CN102240268A - Sustained/controlled release microsphere of biological extract Genipin cross-linked chitosan coated stilbene compound and preparation method thereof - Google Patents

Abstract

The invention discloses a slow-controlled release microsphere prepared by using biological extract genipin as a crosslinking agent to prepare chitosan-coated stilbene compounds and a preparation method thereof. The microspheres use the cross-linked condensate layer of chitosan and genipin as the capsule shell, and are coated with drugs such as 3,5-dihydroxy-4-isopropylstilbene, resveratrol, pterostylbene, and oxidized resveratrol Retrol or picatanol, the drug stability of the product is improved, and the sustained and controlled release of the drug is realized; the genipin released after the capsule shell is metabolized in the body has the characteristics of biocompatibility and non-cytotoxicity. The invention is suitable for coating 3,5-dihydroxy-4-isopropyl stilbene, resveratrol, pterostilbene, oxidized resveratrol, piceatanol and others on genipin cross-linked chitosan Sustained and controlled release microspheres of stilbene compounds and preparation thereof, the preparation method requires low control conditions, is easy to operate, the quality of the microspheres is uniform, and the particle size is 0.1 to 100 microns. The encapsulation rate is 30-90%.

Description

Sustained and controlled release microspheres of biological extract genipin cross-linked chitosan coated stilbenes and preparation method thereof

technical field

The invention belongs to the field of chemical and pharmaceutical preparation, and in particular relates to a slow-controlled release microsphere coated with stilbene compounds by biological extract genipin cross-linked chitosan and a preparation method thereof.

Background technique

Stilbene compounds 3,5-dihydroxy-4-isopropyl stilbene, resveratrol (3,4',5-trihydroxy stilbene), pterostylbene (3,5-dimethoxy base-4'-hydroxystilbene), oxidized resveratrol (2,4,3',5'-tetrahydroxystilbene), piceatanol (3,4,3',5'-tetrahydroxystilbene) Hydroxystilbene), etc., are active non-flavonoid polyphenols, which are widely found in natural foods or plants. A large number of studies have shown that they have anti-inflammatory, anti-oxidation, anti-bacterial, anti-free radical, inhibition of platelet aggregation, anti-thrombotic , Anti-hyperlipidemia, can be used for the prevention and treatment of fungi, eczema, arteriosclerosis, coronary heart disease, viral hepatitis, AIDS, cancer and other diseases. Since the above stilbene molecules contain different numbers of phenolic hydroxyl groups, they are easily oxidized in the air, resulting in changes in the color and performance of the product, reducing the stability of the product and affecting its efficacy when used as a drug; and The presence of phenolic hydroxyl groups also makes this type of compound susceptible to the influence of pH, and complex changes may occur when it is used as a drug or health product when it is compounded with other drugs, affecting its effective curative effect.

Chitosan (Chitosan, CS) is a natural polymer polyaminopolysaccharide with bioadhesion and various biological activities. It can be biodegraded by various enzymes in the body and completely absorbed by organisms; at the same time, chitosan can also increase The permeability of the drug to the biomembrane is conducive to the better efficacy of the drug in the cell. The preparation of chitosan as slow and controlled release microspheres as a good biological organic carrier to coat drug molecules is a new dosage form in pharmaceutical preparations, which can achieve slow and controlled release of drugs, tissue targeting, and improve drug stability, etc. satisfactory effect.

