CN114601743B

CN114601743B - Dihydromyricetin liposome and preparation and application thereof

Info

Publication number: CN114601743B
Application number: CN202210284468.2A
Authority: CN
Inventors: 屈恋; 贺青; 刘逸华; 李彩萍; 曾茜; 杨楠楠
Original assignee: Baocui Biotechnology Co ltd
Current assignee: Baocui Biotechnology Co ltd
Priority date: 2022-03-22
Filing date: 2022-03-22
Publication date: 2022-09-13
Anticipated expiration: 2042-03-22
Also published as: CN114601743A

Abstract

The invention provides a dihydromyricetin liposome and preparation and application thereof. The dihydromyricetin liposome comprises the following components in parts by weight: 0.1-10 parts of dihydromyricetin, 0.1-10 parts of an oil phase, 2-8 parts of an emulsifier No. 1, 4-12 parts of an emulsifier No. 2, 25-35 parts of a co-emulsifier, 0.1-15 parts of a preservative and 30-45 parts of water; wherein the oil phase comprises cholesterol, sodium stearate, coco-caprylate/caprate. The dihydromyricetin liposome prepared by the invention not only can keep the self-oxidation resistance, bacteriostasis, corrosion prevention and other performances of the raw material dihydromyricetin, but also can obviously improve the water solubility, stability and transdermal absorption capacity of the dihydromyricetin, and can effectively play the effects of oil control and moisture retention when being used for skin.

Description

Dihydromyricetin liposome and preparation and application thereof

Technical Field

The invention belongs to the technical field of liposome preparation. More particularly, relates to a dihydromyricetin liposome and preparation and application thereof.

Background

Dihydromyricetin is mostly extracted from a woody vine plant of Ampelopsis of Vitaceae, and also can be extracted from Hovenia acerba, the main active component of the dihydromyricetin is a flavonoid compound, the dihydromyricetin not only has multiple peculiar efficacies of scavenging free radicals, resisting oxidation, thrombus, tumor, inflammation and the like, but also has bacteriostatic action on bacillus subtilis, staphylococcus aureus, salmonella, Escherichia coli, aerobacter, beer yeast, Rhodotorula glutinis, Penicillium, Aspergillus niger, Aspergillus flavus, Mucor and Rhizopus, and particularly has obvious action on gram-positive and gram-negative cocci or bacilli.

However, because dihydromyricetin has poor water solubility and stability and is easily decomposed under the conditions of high temperature and illumination, the dihydromyricetin cannot be widely applied in the cosmetic industry all the time, in order to overcome the defect of poor water solubility, liposomes are mostly adopted to wrap the dihydromyricetin at present, for example, the prior art discloses a preparation method of multi-vesicle type dihydromyricetin liposome, and specifically, a water phase solution is prepared by using Tween 80 and PEG-4000, an oil phase solution is prepared by using dihydromyricetin, cholesterol and egg yolk lecithin, then the oil phase solution is dripped into the water phase solution, and the multi-vesicle type dihydromyricetin liposome is obtained after treatment, but the liposome prepared by the method is in a white emulsion state at 25 ℃, 50 ℃ and illumination conditions, and has poor stability.

Therefore, a dihydromyricetin liposome with good water solubility and stability is needed to be found, which is necessary for the wide application of dihydromyricetin in the cosmetic industry.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the dihydromyricetin liposome with better water solubility and stability, which effectively plays the roles of oxidation resistance and bacteriostasis.

The invention mainly aims to provide a dihydromyricetin liposome.

The invention also aims to provide a preparation method of the dihydromyricetin liposome.

The invention also aims to provide the application of the dihydromyricetin liposome in preparing oil control products.

The above purpose of the invention is realized by the following technical scheme:

the invention provides a dihydromyricetin liposome which comprises the following components in parts by weight: 0.1-10 parts of dihydromyricetin, 0.1-10 parts of an oil phase, 2-8 parts of an emulsifier No. 1, 4-12 parts of an emulsifier No. 2, 25-35 parts of a co-emulsifier, 0.1-15 parts of a preservative and 30-45 parts of water; wherein the oil phase comprises cholesterol, sodium stearate, coco-caprylate/caprate.

Aiming at the defects of poor water solubility and poor stability of the dihydromyricetin, the invention adopts specific oil phases of cholesterol, sodium stearate, coconut oil alcohol-caprylate/caprate and other auxiliary materials to be compounded with each other to wrap the dihydromyricetin, so that the obtained dihydromyricetin liposome not only can keep the performances of oxidation resistance, bacteriostasis, corrosion prevention and the like of the dihydromyricetin, but also can remarkably improve the water solubility, stability and transdermal absorption capacity of the dihydromyricetin, and can effectively play the effects of oil control and moisture preservation when being used for skin.

