CN112791001B - Preparation method of astaxanthin liposome - Google Patents

Abstract

The invention provides a preparation method of astaxanthin liposome, which utilizes the characteristic that liposome components are similar to cell membranes, and the astaxanthin is wrapped in anionic liposome, and the astaxanthin is transferred into cells through endocytosis or membrane fusion, so that the prepared astaxanthin liposome has a good transdermal effect. The astaxanthin liposome provided by the invention has the advantages that the preparation method is simple, the preparation raw materials are easy to obtain, the prepared astaxanthin liposome is always negatively charged, is safe and stable, has good antioxidant effect and transdermal effect, can be used as a cosmetic additive to develop cosmetics containing astaxanthin active substances, and has strong action, good stability, no toxicity, no harm and no stimulation, and has good application prospects in the field of cosmetics with antioxidant functions.

Description

Preparation method of astaxanthin liposome

Technical Field

The invention relates to the technical field of biochemistry or cosmetics, in particular to a preparation method of astaxanthin liposome.

Background

Liposomes (lipomes) are tiny spherical vesicles, and phospholipids rely on their own hydrophobic association and a molecular ordered structure formed when other amphiphilic compounds such as cholesterol are dispersed in an aqueous phase, consisting of an inner aqueous phase and one or more phospholipid bilayer layers surrounding the inner aqueous phase. Initially, phospholipids were dispersed in water by Banghan and standby for electron microscopy. Liposomes have the property of a biological membrane and are therefore also known as artificial biological membranes. Generally, the water-soluble drug is entrapped in the inner aqueous phase of the liposome, and the lipid-soluble drug is in the phospholipid bilayer.

Astaxanthin (ASX) is a dark purple brown crystal with a melting point of 224 ℃. Astaxanthin is fat-soluble, insoluble in water, easily soluble in chloroform, acetone, benzene, carbon disulfide, etc., and slightly soluble in most organic solvents. Astaxanthin is a ketocarotenoid which is not a vitamin A source and can be directly stored in tissues without modification and transformation, so that skin and muscle can be provided with healthy and bright color. Astaxanthin is a terpene unsaturated compound, and has chemical name of 3, 3-dihydroxy-4, 4-diketo-beta, beta-carotene and molecular formula of C ₄₀ H ₅₂ O ₄ The relative molecular mass was 596.86. The existing forms are mainly free and esterified, and the special long-chain conjugated olefin structure in the molecular structure of the antioxidant endows the antioxidant with the function of effectively quenching active oxygen, and the antioxidant is the strongest natural antioxidant in the natural world so far. However, free astaxanthin is unstable and easily oxidized, and astaxanthin is a fat-soluble substance, has poor water solubility, and has poor transdermal effect and low bioavailability when directly used, so that the preparation of astaxanthin liposome solves the application problems.

Beta-cyclodextrin (beta-CD) is a cyclic oligosaccharide linked by seven glucose residues through alpha-1, 4-glycosidic linkages. The lipophilic guest molecules can be dynamically encapsulated by virtue of the hydrophobic cavities of the supermolecular system to form a compound supermolecular system. Thereby realizing the purposes of improving the water solubility, stability and oxidation resistance and photodecomposition capability of guest molecules, or achieving the slow release and three-dimensional separation effects and the like. However, beta-cyclodextrin has the problems of poor water solubility and the like, while hydroxypropyl-beta-cyclodextrin (HPCD) is an etherified derivative of beta-cyclodextrin, the introduction part of hydroxypropyl opens the intramolecular hydrogen bond of beta-cyclodextrin, the water solubility is greatly improved, and experiments prove that the beta-cyclodextrin has higher safety and is considered to be a potential parent cyclodextrin substitute. Therefore, an ASX-HPCD compound can be prepared by adopting a precipitation method, the water solubility and the stability of astaxanthin are improved, and the astaxanthin liposome is prepared by taking the clathrate compound aqueous solution as hydration liquid, so that the astaxanthin liposome is wrapped in the aqueous phase in the liposome, and the stability of astaxanthin is better improved.

Disclosure of Invention

The invention aims to solve the technical problems that: in order to solve the problems in the prior art, an improved preparation method of astaxanthin liposome is provided, so that the defects of poor water solubility, poor stability and low bioavailability in the prior art of astaxanthin application are overcome.

The technical scheme adopted for solving the technical problems is as follows: a method for preparing astaxanthin liposome, comprising the following steps: dissolving a membrane material and astaxanthin in chloroform, removing the chloroform by reduced pressure evaporation, forming a lipid membrane in a round-bottomed flask, adding an ASX-HPCD aqueous solution for hydration and ice bath ultrasound to obtain a liposome suspension, and filtering with a filter membrane to obtain astaxanthin liposome;

the concentration of astaxanthin in the liposome membrane material is 40 mug/mL, and the concentration of astaxanthin in the hydration liquid is 30-70 mug/mL.

