Salvia miltiorrhiza is one of the earliest and most widely used drugs in traditional Chinese medicine, which is widely used in clinic to treat coronary heart disease, angina pectoris, ischemic stroke and other diseases. At present, there are more than 100 kinds of Chinese patent medicines made from salvia miltiorrhiza in China, among which compound Danshen dripping pills have entered the mainstream drug market in 16 countries. In 2005, salvianolic acid salt and salvianolic acid salt for injection were listed as new Chinese patent medicines. The development of these drugs not only fully explored the medicinal value of Salvia miltiorrhiza, but also expanded the market demand of Salvia miltiorrhiza, resulting in a shortage of Salvia miltiorrhiza supply. However, since the 1970s, the output of Salvia miltiorrhiza in China has been in short supply, and there is no unified cultivated variety of Salvia miltiorrhiza in China. It is urgent to cultivate new varieties of salvia miltiorrhiza. Therefore, the triploid provided by this project.
Nankai University has established the first triploid sports species system of Salvia miltiorrhiza in China, and established cultivated and wild Salvia miltiorrhiza germplasm resources gardens in the main producing areas of Salvia miltiorrhiza in China. It has studied Salvia miltiorrhiza from the aspects of morphology, cytology and molecular biology, and cultivated a series of new germplasm and varieties of Salvia miltiorrhiza genome polyploid. The specific research work is as follows:
A total of 17 autotetraploids of Salvia miltiorrhiza were created, and there were about 400 populations in the M2 generation of tetraploid, which could meet the needs of triploid Salvia miltiorrhiza cultivation.
The heterosis rate of 180 triploid salvia miltiorrhiza f 1 was analyzed. The highest yield per plant was 1 150g/ plant, and the heterosis rate among plants was 100%.
Salvia miltiorrhiza Bge. in 12 provinces and cities in China was transformed into triploid, and new triploid varieties of Salvia miltiorrhiza Bge. in 7 regions were selected, which made preparations for popularizing triploid salvia miltiorrhiza Bge. in China.
The natural hybrid seed production method of triploid salvia miltiorrhiza was established, which laid the foundation for large-scale triploid hybrid seed production.
In June, 2007, the site acceptance meeting of "Breeding of New Varieties of Salvia miltiorrhiza Bunge with Polyploid Genome" was held in Jixian Experimental Base, Tianjin. The root growth and yield of triploid salvia miltiorrhiza passed the acceptance test. Among the 42 triploid Salvia miltiorrhiza plants tested, the average yield of root medicinal materials per plant reached 465,438 06.5 g/plant, which was 3.3 times higher than that of diploid control.
Salvia miltiorrhiza is widely distributed in China, which is resistant to thin soil ridges and can grow in various soil types. It is a good helper for building a new countryside and making farmers rich.
The new varieties cultivated by Nankai University have the following characteristics:
The heterosis of triploid salvia miltiorrhiza can be maintained for more than 10 years. Triploid salvia miltiorrhiza has enhanced disease resistance and strong adaptability, and can be planted all over the country. The yield of cultivated salvia miltiorrhiza can be greatly improved through the heterosis produced by new triploid varieties.
The average yield per mu of cultivated salvia miltiorrhiza is 600- 1000 kg, and the yield per mu of the new triploid salvia miltiorrhiza promoted in this project can reach more than 2000 kg, which significantly increases the benefits.
The basic research of this project, "Research on Genetic Improvement and Germplasm Innovation of Salvia miltiorrhiza Bunge", was supported by the National Natural Science Foundation of China. The cultivation method of triploid Salvia miltiorrhiza Bunge has applied for an invention patent, and the patent (application) number is 2007 1006 1227.7.
