CN112791001A

CN112791001A - Preparation method of astaxanthin liposome

Info

Publication number: CN112791001A
Application number: CN202011505988.9A
Authority: CN
Inventors: 樊燕蓉; 冯娇燕; 周奔; 丁亦钦; 赵广义; 徐根兴
Original assignee: Doudou Gene Technology Nanjing Co ltd; Nanjing University of Science and Technology
Current assignee: Doudou Gene Technology Nanjing Co ltd; Nanjing University of Science and Technology
Priority date: 2020-12-18
Filing date: 2020-12-18
Publication date: 2021-05-14
Anticipated expiration: 2040-12-18
Also published as: CN112791001B

Abstract

The invention provides a preparation method of an astaxanthin liposome, which is characterized in that astaxanthin is wrapped in an anionic liposome by utilizing the characteristic that liposome components are similar to cell membranes, 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 disclosed by the invention is simple in preparation method, the preparation raw materials are easy to obtain, the prepared astaxanthin liposome is always negatively charged, safe and stable, has better antioxidant effect and transdermal effect, can be used as a cosmetic additive to develop a cosmetic containing astaxanthin active substances with strong action, good stability, no toxicity, harmlessness and no irritation, and has a good application prospect in the field of cosmetics with antioxidant function.

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 an astaxanthin liposome.

Background

The liposome (liposome) is a tiny spherical vesicle, phospholipid forms a molecular ordered structure by virtue of self hydrophobic association and other amphoteric compounds such as cholesterol and the like when dispersed in an aqueous phase, and the molecular ordered structure consists of an inner aqueous phase and one or more layers of phospholipid bilayers wrapping the inner aqueous phase. Initially, phospholipids were dispersed in water by Banghan and Standish for observation in an electron microscope. Liposomes have the properties of biofilms and are therefore also called artificial biofilms. Generally, the water soluble drug is encapsulated in the inner aqueous phase of the liposome, and the lipid soluble drug is in the phospholipid bilayer.

Astaxanthin (Astaxanthin, ASX) is a dark purple brown crystal with a melting point of 224 ℃. Astaxanthin is fat-soluble, insoluble in water, soluble in chloroform, acetone, benzene, carbon disulfide and the like, and slightly soluble in most organic solvents. Astaxanthin is a non-vitamin A-derived ketocarotenoid and can be stored in tissues without modification or transformation, and the skin and muscle can be colored healthily and gorgeously. Astaxanthin is a terpene unsaturated compound with chemical name of 3, 3-dihydroxy-4, 4-diketone-beta, beta-carotene and molecular formula of C₄₀H₅₂O₄The 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 is the strongest natural antioxidant in the natural world to date. However, free astaxanthin is unstable and easy to oxidize, astaxanthin is a fat-soluble substance, the water solubility is poor, the transdermal effect is poor when the astaxanthin is directly used, and the bioavailability is low, so that the preparation of the astaxanthin liposome solves the application problems.

Beta-cyclodextrin (beta-CD) is a cyclic oligosaccharide consisting of seven glucose residues linked by alpha-1, 4-glycosidic bonds. The hydrophobic cavity can be used for dynamically wrapping lipophilic guest molecules to form a compound supermolecule system. Thereby realizing the purposes of improving the water solubility, stability and oxidation resistance and photodecomposition capability of the object molecule, or achieving the effects of slow release and stereo separation, and the like. However, beta-cyclodextrin has the problems of poor water solubility and the like, hydroxypropyl-beta-cyclodextrin (HPCD) is an etherified derivative of beta-cyclodextrin, intramolecular hydrogen bonds of beta-cyclodextrin are opened by an introduced part of hydroxypropyl, so that the water solubility is greatly improved, and the hydroxypropyl-beta-cyclodextrin has higher safety proved by experiments and is considered as a potential mother cyclodextrin substitute. Therefore, the ASX-HPCD compound can be prepared by adopting a precipitation method to improve the water solubility and stability of the astaxanthin, and the astaxanthin liposome is prepared by taking the inclusion compound aqueous solution as a hydration solution to be wrapped in the aqueous phase in the liposome, so that the stability of the astaxanthin is better improved.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: in order to solve the problems in the background 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 by the invention for solving the technical problems is as follows: a preparation method of astaxanthin liposome comprises the following steps: dissolving a membrane material and astaxanthin in chloroform, removing the chloroform through 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 an astaxanthin liposome;

