CN113244190A

CN113244190A - Astaxanthin long-acting nano preparation prepared by micelle template method and preparation method thereof

Info

Publication number: CN113244190A
Application number: CN202110382269.0A
Authority: CN
Inventors: 朱源; 廖有武; 李双; 顾正清; 薛媛媛; 彭后平
Original assignee: Jiangsu University
Current assignee: Jiangsu University
Priority date: 2021-04-09
Filing date: 2021-04-09
Publication date: 2021-08-13
Anticipated expiration: 2041-04-09
Also published as: CN113244190B

Abstract

The invention discloses an astaxanthin long-acting nano preparation prepared by a micelle template method and a preparation method thereof, relating to the field of nano medicine preparations. Phospholipid and sodium cholate are taken as micelle templates to provide solubilizing and synergistic carriers for insoluble compound astaxanthin; taking polylactic acid-glycolic acid copolymer (PLGA) as a long-acting carrier, and constructing a novel PLGA nano preparation carrying the astaxanthin micelle by an emulsion solvent volatilization method, wherein the mass ratio of each component is astaxanthin: phospholipid: sodium cholate: PLGA ═ 1:3-7:2-5:10-50, the ratio of total organic phase to total aqueous phase was 1: 5-25. The preparation realizes the long-acting liver protection effect of the astaxanthin on the premise of remarkably improving the solubility of the astaxanthin. The invention has simple process, innovatively applies the micelle template to the preparation of the long-acting preparation of the insoluble drug, and has better application prospect.

Description

Astaxanthin long-acting nano preparation prepared by micelle template method and preparation method thereof

Technical Field

The invention relates to the field of nano-drug preparations, in particular to a long-acting nano-preparation of astaxanthin as an insoluble active ingredient and a preparation method thereof.

Background

Astaxanthin (astaxanthin), 3,3 ' -dihydroxy-4, 4 ' -diketo-beta, beta ' -carotene, a ketocarotenoid of non-vitamin A origin, is a reddish-brown terpene-based unsaturated compound. As a chain-breaking antioxidant, Astaxanthin not only can quench singlet Oxygen and directly scavenge Oxygen free radicals, but also can block the chain reaction of fatty acids, and has extremely strong antioxidant activity (see: Zuuaga, M., V. Gueguen, D.Letourreur, et al., Astaxan-antioxidant impact on exogenous Reactive Oxygen Species generation induced by ischemia and dispersion in J]chemical-Biological Interactions,2018.279: 145-158). Astaxanthin is a natural pigment carotenoid approved by the U.S. Food and Drug Administration (FDA) and has a variety of biological potentials (Shanmugaprya, K., Kim, H., Saravana, P.S., Chun, B.S., Kang, H.W.,2018, Astaxanthin-alpha tophenophil nanoemulsions for use by emulsion methods: Investigation on anti-reagent, around health, and antibiotic effects.Coloids Surf.B.biointerfaces 172, 170- & 179.). Numerous animal studies have demonstrated the benefits of astaxanthin in immune responses (Meghani N, Patel P, Kansara K, et al.A formulation of vitamin D encapsulated citrus oil emulsion nanoparticles: Its potential anti-cancer activity in human animal vehicle cells [ J]Clones and surfaces B: Biointerfaces,2018: 349-357.). Their safety, bioavailability and effectiveness against human oxidative stress were also evaluated (Zhang, L., Wang, H.,2015.Multiple mechanisms of anti-cancer effects experienced by astaxanthanthin. Mar. drugs 13, 4310-. It reduces oxidative stress(Manca, M.L., Mir-Palomo, S., Caddeo, C., Nacher, A., D i ez-salts, O., Peris, J.E., Pedraz, J.L., Fadda, A.M., Mancon, M.,2018. Sorbitol-linkage derived from cells attached with a nucleic acid for the protection and regeneration of skin-attached with reactive oxygen species (Busett, R.G., Cobes, J.S. 2009, active tissue, oxygen tissue, intake tissue, S.D., S.S. 2009, S.S. 13, S.S. 2009, S.3, S.D., S. 3, S.S. 10, S.S. 2009, S.11, oxygen tissue, S. 13, S.S. 3, S. 5, S.S. 3, S. 1, S. 3, cell, S. 3, cell, proliferation, cell, proliferation, cell, proliferation of cell, proliferation, cell, proliferation, cell, proliferation, cell, proliferation, cell, proliferation, cell, proliferation of cell, proliferation, cell, proliferation, cell, proliferation of cell, proliferation, cell, proliferation, cell, proliferation, cell, proliferation, cell,

j., Juvonen, R.O., Mahadik, K.R., Paradkar, A.R.,2010. Transferrin-mediated soluble peptides associating cure in enhanced in vitro activity by indication of apoptosis, int.J.Pharm.398, 190-203), preventing cancer spread (Yasui, Y. Hosokawa, M.J., Mikami, N.Miyashita, K., Tanaka, T.2011. acquisition activity peptides encoding and diagnosis-assisted collagen synthesis of infection of cells, expression of expression cells, chemistry. 193.193.12. infiltration of cells and expression of cells of expression of infection, 2. expression of cells and expression of infection, 2. expression of cells and expression of cells of infection, 2. expressing and expression of cells of infection, 2. secretion of cells, 2. secretion, and 2. secretion of cell death of cells of infection.