New production technology of anticancer drug paclitaxel
Paclitaxel is a broad-spectrum natural anticancer drug, which exists in the bark of Taxus chinensis. Since the listing of 1992, paclitaxel and its derivative docetaxel have been the most popular anticancer drugs, with annual sales of about 2-3 billion dollars and increasing demand. In addition to anti-tumor, new uses of paclitaxel have been found in clinic, such as anti-rheumatoid arthritis. Anti-malaria drugs are also effective for stroke, Alzheimer's disease and congenital polycystic kidney disease, which further expands the market. Paclitaxel is already a generic drug product, and the barriers to intellectual property protection have been lifted.
Existing industrial production technology
The global taxol producers represented by Bristol-Myers Squibb, the largest pharmaceutical company in the United States, all extract and purify taxol from the bark of Taxus chinensis. Because of its extremely low content, the global total output is far from meeting the market demand, so paclitaxel is expensive. Many manufacturers plant a large number of yew to solve the shortage of raw materials, and dozens of small-scale manufacturers in China produce taxol by planting yew.
The chemical synthesis method was completed by two American laboratories in 1994. However, paclitaxel has complex structure, low synthesis efficiency and high cost, so it can not be used in industrial production. Although the cell culture technology of Taxus chinensis has been spared no effort for more than ten years, the possibility of application is unpredictable because it can not solve the problem of expansion (Taxus chinensis cells die after expansion).
Production new technology
Nankai University has independent intellectual property rights in the new technology of taxol production, that is, large-scale fermentation of fungi to produce taxol, with high-yield strains, and its technical level is in the leading position in the world, which has met the economic requirements of industrial production.
Research foundation
This project is supported by the 863 key project in the 11th Five-Year Plan (project number: 2007AA02 150 1), and the pilot test under the best conditions has been completed, with low investment cost and preparation cost.
Figure) Control of Taxol-producing Fungi
Biological activity detection: A. Taxol produced by fungi, B. standard sample, C. solvent control.
Preparation of high purity monomer alkaloids from tetrandrine
Background and main uses of results and projects
Tetrandrine is an important Chinese herbal medicine, which has the effects of diuresis, detumescence, expelling wind and relieving pain. Tetrandrine is the main component of tetrandrine, which has the functions of diminishing inflammation, relieving pain, lowering blood pressure and resisting silicosis. With the rapid development of cell separation technology and molecular biology research, pharmacological research has gradually penetrated into the cellular, subcellular and molecular levels. Tetrandrine (Tet) is a natural non-selective calcium channel blocker and a natural antagonist of calmodulin, which affects the transmembrane transport and intracellular distribution of calcium ions. Therefore, Tet has a wider pharmacological action and clinical application prospect, and has attracted great attention in preventing and treating hepatocyte fibrosis and hypoxic pulmonary hypertension, especially in reversing multidrug resistance in tumor chemotherapy.
At present, the preparations of tetrandrine in the market include tablets and water injections, both of which are symbolic varieties upgraded to the national standard, which have the shortcomings of low quality standards and simple quality control methods. In addition, in order to ensure the stability of the main drug, a large number of antioxidants and complexing agents are added to the water injection, which affects the safety of clinical medication. Therefore, we adopted a new raw material extraction process and established a high-purity tetrandrine quality standard. The traditional water injection form was changed, and a new freeze-dried powder injection was developed, which made the storage of the main drug more stable, did not contain any stabilizer, and increased the safety of clinical medication.
Stephania tetrandra contains alkaloids such as tetrandrine, tetrandrine, tetrandrine, berbamine and tripterine alkaloid. Because of their similar structures and properties, it is difficult to separate and purify them. The existing methods have the following problems: 1) most of the extraction processes need to use a large number of highly toxic solvents such as benzene and chloroform in an open environment, which is very harmful to operators and the environment; 2) The sample loss is more when the activated carbon is decolorized; 3) The method is complicated, the operability is poor and the production cost is high. Therefore, we established a resin separation and purification process, changed the traditional method of synthesizing macroporous adsorption resin, and introduced the interaction of hydrophobic interaction, dipole interaction, hydrogen bonding interaction and chemical interaction of functional groups to synthesize a weak polar adsorption resin, which was used to separate tetrandrine and tetrandrine alkaloids, and prepared tetrandrine bulk drug with a content of more than 98%.