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

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

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

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

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

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

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

The invention has the beneficial effects that:

(1) the main components of the membrane material of the astaxanthin liposome in the preparation method of the astaxanthin liposome are similar to those of a biological membrane, and the astaxanthin liposome has the advantages of no toxicity, no immunogenicity, safety and reliability in vivo, can improve the stability of astaxanthin, solve the problem of water solubility, improve the transdermal effect of astaxanthin on skin, exert the effects of antioxidation and aging delaying, can be used as a cosmetic additive to develop a cosmetic containing astaxanthin active substances with strong action, good stability, no toxicity, no harm and no irritation, and has a 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, thereby preventing organisms from being hurt and even preventing cancers, and in addition, the astaxanthin can promote human bodies to generate immunoglobulin, so that the human bodies have higher immunoregulation activity. Therefore, the astaxanthin can be added into cosmetics, and the functional cosmetics capable of delaying aging can be developed through the functions of reducing lipid peroxidation, scavenging free radicals and the like of the astaxanthin;

(2) the astaxanthin used in the invention can exert the antioxidation effect only by being transferred into cells, the astaxanthin is wrapped in the anionic liposome by utilizing the characteristic that liposome components are similar to cell membranes, and is transferred into the cells by endocytosis or membrane fusion, so that the problems of poor water solubility and short transdermal retention time of the astaxanthin can be effectively solved, the astaxanthin slow-release effect is good, the action time of the astaxanthin is greatly prolonged, the astaxanthin is more effectively transferred into the 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 is further illustrated with reference to the following figures and examples.

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

FIG. 2 shows the DPPH radical scavenging results of astaxanthin liposomes (No. 1) in example 1 of the present invention.

FIG. 3 shows the result of scavenging hydroxyl radicals from astaxanthin liposome (No. 1) in example 1 of the present invention.

FIG. 4 shows the results of scavenging superoxide anions from astaxanthin liposomes (No. 1) in example 1 of the present invention.

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

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

FIG. 7 shows the hydroxyl radical scavenging results of astaxanthin liposomes (Nos. 2-6) in example 2 of the present invention.

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

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

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

FIG. 11 shows the result of scavenging hydroxyl radicals of astaxanthin liposomes (Nos. 7 to 8) in example 3 of the present invention.

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

Fig. 13 shows the results of the transdermal test of the liposome of example 1 and astaxanthin solution according to the present invention.

Fig. 14 is a result of transdermal experiment of liposomes nos. 2 and 7 in examples 2 and 3 of the present invention.

Detailed Description

The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic views illustrating only the basic structure of the present invention in a schematic manner, and thus show only the constitution related to the present invention.

Example 1 ASX Liposome protocol

Preparation of ASX liposomes

Dissolving soybean lecithin and cholesterol in appropriate amount of chloroform at a mass ratio of 5:1 by thin film dispersion method, adding 0.4mg astaxanthin, evaporating at 30 deg.C under reduced pressure to remove chloroform, forming lipid membrane in round bottom flask, and adding 10mL PBS solution with pH of 7.4 when hydrating at 50 deg.C. 300W (2s, 2s), after 2min ice bath ultrasound, filtering with 0.45 μm and 0.22 μm filter membranes in sequence to obtain astaxanthin liposome (number 1). Charging nitrogen, and storing at 4 deg.C in dark.

