CN108774330B

CN108774330B - Preparation method of crosslinked hyaluronic acid gel, obtained product and application

Info

Publication number: CN108774330B
Application number: CN201810999791.1A
Authority: CN
Inventors: 刘建建; 杨莹莹; 张燕; 吴万福; 郭学平
Original assignee: Bloomage Biotech Co Ltd; Shandong Huaxi Haiyu Biological Medicine Co Ltd
Current assignee: Huaxi Biotechnology Tianjin Co ltd; Bloomage Biotech Co Ltd
Priority date: 2018-08-30
Filing date: 2018-08-30
Publication date: 2020-11-10
Anticipated expiration: 2038-08-30
Also published as: CN108774330A

Abstract

The invention discloses a preparation method of cross-linked hyaluronic acid gel, an obtained product and application, comprising the following steps: standing HA and cross-linking agent in alkaline solution at 1-4 deg.C for 8-16h, standing at 50-80 deg.C for 5min-30min, and standing at 1-4 deg.C for 0.5-5 h; repeating the steps of standing and reacting at 50-80 ℃ for 5-30 min and then standing and reacting at 1-4 ℃ for 0.5-5h for 3-5 times to obtain the product. The highly crosslinked hyaluronic acid gel particles are prepared by repeating the conditions of high temperature and low temperature, the obtained product has moderate hardness, the interior of the particles has a special three-dimensional reticular molecular structure, the particles have adsorbability and elasticity, and the particles are environment-friendly, can be used as abrasive particles or massage particles of washing and caring products, play roles in stimulating and promoting blood circulation, promoting new cell regeneration, mildly removing cutin and the like, and enable the skin to be smooth, soft and healthy.

Description

Preparation method of crosslinked hyaluronic acid gel, obtained product and application

Technical Field

The invention relates to a preparation method of cross-linked hyaluronic acid gel, and also relates to a product obtained by the method and application of the product as scrub particles or massage particles for washing and caring products, belonging to the technical field of cross-linked hyaluronic acid preparation.

Background

The human skin metabolic cycle is 28 days. However, if the skin is dry due to lack of water, the process becomes slow, and old waste cells accumulate on the surface of the skin, so that the skin cannot breathe normally. The skin becomes dull and rough. This condition can be changed by periodic use of the abrasive product. The common frosted particles in the traditional frosted products comprise micro soft beads, almonds, walnuts, bone meal, pearl layer powder, calcium carbonate and the like. With the increasing development of abrasive products, new abrasive particulate materials have come into play, such as passion fruit seeds, cellulose, chemically synthesized wax beads, and the like.

The traditional micro flexible beads which are most commonly used are micro plastic particles, and the substances are insoluble in water and cause serious environmental pollution. After people use cosmetics and nursing products, the micro soft beads can be directly discharged into a sewage system, and water treatment facilities in a sewage treatment plant have difficulty in filtering fine plastic particles. The waste water is then discharged to the ocean where fish ingest toxic plastic particles, and when ingested by other fish or humans, the toxins in the plastic beads enter the food chain. Researchers have shown that "micro flexible beads" can cause symptoms of "cell necrosis, inflammation and tissue laceration" after entering the human body. According to the law of the national academy of public health, the use of environmentally hazardous beads was prohibited from 7/1 in 2017. Australia et al also listed a list of products containing "mini-beads" by participating in the "beathemimrcobead" activity. Mineral powder such as calcium carbonate is hard in texture, not suitable for common use, and not suitable for sensitive skin. The problems of dust pollution, large energy consumption and the like easily exist in the production and preparation of micro-particles by natural plants such as almonds, walnuts, passion fruit seeds and the like, and the particle texture is also hard.

Patent CN 104194982a provides a preparation method of sea sand soap, wherein sea sand is added as frosted particles, which has the defects of hard particles and large skin friction irritation. The frosted bath salt and the bath soap mentioned in the patents CN106726754A and CN106675890A adopt polyethylene frosted particles and mineral salt as the frosted particles, and have the defect that the polyethylene frosted particles are easy to cause environmental pollution. Patent nos. CN107280990A and CN107280991A propose that sugar (brown sugar, etc.) is used as the abrasive grains, which have a defect that the abrasive grains have a large friction force and are not suitable for frequent use.

Hyaluronic Acid (HA) is a linear polysaccharide composed of (1 → 3) -2-acetamido-2-deoxy- β -D-glucose- (1 → 4) -O- β -D-glucuronic acid disaccharide repeating units, widely exists in many connective tissues such as skin, vitreous body of eye, cartilage and synovial fluid of joint, and plays physiological roles such as moisture retention, nutrition and repair. The cross-linked HA HAs good physical and chemical properties and biocompatibility, and is often applied to the fields of cosmetic reshaping, isolation protection, ophthalmic surgery, joint cavity injection, surgical protection, drug carriers, cosmetic raw materials and the like. At present, the research on hyaluronic acid mainly focuses on enzymolysis resistance, aging resistance, biocompatibility and wettability, and there is no report on hyaluronic acid or cross-linked hyaluronic acid as the ground particles.

