CN108478540B - Preparation method of tripterygium wilfordii slow-release microcapsule - Google Patents
Preparation method of tripterygium wilfordii slow-release microcapsule Download PDFInfo
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- CN108478540B CN108478540B CN201810487302.4A CN201810487302A CN108478540B CN 108478540 B CN108478540 B CN 108478540B CN 201810487302 A CN201810487302 A CN 201810487302A CN 108478540 B CN108478540 B CN 108478540B
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- triptolide
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- tripterygium wilfordii
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
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- A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
- A61K9/5042—Cellulose; Cellulose derivatives, e.g. phthalate or acetate succinate esters of hydroxypropyl methylcellulose
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
- A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/37—Celastraceae (Staff-tree or Bittersweet family), e.g. tripterygium or spindletree
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J73/00—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms
- C07J73/001—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom
- C07J73/003—Steroids in which the cyclopenta[a]hydrophenanthrene skeleton has been modified by substitution of one or two carbon atoms by hetero atoms by one hetero atom by oxygen as hetero atom
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
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- A61K2236/30—Extraction of the material
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- A61K2236/55—Liquid-liquid separation; Phase separation
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids
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Abstract
The invention discloses a preparation method of tripterygium wilfordii slow-release microcapsules, which comprises the steps of adopting ultrasonic wave to cooperate with supercritical CO2Extracting and separating by high pressure liquid chromatography to purify triptolide extract with purity of about 95% to make its purity reach more than 99%; then preparing the triptolide purified extract with the purity of more than 99 percent into the sustained-release microcapsule. The invention reduces the influence of impurities on triptolide extract in drug treatment, and is beneficial to researchers to take effective measures to treat the toxicity of the triptolide extract in a targeted manner; the invention carries out microencapsulation treatment on the purified triptolide extract, has high embedding property and good slow release speed of the medicine, can slowly release the active ingredients in the medicine, prolongs the release, absorption and distribution of the medicine in vivo, and thus reduces the toxicity of the triptolide to human bodies.
Description
Technical Field
The invention belongs to the technical field of medicine preparation, and particularly relates to a preparation method of a tripterygium wilfordii slow-release microcapsule.
Background
Tripterygium wilfordii is root, leaf and flower of Tripterygium wilfordii hook F.H. Chen et C.A. and also called Fibraurea recisa and gelsemium elegans, and it is pungent and cool in nature, with strong toxicity, entering liver channel, and spleen and kidney channels. Modern pharmacological studies show that tripterygium wilfordii has the effects of anti-inflammation, analgesia, anti-tumor, immunoregulation and the like, is commonly used for treating skin inflammation, immunoregulation, kidney diseases and respiratory diseases, but has larger toxic reaction so as to limit the clinical application of the tripterygium wilfordii.
The active ingredients of tripterygium wilfordii are mainly Triptolide, tripterygium triterpenes, tripterygium alkaloids and the like, wherein Triptolide (Triptolide), also called Triptolide, which belongs to the diterpene lactones is regarded as the most important physiologically active ingredient, so that the purification of Triptolide from tripterygium wilfordii has important value. The purity of triptolide extracted by the existing extraction method can only reach about 95 percent, for example, the Chinese patent invention with the publication number of CN 101445545B provides a method for separating triptolide from tripterygium wilfordii leaves, and the purity of the triptolide extracted by the existing extraction method is between 94.3 percent and 98.7 percent. Since the effective components of triptolide are basically toxic, when triptolide is added into the medicine, various measures are needed to reduce the toxicity of triptolide, but if triptolide is not purified to high purity, the drug effect is influenced on one hand, and on the other hand, the interaction between impurities and other components is difficult to judge after the impurities are mixed into the components of the formula, so that the toxicity of triptolide is difficult to reduce by adopting targeted measures.