Brief introduction of technical principle and process flow
By changing the types and amounts of monomers, pore-forming agents and crosslinking agents, a macroporous adsorption resin with non-polystyrene skeleton was synthesized, and the required functional groups could be introduced into the resin skeleton during polymerization. Because this resin skeleton avoids the introduction of benzene rings, the π -π interaction between resin and adsorbate molecules is greatly weakened, and the adsorption of tetrandrine is weak. On the other hand, due to the introduction of special functional groups into the resin skeleton, it can interact specifically with phenolic hydroxyl groups in tetrandrine molecules. Under the synergistic effect of various adsorption forces, the adsorption capacity of the resin for tetrandrine is greatly improved, and tetrandrine with weak binding force can be separated from the resin column by selecting appropriate eluent.
Technical level and patents
The effects of operating conditions such as adsorption solution concentration, adsorption speed, eluent type and elution speed on the separation and purification effect were investigated in detail, and the best extraction process was established, so that the quality of tetrandrine extract was stable and the content reached over 99%. Obtained the approval of raw material drug production issued by China Drug Administration, and developed the freeze-dried powder injection. Two invention patents were applied: patent (application)No.: 2007 100568.
Application fields and key technologies that can be solved for the industry
The application field is medicine and material industry, which can provide a large number of low-cost monomer alkaloid samples, conduct more in-depth pharmacological and pharmacodynamic research and chemical modification, and prepare natural drugs with anticancer activity.
The separation problem of two alkaloids with similar structures in tetrandrine was solved, and the production process of high purity tetrandrine with low cost, simplicity and environmental protection was established.
Application prospect analysis
In recent years, it has been found that tetrandrine, an effective alkaloid component in tetrandrine, as an effective natural calcium antagonist, not only can directly inhibit tumor cells, but also can be used as an effective tumor drug resistance reversal agent in clinic, which has important clinical application value and broad application prospects for overcoming multidrug resistance of tumor cells and improving the efficacy of anticancer drugs.
Preparation of high purity ginkgolides
Background and main uses of results and projects
In the development of natural plant medicine, the modern medicinal research of Ginkgo biloba leaves is undoubtedly one of the hot spots. In the early 1970s, Ginkgo biloba extract EGb76 1 with clear quality standards, that is, a mixture of flavonoid glycosides (with a content of more than 24%) and terpene lactones (with a content of more than 6% as the sum of ginkgolides and ginkgolides), was produced on a large scale for the first time in Germany, and developed into a stable and effective drug for the treatment of cardiovascular and cerebrovascular diseases.
At present, the raw materials of Ginkgo biloba preparations listed at home and abroad meet the quality standard of EGb76 1. However, with the deepening of research, a large number of pharmacological and clinical experiments have proved that the pharmacological effects of flavonoid glycosides and terpene lactones, the main effective components in Ginkgo biloba extract, are not exactly the same. Therefore, new drugs with single active ingredient have become the goal pursued by developed countries in Europe and America in recent ten years. The research group led by French scientist P. Braquet studied the pharmacological activities of ginkgolides, and found for the first time that ginkgolides are a very effective natural antagonist of platelet activating factor (PAF), which is an endogenous phospholipid produced and secreted by platelets and various inflammatory cells, and is the most effective platelet aggregation inducer found so far, with a wide range of biological activities. PAF not only causes thrombosis and participates in the occurrence and development of cardiovascular diseases, but also is closely related to the occurrence of many other diseases, such as asthma, shock, inflammation and rejection during organ transplantation. Therefore, since the 1980s, the research on PAF antagonists has been a hot spot in the search for effective and efficient drugs for these diseases. In addition, recent studies have found that besides ginkgolides, ginkgolides also include another kind of compounds, namely ginkgolides. It can effectively resist the aging of nerve endings, and has obvious curative effect on organic nervous system diseases, especially depression, and has no toxic or side effects. Ginkgolide has high bioavailability when taken orally, and can quickly enter the blood within 1-2 hours. It is no longer a problem to treat general diseases, but its efficacy is slow when used in first aid. Therefore, in recent years, the international community is keen on the development of ginkgolide injection, which is beneficial to the development of ginkgolide injection.