The mass volume ratio of the membrane material in the liposome suspension is 3.0mg/mL.

The membrane material consists of lecithin and cholesterol, wherein the lecithin is as follows: the mass ratio of cholesterol is 5:1.

The concentration of the membrane material in the chloroform is 9.0mg/mL.

The conditions of hydration and ice bath ultrasound are as follows: hydrating in water bath at 50deg.C, and performing ultrasonic treatment in 300W ice bath for 2-4min.

The particle size of the astaxanthin liposome is 135-155nm, the potential is-21 to-45 mV, and the encapsulation rate is 60-85%.

The invention also provides application of the astaxanthin liposome in daily chemical products, so that the astaxanthin liposome has an antioxidant function; preferably, the astaxanthin liposome is used as a raw material to be added into skin care products or cosmetics for delaying aging.

The beneficial effects of the invention are as follows:

(1) The membrane material of the astaxanthin liposome in the preparation method of the astaxanthin liposome is similar to a biological membrane, has the advantages of no toxicity, no immunogenicity, safety and reliability in vivo, can improve the stability of the astaxanthin, solve the problem of water solubility, improve the transdermal effect of the astaxanthin on skin, exert the effects of resisting oxidization and delaying aging, can be used as a cosmetic additive to develop cosmetics containing astaxanthin active matters, and has strong action, good stability, no toxicity, no harm and no stimulation, and has good application prospect in the field of cosmetics;

(2) The astaxanthin used in the invention can quench singlet oxygen, remove free radicals and inhibit lipid peroxidation, so that the organism is prevented from being damaged and even cancer can be prevented. Therefore, astaxanthin can be added into cosmetics, and the functions of reducing lipid peroxidation, scavenging free radicals and the like through astaxanthin are utilized to develop the functional cosmetics capable of delaying aging;

(2) The astaxanthin used in the invention can exert the antioxidation effect by being transferred into cells, and the astaxanthin is wrapped in anionic lipid bodies by utilizing the characteristic that liposome components are similar to cell membranes, and is transferred into cells through endocytosis or membrane fusion, so that the problems of poor water solubility and short transdermal residence time of the astaxanthin can be effectively solved, the astaxanthin has good slow release effect, the action time of the astaxanthin is greatly improved, the astaxanthin is more effectively transferred into cells, the stability of the astaxanthin is improved, the irritation is reduced, and the activity and the application universality of the astaxanthin are further improved.

Drawings

The invention will be further described with reference to the drawings and examples.

FIG. 1 is a graph showing the particle size distribution of astaxanthin liposome (No. 1) in example 1 according to the present invention.

FIG. 2 shows the DPPH radical scavenging result of astaxanthin liposome (No. 1) in example 1 according to the present invention.

FIG. 3 shows the results of scavenging hydroxyl radicals in astaxanthin liposomes (No. 1) according to example 1 of the present invention.

FIG. 4 shows the result of scavenging superoxide anions in astaxanthin liposome (No. 1) of example 1 according to the present invention.

FIG. 5 is a graph showing the particle size distribution of astaxanthin liposome (No. 2) according to example 2 of the present invention.

FIG. 6 shows the DPPH radical scavenging results of astaxanthin liposomes (Nos. 2-6) in example 2 according to the present invention.

FIG. 7 shows the results of scavenging hydroxyl radicals in astaxanthin liposomes (No. 2-6) according to example 2 of the present invention.

FIG. 8 shows the results of scavenging superoxide anions by astaxanthin liposomes (numbered 2-6) in example 2 of the present invention.

FIG. 9 is a graph showing the particle size distribution of astaxanthin liposome (No. 7) in example 3 according to the present invention.

FIG. 10 shows the DPPH radical scavenging results of astaxanthin liposomes (Nos. 7-8) in example 3 according to the present invention.

FIG. 11 shows the results of scavenging hydroxyl radicals in astaxanthin liposomes (No. 7-8) according to example 3 of the present invention.

FIG. 12 shows the results of scavenging superoxide anions by astaxanthin liposomes (Nos. 7-8) in example 3 of the present invention.

FIG. 13 shows the results of a transdermal test of liposome No. 1 and astaxanthin solution in example 1 of the present invention.

FIG. 14 shows the results of transdermal experiments on liposomes No. 2 and No. 7 of examples 2 and 3 according to the present invention.