Wherein, the coco-caprylate/caprate in the oil phase can better dissolve and disperse emulsifier No. 1 (such as soybean lecithin), so that the liposome can still ensure better stability under the condition of not adding an organic solvent; in order to better emulsify the coconut oil alcohol-caprylate/caprate, the dihydromyricetin liposome is also added with an emulsifier No. 2 (such as PEG-40 hydrogenated castor oil), and the dihydromyricetin liposome and the emulsifier No. 1 are emulsified together to play a complementary effect. In addition, the addition of the other two components (cholesterol and sodium stearate) in the oil phase can make the structure of the liposome more stable and not easy to agglomerate and deposit.

Wherein, the structural formula of the dihydromyricetin is as follows:

preferably, 4-8 parts of dihydromyricetin, 2-6 parts of an oil phase, 4-6 parts of an emulsifier No. 1, 6-10 parts of an emulsifier No. 2, 28-32 parts of a co-emulsifier, 4.6-12 parts of a preservative and 30-45 parts of water.

Most preferably, 5 parts of dihydromyricetin, 3.7 parts of oil phase, 5 parts of emulsifier No. 1, 8 parts of emulsifier No. 2, 30 parts of co-emulsifier, 10.6 parts of preservative and 37.7 parts of water.

Further preferably, in the oil phase, the mass ratio of the coco-caprylate/caprate to the cholesterol to the sodium stearate is 1-5: 0.1-0.8: 0.1 to 0.7.

More preferably, the mass ratio of the coconut oil alcohol-caprylate/caprate to the cholesterol to the sodium stearate is 2-4: 0.2-0.5: 0.3 to 0.7.

Most preferably, the mass ratio of coco-caprylate/caprate, cholesterol, sodium stearate is 3: 0.3: 0.4.

preferably, emulsifier No. 1 is soy lecithin and emulsifier No. 2 is PEG-40 hydrogenated castor oil.

Preferably, the co-emulsifier comprises 1, 3-propanediol and 1, 2-pentanediol.

Further preferably, in the co-emulsifier, the mass ratio of 1, 3-propylene glycol to 1, 2-pentanediol is 11-19: 11 to 19, more preferably 13 to 17: 13-17, most preferably 15: 15.

preferably, the preservative comprises p-hydroxyacetophenone, 1, 3-propanediol, and 1, 2-pentanediol.

Further preferably, in the preservative, the mass ratio of the p-hydroxyacetophenone to the 1, 3-propanediol to the 1, 2-pentanediol is 0.4-0.8: 2-8: 2 to 8, more preferably 0.5 to 0.7: 4-6: 4-6, most preferably 0.6: 5: 5.

in addition, the invention also provides a preparation method of the dihydromyricetin liposome, which comprises the following steps:

s1, mixing and homogenizing dihydromyricetin, an oil phase, an emulsifier No. 1 and a co-emulsifier according to the formula amount to obtain a mixture A;

s2, dissolving the preservative with the formula amount in water, adding the dissolved preservative into the mixture A of S1, and stirring;

s3, cooling to 50-60 ℃, adding the emulsifier No. 2, and stirring for 25-35 min to obtain the dihydromyricetin liposome.

S1, homogenizing to prevent emulsifier No. 1 from agglomerating and avoid the problem of uneven particle size of the prepared liposome and to provide particles, S2, stirring to make the emulsifier added in S3 better connect the oil phase and the water phase, and S3, stirring to make the liposome more uniformly distributed and smaller in particle size.

Preferably, the mixing of S1 is at 75-85 deg.C and 400-600 rpm/min, and most preferably at 80 deg.C and 500 rpm/min.

Preferably, the homogenization is carried out for 3-7 min at the rotating speed of 10-15 rpm/min, and most preferably for 5min at the rotating speed of 12 rpm/min.

Further preferably, the homogenization in S1 is followed by stirring.

More preferably, the stirring is carried out at 70-90 ℃ and 450-550 rpm/min for 25-35 min. Most preferably, the mixture is stirred at 80 ℃ and 500rpm/min for 30 min.

The mixing, homogenizing and stirring are used for uniformly dispersing the dihydromyricetin in the emulsifier.

Preferably, the dissolving temperature of S2 is 75-85 ℃, and most preferably 80 ℃.

Preferably, the stirring of S2 is at 700-900 rpm/min for 0.8-1.2 h, and most preferably at 800rpm/min for 1 h.

The dihydromyricetin liposome prepared by the invention not only can keep the self performances of oxidation resistance, bacteriostasis, corrosion prevention and the like of the raw material dihydromyricetin, but also can remarkably improve the water solubility, stability and transdermal absorption capacity of the dihydromyricetin, and can effectively play the effects of oil control and moisture retention when being used for skin, so that the dihydromyricetin liposome is applied to the preparation of cosmetics.

Preferably, the cosmetic comprises one or more of gel, lotion, essence and facial mask.