Therefore, according to the structural characteristics of flavonoids and terpenoids, we designed and synthesized an adsorption resin with hydrogen bonding, hydrophobicity and screening synergy. Flavonoids and lactones were successfully separated, and ginkgolides with a content of more than 90% could be obtained by one step of adsorption and elution.
Brief introduction of technical principle and process flow
By changing the reactive monomer and crosslinking agent, the required functional groups are introduced into the resin skeleton during the resin polymerization process, and the content of functional groups on the resin is controlled by changing the amount of reactive monomer containing the required functional groups, so that it can specifically adsorb ginkgo flavonoids. Because the introduction of benzene ring is avoided, the polarity of the resin is large, and the adsorption capacity of ginkgolides is greatly weakened, so that ginkgolides and flavonoids can be effectively separated. A series of resins with adjustable pore size can be prepared by changing the initial crosslinking degree of the resin and making it undergo post-crosslinking reaction at different swelling degrees. Through the screening of adsorption experiments, the resin with suitable pore size can be obtained to remove unknown impurities from the crude extract of ginkgolides, so that the content of ginkgolides can reach 90%.
The effects of operating conditions such as adsorption concentration, adsorption speed, eluent concentration and elution speed on the purification effect were investigated in detail, and the best extraction process was established.
Application field, technical level and key technologies and patents that can be solved for the industry.
The application field is medicine and material industry, which can provide low-cost and high-purity ginkgolide extract (the purity of total lactone is higher than 90%), and can further develop ginkgolide freeze-dried powder injection for clinical treatment of cerebral infarction (cerebral thrombosis and cerebral embolism) with phlegm and blood stasis blocking meridians. Patent (Application)No.: 2007 1005753.6.
Application prospect analysis and benefit prediction
Using this new adsorption resin to prepare ginkgolide extract has simple process and can be directly used in industrial production. Compared with the solvent extraction method, the production cost of this method is greatly reduced, so the extract itself has strong market competitiveness, and the ginkgolides injection developed by this method should undoubtedly have stronger competitiveness and broader application prospects.
Ultra-efficient nano-polymer adsorption material and its application in pharmacy
Background and main uses of results and projects
This project has developed a new technology for preparing polymer nanoparticles and a platform for preparing ultra-efficient nanoparticles. The types of polymer nanoparticles prepared include: a series of polymer nanoparticles with monoene and diene compounds as monomers. The morphology of microspheres is spherical, saccular, spindle-shaped and core-shell structure. The characteristics of ultra-efficient microspheres obtained by this platform are that they do not contain any surfactants and ionic groups, and nanoparticles can exist stably in aqueous solution without agglomeration. The particle size of the sphere can be controlled between 30 and 800.
Ultra-efficient nano-adsorption materials have important application prospects in the pharmaceutical field. The experimental results of adsorption of various drugs show that these nanoparticles have extraordinary swelling and adsorption ability. In addition, they have super purification ability for the remaining mother liquor of drug crystallization, and can be used for the extraction of precious drugs and the purification of highly toxic substances. At the same time, because of its uniform and adjustable particle size, it can also be used as a polishing agent in chemical mechanical planarization technology of computer chips.
Brief introduction of technical principle and process flow
Preparation of polymer nano-particles and nano-particles by self-assembly of polymers or their monomers. The method is suitable for preparing various polymer materials, such as polystyrene, polyacrylate, polypyrrolidone and biodegradable polymer materials.