Detailed Description

The invention will now be described in further detail with reference to the accompanying drawings. The drawings are simplified schematic representations which merely illustrate the basic structure of the invention and therefore show only the structures which are relevant to the invention.

EXAMPLE 1 ASX Liposome protocol

Preparation of ASX liposomes

By a film dispersion method, soybean lecithin and cholesterol were dissolved in a proper amount of chloroform at a mass ratio of 5:1, 0.4mg of astaxanthin was added, chloroform was removed by evaporation under reduced pressure at 30℃to form a lipid film in a round bottom flask, and 10mL of PBS solution at pH7.4 was added at 50℃for hydration. 300W (2 s,2 s), 2min ice bath ultrasound, and then using a filter membrane with a pore size of 0.45 μm and 0.22 μm to obtain astaxanthin liposome (No. 1). Nitrogen is filled in, and the mixture is preserved in dark at 4 ℃.

2. Astaxanthin liposome encapsulation efficiency determination:

(1) Determination of astaxanthin-chloroform standard curve:

and (3) measuring the astaxanthin content in the astaxanthin liposome prepared in the step (1) by adopting a visible light spectrophotometer at the wavelength of 492 nm. Firstly, preparing an astaxanthin chloroform solution with the concentration of 8 mug/mL, carrying out gradient dilution by chloroform, measuring the absorbance value at 492nm to obtain the proportional relation between the astaxanthin concentration and the absorbance value, and drawing a standard curve. The regression equation for the standard curve is y= 0.2164x-0.0035, where x is the concentration μg/mL of the astaxanthin solution and y is the absorbance value of the astaxanthin solution at 492nm, regression coefficient 0.9999.

(2) The encapsulation efficiency of astaxanthin liposome is measured by petroleum ether extraction method:

taking 0.5mL of prepared astaxanthin liposome, adding 5mL of petroleum ether, sufficiently shaking and stirring for 5min at 30 ℃, standing for 30min, transferring the upper layer liquid into a rotary steaming bottle, adding 5mL of petroleum ether for extraction, and repeating twice. The upper liquid is subjected to vacuum rotary evaporation at 50 ℃ to remove petroleum ether, so that the free astaxanthin is separated out. The absorbance was measured at the maximum absorption peak of astaxanthin by redissolving with an appropriate amount of chloroform. Three parallel experiments were performed and the average was taken.

The encapsulation efficiency calculation formula:

TABLE 1 astaxanthin liposome results

Numbering device	Particle size (nm)	PDI	Potential (mV)	Encapsulation efficiency%
					1	135.7	0.253	-21.6	84.9±0.9

Astaxanthin liposome was prepared under this condition and its encapsulation efficiency was measured, yielding an encapsulation efficiency of 84.9.+ -. 0.9%. The particle size potential measurement is carried out on the liposome No. 1, and a ZS90 (Malvern, UK) potentiometer is adopted to measure, so that the particle size is 135.7nm (see figure 1), the potential is-21.6 mV, the PDI value is 0.253, and the particle size of the liposome is proper, the potential is high, the system is stable, the PDI value is small, and the particles are uniform and are in normal distribution.

3. Antioxidant capacity assay

(1) DPPH radical scavenging Capacity determination

The characteristic absorption peak of the DPPH-absolute ethanol solution with purple groups at 517nm is utilized, and after the antioxidant is added by spectrophotometry, the decrease of the absorption value at 517nm indicates the scavenging capability of the DPPH free radical.

The results are shown in FIG. 2, which shows the comparison of ASX-ethanol solution with corresponding concentration and 10. Mu.g/mL VC aqueous solution.

(2) Determination of the ability to scavenge hydroxyl radicals

Salicylic acid-FeSO ₄ Fe for measurement by method ²⁺ Mixing with hydrogen peroxide to react to generate high-activity hydroxyl radical, and adding salicylic acid to captureHydroxyl radicals produce colored products and have strong absorption at 510 mm. When the test substance is added, the salicylic acid competes with the salicylic acid for binding to the hydroxyl radical, thereby reducing the generation of colored substances and lowering the absorbance.

The results are shown in FIG. 3, which shows the comparison of ASX-ethanol and 10. Mu.g/mL VC aqueous solution at the corresponding concentrations.

(3) Determination of superoxide anion scavenger Capacity

The scavenging rate of the sample to be tested on the superoxide anion free radical is determined by adopting a pyrogallol autoxidation method. The o-trimellitic acid is rapidly autoxidized under alkaline condition, and superoxide anion free radical generated in the autoxidation process can accelerate the autoxidation rate of the o-trimellitic acid, and simultaneously a colored intermediate product is generated and has strong absorption at 325 nm. The superoxide anions are removed after the test sample is added, so that the autoxidation reaction can be inhibited, the generation of intermediate products is prevented, and the absorbance of the solution at 325nm is reduced. The results are shown in FIG. 4, with 10. Mu.g/mL VC in water.