As a preferred implementation mode, the jelly comprises the following components in percentage by mass: 1-5% of dihydromyricetin liposome, 0.1-1.0% of emollient, 0.1-1.0% of emulsifier, 0.1-3.0% of preservative, 0.1-3.0% of thickening agent, 0.1-1.0% of pH regulator, 0.1-2.0% of humectant, 0.01-0.05% of chelating agent and the balance of water.

Preferably, the emollient comprises one or more of hydrogenated polyisobutene, allantoin, and tocopherol acetate.

Preferably, the emulsifier comprises one or more of cetearyl alcohol, cetearyl glucoside.

Preferably, the preservative comprises one or more of butanediol, phenoxyethanol, ethylhexyl glycerol and p-hydroxyacetophenone.

Preferably, the thickening agent comprises one or more of carbomer, acrylic acid/C10-30 alkanol acrylate cross-linked polymer.

Preferably, the pH regulator comprises one or more of sodium hydroxide and arginine.

Preferably, the humectant comprises one or more of glycerin, 1, 2-pentanediol, sodium hyaluronate, trehalose and betaine.

Preferably, the chelating agent comprises disodium EDTA.

The invention has the following beneficial effects:

aiming at the defects of poor water solubility and poor stability of the dihydromyricetin, the invention adopts specific oil phases of cholesterol, sodium stearate, coconut oil alcohol-caprylate/caprate and other auxiliary materials to be compounded with each other to wrap the dihydromyricetin, so that the obtained dihydromyricetin liposome not only can keep the self-oxidation resistance, bacteriostasis, corrosion resistance and other performances of the raw material dihydromyricetin, but also can obviously improve the water solubility, stability and transdermal absorption capacity of the dihydromyricetin, and can effectively play the effects of oil control and moisture preservation when being used for skin.

Drawings

FIG. 1 shows the DPPH radical scavenging results of Experimental example 4.

Fig. 2 is the result of the percutaneous absorption test of experimental example 5.

FIG. 3 shows the results of the human body evaluation test of the oil-controlling jelly of Experimental example 6.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

5% DMY: dissolving dihydromyricetin in polyethylene glycol-8 to obtain 5% Dihydromyricetin (DMY) solution.

EXAMPLE 1 preparation of Dihydromyricetin liposomes

1. The components of 100g of dihydromyricetin liposome are as follows:

5g of dihydromyricetin, 3.7g of oil phase, 5g of soybean lecithin, 8g of PEG-40 hydrogenated castor oil, 30g of auxiliary emulsifier, 10.6g of preservative and the balance of water;

wherein the oil phase is prepared from the following components in percentage by mass 3: 0.3: 0.4 of coconut oil alcohol-caprylic acid ester/caprate, cholesterol and sodium stearate, wherein the mass ratio of the auxiliary emulsifier is 15: 15, 1, 3-propylene glycol and 1, 2-pentanediol, wherein the preservative is prepared from the following raw materials in a mass ratio of 0.6: 5: 5, p-hydroxyacetophenone, 1, 3-propylene glycol and 1, 2-pentanediol.

2. Preparing dihydromyricetin liposome:

s1, mixing dihydromyricetin, an oil phase, soybean lecithin and a co-emulsifier according to the formula amount at 80 ℃ and 500rpm/min, homogenizing at 12rpm/min for 5min, and stirring at 80 ℃ and 500rpm/min for 30min to obtain a mixture A;

s2, dissolving the preservative with the formula amount in water at 80 ℃, adding the preservative into the mixture A obtained in the S1, and stirring for 1h at 800 rpm/min;

s3, cooling to 50 ℃, adding PEG-40 hydrogenated castor oil, and stirring for 30min to obtain the dihydromyricetin liposome.

EXAMPLE 2 preparation of Dihydromyricetin liposomes

1. The components of 100g of dihydromyricetin liposome are as follows:

8g of dihydromyricetin, 2g of an oil phase, 6g of soybean lecithin, 10g of PEG-40 hydrogenated castor oil, 32g of a co-emulsifier, 4.6g of a preservative and the balance of water;

wherein the oil phase is prepared from the following components in percentage by mass: 0.1: 0.1 of coconut oil alcohol-caprylic acid ester/caprate, cholesterol and sodium stearate, wherein the mass ratio of the auxiliary emulsifier is 13: 17, 1, 3-propylene glycol and 1, 2-pentanediol, wherein the preservative is prepared by mixing the following components in a mass ratio of 0.5: 6: 4, p-hydroxyacetophenone, 1, 3-propanediol and 1, 2-pentanediol.

