阅读说明：本技术 一种从红豆杉干废料中提取红豆杉多糖的制备方法及应用 (Preparation method and application for extracting taxus chinensis polysaccharide from dry taxus chinensis waste ) 是由 李晓 于 2019-12-30 设计创作，主要内容包括：本发明公开了一种具有降肌酐、提高内生肌酐清除率作用的红豆杉多糖的制备方法及应用,具体的红豆杉多糖的制备方法包括以下步骤：A)预处理；B)水提醇沉；C)洗涤；D)去蛋白；E)透析；F)冷冻干燥。并且公开了使用该方法制备得到的红豆杉多糖在降肌酐和/或提高内生肌酐清除率方面的应用,以及公开了使用该方法制备得到的红豆杉多糖而制备得到的降肌酐和/或提高内生肌酐清除率的药剂。本发明提取红豆杉多糖成本低,提取率以及纯度高,得到的红豆杉多糖具有降肌酐、提高内生肌酐清除率的功效。(The invention discloses a preparation method and application of taxus chinensis polysaccharide with the effects of reducing creatinine and improving endogenous creatinine clearance rate, and the specific preparation method of the taxus chinensis polysaccharide comprises the following steps: A) pre-treating; B) water extraction and alcohol precipitation; C) washing; D) deproteinizing; E) dialyzing; F) and (5) freeze drying. The application of the taxus chinensis polysaccharide prepared by the method in reducing creatinine and/or improving the clearance rate of endogenous creatinine is also disclosed, and the medicament prepared by the taxus chinensis polysaccharide prepared by the method for reducing creatinine and/or improving the clearance rate of endogenous creatinine is also disclosed. The method for extracting the taxus chinensis polysaccharide has low cost and high extraction rate and purity, and the obtained taxus chinensis polysaccharide has the effects of reducing creatinine and improving the clearance rate of endogenous creatinine.)

1. The preparation method of the taxus chinensis polysaccharide is characterized by comprising the following steps:

A) pretreatment of

Pulverizing dried Taxus chinensis waste after extraction of paclitaxel and 10-DAB III, adding ethanol for Soxhlet extraction, filtering, and collecting residue;

B) extracting with water and precipitating with ethanol

Adding a proper amount of purified water into the medicine residues in the step A), stirring until the medicine residues are soaked by the water, performing Soxhlet extraction, filtering, and concentrating under reduced pressure; adding ethanol into the concentrated solution, standing at low temperature, and filtering to obtain extract;

C) washing machine

The extract obtained in the step B) is sequentially and slowly dripped with absolute ethyl alcohol, acetone and ethyl acetate, and a crude product is obtained after drying;

D) deproteinization

Dissolving the crude product obtained in the step C) in a proper amount of hot water, and removing protein by a Sevage method to obtain a polysaccharide solution after protein removal.

E) Dialysis

Removing small molecular substances in the solution by using a running water dialysis method for the polysaccharide solution obtained in the step D);

F) freeze drying

Concentrating the dialyzed solution in the step E) under reduced pressure, pre-freezing and freeze-drying to obtain crude polysaccharide of the taxus chinensis;

adding ethanol in the step A) to carry out Soxhlet extraction at a material-liquid ratio of 1: 8-12.

2. The process for preparing polysaccharide extracted from dried taxus chinensis as claimed in claim 1, wherein the mesh number of the crushed dried taxus chinensis waste in step a) is 50-100 mesh, the ethanol concentration is 80%, the ethanol is added for carrying out the soxhlet extraction with the ratio of the materials to the liquids of 1:10, the extraction time is 1-5h, the step B) is carried out for carrying out the water extraction twice, the purified water with the ratio of the materials to the liquids of 1:10 is added for the first time, the purified water with the ratio of the materials to the liquids of 1:8 is added for the second time, the concentration in step B) is reduced under pressure until the concentration of the crude drug is 1.0g/m L, the ethanol with the volume of 3-6 times is added to the concentrated solution, the ethanol concentration is 95%, the low-temperature standing is 2-8 ℃, and the standing time is;

the volume of absolute ethyl alcohol, acetone and ethyl acetate added in the step C) is 0.05-0.2 time of the mass of the taxus chinensis, the dialysis time in the step D) is 2-5 days, the cut-off molecular weight of a dialysis bag used in the dialysis method is 8000-14000, and the solution after dialysis is decompressed and concentrated to the crude drug concentration of 3.0g/m L.

3. Use of the taxus chinensis polysaccharide obtained by the preparation method for extracting taxus chinensis polysaccharide from dry taxus chinensis waste as claimed in any one of claims 1-2 in reducing creatinine and/or improving endogenous creatinine clearance.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a preparation method and application of extracting taxus chinensis polysaccharide from dry taxus chinensis waste.

