Extraction method of high-purity resveratrol monomer

Abstract

According to the extraction method of the high-purity resveratrol monomer, the polygonum cuspidatum medicinal material is pretreated by adopting a microbial fermentation engineering technology, effective strains are cultivated, the original metabolic balance is changed, and conversion and generation of resveratrol are promoted; crude fibers in the polygonum cuspidatum medicinal material are greatly destroyed, the extraction rate of effective components is improved, and a foundation is laid for a subsequent purification process. Through selection and proportioning research of a mixed solvent in a two-phase counter-current extraction system, the addition of an auxiliary agent can significantly enhance the action effect of a main solvent, reduce the influence of impurities on extraction as much as possible, and improve the content and yield of effective components. Through research and addition of an antioxidant in product development, the oxidation loss of contained components in the processing process is effectively reduced, and the product quality is improved.

Description

Technical Field

[0001] The invention relates to a resveratrol preparation system, in particular to a method for extracting high-purity resveratrol monomers. Background Art

[0002] With the improvement of living standards and changes in social economy and living environment, people's health concepts and awareness of pursuing quality of life have greatly increased. The spectrum of contemporary human diseases and medical treatment models have also undergone tremendous changes. The demand for natural herbal medicine products with unique efficacy advantages in the treatment of chronic diseases, immune diseases, health care, and longevity in the international pharmaceutical market has risen sharply. In comparison, chemical synthetic drugs have the defects of difficult development, high cost, long development cycle, and obvious toxic and side effects. In recent years, the herbal medicine market in Western developed countries such as Europe and the United States has grown at a high rate of 20% per year, and has begun to consider using traditional Chinese medicine as a therapeutic drug, providing unprecedented opportunities for Chinese medicine to enter the mainstream international pharmaceutical market. Especially after my country joined the WTO, a considerable number of chemical synthetic drugs are facing the problem of further development of intellectual property rights. Counterfeiting drugs within the patent period will constitute illegal infringement. Chinese medicine will surely become the product with the most national characteristics and technical advantages for my country's pharmaceutical industry to participate in domestic and foreign market competition, and the market prospects will be broader.

[0003] Resveratrol (3,4',5-trihydroxystilbene) is a kind of active polyphenolic substance with two conformations, cis and trans, of which the trans is a stable structure with biological activity. Modern medical research shows that resveratrol has the following pharmacological functions: anti-platelet aggregation, reducing the oxidation of low-density lipoprotein, thereby preventing atherosclerosis and coronary heart disease; reducing serum and liver lipids, protecting the liver; inhibiting the three main stages of cell and tissue variation in the process of carcinogenesis (induction, initiation and development), and inhibiting five human tumor cell models A-549, SK-OV-3, SK-MEI-2, XF-498 and HCT-15, thus becoming one of the most potential natural drugs for inhibiting and treating tissue carcinogenesis and tumor occurrence. In addition, resveratrol can be used in combination with carnitine to treat cardiovascular diseases, peripheral vascular diseases and vascular and neurological complications of diabetes, and can also be used as a health food additive.

[0004] Resveratrol is widely found in plants such as Polygonum cuspidotem, grapes, peanuts, and pine trees. Among them, the content in Polygonum cuspidotem Sied. et Zucc is relatively rich, but the content is also very low, <1%, and the impurity components are similar, so it is very difficult to separate and purify to obtain high-content extracts and resveratrol monomers.

[0005] There are currently two ways to obtain resveratrol: one is through chemical synthesis; the other is extraction and separation from plants. The core reaction process of chemical synthesis has not been completely solved, and the biological activity of the synthetic product is weaker than that of natural resveratrol extracted from plants. Therefore, extraction and separation of resveratrol from natural plants is currently the main way to obtain resveratrol.

