Extraction And Separation Of Effective Components Of Scutellariae Radix With Aqueous Two-phase System

Scutellariae Radix has pharmacological effects such as antibacterial,anti-oxidation,anti-tumor,neuroprotective and immunological functions,and is used as a variety of compound or chinese patent medicine ingredients.The main active ingredients are flavonoids,terpenoids,volatile oil,etc.The effective extraction of baicalin or saccharides from Scutellariae Radix has become a hotspot in recent years.The simultaneous extraction and separation of these two components has advantages in terms of energy and cost.The aqueous two-phase system composed of hydrophilic small organic solvent and inorganic salt has the advantages of high water content,low amount of organic solvent,high water content,biocompatibility,low cost,green environmental protection,etc,and fast phase separation and good selectivity.It is of great significance to develop a new liquid-liquid extraction system for the effective extraction and separation of baicalin,saccharides and other active ingredients in Scutellariae Radix.In this paper,two aqueous phase systems（ATPSs）ethanol-ammonium sulfate-water（EtSO）and ethanol-co-solvent-ammonium sulfate-water（EtSO-co-solvent）were used to efficiently extract baicalin and saccharides.Factors such as p H,ammonium sulfate concentration,ethanol volume fraction,co-solvent components and volume fraction,extraction time,extraction temperature and other factors were optimized,and the optimized system was compared with the traditional ethanol extraction method;then the thermodynamic behavior of baicalin distribution in cosolvent system was discussed;through dialysis,explored whether the saccharides extracted with ATPS had a similar molecular weight distribution as the saccharides extracted from other systems;finally,after extraction with the EtSO-MtOH system,the residue was removed,and baicalin and saccharides were redistributed by adding water,ammonium sulfate and ethyl acetate to improve the distribution coefficient to increase the partition coefficient.The results show that:（1）Baicalin was mainly distributed in the top phase,and saccharides was mainly distributed in the bottom phase.In ethanol-ammonium sulfate-water ATPS,when the salt concentration was 0.250 g/m L,p H was 4.00,ethanol volume fraction was 35.0%,shook at constant temperature water bath at 48℃for 40.0 min,the total output（Y₁’）was 21.4%higher than that of baicalin in the extraction system of 70%ethanol（ethanol dosage was 14.0 m L,water 6.00 m L）.Compared with the saccharides system extracted with water（20.0 m L）,the saccharides yield（Y₂’）was 5.34%lower.（2）In the co-solvent system,under the optimal conditions,the amount of total baicalin extracted Y₁’in ethanol-methanol-ammonium sulfate-water（EtSO-MtOH）and ethanol-acetone-ammonium sulfate-water（EtSO-Ac）aqueous phases than the traditional 70%（v/v）ethanol system increased by 24.8%and 26.2%,while the baicalin production in ethanol-ethyl-acetate-ammonium sulfate-water（EtSO-EA）was declining;All EtSO,EtSO-MtOH,EtSO-Ac and EtSO-EA displayed similar saccharides yield,but far higher than that with ethanol system（70%,v/v）.Comprehensive comparison,among the three co-solvent systems,EtSO-MtOH ATPS was the better system for extracting and separating baicalin and saccharides.However,this system had a better baicalin partition coefficient（K₁）,but the saccharides partition coefficient was poor（K₂）.（3）In the redistribution experiment,we tried to increase the partition coefficient of saccharides and concentrated the extraction amount of saccharides in the bottom phase.Therefore,the combination of ammonium sulfate,ethyl acetate and water was added to EtSO-MtOH ATPS for verification.It was proved that when the EA volume fraction was 8.33%,the water concentration was 2.00 m L and the ammonium sulfate concentration was 0.327g/m L in the system,baicalin and saccharides were well distributed,the distribution coefficients were 30.6 and 4.45,respectively.At this time,25.5%of the saccharides were distributed in the top phase,which meant that 74.5%of the saccharides were distributed in the bottom phase,and the advantage of simultaneously extracting and separating different chemicals in the two aqueous phase has been realized.（4）Three cosolvent two aqueous phase systems（EtSO,EtSO-Ac,EtSO-MtOH）were studied to investigate the thermodynamic behavior of baicalin partitioning between the two phases.It was found that theΔG value of each system was negative,and the negative value of the EtSO-MtOH system was the largest.Further illustrated the effect of extraction of baicalin in the improved system was good,and baicalin was relatively spontaneously distributed from rich salt to the organic phase.（5）Using EtSO and EtSO-MtOH as the ATPS model,the two-phase extracts were dialyzed to remove carbohydrates with small molecular weight（<2000 Da or<7000 Da）,and the saccharides captured in the dialysis bag were quantitatively analyzed.It was found that more than 90%of the saccharides in the top and bottom phase solutions extracted by the two-aqueous phase had a molecular weight less than 2000 Da.It also proved that high molecular weight saccharides were easily distributed in the weak polar phase,while low molecular weight saccharides tended to be distributed in the high polar phase.The results show that the saccharides extracted with ATPS has a molecular weight distribution similar to that extracted by other systems.（6）After subsequent macroporous resin and silica gel column chromatography purification,two less polar compounds were isolated,namely oroxylin A and wogonin.It is fully proved that the ATPS extraction has the advantages of continuous operation.