Compared with free ASX and free VC, the ASX liposome has better effect of scavenging free radicals, which indicates that encapsulation does not influence the scavenging capacity of ASX to free radicals.

Example 2: ASX-HPCD liposome preparation scheme

ASX-HPCD Liposome preparation

(1) Preparation of ASX-HPCD liposomes

ASX-HPCD inclusion compound is prepared by precipitation method, and the inclusion rate is 46.07%. By a film dispersion method, soybean lecithin and cholesterol were dissolved in a proper amount of chloroform according to a mass ratio of 5:1, chloroform was removed by evaporation under reduced pressure at 30 ℃, a lipid film was formed in a round bottom flask, and 10mL of ASX-HPCD aqueous solution was added at 50℃for hydration. 300W (2 s,2 s), 4min ice bath ultrasound, and 0.45 μm and 0.22 μm filter membrane, to obtain ASX-HPCD liposome No. 2-6 (No. 2 liposome particle size diagram see FIG. 5) in Table 4, and performing encapsulation efficiency determination.

Table 4.ASX-HPCD Liposome formulation table and results

ASX-HPCD liposome with different concentrations has different particle size, potential, encapsulation efficiency and other factors. Wherein the particle size is the largest No. 5 and the particle size is the smallest No. 2. The PDI is lower, and the particle size is relatively uniform. The highest potential number 4 and the lowest potential number 5 are both greater than 35, which indicates that the system is stable. Encapsulation efficiency No. 4 is highest and No. 6 is lowest, but all remain above 60%.

ASX-HPCD Liposome Oxidation resistance assay

The DPPH radical scavenging ability measurement results are shown in FIG. 6; the results of the determination of the scavenging ability of hydroxyl radicals are shown in FIG. 7; the results of the superoxide anion-scavenging capacity measurement are shown in FIG. 8.

The three free radical scavenging conditions of ASX-HPCD liposome with different concentrations are analyzed, the DPPH free radical scavenging capacity of the No. 2 liposome is highest, the hydroxyl free radical scavenging capacity and the superoxide anion scavenging capacity are not optimal, but the scavenging rate of other liposome is not large compared with the scavenging rate of other liposome. And the particle size, the potential and the encapsulation efficiency are comprehensively considered, and the liposome No. 2 in the liposome No. 2-6 meets the practical requirements most.

Example 3: preparation scheme of ASX double-encapsulated liposome

Preparation of ASX double-coated liposomes

By a film dispersion method, soybean lecithin and cholesterol were dissolved in a proper amount of chloroform at a mass ratio of 5:1, 0.4mg of astaxanthin was added, chloroform was removed by evaporation under reduced pressure at 30℃to form a lipid film in a round bottom flask, and 10mL of ASX-HPCD aqueous solution was added at 50℃for hydration. 300W (2 s,2 s), 2min ice bath ultrasound, and 0.45 μm and 0.22 μm filter membrane, get No. 7-8 astaxanthin liposome (No. 7 liposome particle size diagram see FIG. 9), and encapsulation efficiency determination.

Table 5.ASX double-coated Liposome formulation Table and results

2. Determination of the antioxidant properties of clathrate liposomes

The DPPH radical scavenging ability measurement results are shown in FIG. 10; the results of the determination of the scavenging ability of hydroxyl radicals are shown in FIG. 11; the results of the superoxide anion-scavenging capacity measurement are shown in FIG. 12.

And the indexes such as the comprehensive particle size, the potential, the encapsulation efficiency, the antioxidant capacity and the like are analyzed, compared with the liposome No. 8, the liposome No. 7 has the advantages of lower particle size, higher potential, better stability, less difference between the encapsulation efficiency and the antioxidant capacity and better economic benefit.

Example 4: astaxanthin liposome transdermal effect study

(1) Skin treatment of mice:

the skin model was isolated from spf grade ICR mice (purchased from Jiang Ningou green dragon mountain animal breeding farm, south kyo city). The cervical dislocation of 6 healthy spf grade male ICR mice is killed, the skin of the abdomen of the mice is dehaired by using a dehairing agent, the dehairing agent remained on the abdomen of the mice is washed by using normal saline, the skin of the abdomen of the mice is peeled off, the tissue on the inner side of the isolated skin is scraped by using forceps, and the isolated skin is soaked in 10mM PBS (phosphate buffered saline) with pH of 7.4 for standby.