Background

Chronic Kidney Disease (CKD) has a high prevalence rate, a poor prognosis and a high medical cost, and is now an important disease that endangers human health. Chronic Renal Failure (CRF), also known as chronic renal insufficiency, is a clinical syndrome in which CKD progresses to a later stage, with many complications, and seriously affecting the quality of life and the lifespan of patients.

The traditional Western medicine classical diagnosis standard is divided into four stages according to the pathogenesis stages, namely the creatinine clearance rate in the renal insufficiency compensation stage (stage 1) is 80-51 ml/min, the blood creatinine 133-177 mu mol/L, clinically asymptomatic, the creatinine clearance rate in the renal insufficiency decompensation stage (stage 2) is 50-22 ml/min, the blood creatinine 186-442 mu mol/L, diuresis and nocturia can be caused, and mild anemia but no obvious symptoms exist, the creatinine clearance rate in the renal failure stage (stage 3) is 20-10 ml/min, the blood muscle is 451-707 mu mol/L, the anemia is obvious, the diuresis such as nocturia is caused, the water electrolyte disorder is caused, the mild or moderate metabolic acidosis, the water sodium retention is caused, the low calcium and high phosphorus are caused, the high potassium is not generated generally, the gastrointestinal tract, cardiovascular and central nervous symptoms are caused, the creatinine clearance rate in the uremia stage (stage 4) is less than 10ml/min, the blood creatinine > L, the gastrointestinal tract, cardiovascular and the metabolic nervous system is particularly obvious in water electrolysis.

Taxus chinensis is a plant of Taxus genus of Taxaceae family, and is reported in "Chinese plant-Material^[1]It is recorded that the Chinese yew can be divided into 4 varieties of 1 variety, namely, Tibetan yew, Yunnan yew, Chinese yew, northeast yew and southern yew. The taxus chinensis var mairei is produced in the south of the Yangtze river basin in China, and is usually grown in places with the elevation of 1000-1200m or below, and large-scale artificial propagation and breeding plants are arranged in places such as Zhongxiang, Enshi and Shiweir of Hubei province. Yew, also named Yew, Zhong Hua Ben Cao^[2]Chinese medicine dictionary^[3]The Chinese record shows that the taxus chinensis has the effects of inducing diuresis and reducing edema and mainly treats nephritis edema and dysuria; zhejiang province Chinese medicine decoction piece processing standard^[4]The nature, flavor, meridian tropism and main function of the taxus mairei are recorded: "it is slightly sweet, bitter and neutral, and enters kidney and heart meridians", and has the effects of relieving swelling, resolving hard mass, dredging meridians and inducing diuresis, and can be clinically used for abdominal mass, edema, dysuria, rheumatalgia, etc. The traditional Chinese medicine mainly treats the chronic renal failure by water decoction, and researches show that the taxus can extract the anticancer medicine taxol, and the main component of the water decoction is taxus polysaccharide, so the invention is just based on the point, effectively utilizes the taxus branch and leaf which has been extracted with the taxol and 10-DAB III in industry as the raw material to extract the taxus polysaccharide and carry out purification treatment, thereby not only saving resources, but also further developing the medicinal value of the taxus polysaccharide,in order to realize reasonable and maximized effective application of rare taxus chinensis plant resources and provide more possibilities for developing resources in the field of human medicine health.

Disclosure of Invention

The invention aims to provide a taxus chinensis polysaccharide and a preparation method thereof, and the aim of the invention is to realize the preparation of the medicine for treating the increase of blood creatinine and the reduction of endogenous creatinine clearance rate caused by various reasons, such as: the application of the medicine in treating chronic kidney disease, chronic renal failure, etc.

The invention provides a preparation method of taxus chinensis polysaccharide, which comprises the following steps:

A) pretreatment of

Pulverizing the dried Taxus chinensis waste after extraction of paclitaxel and 10-DAB III, adding ethanol, performing Soxhlet extraction, filtering, and collecting the residue.

B) Extracting with water and precipitating with ethanol

Adding a proper amount of purified water into the medicine residues in the step A), stirring until the medicine residues are soaked by the water, performing Soxhlet extraction, filtering, and concentrating under reduced pressure; adding ethanol into the concentrated solution, standing at low temperature, and filtering to obtain extract.

C) Washing machine

And B), sequentially and slowly dripping absolute ethyl alcohol, acetone and ethyl acetate into the extract obtained in the step B), and drying to obtain a crude product.

D) Deproteinization

Dissolving the crude product obtained in the step C) in a proper amount of hot water, and removing protein by a Sevage method to obtain a polysaccharide solution after protein removal.

E) Dialysis

And D), removing small molecular substances in the solution of the polysaccharide solution obtained in the step D) by adopting a running water dialysis method.

F) Freeze drying

And E), concentrating the dialyzed solution in the step E) under reduced pressure, pre-freezing and freeze-drying to obtain the crude polysaccharide of the taxus chinensis.

