Study On The Optimization Of Extraction And Purification Process For Steamed Panax Notoginseng Saponins And Its Bioactivities

[Objectives]Panax notoginseng(Burk.)F.H.Chen(PN),a traditional Chinese herbal medicine(CHM)used to treat blood disorders for thousands of years,is known as "the raw materials eliminate and the steamed ones tonify".The so-called "eliminate" means that the raw PN can stop bleeding,promote blood circulation,diminish swelling,and ease pain.The "tonify" means that the steamed PN(SPN)performs better efficacies on improving the immunity and nourishing the blood Numerous studies have shown that saponins are the major active components of PN,the variation in the types and contents of saponins results in the efficacy difference between raw PN and SPN.At present,studies on the preparation and biological activity of PN saponins are mainly focused on the raw PN.There are much fewer reports on those of SPN saponins,which greatly impedes the development of this valuable CHMAccording to our previous studies,the major active constituents of SPN include ginsenosides Rh1,Rk3,Rh4,20(R)-Rg3,and 20(S)-Rg3,which were associated with its hematopoiesis,antioxidation and other pharmacological effects.In this research,the efficacy-related constituents were taken into consideration to optimize the extraction and purification technology for SPN saponins.Meanwhile,The bioactivities and toxicity of the obtained SPN saponins were evaluated,which might lay a foundation for the development and utilization of SPN saponins and their products[Methods]The methods were shown as follows1.The root and rhizome of PN were collected.According to our previous result,the SPN samples were prepared by steaming the crushed raw PN in an autoclave at 120? for 2 h2.On the basis of single factor screening,Box-Behnken experimental design was employed,With the yield,content,and antioxidant activity(EC50 value)of saponins as the evaluation indices.the extraction time,ethanol concentration,water to solid ratio,and times of extraction were investigated to optimize the extraction process of saponins in SPN3.The main factors affecting the purification process were screened by the failure mode and effects analysis(FMEA).The purification method with macroporous resin was optimized by the central composite design-response surface methodology(CCD-RSM)based on the recovery and purity of saponins.In this experiment,the concentration of sample solution,loading volume,washing volume,ethanol concentration,and ethanol elution volume were investigated to optimize the purification technology of saponins in SPN.4.The antioxidant,immunomodulatory and anti-anemic activities of SPN saponins were evaluated to confirm its effects.And the acute toxicity effect was studied to validate its safety.[Results]1.The optimum conditions to maximize the yield(31.96%),content(70.49 mg/g),and antioxidant activity(EC50 value of 0.0421 mg/mL)for saponins extracted from SPN were obtained with a extraction time of 1.51 h,ethanol concentration of 60%,extraction done 3 times,and W/S of 10.2.The optimum conditions to maximize the recovery(82.81%)and purity(77.24%)of saponins were obtained with the concentration of saponin sample solution of 11.22 mg/ml,loading volume of 4.97 BY,washing volume of 2 BY,ethanol concentration of 70%,and ethanol elution volume of 3.31 BY.3.Compared with the model group,SPN saponins increased significantly the levels of blood routine parameters including WBC,RBC,HGB,and PLT in a dose-dependent way(p<0.01).Meanwhile,there was no significant difference in the anti-anema effect between the SPN group and the SPN saponins group,of which the dose was 15 times lower than the former one.4.Compared with the model group,SPN saponins increased significantly the organ coefficients of spleen and thymus,carbon clearance index-k,and phagocytic index-a of mice with immunosupression and enhanced the macrophage function in a dose-dependent manner5.Compared with the model group,SPN sapomns significantly increased the superoxidase dismutase(SOD),catalase(CAT),and glutathione preoxidase(GSH-Px)levels and the total antioxidant capacity(T-AOC)of mice with immunosupression.And the malondialdehyde(MDA)level in the mice was decreased in vivo after treated with SPN saponins.Also,there was a dose-dependent relationship between anti oxidation activity and the concentration of SPN sapomns.6.In the acute toxicity test,SPN saponins did not cause the death or toxic symptom of mice during the observation period.There was no significant difference in the absolute and relative weights of vital organs(spleen and liver)between the experimental groups and the control group of mice.The LD50(half lethal dose)values of SPN and its saponins were both higher than 5000 mg/kg.[Conclusion]The optimized extraction and purification process was stable,with high recovery and purity of saponins.The SPN saponins prepared showed better hematopoiesis,immune-modulation and antioxidation activities than SPN.This study provides some evidences for the reasonable use and future development of SPN projects.