For example, patent publication No. CN 101107270A discloses a method of cross-linking HA with divinylsulfone, which mentions that DVS is cross-linked with HA at three temperatures of 5 to 50 deg.C (preferably 20 to 30 deg.C), 20 to 100 deg.C (preferably 30 to 60 deg.C), and 0 to 40 deg.C (preferably 10 to 30 deg.C), but the hydrogel produced by this method is mainly used for pharmaceutical carriers, excipients, or diluents, and HAs the advantage of having good homogeneity, softness, and injectability, but the hardness is not suitable for skin cleansing. Patent publication No. CN 107936272A discloses a method for three-step crosslinking of HA under mild conditions, in which HA and a crosslinking agent are crosslinked at three temperatures of 0-10 deg.C, 30-60 deg.C and 10-40 deg.C, and the obtained crosslinked HA gel HAs good anti-enzymatic and anti-radiation properties, but the hardness is not suitable for cleaning skin.

Disclosure of Invention

In order to solve the defects of the existing frosted particles for the washing and caring product, the invention provides a preparation method of cross-linked hyaluronic acid gel and the obtained product.

The invention also provides application of the cross-linked hyaluronic acid particles as abrasive particles or massage particles of washing and caring products, the cross-linked hyaluronic acid particles have a special three-dimensional reticular molecular structure, moderate texture and hardness, can freely adjust the particle size, can effectively remove aged cells and clean the skin, also have adsorbability and elasticity, can massage the skin and hair follicles, promote blood circulation, can meet the requirements of various washing and caring products, are derived from natural substances, are environment-friendly, have low production energy consumption, have small skin irritation, and are suitable for large-scale use.

The specific technical scheme of the invention is as follows:

a method for preparing a cross-linked hyaluronic acid gel, the method comprising the steps of:

(1) uniformly mixing hyaluronic acid and a cross-linking agent in an alkaline solution, and standing for 8-16h at 1-4 ℃;

(2) standing the reaction solution obtained in the step (1) at 50-80 ℃ for 5-30 min, and then standing at 1-4 ℃ for 0.5-5 h;

(3) and (3) after the step (2), repeating the step of standing and reacting the reaction liquid obtained in the step (2) at 50-80 ℃ for 5-30 min and then standing and reacting at 1-4 ℃ for 0.5-5h for 3-5 times to obtain gel C, wherein the gel C is the cross-linked hyaluronic acid gel.

Further, in step (3), after repeating step (2) 3-5 times, the final reaction became gel C without filtration. And (3) after the step (3), the steps of dialyzing and granulating the gel C to obtain the cross-linked hyaluronic acid gel particles. Dialysis removes unreacted crosslinker. The granulation may be performed by a conventionally disclosed granulation method such as screen granulation, and the size of the prepared granules is in the range of 30 μm to 2000 μm for convenience of use as abrasive granules and massage granules, and the shape of the granules is preferably spherical.

Further, in the step (1), the hyaluronic acid refers to hyaluronic acid and salts thereof, preferably hyaluronate. The hyaluronate is any soluble salt of hyaluronic acid which can be used for cosmetics and washing and caring products, and comprises but is not limited to sodium hyaluronate, calcium hyaluronate, magnesium hyaluronate, potassium hyaluronate, zinc hyaluronate and the like, the performance of each hyaluronate is similar, and the most common hyaluronate is sodium hyaluronate. The molecular weight of the hyaluronic acid and the salt thereof is 50-350 ten thousand Da, the molecular weight has an influence on the swelling performance (namely, the crosslinking degree) of the crosslinked hyaluronic acid gel particles, the larger the molecular weight is, the smaller the swelling coefficient of the crosslinked hyaluronic acid gel is, namely, the higher the crosslinking degree is, and the preferable molecular weight range is 150-250 ten thousand Da.

Further, in the step (1), the crosslinking agent is any one of an epoxide crosslinking agent, divinyl sulfone, polyethylene glycol crosslinking agent or carbodiimide, the epoxide crosslinking agent includes 1, 4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether and the like, and the polyethylene glycol crosslinking agent includes polyethylene glycol diglycidyl ether, four-arm star polyethylene glycol epoxide and the like. The cross-linking agent has an effect on the viscoelasticity of the final cross-linked hyaluronic acid gel, and a preferred cross-linking agent is BDDE. The amount of the crosslinking agent is generally 2 to 10% by mass, preferably 4 to 6% by mass, of the hyaluronic acid or a salt thereof.

Further, in the step (1), the cross-linking agent reacts with HA in an alkaline environment, the HA is not easily dissolved due to too small alkali concentration, and the HA with too large alkali concentration is degraded in strong alkalinity. The alkaline solution is any one of sodium carbonate, sodium bicarbonate or sodium hydroxide solution. The mass concentration of the alkaline solution is preferably 0.5 to 5 wt%. The concentration of hyaluronic acid and its salt in the alkaline solution should not be too high or too low, and is generally 10-30% (w/v), preferably 20% (w/v).

