CN101507733A

CN101507733A - Nano micromolecule hyaluronic acid and preparation method thereof

Info

Publication number: CN101507733A
Application number: CNA2009101317766A
Authority: CN
Inventors: 杜以波; 胡如桂; 杜铁柱
Original assignee: BEIJING INSTITUTE OF OLIGO PEPTIDE BIOLOGY
Current assignee: BEIJING INSTITUTE OF OLIGO PEPTIDE BIOLOGY
Priority date: 2009-04-02
Filing date: 2009-04-02
Publication date: 2009-08-19
Anticipated expiration: 2029-04-02
Also published as: CN101507733B

Abstract

The invention relates to a method for preparing nanometer low molecular hyaluronic acid, which comprises the following steps that: ultralow molecular weight hyaluronic acid with an average molecular weight less than 1,000D, namely nanometer hyaluronic acid oligomer, is obtained from hyaluronic acid with a molecular weight from tens of thousands to millions through hydrolysis methods such as enzyme and the like. The nanometer low molecular hyaluronic acid obtained by the method has functional characteristics of promoting the formation of capillary vessels, promoting the healing of wounds, having remarkable skin affinity and permeability and the like which are remarkably different from common hyaluronic acid, and can be taken as a high-grade raw material for cosmetology, health care food, and medicine.

Description

Nano micromolecule hyaluronic acid and preparation method thereof

Technical field

The present invention relates to a kind of preparation method of nano micromolecule hyaluronic acid, be that hyaluronic acid with macromolecule is by method for hydrolysis such as enzymes specifically, obtain the following ultra-low molecular amount hyaluronic acid of mean molecule quantity 1000 dalton, i.e. nanoscale oligomerization hyaluronic acid (NOHA).The nano micromolecule hyaluronic acid that obtains with this method has the promotion vascularization significantly different with the acid of normal transparent matter, promotes functional characteristics such as wound healing, significant skin affinity and permeability, can be used as good medicine, health product, cosmetic material.

Background technology

Hyaluronic acid (HA) is to be present in the particularly hypodermic mucopolysaccharide of organism, because its high moisture-keeping functions is widely used in the cosmetic material as hyaluronic acid.In addition, by orally ingestible hyaluronic acid or its salt, the reduction of the hyaluronic acid contents that has originally of compensation organism can be seen the effect of improving the preserving moisture of skin, elasticity and flexibility.Therefore, hyaluronic acid or its salt can be used for food or medicine.

Hyaluronic acid is the polysaccharide of high molecule, molecular weight from 500,000 to 3,000,000 dalton, have high viscosity, 1% aqueous solution has been heavy-gravity, so, there is defective in hyaluronic use, be used for cosmetics, because its ultrahigh molecular weight, its skin care effect is mainly reflected in the outside and preserves moisture, it almost is difficult to the inside that penetrates to the skin, and does not have what effect substantially for intrinsic nutrition of skin and maintenance; As food or food additive, because the viscosity height hinders addition, mouthfeel is poor, digests and assimilates poor performance, and cosmetology function is difficult to realize.

Studies show that hyaluronic biological activity is relevant with its degree of polymerization, degree of polymerization atom activity more is high more.The degree of polymerization reduces, molecular weight reduces, viscosity reduces, serviceability improves greatly, and in the time of near molecular weight drops to 1000 dalton, (degree of polymerization is less than 10 promptly to obtain the oligomerization hyaluronic acid, molecular weight is less than 4000), its function strengthens greatly, and shows the not available functional characteristic of normal transparent matter acid, as promoting vascularization, promoting biological activitys such as wound healing, antitumor, immune transfer.And nano micromolecule hyaluronic acid (degree of polymerization is less than 5, and mean molecule quantity is less than 1000), performance especially is better than the O-HA of high polymerization degree, on skin, show as fabulous skin affinity, fast permeability, the beauty treatment repair function.

