Quality Evaluation And Effective Substances On Immune Regulation And Hematopoietic Improvement Of Codonopsis Radix

Genus Codonopsis Wall.(Campanulaceae)has important medicinal and edible value with a long history.Codonopsis Radix,the dried root of Codonopsis pilosula(Franch.)Nannf.,Codonopsis pilosula Nannf.var.modesta(Nannf.)L.T.Shen or Codonopsis tangshen Oliv.,is definitely recorded as traditional Chinese medicine dating back to Qing Dynasty in Ben Cao Cong Xin,with indications for strengthening spleen and tonifying lung,as well as nourishing blood and engendering liquid.At present,no index component of Codonopsis Radix was stipulated in Chinese pharmacopoeia(2015 edition),with only lobetyolin specified as the reference substance in the TLC identification item.In order to explore the bioactive markers reflecting the traditional efficacy and improve the quality standard of Codonopsis Radix,traditional uses,phytochemistry,pharmacology and toxicology of Codonopsis were reviewed on the basis of systematic summary of literatures and studies.Systematic study on chemical constituent analysis(fingerprint chromatography and content determination),isolation and identification of compound,in vivo experiment of extract,in vitro validation of potential bioactive compound,bioinformatics analysis,etc.was conducted.Thus,the quality of Codonopsis Radix was evaluated and the efficacious material basis on immune regulation and hematopoietic improvement of Codonopsis Radix was elucidated.The specific research content and result were as follows:1.The fingerprint of Codonopsis Radix and the determination of lobetyolin were established based on HPLC-DAD method.The determination of polysaccharide was adopted by UV-vis method.The determination of sucrose,glucose and fructose was set using HPLC-ELSD method.The chemical quality evaluation system was established based on above methods to evaluate the effect of different cultivation measures,processing methods and storage methods of Codonopsis Radix and evaluate the chemical quality of Codonopsis Radix slices.For the cultivation measures,the appropriate measure was not using Zhuanggenling,not pinching and shelving based on the content of lobetyolin and polysaccharide.For the processing methods,sun-drying or shade-drying was recommended,and sulfur fumigation should be avoided.And dryer-drying at 40-50? was suggested to meet the requirement of large scale processing.In addition,based on the content of polysaccharide,glucose and fructose,kneading was advised.As for lobetyolin and sucrose,kneading should be avoided.For the storage methods,the content of lobetyolin showed a decrease tendency,while that of polysaccharide,sucrose,glucose and fructose showed a tendency of first rise then fall during 6 months at different storage methods.Low temperature and humidity were beneficial for the quality stability of Codonopsis Radix.For Codonopsis Radix slices,the consistency of chemical quality of Codonopsis Radix slices from the production base in Min county,Gansu province was consistent,while consistency of chemical quality of slices from the Anguo medicine market in Hebei province and pharmacy in Beijing was poor.2.The bioactive compounds reflecting the immune regulation and hematopoietic improvement activities of Codonopsis Radix were explored by spectrum-effect relationship analysis.Firstly,UPLC-ESI-Q-TOF-MS/MS analysis was conducted to identify the chemical constituents in Codonopsis Radix and 30 compounds were deduced.UPLC fingerprint of Codonopsis Radix prossessed with different methods was established.There were 31 common peaks in total and 13 of them were pointed out.Similarity analysis and heatmap analysis indicated that chemical constituents in Codonopsis Radix prossessed with shade-drying were higher than those prossessed with sun-drying or dryer-drying.Secondly,immune and hematopoietic function of Codonopsis Radix was investigated based on traditional efficacy.The results indicated that Codonopsis Radix methanol extract could ameliorate the immune and hematopoietic function inhibited by cyclophosphamide in mice.And the function of Codonopsis Radix methanol extract prossessed with shade-drying was better than that prossessed with sun-drying or dryer-drying.Then,the spectrum-effect relationship analysis between UPLC fingerprint and pharmacological effect was carried out.According to the results