| The commodity grade of Chinese medicinal materials（CMM）is one of the standards for using in clinical since ancient times.According to their size,weight,color and number,CMM are divided into different commodity grades,which is conducive to evaluating the quality of CMM in market circulation.Danggui is the dried root of Angelica sinensis（Oliv.）Diels（RAS）,which was first recorded in Shennong herbal Scripture.RAS is mainly used to replenish and invigorate blood circulation,regulate menstruation and relieve pain according to the 2020 edition of the Chinese Pharmacopoeia.The quality control constituent of RAS is ferulic acid.The different commodity grades of RAS are only divided according to weight,there is a lack of connection between efficacy and commodity grades.RAS not only has high medicinal value,but also plays an important role in health care products,diet therapy and cosmetics.It is important to clarify the difference in efficacy between commodity grades.The purpose of this study was to conduct in-depth research on the chemical composition,biological activity,and pharmacodynamic substances of different commodity grades of RAS.The quality marker（Q-marker）of RAS was screened out by the spectrum-effect correlation method.The quantitative analysis of multi-components by single marker（QAMS）technical was established to detect ligustilide and senkyunolide I by using ferulic acid as the reference substance.Moreover,the mechanism of blood-tonifying of different commodity grades of RAS by the metabolomics and lipidomics methods.Firstly,high performance liquid chromatography（HPLC）was used to study the fingerprints and Q-markers on anti-platelet aggregation activity of different grades of RAS.（1）A total of 17 HPLC common peaks were obtained in the water extract of RAS,and 9 metabolites were identified,including ferulic acid,uridine,guanine,ligustilide,senkyunolide I,chlorogenic acid,eusinolide A,adenosine,and L-tryptophan.The content of ligustilide in the third grade of RAS（26.27±0.23 mg/g）was better than other grades.Subsequently,re-purchased different commodity grades of RAS for verification,again proved that the content of ligustilide in the third grade of RAS was higher（23.14±0.17 mg/g）.（2）The third grade of RAS showed a good inhibitory activity on platelet aggregation induced by adenosine diphosphate（ADP）or arachidonic acid（AA）in vitro.The IC50value of different commodity grades of RAS on ADP-induced platelet aggregation activity was ranked as follows:third grade（27.87 mg/m L）>second grade（83.14 mg/m L）>first grade（96.33 mg/m L）>fourth grade（137.19 mg/m L）.The IC50value on AA-induced platelet aggregation was ranked as follows:third grade（42.81 mg/m L）>second grade（46.81 mg/m L）>first grade（86.03 mg/m L）>fourth grade（132.45 mg/m L）.（3）The Q-markers of RAS that contributed significantly to platelet aggregation activity.The IC50values of ligustilide,uridine,guanine,senkyunolide I and ferulic acid in inhibiting ADP-induced platelet aggregation were 1.18,2.21,4.37,5.38 and 7.02 mg/m L,respectively.The IC50values for inhibiting AA-induced platelet aggregation were 1.07,1.89,3.25,6.88 and 9.07mg/m L,respectively,which could be used as a Q-marker for anti-platelet aggregation activity of RAS.（4）The QAMS method was established to detect ligustilide and senkyunolide I with ferulic acid as reference substance,the relative retention time of ligustilide and senkyunolide I was 1.23 and 1.79,and the correction factor was 0.512and 2.761,respectively.Secondly,the pharmacodynamic differences and metabolomics studies of different commodity grades of RAS in the treatment of blood deficiency（BD）model rats were explored.（1）The BD rat model was induced by acetylphenylhydrazine（APH）and cyclophosphamide（CTX）.The third grade of RAS was superior to other commercial grades of RAS in restoring spleen index,recovering the damage of organ,increasing the peripheral blood and erythrocyte membrane energy metabolism enzyme activity.