At present, there is a technology of preparing slow and controlled release microspheres by emulsification, cross-linking and curing of chitosan. The cross-linking agent used is usually an aldehyde compound. The positive charge of the carbonyl carbon of the aldehyde makes it particularly sensitive to nucleophiles. Sensitive, its characteristic reaction is the nucleophilic addition reaction of the carbon-oxygen double bond, and the amino group in the chitosan molecule can undergo a Schiff reaction with the carbonyl carbon of the aldehyde, linking the chitosan and the aldehyde to form an interaction network. The commonly used cross-linking agents in the past include formaldehyde, glyoxal, glutaraldehyde, etc. Formaldehyde has high activity, but the formaldehyde released by the cross-linked product after metabolism in the body is relatively toxic, and the two aldehyde groups of glyoxal are directly connected. The cross-linked product formed with chitosan has a tighter crystal structure, and the hardness of the obtained microspheres is high, which is not suitable for drug coating. There are three methylene groups between the two aldehyde groups of glutaraldehyde. After linking, due to the bondage of the cross-linking point and the steric hindrance effect, the strength of the formed cross-linked product is lower than that of the cross-linked product of glyoxal and chitosan, which is more suitable as a drug carrier. For example, the applicant's patent No. 200910075128.3 Chinese invention patent discloses a "chitosan-coated drug sustained-release microspheres and preparation method thereof", involving resveratrol as the core drug, chitosan The slow-release microsphere drug whose condensate with glutaraldehyde is capsule shell and its preparation method. In this patent, the strength of the microspheres prepared by using glutaraldehyde as a crosslinking agent is moderate, but because glutaraldehyde is toxic to human cells, although the microspheres were thoroughly cleaned during the preparation process to remove the residual glutaraldehyde on the surface of the microspheres , but after the microsphere enters the human body and is metabolized by enzymes, it will still release glutaraldehyde, which is harmful to the human body, so its application is subject to certain restrictions.

Genipin is one of the active ingredients of the traditional Chinese medicine Eucommia ulmoides. It is a natural product obtained by separating and purifying geniposide. It belongs to the iridoid heterocyclic compound and is often used as an anti-inflammatory and choleretic Treatment of various hepatobiliary diseases (can promote the secretion of bile) and infection symptoms. Genipin has multiple active functional groups such as hydroxyl group, carboxyl group and acetal (hidden aldehyde group), and can undergo condensation reaction with chitosan under certain conditions to form a cross-linked network. Compared with traditional cross-linking agents, slow-release microspheres formed by natural biological extracts of genipin and chitosan enter the human body to release genipin and chitosan through enzymatic metabolism, because both have good biological compatibility. Capacitance and no cytotoxicity are beneficial to the application of chitosan sustained and controlled release formulations in the field of biomedicine.

Contents of the invention

The technical problem to be solved in the present invention is to provide a slow and controlled release microsphere of biological extract genipin cross-linked chitosan coated stilbenes and a preparation method thereof, using chitosan as a base material, Genipin, a natural biological extract, is used as a cross-linking agent to coat stilbene compounds 3,5-dihydroxy-4-isopropyl stilbene, resveratrol, pterostilbene, oxidized resveratrol or white One of Pitetanol. In the preparation process of the microspheres, the natural biological crosslinking agent genipin is used as the crosslinking agent, and the formed chitosan slow-release microspheres are non-toxic to the human body, which significantly improves the stability of the coated compound, and at the same time realizes The purpose of slow and controlled release: the preparation method provided by the present invention has low requirements on control conditions and is easy to operate. The quality of the prepared slow-release microspheres is uniform, and the encapsulation rate of stilbene compounds by the capsule shell is 30% to 90%.

In order to solve the above-mentioned technical problems, the present invention is achieved through the following technical solutions:

A slow-controlled release microsphere of biological extract genipin cross-linked chitosan coated stilbene compound, the microsphere is a core-shell structure, wherein the core is the stilbene compound coated by the shell , which is special in that the shell is a cross-linked condensate layer of chitosan and natural natural extract genipin.

The microspheres are spherical, the diameter of the microspheres is 0.1-100 microns, and the surface of the microspheres has a porous structure.

As a limitation of the present invention, the stilbene compound is 3,5-dihydroxy-4-isopropyl stilbene, resveratrol, pterostilbene, oxidized resveratrol or piceatanol One of.