Engineering level
The granular structure can form capsules, spheres and spindles; The particle size is between 30 and 800 nm, and the particle size is adjustable; The size can be highly uniform and the size distribution coefficient can be within 1.005. This kind of particle has high solvent retention, high swelling and high adsorption. Patent (authorization)No.: ZL 200310119366.2.
Application prospect analysis and benefit prediction
Ultra-efficient spherical particles are characterized by not containing any surfactants and ionic groups, and nanospheres can exist stably in aqueous solution without agglomeration. The spherical particle size can be controlled at 30-800nm, and the spherical surface is smooth, with single component and monodisperse. They can be used to prepare biodegradable materials and have important application prospects in the pharmaceutical field. The experimental results of many drugs show that these nanoparticles have extraordinary swelling and adsorption ability and can be used as drug sustained-release carriers. It also has strong purification ability for the residual mother liquor of drug crystallization, and can be used for the extraction of precious drugs and the purification of highly toxic substances. Because of its uniform and adjustable particle size, it can be used as polishing agent in chemical mechanical leveling technology of computer chips. The special properties of ultra-efficient materials are expected to bring considerable economic benefits to the application industry.
Application fields and key technologies that can be solved for the industry
Sustained-release drug carriers have low drug loading.
The particle size of computer chip polishing agent is uneven and the particle size is not adjustable.
Separation of Monomer Components of Ginsenoside
Background and main uses of results and projects
Chinese herbal medicines containing ginsenoside have shown remarkable biological activities in traditional folk medicine and modern drug development research, such as ginseng, American ginseng, notoginseng and so on. The pharmacological components of ginsenoside mainly contain several monomer saponins with similar structuRes, such as ginsenoside Rb 1, ginsenoside Rg 1 and ginsenoside re. In addition, Panax notoginseng also contains a special monomer saponin R 1. Generally, monomer saponins can reproduce almost all the biological activities of total saponins, but with the deepening of research, it is found that some monomer saponins have unique pharmacological effects, and even some monomer saponins have opposite pharmacological activities, so different proportions of monomer saponins can achieve different pharmacological effects. More importantly, Rb 1 can obtain some pure secondary glycosides through chemical transformation. Secondary glycosides generally have obvious pharmacological activities. For example, Rh2, a newly discovered natural plant component with anti-tumor and anti-metastasis, is the first choice for enhancing efficacy and reducing toxicity in chemotherapy, with high medicinal value and application prospect. However, the content of these secondary glycosides is generally small or not contained in plants, and they are just metabolites, so their application and development are greatly limited. Therefore, if the required secondary glycosides can be obtained from ginsenoside monomer with high content through chemical structure modification or transformation, it will certainly play a more active role in the medicinal research of ginsenoside.
At present, the main source of monomer saponins is still extraction and separation from natural plants. Generally, solvent extraction method is adopted, and silica gel column or dextran gel column is repeatedly used, which is cumbersome and consumes a lot of solvents, and the separation degree of compounds with small separation properties is poor, and the operation time is long and the solvent consumption is large. In addition, monomer saponins can be separated by normal phase or reversed phase HPLC, and the obtained products have high purity, but the general dosage is small, which is limited to analysis and the cost is very high.
In order to solve the problems existing in the prior art, a highly selective adsorption resin with uniform pore size was designed and synthesized in this project, and it was used to separate ginsenoside Rb 1 from ginsenoside and panax notoginseng saponin extract. Ginsenoside Rb 1 can be completely separated from other monomer saponins by a simple one-step process of "adsorption-desorption". At the same time, the established separation process is suitable for large-scale separation of saponins.
Brief introduction of technical principle and process flow
Instead of the traditional pore-forming method of bad solvent phase separation, a new type of macroporous adsorption resin with small average pore size and uniform pore size distribution was synthesized by Fourier alkylation crosslinking method. This kind of resin has dual functions of screening and adsorption, and can realize the separation of macromolecules monomer saponin Rb 1 from total saponins.