An emulsification-solvent volatilization method is a common method for preparing PLGA nanoparticles, but for active ingredients which are difficult to dissolve in water, in-vitro release investigation of the medicine finds that the medicine is difficult to release from the PLGA nanoparticles, and often causes the contradiction between slow release effect and solubilization synergy. The invention adopts a micelle template method, selects lecithin and sodium cholate to prepare micelles to load astaxanthin, and uses PLGA to wrap the micelle particles loaded with astaxanthin to obtain the PLGA nano-particles of the astaxanthin-loaded micelle particles with a certain long-acting effect.

The research reports of the literature show that astaxanthin is the strongest antioxidant substance, but the application of astaxanthin, an antioxidant, in the industries of medicine, cosmetics, food and the like is greatly limited, and the problem is caused by the extremely poor water solubility of astaxanthin. Therefore, the insoluble substance with certain medical application value is prepared into a micelle particle system with a tiny structure to increase the water solubility of the substance, and the substance is wrapped in a PLGA slow-release capsule material to be designed into a long-acting preparation due to the insolubility and the potential application in the aspects of clinical cardiovascular system diseases, cancers and the like. In addition, the research on astaxanthin long-acting preparations is rarely reported, and the research on the liver protection effect of astaxanthin and the preparation thereof is also less.

Disclosure of Invention

The invention aims to provide a long-acting astaxanthin PLGA nano preparation prepared by a micelle template. The low molecular weight micelle in the drug-carrying system is used as a drug-carrying template to improve the solubility and bioavailability of the astaxanthin, and PLGA is used as a capsule material to encapsulate the drug-carrying micelle to provide a certain slow release effect, so that the preparation has a certain long-acting effect.

The invention also aims to provide a preparation method of the astaxanthin long-acting preparation. The preparation is constructed by taking the drug-loaded micelle as a template and utilizing an emulsification-solvent volatilization method, so that the solubilization and synergism of the carrier component are achieved, the slow-release long-acting effect of the drug is realized, the astaxanthin long-acting solid nano preparation is developed, and the technical innovation is provided for the development of the long-acting preparation of the active component with the related property. The micelle template method can improve the solubility of a difficultly soluble medicament (such as astaxanthin) in water, wherein phospholipid and sodium cholate are used as solubilizing and absorption-promoting auxiliary materials to improve the bioavailability in vivo; PLGA is used as a packaging capsule material and can provide slow-release long-acting effect for the PLGA. On the other hand, the nanoparticles prepared by the method have a double-layer structure, and can prevent the drug burst effect of the drug-loading system, so that the drug is more stable.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

an astaxanthin long-acting nanometer preparation prepared by a micelle template method is prepared by taking phospholipid and sodium cholate as micelle templates and dissolving the micelle templates in a mixed solvent of dichloromethane and methanol; polylactic acid-glycolic acid copolymer (PLGA) is a long-acting carrier and is dissolved in dichloromethane; polyvinyl alcohol (PVA) is used as a dispersant and is dissolved in water; the weight ratio of the components is astaxanthin: phospholipid: sodium cholate: the polylactic acid-glycolic acid copolymer (PLGA) is 1:3-7:2-5:10-50, wherein the mass volume concentration of the astaxanthin in the dichloromethane is 1.5-6 mg/mL.

The phospholipid used in the micelle template of the astaxanthin long-acting nano preparation prepared by the micelle template method can be egg yolk lecithin and soybean lecithin, wherein the content of phosphatidylcholine is more than 80%.

The long-acting carrier of the astaxanthin long-acting nano preparation prepared by the micelle template method is PLGA, the types of the PLGA long-acting carrier can be PLGA75/25, PLGA50/50 and the like, and the viscosity of the long-acting carrier can be 0.09dL/g,0.21dL/g and 0.23 dL/g.

The dispersant of the astaxanthin long-acting nano preparation prepared by the micelle template method is PVA, the type of the dispersant can be PVA403 or PVA 1788, and the concentration of the dispersant is 0.5-4% (w/v).

The mixed solvent of the astaxanthin long-acting nano preparation prepared by the micelle template method is a mixed solvent of dichloromethane and methanol, and is mainly used for dissolving phospholipid and sodium cholate, wherein the volume content (v%) of dichloromethane is 0-30%.

The astaxanthin nano preparation of the astaxanthin long-acting nano preparation prepared by the micelle template method is pink or orange dry powder, and the particle size of the astaxanthin long-acting nano preparation after being redispersed in water is 40-200nm and is in a regular spherical shape.

The astaxanthin nano preparation of the astaxanthin long-acting nano preparation prepared by the micelle template method can be filled in capsules to be prepared into capsules or pressed into tablets.