(2) In vitro transdermal experiments:

in vitro transdermal experiments were performed using a TP-6 transdermal diffusion apparatus, and the transdermal effects of astaxanthin liposomes of numbers 1, 2, and 7 prepared in examples 1, 2, and 3 and astaxanthin solutions were examined and compared. The Franz diffusion cell is formed by an upper cup-shaped ground glass container assembly, a lower cup-shaped ground glass container assembly, a supply chamber and a receiving chamber Chi Duige, and the medicine release area is 1.13cm ² The receiving tank volume was 15mL. During experiments, the skin of the mice is clamped between a supply chamber and a receiving pool, the skin outside the abdomen faces the supply chamber, the skin inside the abdomen faces the receiving pool, 15mL of 8% DMSO-PBS solution is added into the receiving pool as receiving liquid, a magnetic rotor is added, subcutaneous bubbles are removed, and the mice are fixed by a stainless steel clamp. 1mL of astaxanthin liposome was added to the supply chamber, stirred at (37.+ -. 1) C.by a magnetic stirrer at 350rpm, 0.5mL was sampled from the sampling port of the receiving cell every three hours, and the volume was then made up with 0.5mL of the receiving solution (note that there was no possibility of bubbles under the skin), and the experiment was terminated after 24 or 48 hours. The control group replaced the liposomes in the experimental group with an equal volume of astaxanthin solution, and the rest of the operations were identical.

The calculation formula of the transdermal rate is shown below, and the experimental results are shown in fig. 13 and 14.

As can be seen from fig. 13, the transdermal rate of liposome No. 1 and astaxanthin solution reached the highest point within 24 hours, and then gradually decreased. Liposome No. 1 reached the highest point at 18h, with a maximum transdermal rate of 29.32%, whereas the maximum transdermal rate of astaxanthin solution was 15h, at which time the transdermal rate was 25.74%. This shows that compared with astaxanthin solution, liposome No. 1 has a certain slow release effect, and can improve the transdermal rate of astaxanthin and promote the transdermal absorption of astaxanthin.

As can be seen from fig. 14, the highest point of the transdermal rates of liposome nos. 2 and 7 was about 24 hours, and then gradually decreased, so that a 48-hour transdermal rate curve was measured. As can be seen from fig. 13 and 14, the transdermal rates of liposome No. 2 and liposome No. 7 reach the highest point at 24h, and compared with liposome No. 1 and astaxanthin solution, liposome No. 2 and liposome No. 7 have better slow release effect, which can help astaxanthin to slowly play a role in skin. Meanwhile, the transdermal rate of the No. 7 liposome is larger than that of the No. 2 liposome at 24 hours, and the transdermal rate reaches 32.98%, which shows that the effect of the No. 7 liposome for promoting the transdermal absorption of astaxanthin is better. In combination, liposome No. 7 promotes more astaxanthin to permeate the skin and maintain the skin for a longer period of time, so that the application of the liposome to cosmetics is easier to continuously and stably exert the effect.

With the above-described preferred embodiments according to the present invention as an illustration, the above-described descriptions can be used by persons skilled in the relevant art to make various changes and modifications without departing from the scope of the technical idea of the present invention. The technical scope of the present invention is not limited to the description, but must be determined according to the scope of claims.

Claims (1)

1.A preparation method of astaxanthin liposome is characterized by comprising the following steps: the method comprises the following steps: dissolving a membrane material and astaxanthin in chloroform, removing the chloroform by reduced pressure evaporation, forming a lipid membrane in a round bottom flask, adding an ASX-HPCD aqueous solution for hydration and ice bath ultrasound to obtain a liposome suspension, and filtering with a filter membrane to obtain astaxanthin liposome;

the concentration of astaxanthin in the liposome phospholipid bilayer is 40 mug/mL, the concentration of astaxanthin in the hydration liquid is 30-70 mug/mL, and the mass volume ratio of the membrane material in the liposome suspension is 3.0mg/mL;

using the characteristic that liposome components are similar to cell membranes, coating astaxanthin in anionic liposome, and transferring astaxanthin into cells through endocytosis or membrane fusion;

the membrane material consists of lecithin and cholesterol, wherein the lecithin is as follows: the mass ratio of the cholesterol is 5:1;

the concentration of the membrane material in the chloroform is 9.0mg/mL;

the conditions of hydration and ice bath ultrasound are as follows: hydrating in water bath at 50deg.C, and performing ultrasonic treatment in 300W ice bath for 2-4min;

the particle size of the astaxanthin liposome is 135-155nm, the potential is-21 to-45 mV, and the encapsulation rate is 60-85%.