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Geoherbs model Salvia miltiorrhiza hairy root \

Author: LvDongMei
Tutor: HuangZuoZuo;LiangRiXin
School: China Academy of Traditional Chinese Medicine
Course: Of Pharmacy
Keywords: Salvia miltiorrhiza hairy root Model Reproducibility Stability Practicality
CLC: S567.53
Type: PhD thesis
Year: 2008
Downloads: 439
Quote: 1
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Abstract


As the essence of Chinese medicine, herbs genuineness both from the historical and cultural attributes related to all aspects of genetic, environmental, and production practice. Said the the formation of Geoherbs is a product of the interaction between genotype and environment, can be formulated: phenotype = genotype environment decorated changed from biology. Genuine medicine is a complex system, each of these factors and their interactions can not all be investigated, the study must have a choice, subjects reduced-order dimension reduction, will it be possible to achieve a breakthrough. What is the relationship whether environmental factors by modifying the genotype of the genuine medicine play a role, the ecological factors Geoherbs secondary metabolites accumulated since the lack of direct and systematic experimental study. At this point, the scientific model because of its features to simplify complex subjects, begun to attract the attention of researchers of the genuine medicine, build genuine medicine research model (vector), the key to become a genuine medicine biology research. Model a wide range of applications in biology. Established on the basis of the basic prototype model, and the use of model analysis and interpretation of biological phenomena reveal the characteristics of the biological nature of law, and to study the relationship between the various factors in a dynamic living systems. As an important means of modern life science research is indispensable, models and model approach is already penetrated into all aspects of life science research, such as model species for genetic engineering research, animal models for medical experiments. Hairy root culture is a combination of a new technology developed in the 1980s, genetic engineering and cell engineering, it is the hair root Ri plasmid of Agrobacterium T-DNA into plant cells containing DNA induced plant cell hairy root formation. Hairy root as an effective long-term good traits of the species, more and more people as the experimental material, while for secondary metabolites. Hairy roots with fast growth and climate, pests and diseases, and other characteristics of geographic and seasonal restrictions. According to incomplete statistics, at home and abroad has 29 families and 52 species of medicinal plants were hairy root induction of long-term hairy root culture system, secondary metabolites. In this thesis, the genuine medicine biological nature study need to be resolved on the basis of the laboratory work, Salvia miltiorrhiza hairy root is selected as the object of study, whether they have the characteristics of the genuine medicine research model of the system evaluation. That hairy roots in the end can not be used as a model to study the Geoherbs? Can reproduce the original botanical characteristics, whether as a stable, repeatable research tool. The model evaluation study, involving many aspects of plant chemistry, molecular biology, pharmacology. Mainly includes three aspects: (1) reproducibility, as a model to study Geoherbs hairy root and its prototype (raw herbs) to examine the similarities between, including chemical composition, toxicity, and efficacy, to clarify the hairy The root of the reproduction of the original medicinal properties. ② stability, the stability of hairy root nuclear DNA, the rol gene genetic stability, chemical composition stability. ③ practicality of hairy roots model is the basis of stability and reproducibility, its usefulness as a model to study investigated whether the hairy root easy drawn, whether they have an excellent experimental physical properties, as the model is easy to prove create and use. Salvia miltiorrhiza hairy root reproducibility of the model (and the similarity of the original plant) the the similarity selection Pharmacopoeia 1.1 chemical composition of index components Tanshinone Ⅱ A , and Dan acid B, Salvia miltiorrhiza hairy roots and Hebei Salvia miltiorrhiza content determination, Salvia of Tanshinone Ⅱ A, herbs content of 0.12%, Dan acid B content of 5.4%; each batch of hairy root of Tanshinone Ⅱ A content of between 0.04% to 0.10%, compared to the average herbs lower; the Sal B content of 3.21% to 7.25%, higher than average herbs. The same time, the similarity analysis of chromatographic fingerprint evaluation system software version 2004A, the fingerprints of the hairy roots and herbs also compare the similarity of 0.948 or more, Salvia miltiorrhiza hairy root chemical composition composition 2 production locations (Hebei lingshou, Sichuan Jiang) Salvia miltiorrhiza compared to no significant difference. Thus, Salvia hairy roots of Salvia herbs have similar chemical composition. 1.2 Acute toxicity Salvia hairy root extract LD 50 63.94g of crude drug / kg, the 95% confidence interval (58.82 to 69.51) g crude drug / kg. Body weight in kilograms is equivalent to the adult daily intake (Salvia clinical dose of 9 to 15 g / day, 0.25g crude drug / kg) 255.76 times. The results showed that the maximum non-toxic dose of Salvia miltiorrhiza hairy root has more than 100 times the human clinical dosage. 