2. The preparation method of dihydromyricetin liposome is the same as that described in example 1.

EXAMPLE 3 preparation of Dihydromyricetin liposomes

1. The components of 100g of dihydromyricetin liposome are as follows:

4g of dihydromyricetin, 6g of an oil phase, 4g of soybean lecithin, 6g of PEG-40 hydrogenated castor oil, 28g of a co-emulsifier, 12g of a preservative and the balance of water;

wherein the oil phase is prepared from the following components in a mass ratio of 1: 0.8: 0.7 of coconut oil alcohol-caprylic acid ester/caprate, cholesterol and sodium stearate, wherein the mass ratio of the auxiliary emulsifier is 17: 13, 1, 3-propylene glycol and 1, 2-pentanediol, wherein the preservative is prepared by mixing the following components in a mass ratio of 0.7: 4: 6, p-hydroxyacetophenone, 1, 3-propylene glycol and 1, 2-pentanediol.

EXAMPLE 4 preparation of Dihydromyricetin liposomes

1. The components of 100g of dihydromyricetin liposome are as follows:

0.1g of dihydromyricetin, 0.1g of oil phase, 8g of soybean lecithin, 4g of PEG-40 hydrogenated castor oil, 25g of co-emulsifier, 0.1g of preservative and the balance of water;

wherein the oil phase is prepared from the following components in percentage by mass: 0.5: 0.7 of coconut oil alcohol-caprylic acid ester/caprate, cholesterol and sodium stearate, wherein the mass ratio of the auxiliary emulsifier is 19: 11, 1, 3-propylene glycol and 1, 2-pentanediol, wherein the preservative is prepared by mixing the following components in a mass ratio of 0.4: 2: 2, p-hydroxyacetophenone, 1, 3-propylene glycol and 1, 2-pentanediol.

Example 5 preparation of Dihydromyricetin liposomes

1. The components of 100g of dihydromyricetin liposome are as follows:

10g of dihydromyricetin, 10g of an oil phase, 2g of soybean lecithin, 12g of PEG-40 hydrogenated castor oil, 35g of an auxiliary emulsifier, 15g of a preservative and the balance of water;

wherein the oil phase is prepared from the following components in percentage by mass 4: 0.2: 0.3 of coconut oil alcohol-caprylic acid ester/caprate, cholesterol and sodium stearate, wherein the mass ratio of the auxiliary emulsifier is 11: 19, 1, 3-propylene glycol and 1, 2-pentanediol, wherein the preservative is prepared by mixing the following components in a mass ratio of 0.8: 8: 8, p-hydroxyacetophenone, 1, 3-propylene glycol and 1, 2-pentanediol.

EXAMPLE 6 preparation of Dihydromyricetin liposomes

1. The components of dihydromyricetin liposome described in example 1.

2. Preparing dihydromyricetin liposome:

s1, mixing dihydromyricetin, an oil phase, soybean lecithin and a co-emulsifier according to the formula amount at 75 ℃ and 600rpm/min, homogenizing at 10rpm/min for 7min, and stirring at 90 ℃ and 450rpm/min for 25min to obtain a mixture A;

s2, dissolving the preservative with the formula amount in water at 75 ℃, adding the preservative into the mixture A obtained in the S1, and stirring at 700rpm/min for 1.2 h;

s3, cooling to 55 ℃, adding PEG-40 hydrogenated castor oil, and stirring for 35min to obtain the dihydromyricetin liposome.

Example 7 preparation of Dihydromyricetin liposomes

1. The components of dihydromyricetin liposome are the same as those described in example 1.

2. Preparing dihydromyricetin liposome:

s1, mixing dihydromyricetin, an oil phase, soybean lecithin and a co-emulsifier according to the formula amount at 85 ℃ and 400rpm/min, homogenizing at 15rpm/min for 3min, and stirring at 70 ℃ and 550rpm/min for 35min to obtain a mixture A;

s2, dissolving the preservative with the formula amount in water at 85 ℃, adding the preservative into the mixture A obtained in the S1, and stirring at 900rpm/min for 0.8 h;

s3, cooling to 60 ℃, adding PEG-40 hydrogenated castor oil, and stirring for 25min to obtain the dihydromyricetin liposome.

Comparative example 1

The composition and preparation method of dihydromyricetin liposome of example 1 are the same, except that caprylic/capric triglyceride is used instead of coco-caprylic/capric acid ester.

Comparative example 2

The composition and preparation method of dihydromyricetin liposome of example 1 are the same except that the cocoalcohol-caprylate/caprate is replaced by butanediol dicaprylate/dicaprate.

Comparative example 3

The composition and preparation method of dihydromyricetin liposome of example 1 are the same, except that S1 is not homogenized, i.e., S1 is: mixing dihydromyricetin, oil phase, soybean lecithin and auxiliary emulsifier at 80 deg.C and 500rpm/min, and stirring at 500rpm/min for 30min to obtain mixture A.

Comparative example 4

The composition and preparation method of dihydromyricetin liposome of example 1 are the same, except that 2g of hydrogenated castor oil is used as Peg 40.