An astaxanthin long-acting nano preparation prepared by a micelle template method comprises the following steps:

(1) dissolving astaxanthin in certain volume of dichloromethane, and performing ultrasonic treatment for 2-15min to obtain solution A; dissolving phospholipid and sodium cholate in mixed solvent of dichloromethane and methanol, and performing ultrasonic treatment for 2-15min to obtain solution B; dissolving PLGA in certain volume of dichloromethane, and performing ultrasonic treatment for 2-15min to obtain a solution C. Wherein the volume ratio of A to B to C between the solutions is equal to 1:1-8: 4-8.

(2) Dispersing the solution A into the mixed solution of B and C, performing ultrasonic treatment for 10-30min to fully disperse, dispersing the solution into PLGA solution, and performing ultrasonic treatment for 10-30min to fully disperse to obtain a total organic phase.

(3) Dispersing the organic phase in a certain volume of 2-4% PVA aqueous solution under ultrasonic condition, continuing ultrasonic treatment for 1-5min to emulsify the organic phase, dispersing the emulsified liquid in a certain volume of 0.5-1% PVA aqueous solution, wherein the volume ratio of the total organic phase to the total aqueous phase is 1:5-25, and stirring overnight to volatilize the organic solvent. Centrifuging at 3500rpm/min of 1500-.

Advantageous effects

The invention has the following beneficial effects:

1. the invention solves the problem of insolubility of astaxanthin, prepares nano particles which can be uniformly dispersed in a water system and can slowly release astaxanthin in vitro, and in vitro experimental studies show that the astaxanthin nano preparation can release 62.81% in 5 days of a buffer salt medium with pH of 6.8. The solid nanometer preparation loaded with astaxanthin prepared by the method has good dispersibility in water and can be slowly released in vitro.

2. According to the invention, the micelle template system has solubilization and synergism effects, the solubility of the astaxanthin in water is improved, the raw material drug is only 87.35ng/mL in water, and the maximum dissolving capacity of the astaxanthin nano preparation is about 2000 times of that of the raw material drug under the condition of maintaining the precursor of the clarification system. CCl is adopted to evaluate the drug effect in mice₄And evaluating the liver protection effect of the induced acute liver injury mouse model. The results show that: the shrimp meat was compared with the model groupThe nanometer preparation can raise SOD activity in liver tissue, lower MDA level in liver, lower ALT and AST level in blood plasma, and has liver protecting effect superior to that of medicine material. In addition, the astaxanthin nano preparation is administrated once in 3 days, and has obvious long-acting liver protection effect compared with a positive control drug and a raw material drug administrated once in 1 day.

3. The invention provides a preparation method of an astaxanthin long-acting solid nano preparation with a prospect, the nano preparation is dry orange powder prepared by taking a drug-loaded micelle as a template and utilizing an emulsification-solvent volatilization method, the average particle size of the nano preparation after being dissolved in water is 100nm, the zeta potential is-38.59 mV, the PDI index is 0.293, and a transmission electron microscope shows that the particles are uniformly distributed and have a double-layer structure.

Description of the drawings:

FIG. 1 is an appearance morphology of the astaxanthin nano-preparation prepared in example 7 (A, appearance of an aqueous solution of the astaxanthin nano-preparation; B, Didahl phenomenon of the aqueous solution of the astaxanthin nano-preparation; C, appearance of a powdery freeze-dried astaxanthin nano-preparation);

FIG. 2 is a transmission electron micrograph (18.5K times; B, partial enlarged view; C, 59K times) of the astaxanthin nanoformulation prepared in example 7;

fig. 3 is a graph showing in vitro release of the astaxanthin nanoformulation prepared in example 7;

Detailed Description

The examples set out below are intended to facilitate a better understanding of the invention by a person skilled in the art and are not intended to limit the invention in any way.

Main instruments and materials used in the following examples

Experimental materials:

astaxanthin (bio-technologies ltd, tokoron, south china); egg yolk lecithin (national drug group chemical agents limited) sodium cholate (alatin); PLGA75/25 (Shenzhen Boli Limited); positive control drug: silibinin capsule (Tianjin Tianshili saint pharmaceutical Co., Ltd.); SOD kit, MDA kit, ALT kit, AST kit, BCA kit (Nanjing Biotechnology engineering Co., Ltd.)

