| How to evaluate the quality of Chinese herbal medicines scientifically has always been the focus. Chinese herbal medicines are different from the chemicals, it is a complex system with multi-function and multi-component, at the same time affected by the biodiversity, even the same medicinal plants, also has the different efficacy components and proportion. We call this phenomenon as the efficacy component type differentiation. Therefore, only by measuring a certain or some sort of component content to evaluate the quality of Chinese herbal medicines have certain one-sidedness and limitations, but combined with efficacy components content determination and efficacy component type study to evaluate the quality of Chinese medicinal materials is a better way. Rhubarb is commonly used in Chinese herbal medicines which has many Pharmacologic actions, such as purgation, anticoagulation, hemostasis and so on. Rhubarb is not only a typical represent of the "multi-function, multi-component" Chinese herbal medicines, but also a represent of the Chinese herbal medicines which have a relatively clear efficacy component.In our study, we selected cultivated rhubarb which grew up in the same environment as the materials for study. First, we used HPLC-DAD method to determinate the content of efficacy components, then divided the efficacy component type based on the difference of efficacy components content and proportion, and received the core sequence of Chalcone synthase, Aloesone synthase and Leucoanthocyanidin reductase by using homologous cloning method. Functional gene expression was evaluated by real time fluorescence quantitative method, finally we interpreted the molecular mechanism of the efficacy component type’s formation in rhubarb from the perspective of the functional gene expression difference. We lay the foundation of theory and technology for breeding special efficacy rhubarb varieties and high-quality varieties of rhubarb.The main research results of our study are as follows:1. We established the method for simultaneous determination of twelve active constituents content in rhubarb by HPLC-DAD, including five free anthraquinone compounds (antibacterial constituents),three anthraquinone glycosides and two anthrone glycoside compounds (purgative constituents), one flavan-3-alcohol compound (anti-clotting constituent) and one tannins compound (styptic constituent). Use0.05%phosphoric acid water and acetonitrile as mobile phase in gradient elution, can obtain good separation effect within115min.2. Base on rich intraspecific variation of rheum tanguticum, we devided into eight plant phenotypes according to their leaf morphology, flower color and bark color. 3. Twelve active constituents’content of forty rhubarb including eight plant phenotypes were determined by HPLC-DAD method. SPSS software was used for statistical analysis, the results of One-way ANOVA showed that, the content of aloe emodin, chrysophanol and physcion had a significant difference (p<0.05) while the content of free anthraquinone had a significant difference(p<0.1) in eight plant phenotypes. Based on the results of One-way ANOVA, could indicated that the difference of plant phenotypic had a correlation with the difference of active constituent content.4. Combined with the difference of efficacy component content and proportion of rhubarb in eight plant phenotypes, we divided into four kinds of efficacy component type. E-type and F-type sample could be used as potential purgative herbs; J-type sample could be used as potential antibacterial herbs; C-type and J-type sample could be used as potential anti-clotting herbs; B-type and J-type sample could be used as potential styptic herbs.5. According to the statistical analysis results for four kinds of efficacy components content of rhubarb which grew in the same environment, we found that the anthracene glycosides content difference about10times between the different individuals,and the free anthraquinone content difference of about10times, catechins difference of about2times, gallic acid content difference of about11times. In addition, based on the analysis results of pie chart, we found that the content of anthracene glycosides was the highest, followed by catechins, and the free anthraquinone compounds, the content of gallic acid was the lowest. This result was conformed to the traditional views which considered rhubarb as the best drugs for treating constipation.6. According to the results of PCA analysis, we screened fifteen samples for subsequent molecular biology experiments. In addition, combined with the difference of efficacy component content and proportion, we divided into four kinds of efficacy component type. Sample B-1,B-2,D-2,I-1,J-1could be used as potential purgative herbs; Sample I-1, J-1could be used as potential antibacterial;Sample D-1, F-3could be used as potential anti-clotting herbs; Sample B-2,I-1, J-3could be used as potential styptic herbs.7. In our study, core fragment of three functional genes and one reference gene were obtained by homologous cloning method. The four core fragments belong to chalcone synthase (CHS), aloe loose synthase (ALS) and leucocyanidin reductase (LAR), respectively. Combined Blast analysis and amino acid sequence analysis, could confirm that the core fragment obtained belong to the objective genes, had a length of493bp,364bp,639bp and333bp, respectively.8. We established the method for determination of CHS, ALS, LAR gene expression by real-time PCR, detected fifteen samples, and used beta-actin as reference gene to correct gene expression. The results showed a larger difference of three functional genes’ expression between different samples. Addition, LAR gene and CHS gene expression was relatively higher than the ALS gene expression, but almost no expression of the ALS gene in some samples.9. The results of correlation analysis between functional gene expression and the content of twelve active constituents or four kinds of effect component showed that CHS gene expression had a moderate negative correlation with the content of physcion, and also had a correlation with emodin-8-β-D-glucopyranoside, emodin, aloe-emodin, rhein, chrysophanol, anthraquinones and catechin; LAR gene expression had little correlation with the content of catechin; ALS gene expression had correlation with the content of emodin-8-β-D-glucopyranoside. Based on the results of correlation analysis, could indicated that the difference of functional gene expression and proportion would cause the difference of active constituent or effect component content. |
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