Comparative example 5

The composition and preparation method of dihydromyricetin liposome of example 1 are the same, except that Peg40 hydrogenated castor oil is used in an amount of 14 g.

Comparative example 6

The components and preparation method of the dihydromyricetin liposome in the example 1 are the same, except that the adding sequence of the soybean lecithin and the PEG-40 hydrogenated castor oil is changed, namely the preparation method of the dihydromyricetin liposome specifically comprises the following steps:

s1, mixing dihydromyricetin, an oil phase, PEG-40 hydrogenated castor oil and a co-emulsifier in a formula amount at 80 ℃ and 500rpm/min, homogenizing at 12rpm/min for 5min, and stirring at 500rpm/min for 30min to obtain a mixture A;

s3, cooling to 55 ℃, adding soybean lecithin, and stirring for 30min to obtain the dihydromyricetin liposome.

Comparative example 7

The components and preparation method of the dihydromyricetin liposome in the example 1 are the same, except that the PEG-40 hydrogenated castor oil is added into S1, namely the preparation method of the dihydromyricetin liposome specifically comprises the following steps:

s1, mixing dihydromyricetin, an oil phase, soybean lecithin, PEG-40 hydrogenated castor oil and an auxiliary emulsifier at a formula ratio of 80 ℃ and 500rpm/min, homogenizing at 12rpm/min for 5min, and stirring at 500rpm/min for 30min to obtain a mixture A;

s3, cooling to 55 ℃, and stirring for 30min to obtain the dihydromyricetin liposome.

Comparative example 8

The components and preparation method of dihydromyricetin liposome of example 1 are the same, except that the stirring speed of S2 is 500rpm/min, that is, S2 is: the amount of preservative was dissolved in water at 80 ℃ and added to mixture A described in S1, stirring at 500rpm/min for 1 h.

Comparative example 9

Reference is made to the preparation method of patent 202010957443.5:

s1, dissolving 40 mu L of Tween 80 and 0.02g of PEG-4000 in 40mL of phosphoric acid buffer solution with pH value of 6.5 to obtain an aqueous phase solution;

s2, dissolving 0.04g of dihydromyricetin, 0.05g of cholesterol and 0.3g of egg yolk lecithin in absolute ethyl alcohol, and stirring to fully dissolve the components to obtain an oil phase solution;

s3, under the rotation action of a magnetic stirrer, sucking the oil phase solution by using an injector and dropwise adding the oil phase solution into the water phase solution to obtain a mixed solution;

s4, after titration is finished, placing the mixed solution in a water bath, stirring for 3-4 hours at 47 ℃ and 100rpm/min to remove absolute ethyl alcohol in the emulsion, and obtaining the multi-vesicle dihydromyricetin liposome after the volume of the solution is constant.

Experimental example 1 stability test of Dihydromyricetin liposome

The dihydromyricetin liposomes obtained in examples 1 to 7 and comparative examples 1 to 8 were allowed to stand at 50 ℃, room temperature (25 ℃), 5 ℃, -18 ℃ and light (natural light) for two months, and the color change, precipitation, delamination and the like of the liposomes were observed, and the prepared particle size (before) and the particle size (after) after standing for 2 months were observed under a marvens particle size meter, with the results shown in table 1.

Table 1 stability test results

As can be seen from table 1, after the dihydromyricetin liposomes obtained in examples 1 to 7 are respectively placed under illumination (natural illumination) at 50 ℃, normal temperature (25 ℃), 5 ℃, to-18 ℃ and for two months, no obvious color change occurs, and no phenomena such as crystallization or oil precipitation occur, and the rate of change of the particle size of the dihydromyricetin liposomes in examples 1 to 7 is small, which indicates that the dihydromyricetin liposomes of the present invention have excellent water solubility and stability; the color change, crystallization, oil precipitation and other phenomena of different degrees appear in each proportion, the particle size change rate is high, and the water solubility and the stability are poor.

In addition, the water solubility and stability of the dihydromyricetin liposome obtained in the embodiments 1 to 7 are significantly superior to those of 5% DMY, which shows that the dihydromyricetin liposome obtained by wrapping dihydromyricetin by the method of the present invention can effectively increase the water solubility and stability of dihydromyricetin.

Experimental example 2 measurement of preservative Effect of Dihydromyricetin liposome

The results of measuring the preservation effect of the dihydromyricetin liposome obtained in examples 1-7 and 5% DMY according to the cosmetic preservation challenge test method of the group standard T/GDCDC 010-2019 approved by the Committee of Japan chemical industry, Guangdong province are shown in Table 2.

TABLE 2 results of measurement of anticorrosive effect

As can be seen from Table 2, the dihydromyricetin liposome obtained in examples 1-7 can reach the standard of bacteria and fungi at day 28, which shows that the dihydromyricetin provided by the invention has an excellent preservative effect.