2. Astaxanthin liposome encapsulation efficiency determination:

(1) measurement of astaxanthin-chloroform standard curve:

and (3) measuring the astaxanthin content in the astaxanthin liposome prepared in the step (1) by using a visible spectrophotometer at the wavelength of 492 nm. Preparing 8 mu g/mL astaxanthin chloroform solution, performing gradient dilution by using chloroform, measuring an absorbance value at 492nm to obtain a proportional relation between the astaxanthin concentration and the absorbance value, and drawing a standard curve. The regression equation of the standard curve is that y is 0.2164x-0.0035, wherein x is the concentration of the astaxanthin solution mu g/mL, y is the absorbance value of the astaxanthin solution at 492nm, and the regression coefficient is 0.9999.

(2) The astaxanthin liposome encapsulation efficiency is determined by adopting a petroleum ether extraction method:

taking 0.5mL of the prepared astaxanthin liposome, adding 5mL of petroleum ether, fully shaking and stirring at 30 ℃ for 5min, standing for 30min, transferring the upper layer liquid into a rotary evaporation bottle, adding 5mL of petroleum ether for extraction, and repeating twice. And (3) carrying out vacuum rotary evaporation on the upper layer liquid at the temperature of 50 ℃, removing petroleum ether and precipitating free astaxanthin. Adding appropriate amount of chloroform for re-dissolution, and measuring the light absorption value at the maximum absorption peak of astaxanthin. Three parallel experiments were performed and the average was taken.

Envelope rate calculation formula:

TABLE 1 astaxanthin liposome results

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

Preparation of astaxanthin liposome was performed according to the conditions and the encapsulation efficiency was measured, giving an encapsulation efficiency of 84.9. + -. 0.9%. The particle size potential of the No. 1 liposome is measured by a ZS90 (British Malvern) potentiometer, and the particle size is 135.7nm (shown in figure 1), the potential is-21.6 mV, and the PDI value is 0.253, which indicates that the liposome has proper particle size, higher potential, more stable system, smaller PDI value, more uniform particles and normal distribution.

3. Determination of antioxidant Capacity

(1) DPPH radical scavenging Capacity determination

The characteristic absorption peak of a purple group exists at 517nm of a DPPH-absolute ethyl alcohol solution, and the decrease of the absorbance at 517nm after the antioxidant is added is measured by a spectrophotometry method to show the DPPH free radical scavenging capacity.

The results are shown in FIG. 2, which is compared with ASX-ethanol solutions of the corresponding concentrations and with 10. mu.g/mL VC aqueous solutions.

(2) Determination of hydroxyl radical scavenging Capacity

Adopting salicylic acid-FeSO₄Method for measuring Fe²⁺The hydroxyl radical reacts with hydrogen peroxide after being mixed to generate high-activity hydroxyl radical, and the salicylic acid is added to capture the hydroxyl radical to generate a colored product which has strong absorption at 510 mm. When added to the test substance, competes with salicylic acid for binding to hydroxyl radicals thereby reducing the formation of color and reducing absorbance.

The results are shown in FIG. 3, which is compared with corresponding concentrations of ASX-ethanol and 10. mu.g/mL of VC in water.

(3) Determination of superoxide anion scavenging ability

And (3) determining the scavenging rate of the sample to be tested on superoxide anion free radicals by adopting a pyrogallol autoxidation method. The pyrogallol is quickly autoxidized under the alkaline condition, the superoxide anion free radical generated in the autoxidation process can accelerate the autoxidation rate of the pyrogallol, and meanwhile, a colored intermediate product is generated and has strong absorption at 325 nm. The superoxide anion can be eliminated after the test sample is added, the autoxidation reaction can be inhibited, the generation of intermediate products can be prevented, and the light absorption value of the solution at 325nm is reduced. The results are shown in FIG. 4, with 10. mu.g/mL VC in water as a control.