By degraded macromolecule hyaluronic acid, can obtain the hyaluronic acid of low polymerization degree.Hyaluronic biodegrading process has multiple, as the physics method, all can make hyaluronic acid degradation by factors such as heating, mechanical shear stress, ultraviolet, ultrasound wave, cobalt irradiations, but the dynamics of its degraded is limited, is difficult to obtain nano micromolecule hyaluronic acid; Chemical degradation method has acid hydrolysis, basic hydrolysis, hypochlorite oxidation degraded etc., and the difficult control of chemical degradation method degradation process is easy to generate brown stain and organic solvent residual.Japan Jitian opens up the method for hydrolysis (number of patent application 200680008997.9) that history adopts acid medium, and it adopts under the acid medium condition of ethanol or acetone solvent, and the hyaluronan molecule amount is dropped to 10000 once; Shortcomings such as biological degradation method is degraded under the effect of hyaluronidase, and degraded intensity is low, the cycle is long, Cui Xiangzhen etc. can be molecular weight control 10000 once in " optimum reaction conditions of hyaluronic acid enzyme catalysis hyaluronic acid hydrolysis " publishes the article.

The present invention is that molecular weight distribution is at the nano micromolecule hyaluronic acid below 1000 by enzyme and the bonded method of acid degradation with macromolecular hyaluronic acid degradation.With the purpose that realizes that its beautifying skin, cell are improved looks fast, are convenient to digest and assimilate.

Summary of the invention

One of purpose of the present invention is by the combining of enzyme process and acid degradation method, and obtains the controlled nano micromolecule hyaluronic acid hydrolysate of molecular weight.

A kind of is raw material with macromolecule hyaluronic acid or its salt, and by the nano micromolecule hyaluronic acid that degraded obtains, its mean molecule quantity is below 1000 dalton, and hyaluronic acid one aggressiveness, dimer, trimer, tetramer content surpass 90%.

A kind of preparation method of nano micromolecule hyaluronic acid is the combination in acid medium hydrolysis and two steps of enzymatic degradation.

A kind of preparation method of nano micromolecule hyaluronic acid, its hyaluronic acid raw material, be meant hyaluronic acid or its salt, hyaluronic acid is meant with glucuronic acid and these two kinds of sugar of N-acetamido glucose to repeating to constitute unitary polysaccharide, its esters is unqualified, for example sodium salt, potassium salt, calcium salt, zinc salt, magnesium salt, ammonium salt etc.It generally is with biological tissue such as cockscomb, umbilical cord, eyeball, skin, cartilage or cultivates hyaluronic acid or its salt that Streptococcus microorganism etc. obtains by fermentation method.

A kind of preparation method of nano micromolecule hyaluronic acid, its two-step reaction used medium is a water, can not with an organic solvent not use oxidant.

A kind of preparation method of nano micromolecule hyaluronic acid, at first hydrolysis under acid medium is meant at pH value 1.5～4, hyaluronic acid concentration 0.1～6%, 70～95 ℃ of hydrolysis temperatures, hydrolysis time are 1～5 hour.

A kind of preparation method of nano micromolecule hyaluronic acid after hydrolysis under the acid medium, is proceeded enzymatic degradation, the used enzyme of enzymatic degradation is a hyaluronidase, and the degradation reaction condition is pH value 4～7, hyaluronic acid concentration 0.1～6%, 20～70 ℃ of hydrolysis temperatures, hydrolysis time are 1～5 hour.

With the nano micromolecule hyaluronic acid that this method obtains, have the skin affinity and the permeability of height, can promote generation, promotion wound healing, the promotion collagen protein synthesis of blood capillary.This product is used for food, has that palatability is strong, mouthfeel good, digestible characteristics.Have the enhance immunity ability, promote the effect that skeleton forms, for the preserving moisture of whole skin, soft, reduce xerosis cutis, treat pachylosis, improve skin elasticity, alleviate wrinkle, effect is remarkable.