Further, in the step (3), repeating the operation of the step (2) 3 to 5 times on the reaction solution obtained in the step (2) means that: and (3) repeating the step of standing and reacting the reaction liquid obtained in the step (2) at 50-80 ℃ for 5-30 min, and standing and reacting at 1-4 ℃ for 0.5-5h for 3-5 times. For example, the step of repeating the operation of step (2) 3 times is: after the step (2), standing and reacting the reaction liquid obtained in the step (2) at 50-80 ℃ for 5-30 min, and standing and reacting at 1-4 ℃ for 0.5-5h (repeating once); then standing and reacting at 50-80 ℃ for 5-30 min, and standing and reacting at 1-4 ℃ for 0.5-5h (repeating twice); and then standing and reacting at 50-80 ℃ for 5-30 min, and standing and reacting at 1-4 ℃ for 0.5-5h to finally obtain the gel C (repeating for three times).

Preferably, in the step (1), the standing reaction time is 10-12 h. In the step (2), the time for the reaction solution to stand and react at 50-80 ℃ is preferably 15-20min, and the time for the reaction solution to stand and react at 1-4 ℃ is preferably 2-3 h.

The cross-linked hyaluronic acid gel or gel particles obtained by the method have a special three-dimensional reticular molecular structure inside, are insoluble in water, have moderate hardness, have adsorbability and elasticity, have good effects of removing aged cells and cleaning skin, and can massage skin and hair follicles and promote blood circulation. Therefore, the cross-linked hyaluronic acid gel or gel particles prepared by the method and the application of the cross-linked hyaluronic acid gel or gel particles as scrub particles or massage particles of washing products are also within the protection scope of the invention. When the gel or gel particles are used as abrasive particles or massage particles, the gel or gel particles can mildly remove cutin, promote skin blood circulation, have good compatibility with skin and small irritation to the skin, are suitable for sensitive skin, and can achieve deep cleaning and massage effects.

Further, when the cross-linked hyaluronic acid gel or gel particles are used as the abrasive particles or the massage particles, the dosage of the abrasive particles or the massage particles is 0.01-10% of the total mass of the washing and caring product.

Further, the washing and caring products comprise facial cleanser, facial milk cleanser, facial soap, facial cream, facial lotion, facial honey, flour, facial gel, scrub cream, massage cream, exfoliating powder, toothpaste, tooth powder, soap, bath foam, bath salt, shampoo, hair conditioner and the like. Can be added into facial cleanser, face cleaning, cutin removing, and bath product as scrub granule for gently removing cutin, or added into massage, hair washing, and hair care product as massage granule for massaging skin and hair follicle and promoting blood circulation.

The invention has the following advantages:

according to the invention, a special crosslinking means is adopted, the high-crosslinking hyaluronic acid gel particles are prepared under the repeated high-temperature-low-temperature condition, the preparation process is easy to control, the environment is friendly, the production energy consumption is low, the obtained crosslinked hyaluronic acid gel particles are insoluble in water, the texture is moderate in hardness, the particles have special three-dimensional reticular molecular structures inside, the particles have adsorptivity and elasticity, and the particles are environment-friendly, on one hand, the gel particles can be used as abrasive particles of washing care products, dead cells are gently removed from the surface of the skin by physical friction, and the purpose of deep cleaning is achieved; on the other hand, the gel particles can be used as massage particles of washing and caring products, and can stimulate and promote blood circulation and promote new cell regeneration through moderate squeezing massage; on the other hand, the gel particles have a three-dimensional network molecular structure inside, so that pore garbage can be adsorbed, and the skin is smooth, soft and healthy.

Detailed Description

The present invention is further illustrated by the following specific examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention.

In the following examples, the concentrations are mass concentrations unless otherwise specified.

Example 1 temperature examination

2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.05g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) are uniformly mixed in 10ml of 1% sodium hydroxide solution, 6 parts of the mixture are kept stand in parallel in a refrigerator at the temperature of 1-4 ℃ for 8 hours, then the mixture is respectively kept stand at 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃ for 5 minutes, and then the mixture is kept stand in ice water at the temperature of 1-4 ℃ for 0.5 hour; repeating the steps of standing at 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃ for 5min, and then placing in ice water at 1-4 ℃ for 0.5h to react for 3 times to obtain the cross-linked HA gel block. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

And (3) elasticity examination: the elasticity of the crosslinked HA particles was measured with a Haake RS6000 (seimer feishell science and technology (china)) instrument under the conditions: a rotor: p20Ti L; gap value: 1.00 mm; temperature: 25 ℃; measurement mode: oscillating frequency scan CD; stress: 1 percent; frequency range: 0.01 to 1 Hz. The modulus of elasticity (G') at 0.1Hz was recorded. The results are shown in Table 1.

TABLE 1

Sample numbering	G’(Pa)
		①	1201
②	2015
		③	2519
④	2805
		⑤	2989
⑥	1890

The elastic modulus (G ') can reflect the hardness and elasticity of the crosslinked Hyaluronic Acid (HA) particles, the G ' is too low, the hardness of the crosslinked hyaluronic acid particles is insufficient, and the crosslinked hyaluronic acid particles are not suitable for being used as abrasive particles, and when the G ' is more than 2000, the hardness and elasticity of the crosslinked HA particles meet the requirements. As can be seen from Table 1, the second step crosslinking temperature is between 50-80 ℃ and meets the requirements, and at 40 ℃, the temperature is too low and the elasticity is insufficient; at 90 ℃ the HA is partially degraded due to the high temperature.