Preferably, the ratio of the materials added into the step A) for Soxhlet extraction by 80% ethanol is 1: 10.

According to the scheme, 80% ethanol is used for pretreating the taxus chinensis powder before water extraction and alcohol precipitation, so that the purpose is to remove residual fat-soluble compounds and partial monosaccharides in the medicinal materials, the extraction efficiency of the polysaccharide of the medicinal materials in the step B can be improved, the yield is increased, and the purity of the taxus chinensis polysaccharide is improved. The feed-liquid ratio of the soxhlet extraction of the ethanol can influence the impurity removal degree, and the invention has good impurity removal efficiency and lowest cost after a plurality of attempts, preferably when the feed-liquid ratio of the soxhlet extraction of the 80 percent ethanol is 1: 10.

And B) carrying out water extraction twice, wherein the ratio of purified water to liquid is 1:10 for the first time, the ratio of purified water to liquid is 1:8 for the second time, and the concentration of the crude drug is 1.0g/m L in the step B) through decompression concentration, 3-6 times of volume of ethanol is added into the concentrated solution, the concentration of the ethanol is 95%, the low-temperature standing temperature is 2-8 ℃, and the standing time is 15-36 h.

The water extraction and alcohol precipitation are the most common methods for extracting the polysaccharide from medicinal materials at present, the methods are the same and different, but the extraction processes have great difference, the production efficiency is improved and the cost is reduced under the condition of keeping the extraction rate.

The invention further discloses a method for reducing the concentration of crude drugs to 1.0g/m L by concentrating under reduced pressure, and the scheme discovers that the use amount of alcohol can be effectively reduced under the condition of the concentration, so that only 3-6 times of volume of ethanol is required to be added into the concentrated solution under the condition of alcohol precipitation.

In order to ensure that the washing step can effectively remove impurities and reduce the loss of polysaccharide during washing, the inventor inspects the optimal washing method by observing the color change of the washing liquid in the washing process and the method of polysaccharide content test in the washing liquid, and confirms the washing process.

The finally determined washing method is to add absolute ethyl alcohol, acetone and ethyl acetate, and the guaranteed volume is 0.05-0.2 times, preferably 0.1 times of the feeding mass of the taxus mairei.

In the deproteinization of the step D), the specific steps are as follows: dissolving the obtained crude product in appropriate amount of hot water, adding 1 volume time of n-butanol/chloroform by Sevage method at a volume ratio of 1:4, stirring the mixture for 30 min, and centrifuging.

The cut-off molecular weight of the dialysis bag used in the dialysis method in the step E) is 8000-.

The invention provides a medicament for reducing creatinine and/or improving endogenous creatinine clearance prepared from taxus chinensis polysaccharide, and auxiliary materials for preparing the medicament comprise one or more of a bonding agent, a filling agent, a diluent, a tabletting agent, a lubricant, a disintegrating agent, a coloring agent, a flavoring agent, a wetting agent and a surfactant.

The content of active ingredients of the taxus chinensis polysaccharide in the medicament is 1 to 99.5 percent.

The dosage form of the medicament is various pharmaceutically acceptable dosage forms, and the dosage form of the medicament comprises: enema, tablet, capsule, oral liquid, buccal agent, granule, electuary, pill, dripping pill, powder, ointment, pellet, suspension, powder, solution, suppository, liniment, cream, spray, powder inhalation, aerosol, drop, lozenge, and patch.

The invention particularly provides a medicine prepared from the taxus polysaccharide obtained by the preparation method of the taxus polysaccharide and used for treating chronic kidney disease and chronic renal failure.

The medicament for treating chronic kidney disease and chronic renal failure is various pharmaceutically acceptable preparations. Preferred dosage forms of the medicament include: enema, tablet, capsule, oral liquid, buccal agent, granule, electuary, pill, dripping pill, powder, ointment, pellet, suspension, powder, solution, suppository, liniment, cream, spray, powder inhalation, aerosol, drop, lozenge, and patch.

The invention uses the dry taxus waste after extracting paclitaxel and 10-DAB III as the raw material for extracting the taxus polysaccharide, can greatly reduce the cost according to the difference of the raw materials, such as reducing the use of the extraction raw materials, simultaneously increasing the utilization rate of the taxus, and improving the extraction rate and purity of the taxus polysaccharide through the setting of the process, and simultaneously verifies that the extracted taxus polysaccharide is nontoxic.

The invention discovers that the taxus chinensis polysaccharide has the effects of reducing creatinine and improving the clearance rate of endogenous creatinine, prepares a corresponding preparation, and verifies that a medicament prepared by taking the taxus chinensis polysaccharide as an active ingredient shows excellent effects of reducing creatinine and improving the clearance rate of endogenous creatinine in a chronic renal failure rat animal test.

Detailed Description

In order that the invention may be better understood, it is further illustrated, but is not to be construed as being limited, by the following examples.