In addition, the antiseptic effect of the dihydromyricetin liposome obtained in examples 1 to 7 is compared with that of 5% DMY, and the antiseptic effect of the dihydromyricetin liposome obtained in examples 1 to 7 is equivalent to that of 5% DMY, which shows that the dihydromyricetin liposome obtained by wrapping dihydromyricetin by the method of the present invention can maintain the antiseptic effect of the raw material dihydromyricetin and does not reduce the antiseptic effect of the dihydromyricetin liposome due to the preparation of the liposome.

Experimental example 3 measurement of bacteriostatic Effect of Dihydromyricetin liposome

The minimum inhibitory concentration MIC values for staphylococcus aureus, escherichia coli, clostridium, salmonella, and candida albicans were measured using the dihydromyricetin liposome obtained in example 1 and 5% DMY according to "inhibitory activity of dihydromyricetin and its influencing factor" in 2016, 10, 16, volume 10, respectively, and the results are shown in table 3.

TABLE 3 measurement results of bacteriostatic effect

As can be seen from table 3, comparing the bacteriostatic effects of the dihydromyricetin liposome obtained in example 1 with that of 5% DMY, the bacteriostatic effect of the dihydromyricetin liposome obtained in example 1 is found to be equivalent to that of 5% DMY, which indicates that the dihydromyricetin liposome obtained by coating dihydromyricetin by the method of the present invention can maintain the bacteriostatic effect of the dihydromyricetin as the raw material, and the bacteriostatic effect is not reduced by preparing the liposome.

Experimental example 4 DPPH radical scavenging experiment of Dihydromyricetin Liposome

1. Principle of experiment

DPPH is also called 1, 1-diphenyl-2-trinitrophenylhydrazine, is a very stable free radical with a nitrogen center, and the ethanol solution of DPPH is purple and has strong absorption at the wavelength of 517 nm. When the free radical scavenger exists, the light absorption of the DPPH ethanol solution is weakened due to the pairing of electrons of the free radical scavenger, and the fading degree of the DPPH ethanol solution is in a linear relation with the number of the electrons accepted by the DPPH ethanol solution, so that the capability of a test sample for scavenging free radicals, namely the antioxidant activity, can be evaluated.

2. Preparing solution

(1)0.2mmol/LDPPH in ethanol (as free radical): 78.86mg of DPPH (1, 1-diphenyl-2-trinitrophenylhydrazine) was dissolved in 1000mL of anhydrous ethanol: not less than 98.5% (HPLC), Ron; anhydrous ethanol: and AR.

(2) Sample solution (as radical scavenger): dihydromyricetin liposome of example 1, 5% DMY.

3. Determination of DPPH radical scavenging Rate

(1) Sequentially adding 75 μ L sample solution and 75 μ L0.2mmol/LDPPH alcoholic solution into sample well of enzyme label plate at 25 deg.C, mixing, standing for 30min, measuring light absorption value at wavelength of 517nm with enzyme label instrument, and recording data as A ₁ ；

(2) Sequentially adding 75 μ L water and 75 μ L0.2mmol/LDPPH ethanol solution into sample well of enzyme labeling plate, mixing, standing for 30min, measuring light absorption value at wavelength of 517nm with enzyme labeling instrument, and recording data as A ₂ ；

(3) Sequentially adding 75 μ L sample solution and 75 μ L water into sample well of enzyme labeling plate, mixing, standing for 30min, measuring light absorption value at wavelength of 517nm with enzyme labeling instrument, and recording data as A ₀ 。

(4) According to the formula DPPH

DPPH radical clearance was calculated for the liposomes of example 1 and 5% DMY.

4. Results of the experiment

As shown in FIG. 1, it is understood that the liposome of example 1 has an IC50 value (concentration of the sample solution when DPPH radical scavenging rate reaches 50%) of 0.002% and an IC50 value of 0.0014% in 5% DMY, and thus the ability of the liposome of example 1 to scavenge DPPH radicals is comparable to that of dihydromyricetin as a raw material even after the liposome is encapsulated.

Experimental example 5 transdermal absorption experiment of Dihydromyricetin liposome

The transdermal effect of the dihydromyricetin liposome obtained in examples 1 to 7 and comparative examples 1 to 9, and 5% DMY was measured as follows:

(1) skin treatment: taking a fresh, intact pig skin, removing fine hair on the skin with a hair remover, washing with a small amount of clear water, removing subcutaneous fat with a sharp knife, cleaning with PBS solution, wrapping the treated skin with tinfoil, placing in a box, and storing at-20 deg.C for use.