An experimental instrument:

electronic balance (Sartorius, Germany, Sartorius BSA 124S-CW); a numerical control ultrasonic cleaner (Kunshan ultrasonic instruments Co., Ltd.); high performance liquid chromatography (SHIMADZU, japan); a constant temperature oscillator (large instrument factory in the city of Jiangsu Jintan); high-sensitivity Zeta-potential analyzer (laboconco, usa); a freeze dryer (Beckman Coulter, usa); microcentrifuge (Heraeus, germany); a bench-top refrigerated high-speed centrifuge machine (Thermo corporation, usa); full-automatic enzyme linked immunity detector (U.S. Bruk Hai Wen instrument company)

Example 1 preparation of astaxanthin nanoformulation

(1) Astaxanthin 5mg was precisely weighed and dissolved in 0.5mL of dichloromethane, and homogenized by sonication for 10 min. Precisely weighing 15mg of phospholipid and 10mg of sodium cholate, dissolving in 0.5mL of methanol mixed solvent, and performing ultrasonic treatment for 10min to obtain a uniform solution. PLGA50 mg was precisely weighed and dissolved in 2mL of dichloromethane, and homogenized by sonication for 10 min. Dispersing an astaxanthin solution into a mixed solution of phospholipid and sodium cholate, performing ultrasonic treatment for 10min to fully disperse the astaxanthin solution, dispersing the solution into a PLGA solution, performing ultrasonic treatment for 10min to fully disperse the PLGA solution to obtain an organic phase with a total volume of 3mL, dispersing the organic phase into 6mL of 4% PVA403 aqueous solution under ultrasonic treatment to emulsify the organic phase, dispersing the emulsified liquid into 10mL of 0.5% PVA403 aqueous solution, and stirring overnight to volatilize the organic solvent.

(2) And after the organic solvent is volatilized, the organic solvent is respectively filled into 50mL centrifuge tubes, large particles and part of free drug precipitates are removed by centrifugation at 2000rpm, the supernatant is centrifuged at 12000rpm to obtain solid precipitates, the solid precipitates are resuspended and washed for 3 times by 30mL double-distilled water, and then the solid nanoparticles of the powdered astaxanthin are obtained by freeze drying.

Example 2 preparation of astaxanthin nanoformulation

(1) 5mg of astaxanthin is precisely weighed and dissolved in 2mL of dichloromethane, and the solution is subjected to ultrasonic treatment for 10min to be uniform. Phospholipid 35mg and sodium cholate 25mg are precisely weighed and dissolved in a mixed solvent of 1mL of dichloromethane and 3mL of methanol, and ultrasonic treatment is carried out for 10min to ensure that the solution is uniform. PLGA 250mg was precisely weighed and dissolved in 4mL of dichloromethane and homogenized by sonication for 10 min. Dispersing an astaxanthin solution into a mixed solution of phospholipid and sodium cholate, performing ultrasonic treatment for 10min to fully disperse the astaxanthin solution, then dispersing the solution into a PLGA solution, performing ultrasonic treatment for 10min to fully disperse the PLGA solution to obtain an organic phase with the total volume of 10mL, dispersing the organic phase into 15mL of 4% PVA403 aqueous solution under ultrasonic treatment to emulsify the organic phase, then dispersing the emulsion into 50mL of 1% PVA403 aqueous solution, and stirring overnight to volatilize the organic solvent.

Example 3 preparation of astaxanthin nanoformulation

(1) 10mg of astaxanthin is precisely weighed and dissolved in 2mL of dichloromethane, and the solution is homogenized by ultrasonic treatment for 10 min. Phospholipid 70mg and sodium cholate 50mg are precisely weighed and dissolved in a mixed solvent of 1mL of dichloromethane and 3mL of methanol, and ultrasonic treatment is carried out for 10min to ensure that the solution is uniform. PLGA 400mg was precisely weighed and dissolved in 4mL of dichloromethane, and homogenized by sonication for 10 min. Dispersing an astaxanthin solution into a mixed solution of phospholipid and sodium cholate, performing ultrasonic treatment for 10min to fully disperse the astaxanthin solution, then dispersing the solution into a PLGA solution, performing ultrasonic treatment for 10min to fully disperse the PLGA solution to obtain an organic phase with the total volume of 10mL, dispersing the organic phase into 15mL of 4% PVA403 aqueous solution under ultrasonic treatment to emulsify the organic phase, then dispersing the emulsion into 40mL of 1% PVA403 aqueous solution, and stirring overnight to volatilize the organic solvent.

Example 4 preparation of astaxanthin nanoformulation

(1) 10mg of astaxanthin is precisely weighed and dissolved in 2mL of dichloromethane, and the solution is homogenized by ultrasonic treatment for 10 min. Accurately weighing 45mg of phospholipid and 45mg of sodium cholate, dissolving in a mixed solvent of 1mL of dichloromethane and 3mL of methanol, and performing ultrasonic treatment for 15min to ensure that the solution is uniform. PLGA300mg was precisely weighed and dissolved in 4mL of dichloromethane and homogenized by sonication for 15 min. Dispersing an astaxanthin solution into a mixed solution of phospholipid and sodium cholate, performing ultrasonic treatment for 30min to fully disperse the astaxanthin solution, then dispersing the solution into a PLGA solution, performing ultrasonic treatment for 10min to fully disperse the PLGA solution to obtain an organic phase with the total volume of 10mL, dispersing the organic phase into 15mL of 2% PVA403 aqueous solution under ultrasonic treatment to emulsify the organic phase, then dispersing the emulsion into 50mL of 0.5% PVA403 aqueous solution, and stirring overnight to volatilize the organic solvent.

