CN119112797A

CN119112797A - A kind of dragon blood A nano liposome and its preparation method and application

Info

Publication number: CN119112797A
Application number: CN202411292766.1A
Authority: CN
Inventors: 陶春霖; 鲁金春子
Original assignee: Wansu Guangzhou Biopharmaceutical Co ltd
Current assignee: Wansu Guangzhou Biopharmaceutical Co ltd
Priority date: 2024-09-14
Filing date: 2024-09-14
Publication date: 2024-12-13

Abstract

The invention discloses a lordreya A nanoliposome and a preparation method and application thereof, belonging to the technical field of nanoliposomes, and solving the problem of low bioavailability of lordreya A in the prior art. The lordreya A nanoliposome comprises the following components per 100 parts by weight: 1-10 parts of lordreya A, 5-15 parts of emulsifier, 0-1 part of auxiliary emulsifier, 1-3 parts of cholesterol, 5-20 parts of oil, 30-50 parts of polyol, and the balance is water. The lordreya A nanoliposome provided by the invention not only solves the problem of difficulty in adding lordreya A into pharmaceutical preparations and cosmetic preparations, but also improves the affinity of lordreya A with keratinocytes, so that it has better transdermal penetration ability, and is safe, non-toxic and non-irritating.

Description

Dragon blood A nano liposome and preparation method and application thereof

Technical Field

The invention relates to the technical field of nanoliposomes, in particular to a longxuelin A nanoliposome and a preparation method and application thereof.

Background

The dragon's blood is resin prepared by extracting and processing lipid-containing wood of plants of the dracaena cochinchinensis (Dracaena cochinchinensis) of the lily family, is mainly produced in the south of Yunnan and the south of Guangxi of China, and is a traditional Chinese medicinal material and a national characteristic medicinal material of China. The dragon blood A (loureirinA, chemical name 4' -hydroxy-2, 4-dimethoxy dihydrochalcone) is a main free dihydrochalcone component in dragon blood raw plants, is one of main active components of dragon blood, and is generally used for pharmacodynamics research of the dragon blood. In recent years, the active monomer, namely, the longxuexin A, is increasingly valued by scholars at home and abroad in the research field.

The skin is composed of epidermis layer, dermis layer, subcutaneous tissue and its accessory organs (sweat gland, sebaceous gland, blood vessel, etc.), is the first line of defense of human body against infection and injury, as the organ in direct contact with external environment, is vulnerable to external factor injury, and the repair of skin injury is the result of the action of various cells and factors. The longxuexin A has wide application prospect in wound healing and regulation regeneration of skin injury, can promote proliferation and differentiation of hair follicle stem cells, provides more seed cells for skin injury repair, effectively inhibits in-vitro thrombin activity, promotes blood circulation to remove blood stasis, improves wound microcirculation, and regulates cell differentiation and growth-related factor secretion. In the inflammatory reaction stage of wound repair of skin, the longxuein A can play an anti-inflammatory role, and effectively prevent wound infection. The longxuelin A also has extremely strong oxidation resistance, has oxidation resistance higher than that of N-acetyl-L-cysteine (NAC), can increase the activity of superoxide dismutase (SOD), enhance the free radical scavenging efficiency, reduce the oxidative stress of wound surfaces, and is beneficial to the repair of epidermis and skin appendages. In addition, the longxuexin A regulates the balance of fibroblast proliferation and apoptosis, reduces extracellular matrix deposition, prevents the proliferation of connective tissue after skin injury, and inhibits the formation of pathological scars.

However, as a flavonoid compound, the longxuelin A has the problems of poor water solubility, poor permeability, low bioavailability and the like.

Disclosure of Invention

The invention provides a longxuexin A nano liposome and a preparation method and application thereof, which are used for solving the problem of low bioavailability of longxuexin A in the prior art.

In the first aspect, the invention provides a longxuelin A nanoliposome, which comprises, by weight, 1-10 parts of longxuelin A, 5-15 parts of an emulsifying agent, 0-1 part of a co-emulsifying agent, 1-3 parts of cholesterol, 5-20 parts of grease, 30-50 parts of polyol and the balance of water.

As one possible implementation manner, the emulsifier is any one of hydrogenated lecithin, egg yolk lecithin, soybean lecithin, sunflower lecithin, distearoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, and/or the auxiliary emulsifier is one or a combination of more of PEG-7 glycerol cocoate, PEG-30 dimer hydroxy stearate, polyglycerol-10 laurate, polyglycerol-10 oleate, polyglycerol-10 myristate and polyethylene glycol fatty acid glyceride, and/or the oil is any one of caprylic/capric triglyceride, C12-15 alcohol benzoate, glyceryl monostearate, squalane and acetylated monoglyceride, and/or the polyol is any one of glycerol, 1, 3-propanediol, butanediol, 1, 2-pentanediol and dipropylene glycol.

The invention provides a preparation method of the longxuelin A nano liposome in any one of possible implementation modes of the first aspect, which comprises the following steps of mixing the emulsifying agent, the auxiliary emulsifying agent, the cholesterin and the grease, performing homogenizing and dispersing treatment under the conditions of sealing and heating at 60-80 ℃ to obtain a mixed solution, adding the longxuelin A into the polyalcohol, performing stirring treatment under the conditions of heating at 40-60 ℃ in an inert atmosphere to obtain an alcohol solution of the longxuelin A, mixing the mixed solution, the alcohol solution of the longxuelin A and the water, performing homogenizing and shearing treatment to obtain mixed colostrum, and performing micro-jet homogenizing treatment on the mixed colostrum under the sealing condition to obtain the longxuelin A nano liposome.