(2) Transdermal experiment: transdermal experiments were performed in a Franz diffusion cell device, with skin (half an hour prior to the experiment, pigskin was removed and placed in a PBS solution for thawing) clamped between a supply room and a receiving room. Wherein the effective penetration area of skin is 1.33cm ² (ii) a The receiving solution was a PBS (pH 7.4) solution containing 5% tween 80 by mass, and the volume was 15 mL; the addition amount of the sample is 500 mu L; the temperature of the receiving chamber was 32 ℃ and the magnetic stirring speed was 600 rpm/min. (wherein, the samples are dihydromyricetin liposome and 5% DMY obtained in examples 1-7 and comparative examples 1-9, respectively.)

(3) Absorption by the skin: after the reaction is finished for 24h, the device is carefully removed, the skin clamped between the two chambers is taken out, the skin is washed by clear water and wiped by ethanol, a sample on the surface is removed, and finally, the water on the surface is adsorbed by filter paper for later use.

(4) Content determination:

(4-1) measuring the content of dihydromyricetin in the epidermis by adopting a tape striping method: spreading the skin in a glass dish, removing 21 layers of the skin by using an adhesive tape (3M, Scotch 600), discarding the first layer in order to avoid the sample remained on the surface of the skin, combining the remaining 20 layers, placing the combined layers in a 10mL centrifuge tube, adding 2mL absolute ethyl alcohol, carrying out vortex for 3min, centrifuging at 2000rpm/min for 5min, taking clear liquid, passing through a 220nm organic membrane, and determining the content of dihydromyricetin by using an HPLC method;

(4-2) determination of dihydromyricetin content in dermis: using scissors to remove 21 layersCutting skin, placing in 10m L centrifuge tube, adding 1mL anhydrous ethanol, vortexing for 3min, centrifuging at 2000rpm/min for 5min, collecting supernatant, repeating twice, mixing supernatants, sieving with 220nm organic membrane, and measuring content of dihydromyricetin (μ g/cm) in dermis by HPLC method ² ) And dihydromyricetin content (μ g/cm) in epidermis ² ) The results are shown in fig. 2 and table 4 (in which the dihydromyricetin content in the skin is the sum of the dihydromyricetin content in the epidermis and the dihydromyricetin content in the dermis); in addition, the increase rate of dihydromyricetin in dermis, epidermis and skin was calculated according to the formula "content of dihydromyricetin/control group (5% DMY) dihydromyricetin × 100%", and the results are shown in table 5.

TABLE 4 transdermal absorption test results-Dihydromyricetin content

TABLE 5 transdermal absorption test results-increasing rate of dihydromyricetin

As can be seen from fig. 2, table 4 and table 5, the transdermal absorption effect of the dihydromyricetin liposome obtained in examples 1 to 7 is significantly better than that of comparative examples 1 to 9 and the control group, and the effect of example 1 is the best, which indicates that the dihydromyricetin liposome prepared by the method of the present invention greatly improves the transdermal absorption capacity of dihydromyricetin.

In addition, comparing example 1 with comparative examples 1 and 2, it is shown that the oil is important for the transdermal absorption effect of dihydromyricetin liposome, and only coco oil alcohol-caprylate/caprate can significantly increase the transdermal absorption effect of dihydromyricetin in the liposome of the present invention; comparing example 1 with comparative examples 3 and 8, it is demonstrated that homogenization and a specific stirring speed have an important role in the transdermal absorption effect of dihydromyricetin in the liposome of the present invention; comparing example 1 with comparative examples 4 to 7, it can be seen that the amount and timing of addition of emulsifier No. 2 also play an indispensable role in the transdermal absorption capacity of dihydromyricetin in the liposome of the present invention, and that the higher the emulsifier content, the better the transdermal absorption effect of the liposome.

Experimental example 6 human evaluation test of oil-controlling gel

Preparation of oil-controlling jelly

Taking the dihydromyricetin liposome obtained in example 1, and preparing oil-controlling gel according to the components and the dosage in the table 6 according to the following steps:

s1, mixing the components of phase A according to the formula amount, heating to 80 ℃ to dissolve, and homogenizing at 12rpm/min for 3min to uniformly disperse the components;

s2, heating the components of the phase B according to the formula amount to 80 ℃ for dissolving;

s3, pouring the phase B into the phase A while stirring, uniformly mixing, homogenizing at 12rpm/min for 5min, stirring and cooling;

s4, cooling to 45 ℃, mixing and dissolving the phase C, adding the mixture into S3, cooling to 25 ℃, and adding water to balance to obtain the oil-controlling gel.

TABLE 6 oil-controlling gel compositions

Second, evaluation test of human body

1. Test objects: the skin of 30 healthy test persons of 18-30 years old is mixed oil or oily, wherein 11 male persons and 19 female persons are selected.

