Study On The Pharmacodynamic Substance Basis Of Rehmannia Preparata Based On Blood Deficiency Model

Ratinales:Fresh Rehmannia glutinosa is the fresh root tuber of the Scrophulariaceae plant Rehmannia glutinosa.According to the method of concoction,it is divided into two types: raw and cooked Rehmannlae Radix.Raw Rehmannlae Radix can clear heat and cool the blood,nourish yin and promote the production of body fluid.While cooked Rehmannlae Radix is useful for filling the marrow and nourishing the blood and nourishing yin,and is used clinically to treat various symptoms caused by blood deficiency.It is used clinically to treat various symptoms caused by blood deficiency.Raw and cooked Rehmannlae Radix are used for different symptoms in clinical practice and are among the herbs that are treated differently.In the 2020 edition of the Chinese Pharmacopoeia,it is stipulated that the content of catalpol ≥ 0.20% and diosgenin D ≥0.10% in raw Rehmannlae Radix,and the content of diosgenin D ≥ 0.050% in cooked Rehmannlae Radix.But the efficacy of raw and cooked Rehmannlae Radix is not the same,and only using catalpol and diosgenin D as indicators for content determination does not reflect the differences in their medicinal effects.Therefore,the existing pharmacopoeia standards lack a holistic and specific quality assessment,resulting in the medicinal effects of Rehmannlae Radix not being fully developed.Objectives:(1)To clarify the blood tonic effect of Radix Rehmanniae;(2)To analyze the compound components of raw and cooked Radix Rehmanniae and their differential components as a whole;(3)To screen the potential active components of Radix Rehmanniae for the treatment of blood deficiency and provide new ideas for the quality control of Radix Rehmanniae.Methods:(1)After the adaptation period,ICR female mice were randomly divided into 8 groups(n=8)according to body weight,including normal group(C),model group(M),raw rehmannia group(S),Guangyuyuan Radix Rehmanniae group(G),commercially available Radix Rehmanniae A group(A),commercially available Radix Rehmanniae B group(B),Dingkundan group(D),and Lvjiao Buxue Granule group(L),with doses of 10 m L/kg.This experiment lasted for a total of fifteen days.The modeling method was to inject APH(10 mg/kg)subcutaneously on the day 1 and day 9 of modeling,except for the normal group,and administer it 30 minutes later.On the day 4 and day 11,APH(10 mg/kg)was injected subcutaneously,administered 30 minutes later,and CTX(20mg/kg)was injected intraperitoneally 2 hours later;CTX(20mg/kg)was injected intraperitoneally from day 5 to day 7,and from day 13 to day 15.The blood tonic effect between raw and cooked Rehmannlae Radix and different commercially available cooked Rehmannlae Radix was evaluated and analysed by observing the appearance,body weight,peripheral blood picture,organ index,bone marrow pathological section and biochemical indexes.(2)A more comprehensive analysis of the non-sugar chemical composition,polysaccharides and oligosaccharides of cooked Rehmannlae Radix from different manufacturers by HPLC-UV,HPLC-ELSD and HPLC-RID,and a comparison of the compositional differences between different commercially available cooked Rehmannlae Radix.(3)Using the idea of spectrum-effect correlation,the results of peripheral blood,organ indices and biochemical indices of blood deficient mice were correlated with the chemical composition of Rehmannlae Radix using the grey correlation method and pearson correlation coefficient method,in order to screen the potential active ingredients for improving the blood deficiency in mice,and to reflect the potential medicinal components of the blood tonic effect of Rehmannlae Radix through the above results.Results:(1)Both raw and cooked Rehmannia glutinosa can increase the body weight of mice,improve their appearance,promote the increase of peripheral blood cell content,and improve their bone marrow pathological state.Moreover,Guangyuyuan Rehmannia glutinosa,commercially available Rehmannia A,and Dingkundan can significantly increase the content of G-CSF and IL-3;Guangyuyuan Dihuang,commercially available Dihuang A,Dingkundan,and Lvjiao Buxue Granules can significantly increase the content of EPO and have an impact on TNF-α has a significant callback effect.Guangyuyuan Dihuang and Dingkundan can significantly increase the content of IL-2.According to the pharmacodynamic indicators,raw rehmannia only has significant pharmacological effects on body weight,kidneys,uterus,and red blood cells,while cooked rehmannia significantly improves most pharmacodynamic indicators,indicating a significant difference in blood tonifying effects between raw and cooked rehmannia.