Since triptolide is toxic, measures are taken to reduce its toxicity in the drug. At present, researches on the attenuation method of thunder god vine mainly comprise the modes of traditional Chinese medicine compatibility, preparation attenuation, searching for an active monomer of thunder god vine with high efficiency and low toxicity or carrying out structural modification on the existing active monomer, reducing toxic and side effects by acupuncture and moxibustion and the like, but the effect is not ideal on the whole. Therefore, innovative ideas and methods are needed to solve the problem of toxic and side effects of tripterygium wilfordii. In contrast, the research of Chinese medical workers has found that the tripterygium wilfordii extract is prepared into a slow release capsule, the release amount of the tripterygium wilfordii extract is controlled by controlling the release speed of active ingredients in a body, and the tripterygium wilfordii extract is gradually absorbed by the body, so that the toxicity to the body can be reduced. Such as Zhuhui, etc., and ultrasonic method is selected to prepare triptolide beta-cyclodextrin inclusion compound with inclusion rate of 70%; the preparation method of liposome such as thin film ultrasonic dispersion method, mechanical homogenization method and ethanol injection method is examined by using encapsulation rate of triptolide liposome as index. Therefore, the method for preparing the tripterygium wilfordii microcapsule has certain feasibility for reducing the toxicity of the tripterygium wilfordii. Therefore, only a proper material for coating the microcapsule is found, an effective way for reducing the toxicity of the tripterygium wilfordii can be provided.
The materials commonly used for coating the microcapsule mainly include three types of natural polymer materials, semi-synthetic polymer materials and synthetic polymer materials. The natural high molecular material is a gelatinable colloid material, such as gelatin, Arabic gum, starch and the like, and the material is non-toxic, has good film forming property, but has poor mechanical strength and unstable raw material quality. The semisynthetic polymer material mainly comprises cellulose derivatives, such as sodium carboxymethylcellulose, cellulose acetate phthalate and sodium carboxymethylcellulose, which have the advantages of low toxicity, high viscosity, increased solubility after salt formation and the like, but also has the defects of easy hydrolysis, no high temperature resistance, poor acid resistance and the like. The synthetic high molecular material polyvinyl acetal, polyethylene glycol, polyacrylamide, polyurethane, polyvinylpyrrolidone, polyvinyl alcohol, triethyl citrate, pseudo-polyamino acid synthetic rubber and the like have the characteristics of good film forming property, biodegradability, good chemical property, good stability and high cost. Therefore, there is a need to find a new material or coating method for coating microcapsules to control the release rate and concentration of Tripterygium wilfordii to be above the effective dose and below the toxic dose, so as to further reduce toxicity by changing the dosage form and administration manner of Tripterygium wilfordii, and provide a certain data support for clinical application.
Disclosure of Invention
The invention provides a preparation method of a tripterygium wilfordii slow-release microcapsule, which solves the problem that tripterygium wilfordii in the prior art has larger toxic reaction to limit the clinical application of the tripterygium wilfordii.
The invention provides a preparation method of tripterygium wilfordii slow-release microcapsules, which comprises the following steps:
step 1, purification of triptolide alcohol extract
Step 1.1, raw material pretreatment: collecting alcohol extract of triptolide, adding ethanol 5-10 times the weight of the alcohol extract, stirring to dissolve triptolide completely, adding glycine-hydrochloric acid buffer solution with concentration of 0.05mol/L, and adjusting pH to 4-5 to obtain pretreated alcohol extract of triptolide;
step 1.2, ultrasound wave cooperating with supercritical CO2And (3) extraction: treating the pretreated triptolide extract with ultrasonic wave, and performing supercritical CO treatment on the pretreated triptolide extract with dichloromethane as carrier2Extracting to obtain extractive solution, concentrating, drying to obtain triptolideA product;
wherein the volume ratio of the preprocessed triptolide extract to dichloromethane is 1: 0.1-0.5;
supercritical CO2The extraction conditions were: CO 22The flow is 35-60L/h, the extraction pressure is 15-40MPa, the extraction temperature is 40-60 ℃, and the extraction time is 3-5 h;