[0006] Traditional extraction and separation: In addition to resveratrol and glycosides, Polygonum cuspidatum also contains a large amount of free anthraquinone and anthraquinone glycosides, flavonoids, tannin glycosides, amino acids, trace elements and other chemical components. Therefore, in order to improve the purity and yield of resveratrol, the selection of solvents is the key to whether the effective ingredients can be extracted. The extract must also undergo a series of extraction and separation to preliminarily remove impurities. The commonly used extraction and separation methods are heating reflux extraction and percolation extraction: Heating reflux extraction is to extract with methanol, 98% ethanol (mass fraction), anhydrous ethanol or ethyl acetate, and then extract the extract solution with petroleum ether, chloroform, and ethyl acetate in turn, and then concentrate under reduced pressure and dry in vacuum to obtain crude resveratrol. This method is cumbersome to operate and takes a long time to process, and cannot be applied in industrial scale-up production.

[0007] Percolation extraction is to extract the raw material powder by percolation with 98% ethanol, recover the solvent under reduced pressure, dissolve the extract with 5% tartaric acid, and then extract the product with ether. The ether solvent used in this method has a low boiling point and is toxic, so its application in industrial production is also limited.

[0008] In addition, supercritical CO2 extraction is a modern extraction technology that is particularly suitable for the extraction of heat-sensitive and volatile components. For resveratrol, it has its advantages and can fully retain the pharmacological activity of the product. However, the absolute content of resveratrol in Polygonum cuspidatum is low and the amount of raw materials used is large. If supercritical CO2 extraction is used, it will lack the value of industrial development due to the low input-output ratio.

[0009] Current status of traditional purification technology: The purpose of purification is mainly to separate the cis- and trans-isomers of resveratrol and resveratrol glycosides in the crude Polygonum cuspidatum extract to obtain trans-resveratrol with stable and outstanding efficacy. Silica gel thin layer separation, silica gel column chromatography and high-speed countercurrent chromatography are commonly used.

[0010] Silica gel thin layer separation and silica gel column chromatography mainly consider the selection of developing agent or desorbent based on factors such as the polarity, solubility and activity of the sample. The organic solvent usually adopts one or more ratios of petroleum ether, chloroform, ethyl acetate, acetone and formic acid. The process is complicated, the organic solvent is expensive and highly toxic, and the operating safety is low.

[0011] High-speed countercurrent chromatography is a liquid-liquid distribution chromatography technology that does not use a solid support or carrier. It uses a GS10AZ high-speed countercurrent chromatograph, with an upper and lower phase composition of chloroform-methanol-water (4:3:2), an injection volume of 252 mg, a flow rate of 2.0 ml / min, and a rotation speed of 800 r / min. It can obtain resveratrol products with high purity and yield, with good separation effect and no pollution, and has certain development prospects. However, at present, high-speed countercurrent chromatography technology needs to be improved in theory and practice in terms of how to determine the type and ratio of elution solvents when separating multi-component mixtures, maintain long-term stability of column efficiency, and expand production capacity. Research is limited to the trial production of a small number of standard samples in the laboratory, and there is still a long way to go before industrial-scale production.

[0012] Since modern high-tech separation and purification methods are restricted by the above-mentioned various technical and production factors, the separation and purification of resveratrol from Polygonum cuspidatum in my country is still carried out by using the traditional alcohol extraction, water crystallization and alumina chromatography process, which has a complex process route and low product purity and production rate. Summary of the invention

[0013] The object of the present invention is to provide a method for extracting high-purity resveratrol monomer with good extraction effect and high purity.

[0014] To achieve the above object, the technical solution provided by the present invention is: a method for extracting high-purity resveratrol monomer, the extraction method comprising: The first step: successfully apply microbial fermentation engineering technology to substrate transformation to promote the hydrolysis of resveratrol glycosides into resveratrol; The second step: for the first time, by adding exogenous antioxidants, the oxidation loss of resveratrol during extraction and separation is reduced, and the dissolution of tannins and sugars in the raw materials is inhibited; The third step: Use two-phase countercurrent extraction technology to separate and purify resveratrol, create a mixed solvent extraction system, organically combine the main solvent, dispersant, and antioxidant, and increase the distribution coefficient of resveratrol in the organic phase.