The raw materials for making the active ingredients of the slow and controlled release microspheres include the following components by mass or volume:

Chitosan 3-20 parts by weight,

Stilbene compounds 0.1-5 parts by weight,

Genipin Parts by weight 1,

Dispersion medium Parts by volume 1000-3000;

The ratio relationship between the above parts by weight and parts by volume is gram:milliliter.

The present invention also provides a preparation method of the above-mentioned sustained and controlled release microspheres, which is carried out in the following steps:

(1) preparation of chitosan solution

Mix and stir 2-36% acetic acid solution and chitosan evenly, and let it stand until the air bubbles disappear, so that the ratio of chitosan weight to the volume of the solution is 1-6% (unit is g/mL, or kg/ L) chitosan solution A;

(2) preparation of genipin solution

Take distilled water and genipin respectively, add genipin into the distilled water, stir at 10-60 °C for 0.5-4 h, cool down to room temperature, and obtain a concentration of 0.1-2% (unit is g/mL, or kg/ L) genipin solution B;

(3) Compound dissolution

The stilbene compound is dissolved in an alcoholic solvent, then added to the chitosan solution A, and mixed uniformly to obtain a mixed solution C; wherein the volume-to-weight ratio of the alcoholic solvent to the stilbene compound is 1 to 5:1, The unit is mL/g or L/kg;

(4) Preparation of slow and controlled release microspheres by emulsification

Add C to the dispersion medium containing the emulsifier, stir, and adjust the temperature of the mixture to obtain a uniformly mixed mixed liquid D; wherein the weight part of the emulsifier is 0-10% of the dispersion medium;

Take the above solution B and add it to D. After curing for 1-12 hours, centrifuge, wash with ethyl acetate, filter with suction, and dry to obtain the slow-controlled release microspheres of chitosan-coated stilbene compound.

The concentration of the acetic acid solution used is 2% to 36%, and the volume-to-weight ratio of it to chitosan is 16.5 to 100 milliliters: 1 gram.

Described emulsifying agent is Span-80 (Span-80), Span-85 (Span-85), Twen-80 (Tween-80), Twen-85 (Tween-85), Poloxamer (poloxamer) Mu), OP-10 (alkylphenol polyoxyethylene ether) or a mixture of several.

The dispersion medium is one or a mixture of soybean oil, olive oil, peanut oil, sunflower oil or liquid paraffin.

The alcohol solvent is one or a mixture of methanol, ethanol, propanol, isopropanol or n-butanol.

The reaction temperature in the step (4) of preparing the sustained and controlled release microspheres by emulsification is 0-60°C.

The curing reaction time is 1-12 hours. (Because in the process of preparing microspheres, if other factors (such as temperature, crosslinking agent dosage, etc.) are different, there will be a big difference in the reaction time. If the temperature is low, the reaction time will change accordingly long, otherwise it will be shortened, the length of time is interrelated with other influencing factors, it is reasonable to determine that it is 1-12 hours, and it can ensure the realization of the purpose of the invention.)

In the raw material ratio of the present invention, one of 3,5-dihydroxy-4-isopropyl stilbene, resveratrol, pterostilbene, piceatanol or oxidized resveratrol is used as the core drug; Chitosan is the capsule material, which plays the role of coating the core drug; the biological extract genipin acts as a cross-linking agent, and the cross-linking agent is to connect chitosan molecules through a chemical reaction with chitosan , so that the microspheres are stronger.

Due to the adoption of the above-mentioned technical scheme, the technical progress achieved by the present invention compared with the prior art lies in that in the preparation process of the microspheres, the non-toxic natural biological cross-linking agent genipin is used to replace the more toxic pentamethylene Dialdehyde greatly reduces the toxicity of the metabolites produced by the microspheres after oral administration, making the prepared microspheres more suitable for pharmaceutical dosage forms and health care products. The slow and controlled release microspheres significantly improve the stability of the coated compound properties, while achieving the purpose of sustained and controlled release; the invention is suitable for coating 3,5-dihydroxy-4-isopropyl stilbene, resveratrol, pterostylbene Sustained and controlled release microspheres of stilbene compounds such as resveratrol, oxidized resveratrol, and piceatanol and preparation thereof, and are also applicable to slow and controlled release microspheres and preparation of other stilbene compounds. The present invention provides The preparation method requires low control conditions and is easy to operate, the prepared slow-release microspheres are of uniform quality, and the encapsulation rate of the stilbene compound by the capsule shell is 30-90%.