The first step of standing at 1-4 ℃ and the third step of reacting at 1-4 ℃ are both refrigerated in a refrigerator and placed or ice-water bath, and temperature examination is not carried out.

Example 2 Cross-linking time and number of repetitions

(1) First step of examination of standing time

2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.05g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) are uniformly mixed in 10ml of 1% sodium hydroxide solution, 7 parts of the mixture are placed in a refrigerator with the temperature of 1-4 ℃ respectively for standing for 6h, 8h, 10h, 12h, 14h, 16h and 18h, then the mixture is reacted for 5min at the temperature of 50 ℃ and then placed in ice water with the temperature of 1-4 ℃ for reaction for 0.5 h; repeating the step of reacting at 50 ℃ for 5min and then placing in ice water at 1-4 ℃ for reacting for 0.5h for 3 times to obtain the cross-linked HA gel block. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

The elasticity test method was the same as in example 1. The results are shown in Table 2.

TABLE 2

Sample numbering	G’(Pa)
		①	1889
②	2199
		③	2310
④	2385
		⑤	2200
⑥	2000
		⑦	1982

As can be seen from Table 2, the standing time of the first step is 8-16h, and the obtained crosslinked HA particles G' are above 2000Pa and meet the requirements. Wherein the optimal time is 10-12 h.

(2) Second step examination of the crosslinking reaction time

2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.05g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) are uniformly mixed in 10ml of 1% sodium hydroxide solution, 7 parts of the mixture are placed in a refrigerator at the temperature of 1-4 ℃ for standing for 8 hours, and then the mixture reacts at the temperature of 50 ℃ for 5min, 10min, 15min, 20min, 25min, 30min and 35min, and then the mixture is placed in ice water at the temperature of 1-4 ℃ for reaction for 0.5 hour; repeating the steps of reacting at 50 ℃ for 5min, reacting at 10min, reacting at 15min, reacting at 20min, reacting at 25min, reacting at 30min and reacting at 35min in ice water at 1-4 ℃ for 0.5h for 3 times to obtain the cross-linked HA gel block. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

The elasticity test method was the same as in example 1. The results are shown in Table 3.

TABLE 3

Sample numbering	G’(Pa)
		①	2199
②	2323
		③	2589
④	2617
		⑤	2100
⑥	2001
		⑦	1882

As can be seen from Table 3, when the second-step crosslinking reaction time is 5-30 min, the obtained crosslinked HA particles meet the requirement that G' is more than 2000Pa, and the optimal time is 15-20 min.

(3) Third step crosslinking reaction time examination

2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.05g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) are uniformly mixed in 10ml of 1% sodium hydroxide solution, 7 parts of the mixture are placed in a refrigerator with the concentration of 1-4 ℃ for standing for 8 hours, then the mixture is reacted for 5 minutes at the temperature of 50 ℃, and then the mixture is placed in ice water with the temperature of 1-4 ℃ for reacting for 0.5 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours and 6 hours; repeating the step of reacting at 50 ℃ for 5min, and then placing the mixture in ice water at 1-4 ℃ for 3 times, wherein the step is 0.5h, 1h, 2h, 3h, 4h, 5h and 6h to obtain the cross-linked HA gel block. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

The elasticity test method was the same as in example 1. The results are shown in Table 4.

TABLE 4

Sample numbering	G’(Pa)
		①	2199
②	2323
		③	2489
④	2517
		⑤	2201
⑥	2010
		⑦	1901

As shown in Table 4, the crosslinked HA particles obtained in the third step when the crosslinking reaction time is 0.5-5h all meet the requirement that G' is more than 2000Pa, wherein 2-3h is optimal.

(4) Examination of the number of repetitive reactions

2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.05g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) are uniformly mixed in 10ml of 1% sodium hydroxide solution, 5 parts of the mixture are placed in a refrigerator with the concentration of 1% and kept stand for 8 hours, then the mixture is reacted for 5 minutes at the temperature of 50 ℃, then the mixture is placed in ice water with the temperature of 1-4 ℃ and reacted for 0.5 hour, and the steps of reacting for 5 minutes at the temperature of 50 ℃ and then placing the mixture in the ice water with the temperature of 1-4 ℃ for 0.5 hour are repeated for (2 times, 3 times, three times, 5 times and 6 times), so that the crosslinked HA gel block is obtained. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

The elasticity test method was the same as in example 1. The results are shown in Table 5.

TABLE 5

Sample numbering	G’(Pa)
		①	1503
②	2199
		③	2389
④	2555
		⑤	1986

As can be seen from Table 5, the crosslinked HA particles obtained when the second and third steps are repeated 3-5 times meet the requirement that G' is greater than 2000 Pa.