（2）Nineteen potential biomarkers were screened from plasma.Eleven metabolites including lysophosphatidylcholine Lyso PC（17:0）,sphingomyelin SM（d20:1/20:4（5Z,7E,11Z,14Z）-OH（9））and phosphatidylcholine PC（18:1（9Z）-O（12,13）/P-16:0）were up-regulated,and 8 metabolites such as chenodeoxycholic acid,L-carnitine and citrulline were down-regulated in the model group.Each administration group with RAS could regulate these 19 potential biomarkers.Five major metabolic pathways were screened with Met PA,including AA metabolism,arginine biosynthesis,alanine,aspartate and glutamate metabolism,sphingolipid metabolism and glycerophospholipid metabolism.（3）Twelve metabolites were detected in urine.Four metabolites such as Endoperoxide g2,PA（LTE4/i-15:0）,and PA（i-19:0/PGE1）were up-regulated and eight metabolites such as hippuric acid,citric acid,and Galabiosylceramide were down-regulated in the model group.Each administration group could regulate these 12 potential biomarkers.Four main metabolic pathways were screened out,including TCA cycle,sphingolipid metabolism,AA metabolism,and glyoxylate and dicarboxylate metabolism.Then,the plasma and spleen lipidomics study of RAS in the treatment of BD rats was explored.（1）Seventy three potential biomarkers were screened from plasma.Polyunsaturated fatty acids and glycerophospholipids were the two most obvious changes in the BD rats of plasma lipidomics.Twenty eight metabolites including phosphatidylcholine PC（P-18:0/18:3）,triglyceride TG（16:0/16:0/16:0）,and phosphatidyl ethanolamine PE（20:5/18:0）were up-regulated,and 45 metabolites such as phosphatidylcholine PC（22:2/20:5）,alpha-Linolenic acid,and Linoleic acid were down-regulated in the plasma from the model group.Each administration group with RAS could regulate these 73 potential biomarkers.Three main metabolic pathways were screened out with Met PA,which were alpha-Linolenic acid metabolism,Glycerophospholipid metabolism,and Linoleic acid metabolism.（2）A total of 112potential biomarkers were screened from the spleen.Glycerophospholipids and triglyceride（TG）were the two most obvious substances in the blood deficiency rats of spleen lipidomics.Twenty one metabolites such as phosphatidyl ethanolamine PE（20:4/22:2）,phosphatidylcholine PC（P-18:0/22:5）,and lysophosphatidylcholine Lyso PC（P-18:0/0:0）were up-regulated and 91 metabolites such as lysophosphatidylcholine Lyso PC（20:2/0:0）,triglyceride TG（14:0/16:0/20:4）,and phosphatidyl ethanolamine PE（20:1/0:0）were down-regulated in the spleen from the model group.Each administration group could regulate these 112 potential biomarkers.Five major metabolic pathways were screened out by Met PA,namely glycerophospholipid metabolism,sphingolipid metabolism,Glycerolipid metabolism,glycosylphosphatidylinositol-anchor biosynthesis,and Ether lipid metabolism.Glycerophospholipid metabolism,sphingolipid metabolism,AA metabolism,TCA cycle and amino acid metabolism are the main metabolic pathways of RAS in the treatment of blood deficiency syndrome.RAS may restore the lipid metabolism level by regulating glycerophospholipid and sphingolipid metabolism disorder,and regulating TCA cycle and amino acid metabolism to improve the energy metabolism,and regulating AA metabolism to play a role in promoting blood circulation.Therefore,it is speculated that RAS could reduce the inflammatory promoting factor Lyso PC（17:0）and regulate glycerophospholipid levels to improve energy metabolism.RAS could regulate metabolic pathways including TCA cycle,amino acid metabolism,and AA accumulation to enhance body immunity and exert the effect of nourishing blood and activating blood circulation.In summary,this study for the first time conducted in vivo and in vitro animal experiments on the quality of different commodity grades of RAS.It was found that the therapeutic effect of third grade was better than that of other grades of RAS.Combined with metabolomics and lipidomics techniques,the mechanism of replenish and invigorate blood circulation effects of RAS was elucidated and analyzed,providing a scientific basis for the clinical application of RAS. |
