| Danggui, as one of the traditional Chinese medicinal materials in prescriptions and composite formulae, are used to enrich blood, activate blood circulation, regulate menstruation, relieve pain and relax bowels, etc., which is having the wide application value and development prospects. The group of Danggui roots including Chinese Danggui (CDG, the roots of Angelica sinesis (Oliv.) Diels), Japanese Danggui (JDG, A. acutiloba Katagawa or A. acutiloba Katagawa var. sukiyamae Hikino), Korean Danggui (KDG, A. gigas Nakai), Lovage root (LR, Levisticum officinale Koch.) and Angelica archangelica root (AAR, A. archangelica L.), are widely used herbs owning the similar effects. But their chemical composition and pharmacological action might be different. This thesis focuses on the comparison of chemical consituents and enrich-blood bioactivity among the five species herbs.Firstly, comparing chemical constituent based on high-performance liquid chromatographic (HPLC) fingerprint:48 "Danggui" samples were collected from different countries. HPLC-DAD-MS technique was applied to determinate HPLC chromatogram.12 peaks was selected as the characteristic peak, in which 10 of them was identified by reference the retention time, UV and MS data, respectively. They are (1) ferulic acid, (2) senkyunolide I, (3) senkyunolide H, (5) coniferyl ferulate, (6) senkyunolide A, (7) E-ligustilide, (8) Z-ligustilide, (9) Z-butylidenephthalide, (10) decursin, (12) levistolide A. The average chromatogram of different species was calculated and used as HPLC fingerprint. Result shows that the HPLC fingerprints of CDG, JDG and LR are similar. It indicate they have the common chemicals. JDG has its unique peak, which means that the chemical composition of JDG and CDG are different. KDG has two mains peak named peak 10 (decursin) and 11 (decursenol angelate), which is different with others. The peak of AAR in HPLC fingerprint concentration on 30-50 min, which has not the same compound with CDG Therefore, the chemical in five species are different. And according to the characteristic peak could use to distinguish JDG, KDG and AAR.Secondly, comparing main active components (ligustilide, ferulic acid and polysaccharide):Through the spectral and chromatographic conditions, DNS chromogenic reagent dosage (3,5-Dinitrosalicylic acid) and color color time optimization and method validation, the HPLC method for determination of ferulic acid and ligustilide content and 3,5-dinitmsalicylic acid (DNS) to determine the content of polysaccharide were builded.48 samples were determined and compared. Result showed that the ligustilide in CDG, JDG and LR were estimated to be 13.98 mg·g-1 (n= 14),1.13 mg·g-1(n=13),2.85 mg·g-1 (n=4). Ligustilide was not detected in KDG and AAR. The content of ferulic acid in CDG, JDG, KDG and LR were 1.30 mg·g-1 (n=14),0.14 mg·g-1 (n=13),0.36 mg·g-1(n=8) and 0.32 mg·g-(n=4). AAR did not detect ferulic acid. DNS in this paper for determination of the content of polysaccharide is stable and reliable, simple operation. The content of Polysaccharide in group of Danggui were estimated to be 162.48 mg·g-1 (n=9) in AAR,142.26 mg·g-1 (n=4) in LR,126.78 mg·g-1 (n=13) in JDG,81.38 mg·g-1 (n=8) in KDG, 80.90 mg·g-1 (n= 14) in CDG. The content among these herbal are significant defferent, which may result in the variety of bioactivity and therapeutic effect.Thirdly, comparing enrich-blood activity (EBB) by bioassay:Total of 28 herb samples containing 12 CDG,5 JDG,4 KDG,4 LR and 3 AR samples were collected from China, Japan, Korea and Europe. These samples were first extracted in 95% of ethanol, then in water. The extracts were mixed and dried for pharmacology study. For the assay of enrich-blood effect, cyclophosphamide and phenylhydrazine hydrochloride were used in Kunming mice to make animal model with blood deficiency by intraperitoneal injection. Cyclosporine A (CSA) was chosen as positive drug. Red blood cell (RBC) was chosen as marker to estimate the level of enrich-blood effect. The 0.5 agent’s amount reaction of parallel line analysis (3,3’) method was employed to quantify the enrich-blood effect in testing sample. Definition titer of every milligram (mg) CSA as one unit (U) and the estimate titer of test sample as 1000 U·g-1.The EBB in CDG, KDG, LR and JDG were estimated to be 29.686 U·g-1(n=12),16.585 U·g-1 (n=4),16.140 U·g-1 (n=4),9.884 U·g-1 (n=5), and 3 samples of AAR have not detected EBB. The analysis of variance of EBB showed that: there was a significant difference between the CDG with JDG, KDG and LR (P< 0.01). It indicating that EBB of CDG was significantly stronger than JDG, KDG and LR. Danggui in Chinese medicine, Japan Kampo and traditional medicine of Korean used as enrich-blood medicine, and even the same formula, but the source of the plants are different. At the same time, the EBB of LR was only half of EBB at CDG. In addition, the AAR was not detected in EBB. Therefore, as a tonic medicine, the five species can not substitute each other, mixed herbs. Besides, in order to compare the EBB of different grade CDG, we collected 2 samples of CDG and divided them into three grades depending on the weight, including A, B and C, respectively. The EBB of A, B and C were 33.989,25.201,23.625 U·g-1 and 24.995,24.481,23.625 U·g-1. The results show that the EBB of grade A is higher than B and C. The higher of grade, the stronger of EBB. This result is consistent with the traditional identification of CDG.Fourthly, study on the correlations between chemical component and EBB:One aspect is Profile-Effect relationship between the HPLC fingerprint of extract and EBB, the other is correlations between Danggui raw material and EBB. Grey Incidence Degree Method (GIDM) was employed for study Profile-Effect relationship between EBB and chemical compound including ferulic acid, ligustilide and polysaccharides in extract. It indicating that ligustilide is higher than ferulic acid and polysaccharides to the contributing of EBB. In research of analyzing for extraction, Partial Least Squares Regression (PLSR) was used. Result shows that senkyunolide I, senkyunolide H, senkyunolide A, E-ligustilide, Z-ligustilide, levistolide A are as positive effect in EBB. In contrast, ferulic acid and Z-butylidenephthalide are into negative effect in EBB. It will help to enhance the EBB if increasing the content of E-ligustilide, senkyunolide A, levistolide A and Z-ligustilide. The results of study on extraction ratio, extract and EBB in CDG showed that there is a methematical relationship between extraction ratio and EBB. The correlation coefficient between product moment correlation coefficient and rank correlation coefficient are found 0.587-0.867. The higher extraction rate in the extract, the higher EBB in extract, and the higher EBB in Danggui samples. In the study of the relation between extraction rate (%) and EBB (U·g-1), we established Fisher discriminant model of CDG and no-CDG. Taking sample back to Fisher discriminant model, the result found that CDG’s coincidence rate was 100%, and the non-CDG was 100% too.In addition, the study also summarized the chemical composition and pharmacological effects of group Danggui, and provided scientific basis for the clinical application in. group of Danggui roots. |
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