EXAMPLE 3 Cross-linker dosage examination

2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.02g (1%), 0.04g (2%), 0.08g (4%), 0.12g (6%), 0.16g (8%), 0.20g (10%), and 0.24g (12%) are uniformly mixed in 10ml of 1% sodium hydroxide solution, the mixture is placed in a refrigerator at the temperature of 1-4 ℃ for standing for 8 hours, then the mixture is placed in ice water at the temperature of 50 ℃ for reaction for 5 minutes, then the ice water is placed in ice water at the temperature of 1-4 ℃ for reaction for 0.5 hour, the reaction is repeated for 5 minutes at the temperature of 50 ℃ and then the ice water at the temperature of 1-4 ℃ for reaction for 3 times, and the crosslinked HA gel block is obtained. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

Method for determining residual crosslinker: taking a proper amount of sample, and dissolving and diluting the sample with acetone. The test is carried out by taking 1, 4-butanediol diglycidyl ether as a reference substance according to a method specified by external standard 0521 of the fourth general rules of pharmacopoeia of the people's republic of China 2015 edition, taking a DB-17 capillary column as a chromatographic column, and measuring the column temperature: the initial temperature is 150 ℃, the temperature is increased to 260 ℃ at 30 ℃/min, and the temperature is kept for 10 min; the detector is a hydrogen Flame Ionization Detector (FID), and the temperature of the detector is 300 ℃; the temperature of a sample inlet is 260 ℃; the carrier gas was nitrogen and the flow rate was 5 mL/min. Precisely measuring 1 μ L of the test solution and the reference solution, respectively, injecting into a gas chromatograph, recording chromatogram, and calculating according to external standard method by peak area. The results are shown in Table 6.

The elasticity test method was the same as in example 1. The results are shown in Table 6.

TABLE 6

Sample numbering	G’(Pa)	Crosslinker residue (μ g/g)
			①	1866	0.05
②	2056	0.1
			③	2337	0.5
④	2479	1.0
			⑤	2580	1.8
⑥	2613	1.9
			⑦	2787	3.5

As can be seen from Table 6, G' meets the requirement of more than 2000Pa when the amount of the crosslinking agent is 2% to 12%, but the amount of the crosslinking agent is more than 2. mu.g/G when the amount is 12%. Since BDDE has some toxicity, it is generally required that the crosslinker residue be no greater than 2 μ g/g, with less residue being preferred. And comprehensively considering the residual quantity of G' and the crosslinking agent, and finally selecting the crosslinking agent with the dosage of 2-10%, preferably 4-6%.

Example 4 hyaluronic acid molecular weight examination

Uniformly mixing 2g of sodium hyaluronate with the molecular weight of 10 ten thousand, 50 ten thousand, 100 ten thousand, 150 ten thousand, 200 ten thousand, 250 ten thousand, 300 ten thousand and 350 ten thousand Da and 0.05g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) in 10ml of 1% sodium hydroxide solution, standing for 10 hours in a refrigerator at the temperature of 1-4 ℃, standing for reacting for 15min at the temperature of 50 ℃, and then placing in an ice-water bath at the temperature of 1-4 ℃ for reacting for 2 hours; repeating the step of standing and reacting at 50 ℃ for 15min, and then placing in an ice-water bath at 1-4 ℃ for 2h for 4 times to obtain the crosslinked HA gel block. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

The elasticity test method was the same as in example 1, and the results are shown in Table 7.

And (3) measurement of expansion coefficient: taking about 5g of cross-linked HA gel particles, adding 50mL of 9g/L physiological saline, stirring at room temperature for 20min, filtering with a No. six sieve, standing until no liquid drips within 2min, placing the gel in a culture dish with constant weight, weighing the gel m₁Drying at 105 deg.C to constant weight, weighing m₂. Coefficient of expansion m according to the formula₁/m₂And (4) calculating. The results are shown in Table 7.

TABLE 7

Sample numbering	Coefficient of expansion	G’(Pa)
			①	75.3	768
②	20.0	2010
			③	18.5	2150
④	15.3	2233
			⑤	15.1	2360
⑥	14.8	2487
			⑦	13.5	2591
⑧	11.2	2699

The molecular weight of HA varies, and the expansion coefficient of the prepared samples, i.e., the degree of crosslinking, varies, and the expansion coefficient tends to decrease (the degree of crosslinking increases) as the molecular weight of HA increases. Crosslinked HA particles having a coefficient of expansion of between 10 and 20 (especially around 15) are considered most suitable as abrasive particles, since too high a coefficient of expansion, i.e. too low a degree of crosslinking, affects the abrasive effect of the crosslinked HA particles. In the test, the expansion coefficient and G' of the crosslinked HA particles prepared from the HA with the molecular weight of 50-350 ten thousand meet the requirements, and 150-250 ten thousand of HA particles are optimal in terms of the expansion coefficient.

Example 5

2g of sodium hyaluronate (with the molecular weight of 50 ten thousand Da) and 0.04g of crosslinking agent carbodiimide are uniformly mixed in 10ml of 0.5% sodium carbonate solution, and the mixture is stood for 8 hours at the temperature of 1-4 ℃, then stood for reaction for 30min at the temperature of 50 ℃ and then reacted for 0.5 hour at the temperature of 1-4 ℃; repeating the steps of standing at 50 ℃ for 30min and then standing at 1 ℃ for 0.5h for 5 times to obtain the cross-linked HA gel block. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 20-mesh screen to obtain cross-linked HA gel particles with the particle size of 2000 mu m.