step 1.3, high pressure liquid chromatography separation: mixing the triptolide primary purified product with ethanol according to a ratio of 1 g: 5-8mL of alcohol solution of the preliminary purification product of triptolide is obtained after mixing and dissolving; separating and purifying the ethanol solution of the primarily purified product of triptolide by high pressure liquid chromatography, detecting with ultraviolet detector at wavelength of 218nm, collecting 12.3min fraction, concentrating the fraction after collection, and drying to obtain purified extract of triptolide with purity of more than 99%;
step 2, preparing the tripterygium wilfordii slow release microcapsule
Step 2.1, preparation of organic phase: mixing sodium carboxymethylcellulose, modified chitosan and polyethylene glycol according to the weight ratio of 5: 1: mixing according to the mass ratio of 0.05, adding the mixture into dichloromethane which is 10 times of the total mass of the sodium carboxymethyl cellulose, the modified chitosan and the polyethylene glycol, stirring and dissolving until the solution is clear and transparent, and obtaining a mixed organic phase;
step 2.2, preparation of the aqueous phase: mixing polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester according to the weight ratio of 1: 4: mixing at a mass ratio of 0.05, adding into distilled water 20 times of the total mass of polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester, stirring at 80-90 deg.C, stopping heating when the solution becomes clear and transparent, and cooling to room temperature to obtain mixed water phase;
step 2.3, preparing a colostrum phase: adding the purified triptolide extract obtained in the step 1 into the mixed organic phase obtained in the step 2.1, stirring for 15-30min at the stirring speed of 1000-1500rmp, and obtaining a primary emulsion phase dispersed with the medicine after stirring;
step 2.4, preparing the tripterygium wilfordii slow release microcapsule: and (3) dropwise adding the primary emulsion phase obtained in the step (2.3) into the mixed water phase obtained in the step (2.2) under the condition that the rotation speed is 1000-1500rpm, continuously stirring for 1-2h, heating to 60 ℃ after the surface of the capsule is solidified, simultaneously reducing the stirring speed to 500rpm, stirring for 3-4h, washing, filtering and drying after stirring is finished, and thus obtaining the tripterygium wilfordii slow-release microcapsule.
Preferably, the alcohol extract of triptolide used in step 1.1 has a purity of 95.2%.
Preferably, the ultrasonic power in step 1.2 is 900-.
Preferably, the high pressure liquid chromatography separation conditions in step 1.3 are as follows: 21.2mm × 250mm, 5 μm C18 chromatographic column, flow rate of 20mL/min, mobile phase of methanol, each injection of 500 μ L.
Preferably, the preparation method of the modified chitosan comprises the following steps: dissolving chitosan into an acetic acid solution to prepare a chitosan solution with the mass concentration of 2%;
transferring the chitosan solution into a castor oil solution of tween-80 with the mass concentration of 1%, uniformly stirring, then dropwise adding a glutaraldehyde crosslinking agent, stirring for reacting for 3 hours after dropwise adding to obtain a reaction solution, filtering the reaction solution to remove the solvent, washing with absolute ethyl alcohol, and drying to obtain the modified chitosan;
wherein the volume ratio of the chitosan solution to the Tween-80 castor oil solution to the glutaraldehyde is 25: 5: 1.
the invention firstly adopts glycine-hydrochloric acid buffer solution to process triptolide alcohol extract, increases the solubility of the triptolide alcohol extract in an extraction solvent, and uses ultrasonic wave to cooperate with supercritical CO2The triptolide can be efficiently and quickly extracted by extraction; the extraction liquid is concentrated and then separated by adopting the high-pressure liquid chromatography, the high-pressure liquid chromatography can accelerate the chromatography speed, shorten the elution time, simultaneously complete the gradient development of one solvent, and can be developed again by using another gradient solution after being dried, thereby effectively preventing the intercrossing and the influence among all fractions and greatly improving the separation efficiency. In addition, compared with the conventional normal pressure column chromatography and rapid column chromatography, the high pressure liquid chromatography has the advantages of simple equipment, simple and convenient operation, high separation speed and resolutionHigh efficiency, large separation capacity and low solvent consumption. After the treatment, the purity of the triptolide reaches over 99 percent.