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

Example 2: ASX-HPCD liposome preparation scheme

Preparation of ASX-HPCD liposomes

(1) Preparation of ASX-HPCD liposomes

The ASX-HPCD inclusion compound is prepared by a precipitation method, and the inclusion rate is 46.07%. Dissolving soybean lecithin and cholesterol in chloroform at a mass ratio of 5:1 by thin film dispersion method, removing chloroform by vacuum evaporation at 30 deg.C to form lipid membrane in round-bottomed flask, and adding 10mL of ASX-HPCD aqueous solution when hydrating at 50 deg.C. After ultrasonic treatment in ice bath at 300W (2s, 2s) for 4min, sequentially filtering with filter membranes with the pore diameter of 0.45 μm and 0.22 μm to obtain ASX-HPCD liposomes (particle size diagram of liposome No. 2 is shown in figure 5) with numbers 2-6 in Table 4, and determining the encapsulation rate.

TABLE 4 ASX-HPCD Liposome formulation Table and results

The ASX-HPCD liposome with different concentrations has different factors such as particle size, potential, entrapment rate and the like. Wherein the particle size is No. 5 largest and No. 2 smallest. The PDI is low and the particle size is uniform. The number of potential No. 4 is highest, the number of potential No. 5 is lowest, and the number of potential values is more than 35, which indicates that the system is relatively stable. The encapsulation efficiency is highest in number 4 and lowest in number 6, but all are kept above 60%.

ASX-HPCD liposome antioxidant assay

The result of DPPH radical scavenging ability measurement is shown in FIG. 6; the measurement results of the hydroxyl radical scavenging ability are shown in FIG. 7; the results of the superoxide anion scavenging ability measurement are shown in FIG. 8.

The ASX-HPCD liposome with different concentrations is analyzed for the elimination of three free radicals, the DPPH free radical elimination capability of the liposome No. 2 is the highest, the hydroxyl free radical elimination capability and the superoxide anion elimination capability are not optimal, but the elimination rate of other liposomes has little increase compared with the hydroxyl free radical elimination capability and the superoxide anion elimination capability. The results of particle size, potential and encapsulation rate are comprehensively considered, and the No. 2 liposome in the numbered 2-6 liposomes meets the practical requirement most.

Example 3: preparation scheme of ASX double-coated liposome

Preparation of ASX double-coated liposomes

Dissolving soybean lecithin and cholesterol in chloroform at a mass ratio of 5:1 by thin film dispersion method, adding 0.4mg astaxanthin, evaporating at 30 deg.C under reduced pressure to remove chloroform, forming lipid membrane in round bottom flask, and adding 10mL ASX-HPCD aqueous solution when hydrating at 50 deg.C. 300W (2s, 2s), after 2min ice bath ultrasonic treatment, filtering with 0.45 μm and 0.22 μm filter membrane to obtain astaxanthin liposomes (7-8 in Table 5) (see figure 9 for particle size diagram of liposome 7), and determining encapsulation efficiency.

TABLE 5 ASX double-coated Liposome formulation Table and results

2. Inclusion compound liposome antioxidant determination

The result of DPPH radical scavenging ability measurement is shown in FIG. 10; the measurement results of the hydroxyl radical scavenging ability are shown in FIG. 11; the results of the superoxide anion scavenging ability measurement are shown in FIG. 12.

The indexes of particle size, potential, encapsulation efficiency, oxidation resistance and the like are comprehensively analyzed, and compared with the liposome No. 8, the liposome No. 7 has the advantages of lower particle size, higher potential, better stability, smaller difference between the encapsulation efficiency and the oxidation resistance, and better economic benefit.

Example 4: research on transdermal effect of astaxanthin liposome

(1) Mouse skin treatment:

an in vitro skin model of spf-grade ICR mice (purchased from Qinglong mountain animal breeding farm in Jiangning district of Nanjing) was used. Cervical dislocation of 6 healthy spf male ICR mice was performed, the abdominal skin of the mice was subjected to depilation treatment with a depilatory, the depilatory remaining on the abdominal part of the mice was washed with physiological saline, the abdominal skin of the mice was peeled off, the tissue on the inner side of the excised skin was scraped with forceps, and the excised skin was soaked in PBS (10 mM, pH 7.4) for use.