Example 6

2g of sodium hyaluronate (with the molecular weight of 350 ten thousand Da) and 0.2g of cross-linking agent DVS (divinyl sulfone) are uniformly mixed in 10ml of 5% sodium bicarbonate solution, the mixture is kept stand at the temperature of 1-4 ℃ for 16h, then the mixture is kept stand at the temperature of 80 ℃ for 5min and then is kept stand at the temperature of 1-4 ℃ for 5 h; repeating the steps of standing at 80 ℃ for 5min and then standing at 4 ℃ for 5h for 3 times to obtain the cross-linked HA gel block. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 200-mesh screen to obtain cross-linked HA gel particles with the particle size of 30 mu m.

Example 7

2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.1g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) are uniformly mixed in 10ml of 1% sodium hydroxide solution, and the mixture is stood for 10 hours at the temperature of 1-4 ℃, then stood for reaction for 15 minutes at the temperature of 65 ℃ and then placed for reaction for 3 hours at the temperature of 1-4 ℃; repeating the step of standing at 65 ℃ for 15min and then standing at 1-4 ℃ for 3h for 4 times to obtain the crosslinked HA gel block. And dialyzing the cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

Example 8 facial cleanser with addition of cross-linked HA scrub particles

The crosslinked HA gel particles of example 7 were added to the facial cleanser as scrub particles to make a scrub particle-added facial cleanser. The formula is (wt%): 10% of stearic acid, 10% of palmitic acid, 2% of lanolin, 2% of coconut oil, 2% of glycerol monostearate, 2% of N-acyl-N-methyl sodium taurate, 4% of potassium hydroxide, 0.1% of EDTA disodium, 10% of glycerol, 0.3% of preservative, 0.01% of frosted particles and the balance of deionized water.

The preparation method comprises the following steps: adding stearic acid, palmitic acid, lanolin, coconut oil, glycerin and antiseptic into an oil phase pot, heating and stirring to 70 ℃, filtering, adding into an emulsifying pot, and keeping the temperature at 70 ℃; adding potassium hydroxide into a water phase pot, fully dissolving with deionized water, filtering, adding into an emulsifying pot, keeping at 70 ℃ for reacting for 1h, cooling to 45 ℃ after reaction, adding other raw materials except the frosted particles, uniformly stirring, adding the frosted particles when the temperature is reduced to 30 ℃, and uniformly mixing to obtain the facial cleanser added with the frosted particles.

Example 9

A scrub particle-added facial cleanser was prepared as in example 8, except that: the content of the abrasive grains was 10 wt%.

Example 10

A scrub particle-added facial cleanser was prepared as in example 8, except that: the content of the abrasive grains was 0.5 wt%.

Comparative example 1

(1) And (3) crosslinking under mild conditions: the same procedure was followed as in example 7 except that the reaction temperature and time were varied. The method comprises the following steps: 2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.1g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) are uniformly mixed in 10ml of 1% sodium hydroxide solution, and the mixture is stood at the temperature of 1-4 ℃ for 10 hours and then stood at the temperature of 40 ℃ for reaction for 13 hours. Dialyzing the obtained cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

(2) The three-step crosslinking of low temperature-high temperature-low temperature: 2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.1g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) are uniformly mixed in 10ml of 1% sodium hydroxide solution, and the mixture is stood for 10 hours at the temperature of 1-4 ℃, then stood for reaction for 15 minutes at the temperature of 65 ℃ and then placed for reaction for 3 hours at the temperature of 1-4 ℃. Dialyzing the obtained cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

(3) The three-step crosslinking of low temperature-high temperature-low temperature: 2g of sodium hyaluronate (with the molecular weight of 200 ten thousand Da) and 0.1g of crosslinking agent BDDE (1, 4-butanediol diglycidyl ether) are uniformly mixed in 10ml of 1 percent sodium hydroxide solution, and the mixture is stood at 25 ℃ for reaction for 10 hours, then stood at 50 ℃ for reaction for 15 minutes and then stood at 20 ℃ for reaction for 3 hours. Dialyzing the obtained cross-linked HA gel block, washing off the cross-linking agent which does not participate in the reaction, and granulating through a 40-mesh screen to obtain cross-linked HA gel particles with the particle size of 100 mu m.

Comparative example 2

(1) The facial cleanser added with the mineral calcium carbonate frosted particles comprises the following components: a facial cleanser was prepared by the method of example 10 except that: the crosslinked HA gel particles of example 7 were replaced with calcium carbonate particles having a particle size of 100 μm.

(2) The facial cleanser added with the guava seed frosted particles comprises the following components: a facial cleanser was prepared by the method of example 10 except that: the crosslinked HA gel particles of example 7 were replaced with guava seed particles, the particle size of which was 100 μm.