The triptolide with the purity of more than 99 percent is used for preparing the sustained-release microcapsule, sodium carboxymethylcellulose, modified chitosan, polyethylene glycol, polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester are used as materials for coating the microcapsule, the coating material is used for preparing the sustained-release microcapsule in a mode of combining semisynthetic polymer materials and synthetic polymer materials, and has the advantages of low toxicity, high viscosity and increased solubility after salification, good film forming property, good stability and low cost.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, firstly, the triptolide extract with the purity of 95% is purified by adopting a purification process, so that the purity of the triptolide extract reaches more than 99%, the influence of impurities on the triptolide extract in the drug treatment is reduced, and researchers can be helped to take effective measures to treat the toxicity of the triptolide extract in a targeted manner;
the invention carries out microencapsulation treatment on the purified triptolide extract, has high embedding property and good slow release speed of the medicine, can slowly release the active ingredients in the medicine, prolongs the release, absorption and distribution of the medicine in vivo, and thus reduces the toxicity of the triptolide to human bodies.
Detailed Description
In order to make the technical solutions of the present invention better understood and enable those skilled in the art to practice the present invention, the following embodiments are further described, but the present invention is not limited to the following embodiments.
The test methods described in the examples of the present invention are all conventional methods unless otherwise specified, and the raw materials used are all conventional reagents unless otherwise specified.
Example 1
A preparation method of a thunder god vine sustained-release microcapsule comprises the following steps:
step 1, purification of triptolide alcohol extract
Step 1.1, raw material pretreatment: collecting alcohol extract of triptolide with purity of 95.2%, adding ethanol 5 times of the weight of the alcohol extract, stirring to dissolve the triptolide completely, adding glycine-hydrochloric acid buffer solution with concentration of 0.05mol/L, and adjusting pH to 4-5 to obtain pretreated alcohol extract of triptolide;
step 1.2, ultrasound wave cooperating with supercritical CO2And (3) extraction: while the pretreated triptolide extract is treated by ultrasonic wave with power of 900W, the pretreated triptolide extract is subjected to supercritical CO with dichloromethane as carrying agent2Extracting to obtain extract, concentrating and drying the extract to obtain a triptolide primary purification product;
wherein the volume ratio of the preprocessed triptolide extract to dichloromethane is 1: 0.1;
supercritical CO2The extraction conditions were: CO 22The flow is 35L/h, the extraction pressure is 40MPa, the extraction temperature is 40 ℃, and the extraction time is 5 h;
step 1.3, high pressure liquid chromatography separation: mixing the triptolide primary purified product with ethanol according to a ratio of 1 g: 8mL of the mixture is mixed and dissolved to obtain an ethanol solution of a preliminary purification product of triptolide; separating and purifying the ethanol solution of the preliminary purification product of triptolide by high pressure liquid chromatography, detecting with ultraviolet detector at wavelength of 218nm, collecting 12.3min fraction, concentrating the fraction after collection, and drying to obtain purified triptolide extract with purity of 99.3%;
wherein, the high pressure liquid chromatography separation conditions are as follows: 21.2mm × 250mm, 5 μm C18 chromatographic column, flow rate of 20mL/min, mobile phase of methanol, each injection of 500 μ L.
Step 2, preparing the tripterygium wilfordii slow release microcapsule
Step 2.1, preparation of organic phase: mixing sodium carboxymethylcellulose, modified chitosan and polyethylene glycol according to the weight ratio of 5: 1: mixing according to the mass ratio of 0.05, adding the mixture into dichloromethane which is 10 times of the total mass of the sodium carboxymethyl cellulose, the modified chitosan and the polyethylene glycol, stirring and dissolving until the solution is clear and transparent, and obtaining a mixed organic phase;
step 2.2, preparation of the aqueous phase: mixing polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester according to the weight ratio of 1: 4: mixing at a mass ratio of 0.05, adding into distilled water 20 times of the total mass of polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester, stirring at 80-90 deg.C, stopping heating when the solution becomes clear and transparent, and cooling to room temperature to obtain mixed water phase;
step 2.3, preparing a colostrum phase: adding the purified extract of triptolide obtained in step 1 into the mixed organic phase obtained in step 2.1, stirring at 1000rmp for 30min, and stirring to obtain primary emulsion phase dispersed with the medicine;
step 2.4, preparing the tripterygium wilfordii slow release microcapsule: dropwise adding the primary emulsion phase obtained in the step 2.3 into the mixed water phase obtained in the step 2.2 under the condition that the rotation speed is 1000rpm, continuously stirring for 2 hours, heating to 60 ℃ after the surface of the capsule is solidified, simultaneously reducing the stirring speed to 500rpm, stirring for 3.5 hours, washing, filtering and drying after stirring is finished, and thus obtaining the thunder god vine sustained-release microcapsule.