(2) And after the organic solvent is volatilized, the organic solvent is respectively filled into 50mL centrifuge tubes, large particles and partial free drug precipitates are removed by centrifugation at 2500rpm, the supernatant is centrifuged at 15000rpm to obtain solid precipitates, the solid precipitates are resuspended and washed for 3 times by 30mL double-distilled water, and then the solid nanoparticles of the powdered astaxanthin are obtained by freeze drying.

Example 5 preparation of astaxanthin nanoformulation

(1) 10mg of astaxanthin is precisely weighed and dissolved in 1.5mL of dichloromethane, and the solution is homogenized by ultrasonic treatment for 10 min. Phospholipid 60mg and sodium cholate 75mg are precisely weighed and dissolved in a mixed solvent of 0.5mL of dichloromethane and 2mL of methanol, and ultrasonic treatment is carried out for 10min to ensure that the solution is uniform. PLGA300mg was precisely weighed and dissolved in 3mL of dichloromethane and homogenized by sonication for 11 min. Dispersing an astaxanthin solution into a mixed solution of phospholipid and sodium cholate, performing ultrasonic treatment for 12min to fully disperse the astaxanthin solution, dispersing the solution into a PLGA solution, performing ultrasonic treatment for 30min to fully disperse the PLGA solution to obtain an organic phase with a total volume of 7mL, dispersing the organic phase into 6mL of 2% PVA403 aqueous solution under ultrasonic treatment to emulsify the organic phase, dispersing the emulsified liquid into 30mL of 1% PVA403 aqueous solution, and stirring overnight to volatilize the organic solvent.

(2) And after the organic solvent is volatilized, the organic solvent is respectively filled into 50mL centrifuge tubes, large particles and part of free drug precipitates are removed by centrifugation at 1500rpm, the supernatant is centrifuged at 16000rpm to obtain solid precipitates, the solid precipitates are resuspended and washed for 3 times by 30mL double-distilled water, and then the solid nanoparticles of the powdered astaxanthin are obtained by freeze drying.

Example 6 preparation of astaxanthin nanoformulation

(1) 10mg of astaxanthin is precisely weighed and dissolved in 1.5mL of dichloromethane, and the solution is homogenized by ultrasonic treatment for 10 min. 100mg of phospholipid and 30mg of sodium cholate are precisely weighed and dissolved in a mixed solvent of 0.5mL of dichloromethane and 3mL of methanol, and the mixture is subjected to ultrasonic treatment for 10min to be uniform. PLGA 200mg was precisely weighed and dissolved in 4mL of dichloromethane and homogenized by sonication for 8 min. Dispersing an astaxanthin solution into a mixed solution of phospholipid and sodium cholate, performing ultrasonic treatment for 5min to fully disperse the astaxanthin solution, then dispersing the solution into a PLGA solution, performing ultrasonic treatment for 25min to fully disperse the PLGA solution to obtain an organic phase with the total volume of 9mL, dispersing the organic phase into 8mL of 4% PVA403 aqueous solution under ultrasonic treatment to emulsify the organic phase, then dispersing the emulsion into 35mL of 0.5% PVA403 aqueous solution, and stirring overnight to volatilize the organic solvent.

(2) And after the organic solvent is volatilized, the organic solvent is respectively filled into 50mL centrifuge tubes, large particles and partial free drug precipitates are removed by centrifugation at 3500rpm, the supernatant is centrifuged at 14000rpm to obtain solid precipitates, the solid precipitates are resuspended and washed for 2 times by 30mL double-distilled water, and then the solid nanoparticles of the powdered astaxanthin are obtained by freeze drying.

Example 7 preparation of astaxanthin nanoformulation

(1) 10mg of astaxanthin is precisely weighed and dissolved in 1.5mL of dichloromethane, and the solution is homogenized by ultrasonic treatment for 10 min. Phospholipid 40mg and sodium cholate 30mg are precisely weighed and dissolved in a mixed solvent of 0.5mL of dichloromethane and 2mL of methanol, and ultrasonic treatment is carried out for 10min to ensure that the solution is uniform. PLGA300mg was precisely weighed and dissolved in 2mL of dichloromethane, and homogenized by sonication for 10 min. Dispersing an astaxanthin solution into a mixed solution of phospholipid and sodium cholate, performing ultrasonic treatment for 10min to fully disperse the astaxanthin solution, then dispersing the solution into a PLGA solution, performing ultrasonic treatment for 10min to fully disperse the PLGA solution to obtain an organic phase with the total volume of 6mL, dispersing the organic phase into 10mL of 4% PVA403 aqueous solution under ultrasonic treatment to emulsify the organic phase, then dispersing the emulsion into 40mL of 1% PVA403 aqueous solution, and stirring overnight to volatilize the organic solvent.