(3) Facial cleanser with addition of comparative example 1(1) crosslinked HA gel particles: a facial cleanser was prepared by the method of example 10 except that: the crosslinked HA gel particles of example 7 were replaced with the crosslinked HA gel particles of comparative example 1 (1).

(4) Facial cleanser with addition of comparative example 1(2) crosslinked HA gel particles: a facial cleanser was prepared by the method of example 10 except that: the crosslinked HA gel particles of example 7 were replaced with the crosslinked HA gel particles of comparative examples 1 (2).

(5) Facial cleanser with addition of comparative example 1(3) crosslinked HA gel particles: a facial cleanser was prepared by the method of example 10 except that: the crosslinked HA gel particles of example 7 were replaced with the crosslinked HA gel particles of comparative examples 1 (3).

Application example 1 elasticity test of crosslinked HA particles

The gel particles of examples 5 to 7 and comparative example 1 were subjected to the elasticity test in the same manner as in example 1, and the test results are shown in Table 8.

TABLE 8

Product(s)	G’(Pa)
		Example 5	2209
Example 6	2689
		Example 7	2963
Comparative example 1(1)	330
		Comparative examples 1(2)	295
Comparative examples 1(3)	497

As can be seen from the data in the table above: the G' of the crosslinked HA particles obtained by the high-temperature-low-temperature repeated crosslinking method can meet the requirement of more than 2000Pa when the crosslinking agent selects carbodiimide, DVS and BDDE, wherein the highest crosslinking agent is selected when the crosslinking agent selects BDDE. In comparative example 1, the medium-temperature and conditional crosslinking and the "low-temperature-high-temperature-low-temperature" three-step crosslinking did not give crosslinked HA particles satisfying the conditions.

Application example 2 cleaning effect evaluation

7 pig back skins (8cm multiplied by 8cm) were removed, 0.01g of activated carbon was evenly smeared on each skin, and a skin model for testing was prepared and recorded as A, B, C, D, E, F, G. Model a was washed according to the shampooing step using a normal facial cleanser containing no abrasive particles (i.e., the facial cleanser formulation of example 10 with abrasive particles removed), model B was washed according to the shampooing step using the facial cleanser prepared in example 10, model C was washed according to the shampooing step using the facial cleanser prepared in comparative example 2(1), model D was washed according to the shampooing step using the facial cleanser prepared in comparative example 2(2), model E was washed according to the shampooing step using the facial cleanser prepared in comparative example 2(3), model F was washed according to the shampooing step using the facial cleanser prepared in comparative example 2(4), and model G was washed according to the shampooing step using the facial cleanser prepared in comparative example 2 (5). And observing the cleaning effect and the skin irritation condition by adopting a naked eye observation mode and a 2.5-time magnifying glass observation mode after the cleaning is finished. The main observation indexes of the cleaning effect are skin texture and activated carbon residue in pores, scurf residue and the like, and the main observation indexes of the skin irritation condition are frosted damage and the like. The cleaning effect is counted as 10-0 minutes from good to bad, and the irritation is counted as 10-0 minutes from high to low. The evaluation results are shown in Table 9.

TABLE 9 evaluation of cleansing effect of different cleansing milks

Group of	Cleaning effect	Irritation property
			A	6 minutes	0 point (min)
B	10 minutes	2 is divided into
			C	7 points of	8 is divided into
D	7 points of	6 minutes
			E	6 minutes	1 minute (1)
F	6 minutes	1 minute (1)
			G	7 points of	1 minute (1)

As can be seen from the comparison of A-G, the cleansing milk added with the cross-linked HA gel particles prepared by the invention HAs the best cleansing effect and the lowest irritation, because the cross-linked HA gel particles obtained by the invention are natural substances, have elasticity, moderate hardness and good compatibility with skin, and have small irritation to the skin. In addition, the cross-linked HA gel particles prepared by the invention have a three-dimensional network molecular structure inside, have adsorbability, can adsorb pore garbage, have a mild sanding function and can achieve a deep cleaning effect. From the comparison of B, E, F, G, it can be seen that the crosslinked HA particles prepared by mild crosslinking and "low temperature-high temperature-low temperature" crosslinking have insufficient elasticity and hardness, do not have the efficacy of abrasive particles, and have poor cleaning effect.

Application example 3 evaluation of exfoliating Effect

The cutin removing effect of the facial cleanser is evaluated by using the skin water loss as an index, and the cutin removing effect is stronger when the skin water loss is larger.

The test was performed using a self-control method, with 20 subjects, 5 males, 15 females, and 30 to 45 years of age. The trial areas were marked on the flexed side of the forearm of the left and right arm of the subject for seven trial areas, each 3cm x 3cm, marked A, B, C, D, E, F, G, with the seven trial areas corresponding to the same position for each subject. For each subject, the normal facial cleanser without frosted particles was used in panel a (i.e., the facial cleanser formulation of example 10 was removed from the frosted particles), the facial cleanser prepared in example 10 was used in panel B, the facial cleanser prepared in comparative example 2(1) was used in panel C, the facial cleanser prepared in comparative example 2(2) was used in panel D, the facial cleanser prepared in comparative example 2(3) was used in panel E, the facial cleanser prepared in comparative example 2(4) was used in panel F, and the facial cleanser prepared in comparative example 2(5) was used in panel G. Washing according to the face washing steps: wetting the test area, gently massaging for 1min with the same amount of corresponding facial cleanser, washing, and wiping. The skin water loss increase rate was calculated by measuring the amount of skin water loss in each region before and after washing for 30min, 1h, 3h, and 6h using Corneometer CM825 and Corneometer TM 300. The skin water loss increase rate (skin water loss after washing-skin water loss before washing)/skin water loss before washing is 100%. The results are shown in Table 10.

