation and purification of B and W was performed by liquid liquid extraction, low-pressure preparative silica gel column chromatography, low temperature crystallization and recrystallization to obtain the crystals of B and W. After a series of separation and purification, the total yields of baicalein and wogonin with the purity of over98%were59mg/g, and14mg/g, respectively.