of canonical correlation analysis,common peak 4(Tryptophan),6(Syringin),7(Unknown),8(Tangshenoside 1),10(Codonopyrrolidium A),11(Unknown),17(Lobetyolin)might be the potential bioactive compounds related to the immune regulation and hematopoietic improvement function of Codonopsis Radix.The two unknown compounds were further isolated by chromatographic separation techniques and their structures were identified by spectroscopic techniques.It turned out that Cordifolioidyne A(common peak 7)was isolated firstly in Codonopsis Radix,and Creoside IV(common peak 11)was isolated firstly in Codonopsis.Finally,UPLC-DAD method was applied for the quantitative analysis of 7 potential bioactive compounds in Codonopsis Radix prossessed with different methods.The mechanism of 7 potential bioactive compounds of Codonopsis Radix in treating leukopenia was studied by network pharmacology.Target prediction,biomolecular function analysis,pathway analysis and molecular docking analysis were conducted for compounds and disease,and the network diagram of "bioactive compound-target-pathway"was constructed.The results indicated that 7 compounds in Codonopsis Radix acted on 70 targets and 56 pathways in the treatment of leukopenia.3.Cell model was used to verify the immune regulation function of Codonopsis Radix methanol extract and 7 potential bioactive compounds in vitro.The results indicated that Codonopsis Radix methanol extract and 4 compounds could promote the proliferation of RAW264.7 cells and inhibit the secretion of IL6,TNFa and IFN-y induced by LPS in a dose-dependent manner.In addition,Codonopsis Radix methanol extract and 7 compounds could promote the proliferation of JURKAT E6-1 cells and improve the secretion of IL6,TNFa and IFN-? induced by 5-Fu in a dose-dependent manner.The findings showed that Codonopsis Radix methanol extract and compounds could promote the proliferation of immune-related cells,and regulate the secretion of inflammatory factors,thus playing a role in immune regulation.4.Cell model was used to verify the hematopoietic improvement function of Codonopsis Radix methanol extract and 7 potential bioactive compounds in vitro.The results indicated that Codonopsis Radix methanol extract and 7 compounds could promote the formation of CFU-E,BFU-E,CFU-GM and CFU-GEMM and promote the expression of CD34+of hematopoietic stem/progenitor cells,while inhibit the expression of CD45+,CD14+,CD19+ and CD71+.In addition,after all-trans retinoic acid(ATRA)treatment,the expression of CD34+,CD3+,CD19+and CD71+ was down-regulated,while the expression of CD45+ and CD14+was up-regulated,and the cells showed significant differentiation morphologically.Codonopsis Radix methanol extract and 7 compounds could increase the expression of CD34+,CD3+,CD19+and CD71+,while decrease the expression of CD45+ and CD14+,and the differentiation degree of cells was slowed obviously in morphology.In conclusion,Codonopsis Radix methanol extract and compounds could promote the proliferation and maintain the differentiation function of hematopoietic stem/progenitor cells,thus improving the hematopoietic function.To sum up,the quality evaluation system of Codonopsis Radix was established from multiple dimensions,and the effect of different cultivation measures,processing methods,storage methods and different Codonopsis Radix slices were analyzed by the developed quality evaluation system.The spectrum-effect relationship analysis was conducted to explore the bioactive compounds reflecting the traditional efficacy of Codonopsis Radix.The results showed that Tryptophan,Syringin,Cordifolioidyne A,Tangshenoside I,Codonopyrrolidium A,Creoside ? and Lobetyolin might be the potential bioactive compounds related to the immune regulation and hematopoietic improvement function of Codonopsis Radix.Network pharmacology was applied to explore the mechanism of 7 potential bioactive compounds in treating leukopenia,and the results reflected the characteristics of multi-component,multi-target and multi-pathway.The function of Codonopsis Radix methanol extract and 7 potential bioactive compounds on immune regulation and hematopoietic improvement function was verified by cell model in vitro.In conclusion,the study clarified the effective substances on immune regulation and hematopoietic improvement and laid a foundation for the improvement of quality standard of Codonopsis Radix.