Example 2
A preparation method of a thunder god vine sustained-release microcapsule comprises the following steps:
step 1, purification of triptolide alcohol extract
Step 1.1, raw material pretreatment: collecting alcohol extract of triptolide with purity of 95.2%, adding ethanol 8 times of the weight of the alcohol extract, stirring to dissolve the triptolide completely, adding glycine-hydrochloric acid buffer solution with concentration of 0.05mol/L, and adjusting pH to 4-5 to obtain pretreated alcohol extract of triptolide;
step 1.2, ultrasound wave cooperating with supercritical CO2And (3) extraction: in the adoption ofTreating the pretreated triptolide extract with ultrasonic wave of 1000W, and performing supercritical CO treatment on the pretreated triptolide extract with dichloromethane as carrier2Extracting to obtain extract, concentrating and drying the extract to obtain a triptolide primary purification product;
wherein the volume ratio of the preprocessed triptolide extract to dichloromethane is 1: 0.3;
supercritical CO2The extraction conditions were: CO 22The flow is 45L/h, the extraction pressure is 25MPa, the extraction temperature is 50 ℃, and the extraction time is 4 h;
step 1.3, high pressure liquid chromatography separation: mixing the triptolide primary purified product with ethanol according to a ratio of 1 g: mixing and dissolving in a ratio of 6mL to obtain an ethanol solution of a preliminary purification product of triptolide; separating and purifying the ethanol solution of the primarily purified product of triptolide by high pressure liquid chromatography, detecting with ultraviolet detector at wavelength of 218nm, collecting 12.3min fraction, concentrating the fraction after collection, and drying to obtain purified extract of triptolide with purity of 99.5%;
wherein, the separation conditions of the high pressure liquid chromatography are the same as that of the example 1;
step 2, preparing the tripterygium wilfordii slow release microcapsule
Step 2.1, preparation of organic phase: mixing sodium carboxymethylcellulose, modified chitosan and polyethylene glycol according to the weight ratio of 5: 1: mixing according to the mass ratio of 0.05, adding the mixture into dichloromethane which is 10 times of the total mass of the sodium carboxymethyl cellulose, the modified chitosan and the polyethylene glycol, stirring and dissolving until the solution is clear and transparent, and obtaining a mixed organic phase;
step 2.2, preparation of the aqueous phase: mixing polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester according to the weight ratio of 1: 4: mixing at a mass ratio of 0.05, adding into distilled water 20 times of the total mass of polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester, stirring at 80-90 deg.C, stopping heating when the solution becomes clear and transparent, and cooling to room temperature to obtain mixed water phase;
step 2.3, preparing a colostrum phase: adding the purified extract of triptolide obtained in step 1 into the mixed organic phase obtained in step 2.1, stirring at 1200rmp for 20min, and stirring to obtain primary emulsion phase dispersed with the medicine;
step 2.4, preparing the tripterygium wilfordii slow release microcapsule: dropwise adding the primary emulsion phase obtained in the step 2.3 into the mixed water phase obtained in the step 2.2 under the condition that the rotation speed is 1200rpm, continuously stirring for 1.5h, heating to 60 ℃ after the surface of the capsule is solidified, simultaneously reducing the stirring speed to 500rpm, stirring for 3h, washing, filtering and drying after stirring is finished, and thus obtaining the thunder god vine sustained-release microcapsule.