2. The test method comprises the following steps: cleaning the skin of a tested person, allowing the skin to enter a constant temperature and humidity room (the temperature is 25 ℃ and the humidity is 50%) for sitting for more than 30min, taking circular areas of about 2cm at the forehead of each of the left side and the right side of the tested person, detecting the moisture and oil content of five points of the upper, the lower, the left side and the right side of the skin of the circular areas of the left side and the right side, smearing 10 mu L of water on the skin of the circular area at the left side, smearing 10 mu L of oil-controlling gel on the skin of the circular area at the right side, and detecting the moisture and oil content of the skin in the areas respectively at 30min, 1h and 2 h.

3. The data for each time point was obtained by averaging the remaining three points after removing the highest and lowest two points, and the experimental results are shown in table 7 and fig. 3.

TABLE 7 evaluation test results of human body

In table 7, left and right are significant differences compared with 0h, respectively; ^ is the significant difference of the left and right contrasts at the same time; p <0.001, x/a; 0.001< p <0.01, > x/< Lambda ^; 0.01< p <0.05, x/< Lambda.

Comparing the water content of the forehead on the left side and the forehead on the right side in the table 7 and the graph 3, it can be seen that the water content of the forehead on the left side is 10 muL at 0h, but the water content of the forehead on the left side at 0.5h, 1.0h and 2.0h is obviously lower than that of the forehead on the right side coated with the oil-controlling gel, which indicates that the oil-controlling gel prepared by the liposome can be used for effectively moisturizing and moisturizing the skin.

As can be seen from Table 7 and FIG. 3, the oil content of the right forehead applied with the oil-controlling gel after 1h and 2h is significantly reduced compared with the oil content before application (0h), which is respectively reduced by 20.42% and 9.68%, thus indicating that the oil-controlling gel prepared from the liposome can realize effective oil control when being applied to skin.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. The dihydromyricetin liposome is characterized by comprising the following components in parts by weight: 0.1-10 parts of dihydromyricetin, 0.1-10 parts of an oil phase, 2-8 parts of soybean lecithin, 4-12 parts of PEG-40 hydrogenated castor oil, 25-35 parts of a co-emulsifier, 0.1-15 parts of a preservative and 30-45 parts of water; wherein the oil phase comprises cholesterol, sodium stearate, coco-caprylate/caprate;

the preparation method of the dihydromyricetin liposome comprises the following steps:

s1, mixing the dihydromyricetin, the oil phase, the soybean lecithin and the co-emulsifier according to the formula amount, and homogenizing to obtain a mixture A;

s2, dissolving the preservative with the formula amount in water, adding the dissolved preservative into the mixture A obtained in the step S1, and stirring at the rotating speed of 700-900 rpm/min;

s3, cooling to 50-60 ℃, adding PEG-40 hydrogenated castor oil, and stirring for 25-35 min to obtain the dihydromyricetin liposome.

2. The liposome of claim 1, wherein the liposome comprises 4-8 parts of dihydromyricetin, 2-6 parts of oil phase, 4-6 parts of soybean lecithin, 6-10 parts of PEG-40 hydrogenated castor oil, 28-32 parts of co-emulsifier, 4.6-12 parts of preservative and 30-45 parts of water.

3. The liposome of claim 1, wherein the mass ratio of coco oil alcohol-caprylate/caprate, cholesterol and sodium stearate in the oil phase is 1-5: 0.1-0.8: 0.1 to 0.7.

4. The liposome of claim 1, wherein the co-emulsifier comprises 1, 3-propanediol and 1, 2-pentanediol.

5. The liposome of claim 1, wherein the homogenization at S1 is performed at a rotation speed of 10-15 rpm/min for 3-7 min.

6. The liposome of claim 1, wherein the stirring of S2 is performed at 700-900 rpm/min for 0.8-1.2 h.

7. Use of dihydromyricetin liposome as claimed in any one of claims 1 to 6 for the preparation of cosmetics.

8. The use of claim 7, wherein the cosmetic comprises one or more of jelly, lotion, essence, and facial mask.

9. The application of the jelly is characterized by comprising the following components in percentage by mass: 1-5% of dihydromyricetin liposome, 0.1-1.0% of emollient, 0.1-1.0% of emulsifier, 0.1-3.0% of preservative, 0.1-3.0% of thickening agent, 0.1-1.0% of pH regulator, 0.1-2.0% of humectant, 0.01-0.05% of chelating agent and the balance of water.

10. The use of claim 9, wherein the emollient comprises one or more of hydrogenated polyisobutene, allantoin, and tocopheryl acetate.

11. Use according to claim 9, wherein the emulsifier comprises one or more of cetearyl alcohol, cetearyl glucoside.

12. The use as claimed in claim 9, wherein the preservative comprises one or more of butylene glycol, phenoxyethanol, ethylhexyl glycerol, p-hydroxyacetophenone.

13. The use of claim 9, wherein the humectant comprises one or more of glycerin, 1, 2-pentanediol, sodium hyaluronate, trehalose, and betaine.

14. The use of claim 9, wherein the chelating agent comprises disodium EDTA.