[0015] The above-mentioned method for extracting high-purity resveratrol monomer comprises the following specific steps: The first step: crushing the raw material of knotweed and adding it into the fermentation tank with the prepared thread liquid to ferment and form mature material; The second step: pouring the antioxidant, main solvent, dispersant and clinker into a circulation tank for circulating reflux reaction; Step 3: The liquid after circulating reflux enters the two-phase countercurrent tank for countercurrent extraction, and the drug residue after circulating reflux is uniformly discharged and collected; Step 4: The liquid after countercurrent extraction enters the concentration tank for low temperature concentration, and the concentrated solvent is uniformly recovered; Step 5: The concentrated crystals enter the centrifuge for separation, and the separated impurities enter the oxidation tank for precipitation and recovery; Step 6: The separated crystal powder is dried, dissolved and filtered, and the filtered rhein products are collected uniformly; Step 7: The filtered crude solution is chromatographed through alumina to obtain high-purity resveratrol; Step 8: Degreasing the high-purity resveratrol, followed by crystallization and filtration; Step 9: Dry the filtered crystals to obtain resveratrol monomers. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the overall principle diagram of the present invention. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with all the accompanying drawings. The preferred embodiments of the present invention are as follows: Figure 1 This embodiment, with the help of the achievements of modern biotechnology research such as microbial fermentation engineering, starts from the optimization of Polygonum cuspidatum resources and the metabolic regulation of pharmacologically active ingredients, discovers or exogenously changes the functions of key enzymes in the biosynthesis and decomposition pathways of secondary metabolites, screens specific strains, regulates the synthesis and metabolic directions, and improves the conversion rate of matrix secondary metabolites, thereby significantly increasing the resveratrol content, greatly reducing the cost of raw materials for large-scale production, and effectively reducing the difficulty of subsequent separation and purification processes.

[0018] On the other hand, antioxidants are added to protect and reduce the loss of resveratrol during processing based on traditional separation and purification technology, the leaching, extraction and chromatographic separation modes are improved, and a selective mixed solvent two-phase countercurrent extraction system is used to fully improve the product purity and production rate; To achieve the above purpose, the present invention provides a method for extracting high-purity resveratrol monomer, which comprises: The first step: crushing the raw material of knotweed and adding it into the fermentation tank with the prepared thread liquid to ferment and form mature material; The second step: pouring the antioxidant, main solvent, dispersant and clinker into a circulation tank for circulating reflux reaction; Step 3: The liquid after circulating reflux enters the two-phase countercurrent tank for countercurrent extraction, and the drug residue after circulating reflux is uniformly discharged and collected; Step 4: The liquid after countercurrent extraction enters the concentration tank for low temperature concentration, and the concentrated solvent is uniformly recovered; Step 5: The concentrated crystals enter the centrifuge for separation, and the separated impurities enter the oxidation tank for precipitation and recovery; Step 6: The separated crystal powder (40% resveratrol content) is dried, dissolved and filtered, and the filtered rhubarb products are collected uniformly; Step 7: The filtered crude solution (50% resveratrol content) is chromatographed through alumina to obtain high-purity resveratrol (90-98% resveratrol content); during the chromatography, a mixed filler is added to the alumina, and the medium of the mixed filler is activated carbon (the ratio of activated carbon to alumina is 50-80:20-50); the mixed filler is formed by mixing activated carbon and alumina, thereby ensuring the high purity of resveratrol; Step 8: Degreasing the high-purity resveratrol, followed by crystallization and filtration; Step 9: Dry the filtered crystals to obtain resveratrol monomer (99% resveratrol content).

[0019] The key points of this program are: (1) Microbial fermentation engineering technology was used to pre-treat the Polygonum cuspidatum medicinal material, cultivate effective bacterial strains, change the original metabolic balance, and promote the conversion and production of resveratrol; a large amount of crude fiber in the Polygonum cuspidatum medicinal material was destroyed, the extraction rate of effective ingredients was increased, and the foundation was laid for the subsequent purification process.