(2) And after the organic solvent is volatilized, the organic solvent is respectively filled into 50mL centrifuge tubes, large particles and part of free drug precipitates are removed by centrifugation at 2000rpm, the supernatant is centrifuged at 16000rpm to obtain solid precipitates, the solid precipitates are resuspended and washed for 3 times by 30mL double-distilled water, and then the solid nanoparticles of the powdered astaxanthin are obtained by freeze drying.

Example 8 preparation of astaxanthin nanoformulation

(1) 15mg of astaxanthin is precisely weighed and dissolved in 1.5mL of dichloromethane, and the solution is homogenized by ultrasonic treatment for 10 min. Phospholipid 60mg and sodium cholate 30mg are precisely weighed and dissolved in a mixed solvent of 0.5mL of dichloromethane and 2mL of methanol, and the mixture is subjected to ultrasonic treatment for 10min to be uniform. PLGA300mg was precisely weighed and dissolved in 4mL of dichloromethane and sonicated for 8min to homogenize. Dispersing an astaxanthin solution into a mixed solution of phospholipid and sodium cholate, performing ultrasonic treatment for 15min to fully disperse the astaxanthin solution, then dispersing the solution into a PLGA solution, performing ultrasonic treatment for 20min to fully disperse the PLGA solution to obtain an organic phase with the total volume of 8mL, dispersing the organic phase into 7mL of 3% PVA403 aqueous solution under ultrasonic treatment to emulsify the organic phase, then dispersing the emulsion into 20mL of 1% PVA403 aqueous solution, and stirring overnight to volatilize the organic solvent.

(2) And after the organic solvent is volatilized, the organic solvent is respectively filled into 50mL centrifuge tubes, large particles and partial free drug precipitates are removed by centrifugation at 3000rpm, the supernatant is centrifuged at 10000rpm to obtain solid precipitates, the solid precipitates are resuspended and washed for 2 times by 30mL double-distilled water, and then the solid nanoparticles of the powdered astaxanthin are obtained by freeze drying.

Example 9 preparation of astaxanthin nanoformulation

(1) 15mg of astaxanthin is precisely weighed and dissolved in 1.5mL of dichloromethane, and the solution is homogenized by ultrasonic treatment for 10 min. Phospholipid 65mg and sodium cholate 45mg are precisely weighed and dissolved in a mixed solvent of 0.5mL of dichloromethane and 2mL of methanol, and the mixture is subjected to ultrasonic treatment for 10min to be uniform. PLGA 200mg was precisely weighed and dissolved in 2mL of dichloromethane, and homogenized by sonication for 8 min. Dispersing an astaxanthin solution into a mixed solution of phospholipid and sodium cholate, performing ultrasonic treatment for 15min to fully disperse the astaxanthin solution, then dispersing the solution into a PLGA solution, performing ultrasonic treatment for 20min to fully disperse the PLGA solution to obtain an organic phase with the total volume of 6mL, dispersing the organic phase into 10mL of 3% PVA403 aqueous solution under ultrasonic treatment to emulsify the organic phase, then dispersing the emulsion into 40mL of 0.5% PVA403 aqueous solution, and stirring overnight to volatilize the organic solvent.

(2) And after the organic solvent is volatilized, the organic solvent is respectively filled into 50mL centrifuge tubes, large particles and part of free drug precipitates are removed by centrifugation at 2000rpm, the supernatant is centrifuged at 16000rpm to obtain solid precipitates, the solid precipitates are resuspended and washed for 2 times by 30mL double-distilled water, and then the solid nanoparticles of the powdered astaxanthin are obtained by freeze drying.

Example 10 morphology and particle size distribution studies of astaxanthin nanoformulations prepared in example 7

(1) Morphology observation the solid precipitate prepared in example 7 was selected, and the appearance and the morphology after freeze-drying were observed, and it was found that the re-suspension of the solid precipitate was orange red (fig. 1A), and the laser treatment of the nanoparticle heavy suspension had a significant tyndall effect (fig. 1B), demonstrating that particles with a particle size of 1-100nm were present in the re-suspension of the solid precipitate. The lyophilized preparation has uniform color, is orange red, fluffy and flocculent, and is a powdery solid preparation with good form (figure 1C).

(2) TEM morphology the solid precipitate prepared in example 7 was selected, diluted with distilled water to an appropriate concentration, 20. mu.L of the diluted solution was dropped onto a copper mesh, naturally dried at room temperature, then dyed by dropping 2% phosphotungstic acid, and after 1-2 minutes, the dye solution was removed by blotting with filter paper cut to a sharp corner. A drop of pure water was dropped on the copper mesh, and the solution was removed by suction with filter paper, and the process was repeated twice, and the excess staining solution was washed off, dried at room temperature, and then the morphological characteristics were observed by Transmission Electron Microscopy (TEM), and the results are shown in fig. 2 below.

(3) Particle size distribution and Zeta potential the solid precipitate prepared in example 7 was selected and the particle size distribution of the nanoformulation was determined using a 90Plus PALS particle size analyzer (bruke hei instruments, usa) and the results showed that the particle size of the ASTA-PLGA @ M nanoparticles was 76nm, the Zeta potential was-38.45 mV and the PDI index was 0.941.