(2) In vitro transdermal experiments:

in vitro transdermal experiments were performed using a TP-6 transdermal diffuser, and the transdermal effects of the astaxanthin liposomes and the astaxanthin solutions of Nos. 1, 2 and 7 prepared in examples 1, 2 and 3 were examined and compared. The Franz diffusion cell is formed by joining a supply chamber and a receiving chamber of an upper cup-shaped ground glass container assembly and a lower cup-shaped ground glass container assembly, and the medicine release area is 1.13cm²The volume of the receiving pool is 15mL. In the experiment, the skin of a mouse is clamped between a supply chamber and a receiving pool, the skin on the outer side of the abdomen faces the supply chamber, the skin on the inner side faces the receiving pool, 15mL of 8% DMSO-PBS solution is added into the receiving pool as a receiving solution, a magnetic rotor is added, subcutaneous bubbles are removed, and the receiving pool is fixed by a stainless steel clamp. The supply chamber was charged with 1mL of astaxanthin liposome, stirred at (37. + -. 1) ° C with a magnetic stirrer at 350rpm, 0.5mL of the liposome was sampled from the sampling port of the receiving cell at three-hour intervals, and the volume was then made up with 0.5mL of the receiving solution (note that no air bubbles were present subcutaneously), and the experiment was terminated after 24 or 48 hours. The control group was prepared by replacing the liposomes in the experimental group with an equal volume of astaxanthin solution, and the rest of the procedure was identical.

The formula for calculating the skin permeability is as follows, and the experimental results are shown in fig. 13 and 14.

As can be seen from fig. 13, the skin permeability of both the liposome No. 1 and the astaxanthin solution reached the highest point within 24h and then gradually decreased. The highest point of the No. 1 liposome is reached at 18h, the highest skin penetration rate is 29.32%, and the highest point of the skin penetration rate of the astaxanthin solution is 15h, wherein the skin penetration rate is 25.74%. This shows that the No. 1 liposome has a certain sustained release effect compared with astaxanthin solution, and can improve the skin penetration rate of astaxanthin and promote the transdermal absorption of astaxanthin.

As can be seen from fig. 14, the peak of the permeability of liposomes nos. 2 and 7 was about 24h, followed by gradual decrease, and thus a 48h permeability curve was measured. As can be seen from fig. 13 and 14, the skin penetration rate of the No. 2 liposome and the No. 7 liposome reaches the highest point at 24 hours, and compared with the No. 1 liposome and the astaxanthin solution, the No. 2 liposome and the No. 7 liposome have better sustained release effect, which can help the astaxanthin to slowly play a role in the skin. Meanwhile, the skin penetration rate of the No. 7 liposome is higher than that of the No. 2 liposome at 24h, and the skin penetration rate reaches 32.98%, which shows that the effect of the No. 7 liposome for promoting the transdermal absorption of astaxanthin is better. In conclusion, the No. 7 liposome can promote more astaxanthin to permeate the skin and keep the astaxanthin in the skin for a longer time, so that the astaxanthin can be applied to cosmetics more easily and stably to play a role.

In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.

Claims

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 through 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 then filtering with a filter membrane to obtain an astaxanthin liposome;

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

2. The method for preparing astaxanthin liposome according to claim 1, which is characterized in that: the mass-volume ratio of the membrane material in the liposome suspension is 3.0 mg/mL.

3. The method for preparing astaxanthin liposome according to claim 1, which is characterized in that: the membrane material consists of lecithin and cholesterol, wherein the weight ratio of lecithin: the mass ratio of cholesterol is 5: 1.

4. The method for preparing astaxanthin liposome according to claim 1, which is characterized in that: the concentration of the membrane material in the chloroform is 9.0 mg/mL.

5. The method for preparing astaxanthin liposome according to claim 1, which is characterized in that: the conditions of hydration and ice bath ultrasound are as follows: hydrating in water bath at 50 deg.C, and performing ultrasonic treatment on 300W ice bath for 2-4 min.

6. The method for preparing astaxanthin liposome according to claim 1, which is characterized in that: the particle size of the astaxanthin liposome is 135-155nm, the potential is-21 to-45 mV, and the entrapment rate is 60-85%.