Example 3
A preparation method of a thunder god vine sustained-release microcapsule comprises the following steps:
step 1, purification of triptolide alcohol extract
Step 1.1, raw material pretreatment: collecting alcohol extract of triptolide with purity of 95.2%, adding ethanol 10 times of the weight of the alcohol extract, stirring to dissolve the triptolide completely, adding glycine-hydrochloric acid buffer solution with concentration of 0.05mol/L, and adjusting pH to 4-5 to obtain pretreated alcohol extract of triptolide;
step 1.2, ultrasound wave cooperating with supercritical CO2And (3) extraction: while the pretreated triptolide extract is treated by ultrasonic wave with power of 1200W, the pretreated triptolide extract is subjected to supercritical CO with dichloromethane as carrying agent2Extracting to obtain extract, concentrating and drying the extract to obtain a triptolide primary purification product;
wherein the volume ratio of the preprocessed triptolide extract to dichloromethane is 1: 0.5;
supercritical CO2The extraction conditions were: CO 22The flow is 60L/h, the extraction pressure is 15MPa, the extraction temperature is 60 ℃, and the extraction time is 3 h;
step 1.3, high pressure liquid chromatography separation: mixing the triptolide primary purified product with ethanol according to a ratio of 1 g: 5mL of the mixture is mixed and dissolved to obtain an ethanol solution of a preliminary purification product of triptolide; separating and purifying the ethanol solution of the primarily purified product of triptolide by high pressure liquid chromatography, detecting with ultraviolet detector at wavelength of 218nm, collecting 12.3min fraction, concentrating the fraction after collection, and drying to obtain purified extract of triptolide with purity of 99.1%;
wherein, the separation conditions of the high pressure liquid chromatography are the same as that of the example 1;
step 2, preparing the tripterygium wilfordii slow release microcapsule
Step 2.1, preparation of organic phase: mixing sodium carboxymethylcellulose, modified chitosan and polyethylene glycol according to the weight ratio of 5: 1: mixing according to the mass ratio of 0.05, adding the mixture into dichloromethane which is 10 times of the total mass of the sodium carboxymethyl cellulose, the modified chitosan and the polyethylene glycol, stirring and dissolving until the solution is clear and transparent, and obtaining a mixed organic phase;
step 2.2, preparation of the aqueous phase: mixing polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester according to the weight ratio of 1: 4: mixing at a mass ratio of 0.05, adding into distilled water 20 times of the total mass of polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester, stirring at 80-90 deg.C, stopping heating when the solution becomes clear and transparent, and cooling to room temperature to obtain mixed water phase;
step 2.3, preparing a colostrum phase: adding the purified extract of triptolide obtained in step 1 into the mixed organic phase obtained in step 2.1, stirring at 1500rmp for 15min, and stirring to obtain primary emulsion phase dispersed with the medicine;
step 2.4, preparing the tripterygium wilfordii slow release microcapsule: and (3) dropwise adding the primary emulsion phase obtained in the step (2.3) into the mixed aqueous phase obtained in the step (2.2) under the condition that the rotation speed is 1500rpm, continuously stirring for 1h, heating to 60 ℃ after the surface of the capsule is solidified, simultaneously reducing the stirring speed to 500rpm, stirring for 4h, washing, filtering and drying after stirring is finished, and thus obtaining the thunder god vine sustained-release microcapsule.
It should be noted that the ultrasonic operation mode in step 1.2 is 12s, i.e. 8s for ultrasonic wave, and 4s for intermittent operation.
Further, the preparation method of the modified chitosan comprises the following steps: dissolving chitosan into an acetic acid solution to prepare a chitosan solution with the mass concentration of 2%;
transferring the chitosan solution into a castor oil solution of tween-80 with the mass concentration of 1%, uniformly stirring, then dropwise adding a glutaraldehyde crosslinking agent, stirring for reacting for 3 hours after dropwise adding to obtain a reaction solution, filtering the reaction solution to remove the solvent, washing with absolute ethyl alcohol, and drying to obtain the modified chitosan;
wherein the volume ratio of the chitosan solution to the Tween-80 castor oil solution to the glutaraldehyde is 25: 5: 1.
the tripterygium wilfordii slow release microcapsules with good slow release performance are prepared in the embodiments 1 to 3, and the performance of the tripterygium wilfordii slow release microcapsules prepared in the embodiments 1 to 3 is detected, and specific results are shown in table 1.