TABLE 10 increase in skin moisture loss after washing of various face washes (%)

Test area	30min	1h	3h	6h
					A	17.56	16.23	17.19	14.33
B	28.08	25.34	26.47	24.29
					C	51.55	50.37	48.69	46.01
D	41.03	40.17	39.32	38.04
					E	16.85	15.73	15.79	13.74
F	17.11	16.07	15.55	13.89
					G	17.88	16.93	15.67	13.97

As can be seen from Table 10, the cleansing milk containing the mineral scrub particles and the guava seed scrub particles was very effective in exfoliating skin, but had too much water loss and skin irritation, and was not suitable for daily use. The facial cleanser added with the cross-linked HA gel particles prepared by the invention also HAs the function of exfoliating, but the exfoliating effect is mild, and the facial cleanser is suitable for daily use and deep cleaning. The addition of the crosslinked HA gel particles prepared by the method of comparative example 1 did not provide exfoliating effect and even may block pores, and the increase rate of skin moisture loss was slightly decreased compared to the facial cleanser without the addition of the scrub particles, although there was no significant difference.

The foregoing is merely a preferred embodiment of the present invention and is not intended to limit the invention, and various modifications and variations of the invention, such as the selection of other soluble hyaluronic acid salts, the use of cross-linked HA gel particles in other toiletries as abrasive particles or massage particles, will occur to those skilled in the art. Any modification, equivalent replacement, improvement, simulation and the like made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. A preparation method of a cross-linked hyaluronic acid gel is characterized by comprising the following steps:

(3) after the step (2), repeating the step of standing and reacting the reaction liquid obtained in the step (2) at 50-80 ℃ for 5-30 min and then standing and reacting at 1-4 ℃ for 0.5-5h for 3-5 times to obtain gel C;

(4) dialyzing and granulating the gel C to obtain cross-linked hyaluronic acid gel particles;

the hyaluronic acid refers to hyaluronic acid and salts thereof, and the molecular weight of the hyaluronic acid and the salts thereof is 50-350 ten thousand Da;

the cross-linking agent is any one of epoxide cross-linking agent, divinyl sulfone, polyethylene glycol cross-linking agent or carbodiimide, and the dosage of the cross-linking agent is 2-10% of the mass of the hyaluronic acid and the salt thereof.

2. The method of claim 1, wherein: in the step (4), the size of the cross-linked hyaluronic acid gel particles is 30-2000 μm.

3. The method according to claim 1 or 2, characterized in that: the cross-linking agent is an epoxide cross-linking agent.

4. The method of claim 3, wherein: the hyaluronic acid refers to hyaluronate.

5. The method of claim 3, wherein: the cross-linking agent is 1, 4-butanediol diglycidyl ether.

6. The method according to claim 1 or 2, characterized in that: the molecular weight of the hyaluronic acid and the salt thereof is 150-250 ten thousand; in the step (1), the mass volume concentration of the hyaluronic acid and the salt thereof in the alkaline solution is 10-30%.

7. The method of claim 6, wherein: in the step (1), the mass volume concentration of the hyaluronic acid and the salt thereof in the alkaline solution is 20%.

8. The method of claim 1, wherein: the dosage of the cross-linking agent is 4-6% of the mass of the hyaluronic acid and the salt thereof.

9. The method according to claim 1 or 2, characterized in that: in the step (1), the alkaline solution is any one of sodium carbonate, sodium bicarbonate or sodium hydroxide solution.

10. The method according to claim 1 or 2, characterized in that: in the step (1), the mass concentration of the alkaline solution is 0.5-5 wt%.

11. The method according to claim 1 or 2, characterized in that: in the step (1), standing reaction time is 10-12 h; in the step (2), the reaction solution is kept stand at 50-80 ℃ for 15-20min and kept stand at 1-4 ℃ for 2-3 h.

12. The crosslinked hyaluronic acid gel according to the method for preparing a crosslinked hyaluronic acid gel according to any of claims 1-11.

13. The use of the crosslinked hyaluronic acid gel of claim 12 as a scrub particle or massage particle for a toiletry.

14. Use according to claim 13, characterized in that: the cleaning and caring product comprises facial cleanser, facial milk, facial soap, facial cream, facial lotion, facial honey, flour, facial gel, scrub cream, massage cream, cutin removing powder, toothpaste, dentifrice, soap, bath lotion, bath salt, shampoo, and hair conditioner.

15. Use according to claim 13, characterized in that: the dosage of the crosslinked hyaluronic acid gel is 0.01-10% of the total mass of the washing and caring product.