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Description of drawings

Fig. 1 is a schematic diagram of the longitudinal section structure of the sustained and controlled release microspheres of the present invention.

In the figure: 1—stilbene compound (core); 2—the channel inside the sphere communicating with the surface of the microsphere; 3—the capsule shell formed by the condensation product layer of chitosan and genipin.

Detailed ways

As shown in Figure 1, it is a slow-controlled release microsphere of biological extract genipin cross-linked chitosan coated stilbene compound, which is a core-shell structure, wherein the core is the coated stilbene compound layer , the capsule shell is a cross-linked condensate layer of chitosan and genipin.

The preparation method of the sustained and controlled release microspheres is carried out according to the following steps:

(1) preparation of chitosan solution

Mix and stir 2-36% acetic acid solution and chitosan evenly, let stand until the air bubbles disappear, and prepare chitosan solution A with a concentration of 1-6% (unit is g/mL);

(2) preparation of genipin solution

Take distilled water and genipin respectively, add genipin into the distilled water, stir at 10-60°C for 0.5-4 hours, and cool down to room temperature to obtain genipin with a concentration of 0.1-2% (unit: g/mL). Flat solution B;

(3) Compound dissolution

The stilbene compound is dissolved in an alcohol solvent, then added to the chitosan solution A, and mixed uniformly to obtain a mixed solution C;

(4) Preparation of slow and controlled release microspheres by emulsification

Add C to the dispersion medium containing emulsifier, stir, and adjust the temperature of the mixture to obtain a uniform mixed liquid D;

Take the above solution B and add it to D. After curing for 1-12 hours, centrifuge, wash with ethyl acetate, filter with suction, and dry to obtain the slow-controlled release microspheres of chitosan-coated stilbene compound.

Concrete raw material proportioning and relevant parameters of each embodiment are as follows:

 

 

For example: Example 3 relates to a slow-controlled release microsphere of a biological extract genipin cross-linked chitosan coated stilbene compound pterostilbene, the ingredients are shown in Figure 1, and the preparation method is carried out in accordance with the following steps:

(31) Configure 3.0% chitosan (CS) solution

Take 20 mL of acetic acid solution with a concentration of 10% and 0.6 g of chitosan, mix them, stir them evenly, and let them stand until the air bubbles disappear to prepare chitosan solution A with a weight-to-volume ratio of 3.0% ( W/V );

(32) Configure 1% genipin solution

Add 0.04 g of genipin to 4 mL of distilled water, stir at 60 °C for 30 min, and cool down to room temperature to obtain 1% (W/V) genipin solution B;

(33) Drug dissolution

Take 0.04 g of pterocarpus stilbene and dissolve it with 0.12 g of n-butanol, then add it into the above-mentioned solution A, mix well, and obtain mixed solution C; then

(34) Preparation of chitosan-coated drug sustained and controlled release microspheres by emulsification

Add C to 120mL olive oil containing 5% ( W / V ) Twen-80, stir, and mix well to obtain a mixture D;

Add solution B into D, solidify at 35°C for 6 h, centrifuge, wash with ethyl acetate, filter with suction, and dry to obtain slow-release microspheres of stilbene compounds coated with genipin cross-linked chitosan , The encapsulation rate is 90%.

The difference between the specific operation process of other examples and the embodiment is only that the parameters involved are different and the types of corresponding substances are different, and the actual data reference is shown in the above table.