Table 1 sustained Release Effect measurement results
As can be seen from Table 1, the sustained-release microcapsules prepared in examples 1-3 have high encapsulation efficiency and good sustained-release performance, and can slowly release the active ingredients of the drugs after entering human bodies, so as to prolong the release, absorption and distribution of the drugs in the bodies, thereby reducing the toxicity of triptolide to human bodies.
The effects of the present invention will be further described below by way of animal experiments.
1. Acute toxicity test
Taking 60 NIH mice, with SPF grade, half of each sex and 18-22g of body weight, and carrying out acute toxicity test. The mice were randomly divided into two groups of 30 mice each, i.e. control and administration groups, fasted for 12 hours prior to the experiment; the tripterygium wilfordii slow release microcapsule prepared in the embodiment 1 of the invention is used for gavage of a mouse, the gavage volume is 0.2mL/10g, the same amount of normal saline is given to a control group, the gavage is carried out by one-time administration, the administration is continuously observed for 7 days, and the toxic reaction and the death number of the mouse are recorded.
The experimental results show that: compared with a control group, the mice have no obvious difference after administration, and the mice have normal general conditions, diet, drinking water and weight increase after continuous observation for 7 days in the experiment. Mouse oral gavage sustained-release microcapsule LD50More than 930mg/kg, so the sustained-release microcapsule of the invention has low acute toxicity and safe clinical medication.
2. Long term toxicity test
The tripterygium wilfordii slow release microcapsule prepared in the embodiment 1 of the invention is used for observing and detecting the mice after 0.2mL/10g of the sustained release microcapsule is continuously used for 16 weeks (1 time every 7 days) and the drug is stopped for 4 weeks.
The experimental results show that: the prepared tripterygium wilfordii slow release microcapsule has no obvious influence on indexes such as hair, behavior, defecation, weight of viscera, hemogram, liver and kidney functions, blood sugar, blood fat and the like of a rat, and the viscera have no obvious change as shown by the naked eyes and the histological examination result, and each viscera of the rat has no obvious change after the administration for 16 weeks and the withdrawal for 4 weeks. The sustained-release tripterygium wilfordii microcapsule has low toxicity after long-term administration to rats, has no abnormal reaction after drug withdrawal, and is safe to apply.
It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any value between the two ends can be selected, and since the steps and methods used are the same as those in embodiments 1-3, the preferred embodiments of the present invention have been described for the purpose of preventing redundancy, but once the basic inventive concept is known, those skilled in the art can make other changes and modifications to these embodiments. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (3)
1. A preparation method of a thunder god vine sustained-release microcapsule is characterized by comprising the following steps:
step 1, purification of triptolide alcohol extract
Step 1.1, raw material pretreatment: collecting alcohol extract of triptolide, adding ethanol 5-10 times the weight of the alcohol extract, stirring to dissolve triptolide completely, adding glycine-hydrochloric acid buffer solution with concentration of 0.05mol/L, and adjusting pH to 4-5 to obtain pretreated alcohol extract of triptolide; the purity of the triptolide extract is 95.2%;
step 1.2, ultrasound wave cooperating with supercritical CO2And (3) extraction: treating the pretreated triptolide extract with ultrasonic wave, and performing supercritical CO treatment on the pretreated triptolide extract with dichloromethane as carrier2Extracting to obtain extract, concentrating and drying the extract to obtain a triptolide primary purification product;
wherein the volume ratio of the preprocessed triptolide extract to dichloromethane is 1: 0.1-0.5;
supercritical CO2The extraction conditions were: CO 22The flow is 35-60L/h, the extraction pressure is 15-40MPa, the extraction temperature is 40-60 ℃, and the extraction time is 3-5 h;