(2)In this experiment,the chemical composition of raw and cooked Rehmannlae Radix was analysed and compared by HPLC-UV,HPLC-RID and HPLC-ELSD.A total of79 chemical components were detected in the HPLC-UV chromatograms,among which the chemical components of raw and cooked Rehmannlae Radix differed significantly in content,with 15 chemical components showing an increasing trend from raw to ripe Rehmannlae Radix and about 14 chemical components showing a decreasing trend from raw to cooked Rehmannlae Radix;and there were also different chemical components in cooked Rehmannlae Radix from different manufacturers,such as peaks 17,50,62 and 66 were only Guangyuyuang Rehmannlae Radix;peaks H44,H47,H51 and H58 were only present in commercially available Dihuang A;peaks H30,H35,H41 and H76 were only present in commercially available Dihuang B.In the molecular weight profile of polysaccharides by HPLC-RID,three peaks of different molecular weights appeared,mainly > 133800 Da and 180-2500 Da.The chromatographic peak DT1 shows an increasing trend in content from raw to cooked Rehmannia glutinosa,with the highest content in commercially available Rehmannia glutinosa B.DT3 is a newly discovered chemical component,and the highest content is found in Guangyuyuan cooked Rehmannia.A total of 12 oligosaccharide peaks were analysed by HPLC-ELSD,with significant variations in content.GT1,GT7,GT8,GT10 and GT12 decreased or even disappeared from raw to cooked Rehmannlae Radix.While the content of the remaining components varied,such as GT4,GT5,GT6 and GT9,which were present in greater amounts in Guangyuyuan cooked Rehmannia.The above results indicate that there are significant differences in the chemical composition of raw and cooked Rehmannlae Radix,and there are also significant differences in the chemical composition of non-sugar and sugar components between different cooked Rehmannlae Radix.(3)In the spectral correlation analysis,the grey correlation degree results showed that the non carbohydrate chemical components represented by peaks H8,H11,H13,H14,H25,H26,H60,H67,H72,H73,and H77 in cooked Rehmannia glutinosa.As well as the carbohydrate components represented by DT1,GT1,GT6,and GT9,have a high degree of correlation with the therapeutic effect in blood deficiency mice.The Pearson analysis results indicate that H4,H7,H8,H13,H14,H17,H19,H45,H50,H51,H56,H58,H60,H62,H63,H70,as well as DT3,GT1,GT4,and GT6,have a significant positive correlation with the blood nourishing effect of Rehmannia glutinosa.Based on the above two analysis methods,it is indicated that the medicinal effect of Rehmannia glutinosa is the result of the joint action of its chemical component group.After combining the results of the grey correlation degree method and Pearson correlation coefficient method,it is preliminarily determined that H8,H13,H14,H19,H60,H70,as well as DT3,GT1,GT4,and GT6 are the medicinal components of its blood tonifying effect.Conclusion: 1.There is a significant difference in the chemical composition between raw and cooked Rehmannia glutinosa,but both have a blood tonifying effect in the blood deficiency model,which can improve the peripheral blood count of mice and regulate its blood tonifying related factors.However,the efficacy of cooked Rehmannia glutinosa is better than that of raw Rehmannia glutinosa,and the effects of different factories’ cooked Rehmannia glutinosa are more prominent among Guangyu Yuan cooked Rehmannia glutinosa and commercially available Rehmannia glutinosa A.2.H8,H13,H14,H19,H60,H70,as well as DT3,GT1,GT4,GT6,etc.,may be potential pharmacological components for the treatment of blood deficiency syndrome with raw and cooked Rehmannia glutinosa.