Claims (10)

1. A slow-controlled release microsphere of biological extract genipin cross-linked chitosan coated stilbene compounds, said microsphere is a core-shell structure, characterized in that: wherein the core is coated by shell stilbene compounds, the shell is a cross-linked condensate layer of chitosan and natural extract genipin. 2. the slow-controlled release microsphere of biological extract genipin cross-linked chitosan coating stilbene compound according to claim 1, it is characterized in that: described microsphere is spherical, and microsphere particle diameter is 0.1 to 100 microns, the surface of the microsphere is a porous structure. 3. the sustained and controlled release microspheres of biological extract genipin cross-linked chitosan coated stilbenes according to claim 1 , characterized in that: the core drug is 3,5-dihydroxy- One of 4-isopropylstilbene, resveratrol, pterostilbene, oxidized resveratrol, or piceatanol. 4. a preparation method of the slow-release microspheres of biological extract genipin cross-linked chitosan coated stilbenes compound as described in any one of claim 1-3, it is characterized in that making The raw materials of the active ingredients required for the slow and controlled release microspheres include the following compositions: Chitosan 3-20 parts by weight, Stilbene compounds 0.1-5 parts by weight, Genipin Parts by weight 1, Dispersion medium 1000-3000 parts by volume; The ratio relationship between the above parts by weight and parts by volume is gram:milliliter. 5. the preparation method of the sustained and controlled release microspheres of biological extract genipin cross-linked chitosan coated stilbenes as claimed in claim 4, is characterized in that it is carried out according to the following steps: (1) preparation of chitosan solution Mix and stir 2-36% acetic acid solution and chitosan evenly, let stand until the air bubbles disappear, and prepare chitosan solution A with a weight-to-volume concentration of 1-6%; (2) preparation of genipin solution Take distilled water and genipin respectively, add genipin into the distilled water, stir at 10-60°C for 0.5-4 hours, and cool down to room temperature to obtain genipin solution B with a concentration of 0.1-2% by weight to volume; (3) Compound dissolution The stilbene compound is dissolved with an alcoholic solvent, then added to the chitosan solution A, and mixed uniformly to obtain a mixed solution C; wherein the volume-to-weight ratio of the alcoholic solvent to the stilbene compound is 1 to 5 milliliters: 1 gram; (4) Preparation of slow and controlled release microspheres by emulsification Add C to the dispersion medium containing the emulsifier, stir, and adjust the temperature of the mixture to obtain a uniformly mixed mixture D; wherein the volume fraction of the emulsifier is 0-10% of the dispersion medium; Add the above solution B to D, solidify for 1-12 hours, centrifuge, wash with ethyl acetate, filter with suction, and dry to obtain slow-controlled release microspheres of chitosan-coated stilbene compound. 6. the preparation method of the sustained and controlled release microspheres of biological extract genipin cross-linked chitosan coated stilbene compound according to claim 5, it is characterized in that: the concentration of acetic acid solution used is 2～36 %, its volume-to-weight ratio to chitosan is 16.5 to 100 milliliters: 1 gram. 7. the preparation method of the sustained and controlled release microsphere of biological extract genipin cross-linked chitosan coating stilbene compound according to claim 5, is characterized in that: described emulsifier is Span-80, One of Span-85, Twen-80, Twen-85, Poloxamer, OP-10, or a mixture of at least two. 8. the preparation method of the slow and controlled release microsphere of biological extract genipin cross-linked chitosan coating stilbene compound according to claim 5, it is characterized in that: described dispersion medium is soybean oil, olive One of oil, peanut oil, sunflower oil or liquid paraffin, or a mixture of at least two. 9. the preparation method of the slow and controlled release microsphere of biological extract genipin cross-linked chitosan coating stilbene compounds according to claim 5, is characterized in that: described alcoholic solvent is methyl alcohol, ethanol , propanol, isopropanol or n-butanol, or a mixture of at least two. 10. the preparation method of the slow and controlled release microsphere of biological extract genipin cross-linked chitosan coating stilbene compound according to claim 5, is characterized in that: emulsification prepares slow and controlled release microsphere step ( 4) The reaction temperature is 0-60°C.

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