Example 11 in vitro drug Release assay of astaxanthin Nanodispersions

An equal amount of the astaxanthin nanoformulation prepared in example 7 was precisely weighed, and put into 4mL of EP containing 2mL of water and a buffer salt release medium having a pH of 1.2, 4.5, and 6.8, and placed in a 37. + -. 1 ℃ constant temperature shaking water bath, and shaken at a constant temperature of 100r/min, and at each time point of 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours, an EP tube was taken, a sample was centrifuged at 4 ℃ and 12000rpm, and the supernatant was taken to determine the astaxanthin content, and an astaxanthin release curve was plotted with time as abscissa and release percentage as ordinate, and the results are shown in FIG. 3. As can be seen from fig. 3, the in vitro release of astaxanthin nanoparticies varies greatly in release media of different pH, with the first 24h release being fastest among water, followed by pH 6.8, 4.5, but four days later, release is significantly faster at pH 6.8, with a total release percentage of 62.18%, while 47.52% in water, only 34.75% at pH 4.5, with the worst release at pH 1.2, and a release percentage of only 5.47% within 5 days. Overall, the release rate was faster in the first 24h and slower in the later period. The design concept of the astaxanthin nano preparation is achieved preliminarily, and a theoretical basis is laid for later-period pesticide effect investigation.

Example 12 preliminary evaluation of hepatoprotective Effect of astaxanthin-containing Nanodiamedicine

The liver protection effect and the long-acting effect of the astaxanthin nano preparation prepared in example 7 are evaluated and compared with a positive control drug and a raw material drug. Establishing a mouse model of carbon tetrachloride-induced acute liver injury, and primarily evaluating the liver protection effect and the long-acting effect of the astaxanthin nano preparation by measuring the ALT and AST levels in the plasma of the mouse and the SOD and MDA levels in the liver homogenate tissue of the mouse and combining the pathological changes of liver tissue slices.

(1) Animal administration

Taking 35 SPF-grade KM mice, male, weighing 20 + -2 g, randomly dividing into 5 groups including blank group, model group, astaxanthin raw material medicine group, astaxanthin preparation group and positive medicine control group, and 7 mice in each group. The experimental preposed animals are adapted to the environment in a laboratory for 3 days at the room temperature of 20-25 ℃, and are fed with standard feed during the period, and water is freely drunk. The blank group was gavaged with physiological saline 0.5mL daily for 15 days; the model group is perfused with physiological saline for 0.5mL every day for 15 days; the astaxanthin raw material medicine is 200mg kg^-1The dosage of the composition is 0.5mL per day by taking 0.5 percent sodium carboxymethylcellulose as a suspension medium for dispersion; astaxanthin preparation group with 10 mg/kg^-1The dosage of the traditional Chinese medicine composition is 0.5mL per time of intragastric administration, and the intragastric administration is carried out once every 3 days for 15 days; control group of positive drug at 60 mg/kg^-1The dose of (3) was gavage 0.5mL daily for 15 days. On day 16, 0.2% CCl was administered to each group of mice except the blank group₄Injecting soybean oil solution into abdominal cavity at a ratio of 20g/0.2mL, and performing CCl₄Acute liver injury is induced. During the injury period, mice were fasted and had free access to water.

(2) Sample processing

Plasma sample: blood is collected from the mouse eyeballs and placed in a 0.5% heparin tube, centrifuged at 3700rpm for 10min, and the upper plasma is separated for later use and diluted to a proper ratio by physiological saline according to the kit instructions.

Liver sample: after the mice are killed by dislocation, the whole liver is dissected and taken, the whole weight of the liver is weighed, and the liver is divided into whole liver lobes which are fixed by 10 percent formaldehyde solution to be subjected to HE staining of liver tissue sections. The rest is weighed and added with normal saline, and the mixture is sheared at high speed in ice water bath to prepare 10 percent liver homogenate solution. The homogenate was taken 0.5mL in a 1.5mL EP tube, centrifuged using a microcentrifuge, centrifuged at 4500rpm/min for 10min, the supernatant taken and diluted to the appropriate fold with physiological saline according to the kit instructions.

After sample treatment, the protein content in mouse liver is determined according to the BCA kit instruction, and the biochemical index in plasma is determined according to the requirements of ALT and AST kits. And (4) measuring various biochemical indexes in the liver homogenate according to the requirements of SOD and MDA kits.

(3) Results

After the astaxanthin bulk drug, the astaxanthin nano preparation and the positive drug control group are orally administered for 15 days, the CCl is measured by the kit₄Various biochemical indexes in plasma and liver homogenate of an induced acute liver injury mouse model.

Compared with a blank control group, the ALT activity in the plasma of the model group mice is remarkably improved (P)<0.001), AST activity is obviously improved (P)<0.05) significantly elevated MDA levels in liver tissue (P)<0.05), a significant reduction in SOD levels (P)<0.001), the results all indicate CCl₄The establishment of the induced acute liver injury mouse model is successful.