1.3 Pharmacodynamic Salvia root for the traditional \Its various formulations used in the treatment of coronary heart disease and cardiovascular disease has received good results. In this study, observed from the whole organ and cell level Salvia miltiorrhiza hairy root protective effect on myocardial ischemia-reperfusion injury. Experiments show that (1) the overall Salvia miltiorrhiza hairy root group can significantly reduce ischemia-reperfusion due to the incidence of VT, VF, lower plasma LDH, MDA content, increased SOD activity; Salvia miltiorrhiza was no significant difference. (2) in vitro experiments found that Salvia miltiorrhiza hairy root can increase coronary blood flow, not only reduces the incidence of arrhythmias, and to postpone the incidence of arrhythmias time, shorten the duration and recovery time, release on LDH, MDA and SOD The improvement results suggest that Salvia miltiorrhiza hairy root treatment on the isolated rat heart ischemia-reperfusion injury may have a protective effect; Salvia miltiorrhiza was no significant difference. (3) cell experiments showed that Salvia miltiorrhiza hairy root compared with a significant increase in the survival rate of the H / R myocardial cells, reduce the content of MDA, increased SOD activity. Each group with the dose increased LDH, MDA decreased SOD activity increased, Salvia miltiorrhiza hairy root can antagonize the rat cardiomyocytes H / R injury, may have a protective effect on the rat cardiomyocytes H / R injury; compared with Salvia miltiorrhiza There was no significant difference. These studies have shown that quite Salvia hairy roots of Salvia miltiorrhiza protective effect on myocardial ischemia-reperfusion injury. 2 of Salvia miltiorrhiza hairy root model Stability of the 2.1 nuclear DNA stability by flow cytometry analysis of nuclear DNA concentration Salvia hairy root, select soybean as an internal reference material, the results indicate that the selected five batches of Salvia miltiorrhiza hairy root samples , the average nuclear DNA content 0.18pg/2C average genome size of 87.8 Mbp RSD 6.6%, nuclear DNA concentration is relatively stable. 2.2 rol gene stability from Salvia miltiorrhiza hairy root culture random optional 10 batches, fresh tissue DNA extraction and PCR amplification, the results show that the exogenous gene rolA can be detected in the genomic DNA of the 10 batches of samples and rolA gene as the target gene the recovery sequencing and the GeneBank login sequence (X12579) 100% homology. 10 batch Salvia hairy root samples rol genes is stable. 2.3 The stability of the chemical composition through the fingerprint of Salvia miltiorrhiza hairy root, the stability of its chemical composition was analyzed. Chromatographic fingerprint evaluation system 2004A software similarity analysis, can be seen from the calculation results, the similarity of 10 batches of hairy roots in more than 0.970, indicating that the chemical composition of different batches of Salvia miltiorrhiza hairy root samples the difference was not significant. The same time, by different batches of Salvia miltiorrhiza hairy root growth analysis showed that different batches of no significant difference in growth rates, relatively stable. Salvia miltiorrhiza hairy root model practical research 3.1 Salvia hairy root growth cycle of 6,7-V medium Salvia miltiorrhiza hairy root culture, harvest, measured in weeks 2, 4, 6, 8, 10 the amount of growth. High Performance Liquid Chromatography Salvia hairy root Tanshinone Ⅱ A , and Sal B content. The hairy root culture on solid medium, the harvest should be six weeks more appropriate, the hairy root growth factor, the active ingredient is relatively high, relatively speaking. Thus, Salvia hairy root culture cycle is short, 6-8 weeks to harvest, with rapid propagation characteristics, and rapid accumulation of secondary metabolites. 3.2 mineral nutrient elements in Salvia miltiorrhiza hairy root growth and accumulation of active components present study, addition and subtraction formula of mineral nutrient elements on the basis of 6,7-V medium, medium-depth study of nitrogen, potassium, iron, the concentration of magnesium single mineral nutrients, Salvia miltiorrhiza hairy root growth and accumulation of active components. The results show that the nitrogen on the growth of hairy roots greatest impact, followed by potassium. Iron and magnesium Salvia miltiorrhiza hairy root growth. Judging from the accumulation of the active ingredient content, low nitrogen and fully treated iron and magnesium conducive to of Tanshinone Ⅱ A of cumulative. High nitrogen, high potassium is conducive to the accumulation of Sal B. Four elements concentration in the medium changes will produce different degrees of stimulation of hairy root, thereby affecting the growth and accumulation of the active ingredient, thus proving that Salvia hairy root stimulation sensitivity of mineral nutrients, therefore, hairy roots as a model analysis deeper Road estate area of ??environmental factors. Conclusion The results of this thesis show that: Salvia miltiorrhiza hairy root has the characteristics of a good reproduction of the original herbs can replace the original herbs used as a model, each batch of genetic, chemical stable, easily obtained, growth and reproduction of fast, with excellent test substance , as a model having a certain degree of practicality. Evaluation of Salvia miltiorrhiza hairy root that hairy roots can be used as a model. Hairy roots as a model, the genuine medicine phenotype and habitat and the relationship of the genotype as the starting point, in-depth study of authentic herbs responses to environmental factors, the function of the gene expression regulation mechanism. This paper first proposed the genuine medicine research models, and hairy root system as a model to evaluate experimental platform for genuine medicine, the innovation of this paper.

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