Studies On Quality Control Method Of Codonopsis Radix Based On Core Function Of Strengthening Spleen And Benefiting Lung,Nourishing Blood And Producing Fluid

1 Background and objectivesCodonopsis Radix(CR)is a perennial herb in the family Campanulaceae.It has more than 40 species worldwide and is widely distributed in East Asia,Southeast Asia and Central Asia.Codonopsis pilosula(Franch.)Nannf.,C.pilosula Nannf.var.modesta(Nannf.)L.T.Shen and C.tangshen Oliv,which have been recorded in the Chinese Pharmacopoeia(2020 edition),can invigorate spleen and benefit lung,nourish blood and produce fluid.It is often used to treat the deficiency of Qi and blood,tiredness,yellowish complexion,palpitation,shortness of breath and thirst.CR is a kind of traditional Chinese medicine(TCM)with a long history,but the research on it is not deep enough.The Chinese Pharmacopoeia only uses lobetyolin as the identifying component for the TLC qualitative identification,which leads to the lack of specificity.Effectiveness and safety of TCM are the lifeblood and permanent subject.Scientific quality control methods and quality standard research are the key to ensure the efficacy and safety of TCM.The quality control of TCM must be combined with the safety and effectiveness,and it is meaningless to separate the quality control from the drug effect.There are many chemical components in CR,and the content of saccharides as active components is high.CR has rich pharmacological effects,mainly in the nervous system,endocrine system,immune system.CR has good antifatigue and antihypoxia activities according to core function of invigorating spleen and lung,nourishing blood and fluid,but the main active components are not clear.In addition,there is no report on the analysis and detection of small molecular saccharides in CR.Therefore,this study takes CR as the research object and comprehensively uses the theoretical methods and techniques of analytical chemistry,pharmacology of TCM and other disciplines to clarify its main active parts of the antifatigue and antihypoxia effect.Finally,the correlation between the chemical components and pharmacodynamic effect of CR was introduced directly into the quality evaluation system,so as to establish the quality evaluation related to the core efficacy of CR.It provided reference for the improvement of the quality standard of CR.And this is of great significance to the improvement of economic benefit of CR industry and the development of health products.2 Methods2.1 Research on antifatigue and antihypoxia activities of Codonopsis Radix and its pharmacodynamics evaluation based on core function of strengthening spleen and benefiting lung,nourishing blood and producing fluid:The antifatigue and antihypoxia activities of the ethanol extract(ETH),water extract(WAT),inulin(INU),polysaccharides(POL)and oligosaccharides(OLI)from CR were evaluated by weight-loaded swimming and normobarie hypoxia test.The mechanism of functional components was further studied by biochemical analysis.OLI was divided into monosaccharides(MON)and frutooligosaccharides(FOS)by adsorption chromatography to further determine the antifatigue and antihypoxia activities.Besides,the pharmacodynamics evaluation of active ingredients was further studied.2.2 Establishment of a method for the analysis of monosaccharides and oligosaccharides in Codonopsis Radix and its quality evaluation:A HPLC method for simultaneous determination of fructose,glucose,sucrose,and fructooligosaccharides(GF2-GF6)was established.65 batches of CR from three species of genus Codonopsis and 34 batches of processed samples were analyzed combined with multivariate statistical techniques to research the effects of varieties,cultivation management measures(plant growth regulators supply,harvesting time,and growth period)and primary process(drying methods)in producing areas on the target compounds.The practicability of the new method was verified,and it provided a reference for the quality evaluation of CR.3 Results3.1 Research on antifatigue and antihypoxia activities of Codonopsis Radix and its pharmacodynamics evaluation based on core function of strengthening spleen and benefiting lung,nourishing blood and producing fluid:The results showed that compared with control group,the swimming time of mice in POL group was significantly increased by 78.15%and107.15%.The POL group showed the strongest hypoxia tolerance,followed by OLI group and WAT groups,and the survival time of mice was increased by 11.90%～28.63%compared with control group.POL and OLI can enhance the abilities of antifatigue and antihypoxia in a dose-dependent manner.The levels of liver glycogen(LG)and muscle glycogen(MG)were significantly increased(P<0.01 or P<0.001),blood urea nitrogen(BUN)and lactic dehydrogenase(LDH)were significantly decreased(P<0.05)in the POL-0.5 and POL-1.0 groups compared with control group.Besides,the levels of malondialdhyde(MDA)decreased and glutathione(GSH)increased significantly in POL and OLI groups.POL can efficiently enhance antifatigue and antihypoxia abilities by increasing energy accumulation,reducing detrimental metabolite accumulation,and enhancing antioxidant activity.OLI can improve antihypoxia activity by preventing lipid peroxidation and enhancing antioxidant activity. OLI was successfully divided into MON and FOS by adsorption chromatography. The results of weight-loaded swimming and normobarie hypoxia test showed that the antifatigue and antihypoxia activities of FOS was stronger than that of MON.The contents of LG and MG in the MON and FOS groups were increased,and the high-dose group was the most significant(P<0.001).The content of BUN decreased significantly and showed dose-dependent tolerance(P<0.05).The levels of LDH and MDA in FOS group were significantly decreased(P<0.05).The level of GSH in FOS-0.5,FOS-1.0and MON-1.0 groups was significantly increased(P<0.05).The antifatigue and antihypoxia mechanism of FOS is the same as that of POL.3.2 Establishment of a method for the analysis of monosaccharides and oligosaccharides in Codonopsis Radix and its quality evaluation:A HPLC method for simultaneous determination of fructose,glucose,sucrose,and fructooligosaccharides(GF2～GF6)was established successfully.The data indicated a good correlation was found for all the compounds in the range of concentration(R~2≥0.9996).The accuracy of the method was good,with a RSD≤2.94%for intra-day and3.07%for inter-day precisions.RSDs of repeatability and stability tests were all less than 3%.The average recoveries were between 98.36 and 101.66%with RSDs values1.25%～2.60%.The results showed that the FOS content of C.tangshen was the highest and the average content was 46.59 mg/g.The average content of FOS in C.pilosula was7.45 mg/g,which was only one fifth of that in C.tangshen.The FOS in C.pilosula var.Modesta was just 2.32 mg/g.Besides,cultivation management measures(plant growth regulators supply,harvesting time,and growth period)and primary process(drying methods)can significantly affected saccharides accumulation.The low concentration Zhuanggenling was beneficial to the accumulation of FOS,which increased FOS by117.21%compared with the normal group.The content of FOS in CR collected in October was 194.58%higher than that in August,while the content of MON decreased by 24.08%.The content of FOS increased gradually with the increase of growth years.The content of FOS was the highest in freeze-dried samples(74.40 mg/g),while that in the sun-dried samples was the lowest(53.29 mg/g).4 ConclusionsFOS has good antifatigue and antihypoxia activities,which can not be ignored in CR,and can be used as an index for quality evaluation.The HPLC method established for the first time can simultaneously detect monosaccharides and oligosaccharides with good repeatability and sensitivity.The accumulation of saccharides in CR was affected by many factors,such as cultivation measures and primary processing.The content of active ingredient FOS in CR varies greatly,so it is difficult to form a reasonable quality standard system.The agricultural planting and primary processing methods of CR have not formed standardized standards.It is recommended to use low concentration Zhuanggenling,and it is best to harvest CR in October after planting for four years.And dryer-drying was suggested to meet the requirement for large scale processing.This study provides a theoretical basis for the study of CR planting industry and producing area processing,and also provides a reference for improving the quality evaluation of CR.