Study On Extraction And Purification Process Of Nigellae Semen

Objective:To determine the best extraction and purification technology of the total extract of Nigellae semen,and to investigate the content determination methods of total saponins and total flavonoids.It provides a theoretical basis for its development as natural plant feed raw material and plant extract feed additive.Methods:In this experiment,the L9(34)orthogonal test was used to study the best extraction technology of Nigellae semen by investigating the ethanol concentration,amount of ethanol and extraction times,and the content of total saponins and total flavones as the evaluation indexes for process selection.With crude extract of Semen Nigellae as raw materials,the adsorption rate and resolution rate of total saponins and total flavonoids are used as indexes.To investigate AB-8 macroporous resin on the influnce factors of the adsorption(sample liquid mass concentration,pH,sample quantity and sample on velocity),resolution(eluent volume fraction,the dosage of eluent and elution velocity)of total saponins and total flavonoids by single factor experiment.On the basis of single factor experiment,orthogonal design was used to optimize the purification process of the crude extract of total saponins and total flavonoids,and pH,eluent volume fraction and eluent flow rate of the samples as the evaluation indexes.The monomer content of hederagenin in Nigellae semen was determined by HPLC before and after purification.The content determination methods of total saponins and total flavonoids were investigated by linear relationship test,precision test,stability test,reproducibility test and sample recovery test.Results:1.The optimal extraction conditions of the extract of Nigellae semen were determined as follows:the ratio of 60%ethanol solution to medicinal material by weight 8:1 was used for reflux extraction for 2 times,1 h each time.Under these conditions,the total saponins and flavonoids were 49.39 mg·g-1(RSD=1.93%)and 2.29 mg·g-1(RSD=1.27%)respectively.2.The optimal purification technology was as follows:the mass concentration of the sample solution was 0.04 g· mL-1,the volume of the sample solution was 90 mL,the pH of the sample solution was 5.2,the flow rate of the sample was 2 BV·h-1,the elution solvent was 70%ethanol,the dosage was 5 BV,and the flow rate was 3 BV·h-1.Under these conditions,the purity of total saponins was increased from 30.51%to 65.77%(RSD=1.56%).The purity of total flavonoids was increased from 1.42%to 3.23%(RSD=2.66%).The content of hederagenin was increased from 43.70 mg·g-1 to 144.47 mg·g-1.3.The results of total saponins methodology showed that there was a good linear relationship between the content of hederagenin and the light absorption value in the range of 0.04 mg～0.24 mg(R2=0.9991).RSDs of precision,reproducibility and stability tests were 0.83%,1.31%and 1.07%,respectively.The average recoveries were 99.60%and RSD was 1.43%(n=6).4.The results of total flavonoids showed methodology that there was a good linear relationship between the content of rutin and the light absorption in the range of 0.025 mg～0.240 mg(R2=0.9997).RSDs of precision,reproducibility and stability tests were 1.02%,2.27%and 2.29%,respectively.The average recovery was 93.47%and RSD was 1.04%(n=6).Conclusions:In this study,the optimal extraction and purification technology of the total extract of Nigellae semen was established.The optimized process was stable and feasible,and could be used for the preparation of the total extract of Nigellae semen.This study laid a foundation for the subsequent studies on pharmacology,toxicology,clinical and clinical expansion of extract of Nigellae semen.It provides a theoretical basis for its development as natural plant feed raw material and plant extract feed additive.