Compared with the model group, the SOD level in the liver tissue of the raw medicine group is increased to a certain extent and has certain significant difference (P is less than 0.01) compared with the model group, the MDA in the liver tissue has no significant difference compared with the model group, the ALT activity in the plasma is significantly reduced (P is less than 0.001), and the AST activity in the plasma is significantly reduced (P is less than 0.05), and the result can show that the high-dose raw medicine has certain protection effect on the liver after the preventive administration on the mouse. Compared with the model group, the astaxanthin nano preparation administration group has the advantages that the SOD level significance (P <0.05) in the liver tissue is increased, the MDA level significance (P <0.05) in the liver tissue is reduced, the ALT level significance (P <0.001) in the blood plasma is reduced, and the AST level significance (P <0.05) in the blood plasma is reduced, so that the result shows that the ASTA-PLGA @ M nano particles have a good protection effect on the liver after the administration of the ASTA-PLGA @ M nano particles to mice in a preventive manner. Compared with the model group, the positive medicine group has the advantages that the SOD level in liver tissues is increased (P <0.001), the MDA level is reduced (P <0.05), the ALT level in plasma is reduced (P <0.001), and the AST level in plasma is also reduced (P <0.05), and the results show that the positive liquid medicine silybin has good protection effect on the liver after the preventive administration to mice.

Compared with the raw material medicines, the astaxanthin nano preparation has no significant difference in SOD level in liver tissues, significant difference in MDA level in liver tissues (P <0.01), significant difference in ALT activity in blood plasma (P <0.01) and significant difference in AST activity in blood plasma (P <0.01), which shows that the astaxanthin nano preparation has better liver protection effect than the astaxanthin bulk drug. And the preparation group is orally taken once in 3 days, so that the preparation can play a long-acting role in a certain time, and the change of each index result is shown in the table I.

TABLE 1 astaxanthin drug substance, astaxanthin solid nanoparticles and positive drug for 0.2% CCl₄Effect of induced liver injury

In addition, from the pathological change of liver tissue, the growth state of the liver cells in a normal group is good, the cell nucleuses are clear and visible, and the cells are uniformly distributed and are relatively compact. In the model group, small areas of liver cell nuclei are fragmented and disappeared, fat grains grow on the small areas, and inflammatory infiltration is accompanied, so that successful modeling is proved. The raw material medicine still has damage after the preventive administration treatment, and the damage area is reduced compared with the model group. Compared with the model group, the astaxanthin nano preparation administration group has fewer damaged areas and smaller damage degree; compared with astaxanthin raw material medicines, the preparation has better effect.

Claims

1. An astaxanthin long-acting nano preparation prepared by a micelle template method is characterized in that: phospholipid and sodium cholate are taken as micelle templates and are dissolved in a mixed solvent of dichloromethane and methanol; polylactic acid-glycolic acid copolymer (PLGA) is a long-acting carrier and is dissolved in dichloromethane; polyvinyl alcohol (PVA) is used as a dispersant and is dissolved in water; the weight ratio of the components is astaxanthin: phospholipid: sodium cholate: PLGA is 1:3-7:2-5:10-50, wherein the mass volume concentration of the astaxanthin in the dichloromethane is 1.5-6 mg/mL.

2. The long-acting nano-preparation of astaxanthin prepared by micelle template method according to claim 1, wherein the phospholipid used in the micelle template is egg yolk lecithin or soybean lecithin, wherein the content of phosphatidylcholine is more than 80%.

3. The micelle templated long-acting nano-preparation of astaxanthin as claimed in claim 1 wherein the long-acting carrier is PLGA, which may be PLGA75/25, PLGA50/50, etc., and has a viscosity of 0.09dL/g,0.21dL/g, and 0.23 dL/g.

4. The long-acting nano preparation of astaxanthin prepared by the micelle template method according to claim 1, wherein the dispersing agent is PVA (type: PVA 403) or PVA 1788, and the concentration is 0.5-4% (w/v).

5. The long-acting nano preparation of astaxanthin prepared by the micelle template method according to claim 1, wherein the mixed solvent is a mixed solvent of dichloromethane and methanol, and is mainly used for dissolving phospholipid and sodium cholate, wherein the volume content (v%) of dichloromethane is 0-30%.

6. The long-acting nano preparation of astaxanthin prepared by the micelle template method according to claim 1, wherein the nano preparation of astaxanthin is pink or orange dry powder, has a particle size of 40-200nm after being re-dispersed in water, and is in a regular spherical shape.

7. The long-acting nano preparation of astaxanthin prepared by the micelle template method according to claim 1, wherein the nano preparation of astaxanthin can be filled in capsules to be prepared into capsules or pressed into tablets.

8. The method for preparing the long-acting nano preparation of astaxanthin prepared by the micelle template method according to claim 1, is characterized by comprising the following steps: