| Prunella vulgaris L. is a perennial herb of Labiatae whose dry spike is used for medicine in china. It is a very important Chinese traditional medicine recorded in the Pharmacopecia of People’s Republic of China (ed.2005). It has the effect of antihypertensive, Antihyperglycemic, anti-AIDS, and cancer therapy. There are widely used in mang fields, which contains pharmaceutical, chemical, food, beverages, and other fields. At present, P.vulgaris mainly distributed in the Jiangsu, Anhui, Zhejiang, Henan, and other provinces. But the problem of intermix germplasm has seriouely effected on the stability of quality. It is important that the quality of medicine is a premise of effective Chinese herbal. However, different region of germplasm have been mixed, which have seriously affected the pharmacodynamic stability. Genetic characteristics, which will ensure the qulaiity of internal medicine, decide the quality of medicines, medicinal materials, and the growing environment of medicines. After a whole investigation in ecological distribution areas and growth habits of P. vulgaris populations, which contained 40 typical wild accessions and 1 planting of the collection from the typical habitat were selected as experimental materials, of which contained 15 representational provinces of China. On the base of elucidating the genetic characteristics of P. vulgaris germplasm and comprehensive evaluation of medicinal quality, in order to discuss the relationship between the genetic diversity of species and medicinal quality, and select stable and high quality P. vulgaris populations to ensure safe and effective of drugs in the clinical, It would provide a theoretical basis on the protection of P. vulgaris germplasm and identification of excellent accessions, to promote development and utilization of P. vulgaris germplasm and modern Chinese medicinal industry. The research contents and results were as follows:(1) The study was to utilize 16 morphological indexs, in order to preliminary assessment genetic variation of morphological characteristic among 41 P. vulgaris germplasm populations. The results were as fellows:different populations of P. vulgaris had rich genetic variation on the morphological traits. The Principal Component Analysis (PCA) has elementarily determined the main factors of many phenotypic differences among traits, which contains stem height, leaf index, corolla length, corolla throat width, calyx length, lip width, lip width, and ear length of main ear. The populations of P. vulgaris could be divided into 3 morphological groups by means of 16 traits, that is, The 1st group: the type of prematurity and hight plant; the 2nd group:the type of intermediate; the 3rd group:the type of dwarf and late-maturity.(2) Pollen grains of 26 populations in P. vulgaris were observed and compared by means of Scanning Electron Microscope (SEM). The results were as fellows:the pollen grains were mainly spheroidal to flat spherical in shape, with long elliptical or wide elliptical in equatorial view and suborbicular in polar view. There were 6 germinationg colps, which are radial symmetry and many particles in germinationg colps. The ornamentation of exine showed typical reticulate sculpture. And the lumina shape was from irregulation to round.The characters of P. vulgaris populations were different in pollen size, pollen shape, and pollen exine ultrastructure. The major differences of reticulate ornamentation among different populations lie in ridge width,cellular shape, cellular diameter and cellular density.And its evolution sequence might be from dense to sparse, from uniform to uneven, from regulation to irregulation, from width to narrow.(3) A collection of 26 P. vulgaris populations have been screened with ISSR primers in order to analyze their genetic diversity. PPL for all accessions was 86.9%, He was 0.29 in average. PPL among the different populations ranged from 11% to 50% and average of 45%. PPL for the highest and lowest populations was Jianou, Fujian and Kunming, Yunnan provinces, respectively. A relatively high level of genetic differentiation was detected among populations with Gst (0.5177) analysis, which indicated the genetic variation among populations accounted for 51.77% of the total variation, and genetic variation within populations accounted for 48.23% of total variance. Analysis of cluster showed that all the 26 populations could be distinguished by ISSR markersand divided into 3 groups according to UPGMA method when the similarity coefficient of 0.79. The first group which were collected by Jiangsu province, the second group included Anhui,Guizhou, Jiangxi and Zhejiang provinces, the third group contained the population of the southwest region, The results also showed that the genetic diversity in P. vulgaris populations based on ISSR genetic similarity was correlated with geographic distribution.