The present invention is further described below in conjunction with embodiment:

Embodiment 1: the hyaluronate sodium of molecular weight 1,500,000 (glucuronic acid content 47.3%) 10g adds water 1000g, stirring and dissolving.Hydrolysis under the acid condition: pH value is 2.5,85 ℃ of constant temperature hydrolysis 3 hours; Enzymatic degradation: cool to 50 ℃, add hyaluronidase (enzyme dosage 150,000 U/L), pH value is 5,50 ℃ of constant temperature hydrolysis 3 hours, is heated to 90 ℃ of constant temperature enzyme denaturing then 10 minutes.Use filter paper filtering, obtain clear, colorless, tasteless, transparent micromolecule hyaluronic acid degraded solutions.The said goods by spray drying, obtains the white powder micromolecule hyaluronic acid through 60 ℃ of vacuum concentration.

The aforesaid liquid product is through chemical analysis, and hyaluronic acid disaccharidase labelling unit glucuronic acid content reaches 46.1% in its product molecule, and its effective retention rate of degraded back reaches 97.4%; The aforesaid liquid product is through liquid-phase chromatographic analysis, and the molecular weight distribution that obtains product is as shown in table 1.Table 1 molecular weight distribution analysis result shows that catabolite 90% above molecular weight distribution is 300～1300, and the corresponding hyaluronic acid degree of polymerization is 1～4, belongs to ultra-low molecular amount distribution in the oligomerization hyaluronic acid.

The ultralow micromolecule hyaluronic acid molecular weight distribution of table 1

Embodiment 2: the hyaluronate sodium of molecular weight 1,500,000 (glucuronic acid content 47.3%) 50g adds water 950g, stirring and dissolving.Hydrolysis under the acid condition: pH value is 2,70 ℃ of constant temperature hydrolysis 4 hours; Enzymatic degradation: cool to 40 ℃, add hyaluronidase (enzyme dosage 800,000 U/L), pH value is 6,40 ℃ of constant temperature hydrolysis 3 hours, is heated to 90 ℃ of constant temperature enzyme denaturing then 10 minutes.Use filter paper filtering, obtain clear, colorless, tasteless, transparent micromolecule hyaluronic acid degraded solutions.The said goods by spray drying, obtains the white powder micromolecule hyaluronic acid through 60 ℃ of vacuum concentration.

The aforesaid liquid product is through chemical analysis, and hyaluronic acid disaccharidase labelling unit glucuronic acid content reaches 46.5% in its product molecule, and its effective retention rate of degraded back reaches 98.3%; The aforesaid liquid product is through liquid-phase chromatographic analysis, and the molecular weight distribution that obtains product is as shown in table 2.Table 2 molecular weight distribution analysis result shows that catabolite 90% above molecular weight distribution is 300～1300, and the corresponding hyaluronic acid degree of polymerization is 1～3, belongs to ultra-low molecular amount distribution in the oligomerization hyaluronic acid.

The ultralow micromolecule hyaluronic acid molecular weight distribution of table 2

Claims

1, a kind of nano micromolecule hyaluronic acid is characterized in that, its mean molecule quantity is below 1000 dalton, and hyaluronic acid one aggressiveness, dimer, trimer, tetramer content surpass 90%;

2, according to the preparation method of the described nano micromolecule hyaluronic acid of claim 1, be to be raw material with macromolecule hyaluronic acid or its salt, obtain nano micromolecule hyaluronic acid by degraded.Degradation process is acid degradation and combining that enzymatic degradation two goes on foot.Acid degradation reactant aqueous solution condition pH value 1.5～4, hyaluronic acid concentration 0.1～6%, 70～95 ℃ of hydrolysis temperatures, hydrolysis time are 1～5 hour.Down after the degraded, proceed enzymatic degradation through acid medium, the used enzyme of enzymatic degradation is a hyaluronidase, and the degradation reaction condition is, pH value 4～7, and hyaluronic acid concentration 0.1～6%, 20～70 ℃ of hydrolysis temperatures, hydrolysis time are 1～5 hour.

3, according to claim 2, a kind of preparation method of nano micromolecule hyaluronic acid, its two-step reaction used medium is a water, can not with an organic solvent not use oxidant.