step 1.3, high pressure liquid chromatography separation: mixing the triptolide primary purified product with ethanol according to a ratio of 1 g: 5-8mL of alcohol solution of the preliminary purification product of triptolide is obtained after mixing and dissolving; separating and purifying the ethanol solution of the primarily purified product of triptolide by high pressure liquid chromatography, detecting with ultraviolet detector at wavelength of 218nm, collecting 12.3min fraction, concentrating the fraction after collection, and drying to obtain purified extract of triptolide with purity of more than 99%;
step 2, preparing the tripterygium wilfordii slow release microcapsule
Step 2.1, preparation of organic phase: mixing sodium carboxymethylcellulose, modified chitosan and polyethylene glycol according to the weight ratio of 5: 1: mixing according to the mass ratio of 0.05, adding the mixture into dichloromethane which is 10 times of the total mass of the sodium carboxymethyl cellulose, the modified chitosan and the polyethylene glycol, stirring and dissolving until the solution is clear and transparent, and obtaining a mixed organic phase;
step 2.2, preparation of the aqueous phase: mixing polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester according to the weight ratio of 1: 4: mixing at a mass ratio of 0.05, adding into distilled water 20 times of the total mass of polyvinyl alcohol, hyaluronic acid and polyglycerol fatty acid ester, stirring at 80-90 deg.C, stopping heating when the solution becomes clear and transparent, and cooling to room temperature to obtain mixed water phase;
the preparation method of the modified chitosan comprises the following steps: dissolving chitosan into an acetic acid solution to prepare a chitosan solution with the mass concentration of 2%;
transferring the chitosan solution into a castor oil solution of tween-80 with the mass concentration of 1%, uniformly stirring, then dropwise adding a glutaraldehyde crosslinking agent, stirring for reacting for 3 hours after dropwise adding to obtain a reaction solution, filtering the reaction solution to remove the solvent, washing with absolute ethyl alcohol, and drying to obtain the modified chitosan;
wherein the volume ratio of the chitosan solution to the Tween-80 castor oil solution to the glutaraldehyde is 25: 5: 1;
step 2.3, preparing a colostrum phase: adding the purified triptolide extract obtained in the step 1 into the mixed organic phase obtained in the step 2.1, stirring for 15-30min at the stirring speed of 1000-1500rmp, and obtaining a primary emulsion phase dispersed with the medicine after stirring;
step 2.4, preparing the tripterygium wilfordii slow release microcapsule: and (3) dropwise adding the primary emulsion phase obtained in the step (2.3) into the mixed water phase obtained in the step (2.2) under the condition that the rotation speed is 1000-1500rpm, continuously stirring for 1-2h, heating to 60 ℃ after the surface of the capsule is solidified, simultaneously reducing the stirring speed to 500rpm, stirring for 3-4h, washing, filtering and drying after stirring is finished, and thus obtaining the tripterygium wilfordii slow-release microcapsule.
2. The method for preparing the tripterygium wilfordii slow-release microcapsule according to claim 1, wherein the ultrasonic power in step 1.2 is 900-.
3. The method for preparing the tripterygium wilfordii slow-release microcapsule according to claim 1, wherein the separation conditions of the high pressure liquid chromatography in the step 1.3 are as follows: 21.2mm × 250mm, 5 μm C18 chromatographic column, flow rate of 20mL/min, mobile phase of methanol, each injection of 500 μ L.
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| CN100490833C (en) * | 2006-09-06 | 2009-05-27 | 天津医科大学 | Method of supercritical CO2 for extracting terpenoid of effective component of triperygium wilfordii, and composition |
| CN104473904B (en) * | 2014-11-10 | 2018-06-05 | 苏州蔻美新材料有限公司 | Slow-release microcapsule and its application |
| CN107129505B (en) * | 2017-06-01 | 2019-03-29 | 陕西理工大学 | The industrial production process of lactone element monomers in a kind of ginkgo leaf purification |
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2018
- 2018-05-21 CN CN201810487302.4A patent/CN108478540B/en not_active Expired - Fee Related
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