Studies On The Bio-synthesis Technology Of Arbutin By Scutellaria Baicalensis Georgi Hairy Roots

Studies on the culture system of S. baicalensis Georgi hairy rootsshowed that, S.baicalensis Georgi hairy roots could synthesize thesecondary metabolites baicalin of S. baicalensis Georgi effectively. Onthe basis, this research of synthesis technology of arbutin which wasbio-transformed by hydroquinone through Scutellaria baicalensis Georgihairy roots was carried out.Studies showed that hairy roots of Scutellariabaicalensis Georgi could synthesize arbutin by biological conversion ofhydroquinone, which provided new ideas and methods for the synthesisof arbutin. The major results and conclusions were as follows:1.Methodological research about the analysis of arbutin andhydroquinone from S. baicalensis Georgi hairy roots: HPLC conditionsfor the determination of arbutin content in the hairy roots: waters-2487,Symmetry-C₁₈column （250mm×46mm,5μm）, the flow rate was0.8mL/min, the mobile phase was methanol-water （15:85, v/v）, wavelengthfor detection was287nm, column temperature was25℃, and the injectionvolume was15μL. Under those conditions, the retention time of arbutinwas5.4min.When the concentration of arbutin was between100μg/mLand200μg/mL, there was a good linear relationship （r=0.9991）,and RSDof precision was1.52%, RSD of stability was2.60%.The averagerecovery was98.56%¹00.29%, RSD0.83%. The retention time ofhydroquinone was9.6min. When the concentration of hydroquinone wasbetween0.4μg/mL and1.4μg/mL, there was a good linear relationship （r=0.9991）.2.Researches about the conversion conditions: the medium, theconversion time, and the amount of substrate were explored. When themedium contained improved MS,50g/L sucrose,10g/L glucose, and0.4 mmol/L phenylalanine.0.5g of hairy roots were inoculated in per100mLof medium, cultured for20days.1mL solution of hydroquinone whoseconcentration was20mg/mL was added into per100mL medium, andconversed for40h. At last, content of arbutin was4.43%（Dry weight ofthe hairy roots） and the conversion rate of hydroquinone was55.6%. Asto the S.baicalensis Georgi hairy roots, the biggest tolerant concentrationof hydroquinone was0.2g/L.3. Researches about the impact of ethanol and surfactant on thetransformation of the S.baicalensis Georgi hairy roots: studies of theimpact of ethanol and Tween-80on the transformation showed thatethanol did not promote the synthesis of arbutin, but Tween-80played agood role in hydroquinone passing through the cell wall. When thesubstrate was added,0.1g Tween-80was also added into per100mLmedium at the same time. The content of arbutin and the conversion rateof hydroquinone was5.45%and85.2%respectively. When Tween-80was added, the conversion rate of hydroquinone was22.6%higher.4. Researches about extraction, separation and purification processof arbutin:1)Extraction: extracted by water for4h,1g materials were extractedwith20mL water. The extract was double concentrated, filtrated, and thesample of arbutin was abtained, whose concentration was1g/L.2)Macroporous resin impurity: D101resin column was used,according to1mL concentrated extract per1g resin. Eluted by4BV purewater, and the elution velocity was2BV/h. The part contained arbutinwas collected.3)Silica gel column refined: the column of400mesh silica gel. Thesolution chloroform: methanol （9:1,8:2, v/v） was chosen to elute, and theelution velocity was0.2mL/min. the part contained arbutin was collected,crystallized by the solution chloroform: methanol （8:2, v/v）, finally thepure arbutin could be obtained.5. Sample identification:1H-NMR spectra of samples,δ：3.22-3.76（m,6H）,4.80（1H,d,J=7.41）,6.69（2H,d,J=5.88and2.24Hz）,6.87（2H,d,J=5.85and3.69Hz）,4.70（OH）. The above data is consistentwith those reported, so it will be identified as arbutin.