| The herb of Chinese Danggui?CDG?is come from the root of Angelica sinensis?Oliv?Diels,which is a perennial herb belonging to the family Umbelliferae to own to the function of replenishing blood,promoting blood circulation,regulating menstruation,alleviating pain,and lubricating the bowels to relieve constipation.Diversiform standards in commodity classes of CDG are the symbol of their quality.But the content of the known chemical composition in CDG can't effectively assess ITS quality.In the world herbal market,there is a confusing usage for various Danggui roots including Chinese Danggui,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.?.The differences between the chemical composition and blood tonics bioactivity in CDG samples were quantified and compared,the correlation between the grade,chemical composition and blood tonics bioactivity were analyzed in this thesis.Meanwhile,by DNA barcode and microscopic identification?including micro semi-quantitative technique?,the five various species of Danggui roots were identified and compared.This study is to provide the basis for the classification of CDG and to promote the reasonable uses of various Danggui roots.The main research works as fellows:First,comparison of the main pharmaceutical compositions and blood tonics bioactivity among the different grades of CDG samples:7 CDG samples were collected from various cultivating areas in China and divided into five grades by the weight of single root,i.e.first?5070 g?,second?2540 g?,third?1020 g?,four?69 g?and five grade?15 g?.The contents of ligustilide,free ferulic acid and total ferulic acid in these 22 various grade samples were determined by HPLC.The content of polysaccharide was determined by 3,5-dinitrosalicylic acid method.As the index,the blood tonic bioactivity was measured with cyclosporine A,and the blood coagulation index of these samples was determined.Correlation analysis was used to analyze the correlation coefficient between the content of the active ingredient,the blood tonic bioactivity index and the standards of different grades of CDG Results:?1?blood tonic bioactivity from high to low order:first-grade(33.99 U?g-1,n=2),second-grade(25.98 U?g-1,n=6),fourth-grade(23.90 U?g-1,n=5),fifth-grade(15.38U?g-1,n=4),and the blood tonic bioactivity index of Danggui could evaluate the quality of CDG in different grades and provide the basis for the grading of CDG;?2?Total ferulic acid content from high to low order:first-grade(1.27 mg?g-1,n=2),second-grade(1.21 mg?g-1,n=6),fifth-grade(1.15 mg?g-1,n=4)and fourth-grade(1.04 mg?g-1,n=5),there might be some correlation between grade and Total ferulic acid content,but rank correlation analysis showed no significant difference between them;?3?Free ferulic Acid content from high to low order:first-grade(0.55 mg?g-1,n=2),third-grade(0.34 mg?g-1,n=5),fifth-grade(0.28mg?g-1,n=4)and fourth-grade(0.16 mg?g-1,n=5),and the content of free ferulic acid was significantly correlated with the grades of CDG.But storage and processing were influence on the content of Free ferulic Acid,the Free ferulic Acid was a small part of the Total ferulic acid,so that could not provide the basis for the grading of CDG;?4?Polysaccharide content from high to low order:fourth-grade(85.59 mg?g-1,n=2),fifth-grade(81.94 mg?g-1,n=6),first-grade(75.50 mg?g-1,n=4)and first-grade(66.31 mg?g-1,n=5),there might be some negative correlation between grade and Polysaccharide content,but rank correlation analysis showed no significant difference between them;?5?The different grades of CDG's Ligustilide content from high to low order:first-grade(13.05 mg?g-1,n=2),second-grade(12.91 mg?g-1,n=6),fifth-grade(12.36 mg?g-1,n=4),third-grade(11.05 mg?g-1,n=5)and fourth-grade(10.80 mg?g-1,n=5),and the grades of CDG was not closely related to the content of Ligustilide.Second,identification of various Danggui roots by DNA barcodes molecular technology.A total of 41 samples of Danggui roots were collected from China,Japan,Korea,Austria,Poland and Germany.Using DNA barcodes technology to extract the DNA of these samples.The ITS1,ITS2,psbA-trnH and rbcL sequences were selected for amplification and sequencing.And then the sequences were spliced and collated.Finally the mutation loci analysis,the genetic distance calculation and the construction of the Neighbor-Joining?NJ?tree were carried out and analyzed.Results:?1?CDG and LR were homogeneously clustered on the NJ tree based on ITS1,ITS2and psbA-trnH sequences,and the rbcL sequence was basically the same?2?JDG and the AAR were homogenized in four NJ trees.Based on the ITS1 and ITS2 sequences,the JDG and the AAR were 52%moderate support and 91%High support rate.?3?The extraction rate of ITS2 and psbA-trnH sequences was higher than that of ITS1 and rbcL sequences.The results showed that the applicability of DNA barcodes identification was"ITS2,psbA-trnH,ITS1",and rbcL was not suitable for the identification of the different species of Danggui roots.CDG and AAR The phylogenetic relationship of the medicinal herbs is close to that of the JDG and the AAR.With the length of ITS sequences and rbcL sequences are difficult to maintain intact over a long period of time,while relatively short ITS2 and psbA-trnH sequences are more likely to be preserved.Thirdly,identification of various Danggui roots by microscopic technique:34samples of these Danggui roots were collected from China,Japan,Korea,Austria,Poland and Germany.Microscopic identification method was used to identify the different species of Danggui roots.The starch granule constants of each sample were determined by micro semi-quantitative method.Based on the data of polysaccharide content of these Danggui roots,the correlation between constant and polysaccharide content was studied The results showed that:?1?The microscopic characteristics of various Danggui roots include:vessel,cork cells,fusiform parenchyma cells and starch granules.The type,shape and size of starch granules were the most significant distinguishing features in the microscopic characteristics of various Danggui roots.?2?The average of starch granule constant of different Danggui roots from high to low order:AAR(2.17×109 mg-1,n=7),EDG(1.19×109 mg-1,n=5),JDG(9.17×108 mg-1,n=11),CDG(1.49×103 mg-1,n=5),KDG(7.68×102 mg-1,n=6).?3?The different species of Danggui roots starch granule constant and polysaccharide content Kendall And Spearman correlation coefficients were 0.533?P<0.01?and 0.640?P<0.01?,respectively.The results showed that the starch granule characters and quantity could be used to identify the different species of Danggui roots.The starch granule constants and polysaccharide contents of the different species of Danggui roots had significant positive rank and correlation.In addition,the advancement of DNA barcodes research on Chinese herb medicine is reviewed. |
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