(4) Analysed genetic diversity and genetic structure of P. vulgaris populations using SRAP molecule marker technology. The results showed that at the species level, PPL was of 87.93%, He was 0.21 in average. At the populations level PPL is from 22% up to 67% and average of 32.27%, the highest Huzhou population and Kunming the lowest population. Gst was 0.4850, which indicated that the genetic variation among populations accounted for 48.50% of the total variation, and genetic variation within populations accounted for 51.50% of total variance. Analysis of cluster showed that all the 26 P. vulgaris populations could be distinguished by SRAP markers and divided into 3 groups according to UPGMA method. The results also showed that had consistency, which also had slightly difference, base on clustering analysis of the results with ISSR.(5) To understand variation type within species, we tested the ribosomal ITS base sequences and analysed the characteristics of them. We obtained rDNA ITS complete sequences from 26 P. vulgaris populations. The length of ITS was 669 bp. The length of ITS1,5.8s and ITS2 were 260bp,155bp, and 254bp, respectively. There are a whole ITS site 7 mutation, ITS1 region had four mutation sites, which are information sites. ITS2 region has three variants, and the variants are single nucleotide point.5.8S had very conservative and no variable sites. It constructed the phylogenetic tree using Neighbor-adjacent method, showing that there are at least five variations in P. vulgaris populations. Jinxian’ planting population constituted a monophyletic clade, with all distinction between the other wild populations.(6) Ursolic acid, oleanolic acid, flavonoids, polysaccharides, and the biological active ingredient as the evaluative indicators to evaluate the quality of medicinal herbs P. vulgaris. The results show that ursolic acid as the evaluative indication was a more reasonable, objective, and fully reflect the overall quality and control the level of Prunella vulgaris. Different P. vulgaris populations had similar Chemical constituents, but there were significant differences among contents. The range of contents of ursolic acid, flavonoids, and polysaccharides were from 0.13% up to 0.40%, from 5.29% up to 9.00%, and from 2.16% up to 10.29%, respectively. The highest content was from Luding of Sichuan. Cluster dendrogram reveal that 36 populations were divided into five categories, and category B and E should be strengthen in breeding work. It provided the valuable basis for quality assessment of P.vulgaris and development production of genuine Chinese medicine material.(7) The contents of 13 elements in 35 P.vulgaris samples were determined by means of ICP-AEC. The results showed that different populations of P. vulgaris contained much of macro elements and trace elements, and their distribution characteristics had more consistent among different populations, the highest contents was Ca and K, followed by Mg and P, the lowest contents followed by Fe, Al, Sr, Mn, Zn, Ti, Cu, Pb, and Co. However, there were significantly difference among different populations of P. vulgaris in the contents of all elements, which may be associated with different germplasm genetic factors and the various elements of the soil environment, which is closely related to the abundance in the soil. Elements Pb, Fe, Cu, Ti, Al, and Co were significantly positive correlation with each other, and maybe exist cooperate with different elements in P. vulgaris. Four principal components which accounted for over 68.73% of the total variance were extracted from the original data. The analysis results show that Co, Fe, Cu, Ti, Al, P, and Mg may be the characteristic elements in P. vulgaris.(8) Active ingredient, commodity characteristics, and inspection items as the evaluative indicators to comprehensively evaluate the quality of medicinal herbs of P. vulgaris populations by the means of PCA and Analytical Hierarrchy Process (AHP), in order to provide a theoretical basis of quality control on P. vulgaris. The results show that PCA and AHP can reflect the comprehensive sample information, and be used in the quality of evaluation indicators of traditional Chinese medicine. The two methods showed that the contents of active ingredient of Luding and Mianyang populations were much higher, which can suit for quality production because these had short ears; followed by Xuanwu, Zhenjiang, Yizheng, Xuyi, Hefei Chuzhou population, which had the characteristics of the long ear, high product, and excellent comprehensive traits, these can suit for large-scale cultivation; the lowest contents followed by other populations.(9) To provide a theoretical basis of rational development and utilization of P. vulgaris germplasm, this study had comparatively analysis the contents of active ingredient in different parts of P. vulgaris. The results showed:different parts of P. vulgaris had obvious difference. The contents of ursolic acid in different parts, which followed by ear, the whole plant, leaves, stem, and root, was the most abundant inorganic elements in root, which contained Pb, Cu, Fe, Co, Al, Mn, and Ti, in contrast the ear. Therefore, the ear will be fully utilized, at the same time, the other parts also will be actively used, in order to expand the source of P. vulgaris and achieve sustainable development. |
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