| Objective:Use Chitosan, Chitosan-alcohol, macroporous resin and polydextran gel purification thchnologies alone or together, to study the best optimal purification method on Shenqi compound recipe, in order to establish favourable basis to promote quality for Shenqi aqueous extraction and injection.Method:Use ultraviolet spectrophotometry method to measure polysaccharide and saponin; use HPLC-UV-ELSD allied method to measure the content of astragaloside, biochanin B and fingerprint; use TLC method to analyze and identification. Use precipitation capacity as index, study Chitosan, ZTC1+1Ⅲand juice clarificant to choose the best. Use the total content of polysaccharide and saponin, and astragaloside , biochanin B as index, to study Chitosan, Chitosan-alcohol, macroporous resin-polydextran gel purification thchnology and alcohol precipitation technology by orthogonal test, to compare fingerprint, to choose the best purification technology for Shenqi aqueous extraction.Result:1. Content determination research indicated that, for polysaccharide, used absorbance (Y,A) and concentration (X,mg/mL) to run linear regression, got the standard curve equation: Y=10.905X+0.1394;r=0.999 8,showed linear correlation in the scope 0.08～0.24 mg; for saponin, used absorbance (Y,A) and astragaloside amount (X,mg) to run linear regression, got the standard curve equation: Y=2.3409 X+0.0252,R=0.999 6,showed linear correlation in the scope 0.031～0.188 mg; for astragaloside, used peak area's log and introducion amount's log to run linear regression, got the standard curve equation: Y=1.3351X+2.3674,r=0.999 8,showed linear correlation in the scope 0.057～1.14μg; for biochanin B, used peak area and introducion amount to run linear regression, got the standard curve equation: Y=5152.3X-101.39,r=0.999 9,showed linear correlation in the scope 0.026～0.416μg.2. Fingerprint determination research indicated that, for Shenqi aqueous extraction, based on acetonitrile and water as mobile phase gradient elute, and used biochanin B and astragaloside as index, it could get 11 peaks in 254 nm UV fingerprint ,which the eleventh peak was biochanin B; and get 12 peaks in ELSD fingerprint, which the sixth peak was astragaloside. They all had great resolving power and basal line . The number of theoretical plates exceeded 5000. Through technology investigation, the RSD % of the main peaks'relative retention time and relative peak area were less than 1.4 %.3. Different clarificant screeing research indicated that, physic liquor concentration and the clarificant amount had signifiance effect, Chitosan purification's precipitation capacity was higher than the other two clarificants.4. Chitosan research indicated that, Chitosan amount had signifiance effect to astragaloside, polysaccharide and saponin; Chitosan amount and flocculation temperature had signifiance effect to biochanin B. After aggregate analysis, we could get the best technology A2B2C2D1, whose polysaccharide and saponin content could achieve 51.70 %, biochanin B 0.02614 %, astragaloside 0.1907 %, solids content 40.29 mg/mL. From HPLC-UV-ELSD fingerprint and TLC spectrum, it could be seen that different technology's peak piece and spot similar on the whole, but existed some difference in content.5. Alcohol precipitation technology research indicated that, polysaccharide and saponin content could achieve 50.89 %, solids content 36.95 mg/mL, and ELSD spectrums display that in time 5～10 min, the peak resolution was great.6. Chitosan-alcohol, macroporous resin-polydextran gel allied purification research indicated that, manipulus alcohol had encourage action in precipitation, the solids content was less low than alcohol precipitation technology; we used macroporous resin purification to get saponins, flavonoids and polydextran gel technology to get polysaccharide, and it obviously boosted the result.The content of solids content was low, the flavonoids,polysaccharide, biochanin B, astragaloside were all high. Its polysaccharide and saponin content could achieve 78.42 %, biochanin B 0.03960 %, astragaloside0.483 %, solids content 4.17 mg/mL. Compared HPLC-UV-ELSD fingerprint spectrum, they were similar with Shenqi aqueous extraction respectively, but macroporous resin-polydextran gel allied purification could get more peaks and the base line is good. It could get 16 peaks in 254 nm UV fingerprint ,which the sixteenth peak was biochanin B; and get 14 peaks in ELSD fingerprint, which the fourteenth peak was astragaloside. TLC spectrum indicated that, as V二氯甲烷:V甲醇:V水(13:9:2) developing solvent, could get more spot, and the resolution was good. Different technology's spot exist some difference in size and color, which indicated that different technology had invariably influence.Conclusion:1. By methodological survey, UV of polysaccharides and saponins precision, stability and reproducibility can meet the requirement for the determination of total polysaccharide, the total saponin content in Shenqi. The established determination method of biochanin B, astragaloside and fingerprint analysis of HPLC-UV-ELSD, is precision, stability and reproducibility to meet the requirement, which can be used for HPLC fingerprint analysis of Shenqi water extract and purified liquid composition's changes. The changes of composition of the Shenqi water extracion and clarified liquid on the whole can be analyzed, and can be used to compare the different purification process of the Shenqi water extraction. Comparing different purification process of the water extraction of Shenqi, the established TLC spectrum can be used to analysis the changes of aqueous extract and purified liquid composition.2. Alone and combined purification methods show that, clarification of chitosan has good sedimentation effect, and a small amount of ethanol can increase the sedimentation rate and sediment volume. Alcohol precipitation has good sedimentation effect, but removed a large number of effective polysaccharide which is not conducive to play preparation curative effect. Saponin and flavonoids purified by macroporous resin flavonoids-polysaccharide purified by polydextran gel technology has excellent purification effect, which contain less solid materials, high ingredients, more fingerprint peaks and good baseline level.3. Comprehensive analysis shows that the macroporous resin-polydextran gel technology combined technology is best in purifing Shenqi water extract. The best technology and verification process screened by orthogonal process , has good stability, high content of active ingredients, less foreign matter, the established analysis evaluation methods can be used well for the quality evaluation of the process product. |
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