[0020] (2) Study on the selection and ratio of mixed solvents in the two-phase countercurrent extraction system. The addition of auxiliary agents can significantly enhance the effect of the main solvent, minimize the impact of impurities on the extraction, and increase the content and yield of effective components.

[0021] (3) Research and addition of antioxidants during product development can effectively reduce the oxidative loss of ingredients during processing and improve product quality.

[0022] This scheme successfully applies microbial fermentation engineering technology to substrate matrix transformation, effectively promoting the hydrolysis and conversion of resveratrol glycosides into resveratrol. At the same time, the cis structure is converted into a stable trans structure, which effectively reduces the cost of raw materials and can greatly reduce the emulsification phenomenon in the subsequent extraction and separation process.

[0023] This scheme, for the first time, effectively reduces the oxidative loss of resveratrol during the extraction and separation process by adding exogenous antioxidants, inhibits the dissolution of tannins and sugars in the raw materials, and increases the extraction rate and content of resveratrol by 2-3 times compared with conventional methods.

[0024] This scheme uses two-phase countercurrent extraction technology to separate and purify resveratrol, creates a mixed solvent extraction system, organically combines the main solvent, dispersant, and antioxidant, increases the distribution coefficient of resveratrol in the organic phase, significantly improves the extraction efficiency, and reduces product costs.

[0025] The technical indicators of the resveratrol product prepared by the above scheme include: (1) The effective content of the crude product is increased from 5% in the traditional process to more than 40%; (2) The yield of resveratrol increased from 60% in traditional process to 80%; (3) The solvent recovery rate is increased from 50% in the traditional process to 85%; (4) The production cycle is shortened by nearly 1 / 3; (5) The cost of 50% crude product per kilogram was reduced from RMB 4,800 using traditional technology to RMB 1,200.

[0026] This scheme uses modern bio-fermentation engineering technology to increase the matrix content, and adopts advanced technologies such as two-phase countercurrent extraction in the separation process to break through the bottleneck of traditional processes and realize large-scale industrial production of Polygonum cuspidatum extracts. It transforms the development of plant resources from traditional cheap to modern high-efficiency industries, accelerates the development of border economy and the process of industrialization of traditional Chinese medicine, and can produce significant economic benefits and good social benefits.

[0027] The embodiments described above are only preferred embodiments of the present invention and are not intended to limit the scope of implementation of the present invention. Therefore, all changes made according to the shape and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for extracting high-purity resveratrol monomer, characterized in that: The extraction method comprises: The first step: successfully apply microbial fermentation engineering technology to substrate transformation to promote the hydrolysis of resveratrol glycosides into resveratrol; The second step: for the first time, by adding exogenous antioxidants, the oxidation loss of resveratrol during extraction and separation is reduced, and the dissolution of tannins and sugars in the raw materials is inhibited; The third step: Use two-phase countercurrent extraction technology to separate and purify resveratrol, create a mixed solvent extraction system, organically combine the main solvent, dispersant, and antioxidant, and increase the distribution coefficient of resveratrol in the organic phase.

2. The method for extracting high-purity resveratrol monomer according to claim 1, characterized in that: It includes the following steps: The first step: crushing the raw material of knotweed and adding it into the fermentation tank with the prepared thread liquid to ferment and form mature material; The second step: pouring the antioxidant, main solvent, dispersant and clinker into a circulation tank for circulating reflux reaction; Step 3: The liquid after circulating reflux enters the two-phase countercurrent tank for countercurrent extraction, and the drug residue after circulating reflux is uniformly discharged and collected; Step 4: The liquid after countercurrent extraction enters the concentration tank for low temperature concentration, and the concentrated solvent is uniformly recovered; Step 5: The concentrated crystals enter the centrifuge for separation, and the separated impurities enter the oxidation tank for precipitation and recovery; Step 6: The separated crystal powder is dried, dissolved and filtered, and the filtered rhein products are collected uniformly; Step 7: The filtered crude solution is chromatographed through alumina to obtain high-purity resveratrol; Step 8: Degreasing the high-purity resveratrol, followed by crystallization and filtration; Step 9: Dry the filtered crystals to obtain resveratrol monomers.

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