As a possible implementation manner, the condition of the homogenizing and dispersing treatment is that the speed is 12000-16000 rpm, and/or the condition of the stirring treatment is that the speed is 800-1200 rpm, and/or the condition of the homogenizing and shearing treatment is that the speed is 8000-12000 rpm and the duration is 5-10 min, and/or the condition of the micro-jet homogenizing treatment is that the pressure is 20000-30000 psi and the circulation times are 3-6 times.

As one possible implementation mode, the method comprises the steps of mixing 10g of hydrogenated lecithin, 0.3g of PEG-7 glycerol cocoate, 3g of cholesterol and 15g of caprylic/capric triglyceride, carrying out homogenizing dispersion treatment with the speed of 14000rpm under the condition of heating in a water bath at 70 ℃ to obtain a mixed solution, adding 6.7g of the longxuridine A into 40g of glycerol, carrying out stirring dissolution treatment with the speed of 1000rpm under the condition of heating in a water bath at 50 ℃ in an inert atmosphere until the mixture is uniformly dissolved to obtain an alcohol solution of the longxuridine A, carrying out mixing homogenizing shearing on the mixed solution, the alcohol solution of the longxuridine A and 25g of ultrapure water at the speed of 10000rpm for 7min to obtain mixed primary emulsion, carrying out micro-jet homogenizing treatment on the mixed primary emulsion at the pressure of 25000psi for 5 times, and obtaining the longxuridine A nano liposome.

In a third aspect, the invention provides an application of the longxuridine a nanoliposome prepared by any one of possible implementation manners of the first aspect or the preparation method of any one of possible implementation manners of the second aspect in preparing a skin repair preparation.

As a possible implementation, the preparation is used for promoting fibroblast proliferation, and/or the preparation is used for promoting cell wound repair, and/or the preparation is used for scavenging free radicals.

In a fourth aspect, the invention provides a skin repair preparation, which comprises the longxuridine A nanoliposome according to any one of the possible implementation manners of the first aspect or the longxuridine A nanoliposome prepared by the preparation method according to any one of the possible implementation manners of the second aspect and auxiliary materials.

As one possible implementation manner, the preparation is any one of cream, emulsion, water aqua, gel, oil, powder, block powder or solid, mud, aerosol, organic solvent, wax base, paste, film and freeze-drying.

As one possible implementation manner, the auxiliary material is one or a combination of several of a solvent, a propellant, a solubilizer, a cosolvent, an emulsifier, a colorant, an adhesive, a disintegrating agent, a filler, a lubricant, a wetting agent, an osmotic pressure regulator, a stabilizer, a glidant, a flavoring agent, a preservative, a suspending agent, a coating material, a fragrance, an anti-adhesive agent, an integrating agent, a permeation enhancer, a pH regulator, a buffer, a plasticizer, a surfactant, a foaming agent, a defoaming agent, a thickening agent, an inclusion agent, a humectant, an absorbent, a diluent, a flocculating agent and a deflocculant, a filter aid and a release retarder.

Liposomes are ultra-miniature spherical carriers formed from lipid bilayers, and are widely used as packaging and delivery systems in common use today for protecting, controlling and releasing bioactive substances. The liposome contains skin inherent components such as phospholipid and cholesterol, has good affinity with skin, high safety and certain repairing effect, and can enter deep skin to enhance mobility and permeability of deep cells, thereby enhancing metabolism of cells and activating cells. The entrapment of the liposome can increase the osmotic retention effect of the active ingredient, so that the active ingredient overcomes the barrier function of the stratum corneum, is stored in the deep layer of the skin, is slowly released, continuously acts and improves the acting effect.

The invention provides a nano liposome of the longxuexin A, which not only solves the problem of difficult addition of the longxuexin A in a pharmaceutical preparation and a cosmetic preparation, but also improves the affinity of the longxuexin A with keratinocytes, so that the longxuexin A has better transdermal permeability, and is safe, nontoxic and nonirritating. After the nano-liposome of the longxuexin A is dispersed in water, a uniform colloid solution can be formed, on one hand, the nano-liposome of the longxuexin A can effectively penetrate through the surface layer of the skin, is enriched in high concentration and stays for a long time in skin tissues, realizes the skin targeted delivery of the longxuexin A, enables the longxuexin A to exert a powerful repairing effect, on the other hand, is beneficial to improving the solubility, stability and bioavailability of the longxuexin A, and overcomes the defects of poor physical stability, low encapsulation efficiency and the like of the traditional liposome, so that the longxuexin A stays for a long time in the skin to achieve the optimal slow release effect. In addition, the moisturizing and repairing functions of the liposome are synergistic with those of the longxuelin A, so that the skin repairing is further promoted, and the liposome has excellent nursing and repairing effects on skin wounds, damaged skin barriers and the like.

In the preparation method of the nano liposome of the longxuelin A, stirring and dissolving treatment under inert atmosphere, and homogenizing and dispersing, mixing and homogenizing and shearing and micro-jet homogenizing treatment under sealing conditions are carried out, so that the problems of reduced fluidity of a liposome membrane, poor encapsulation effect, increased particle size and polydispersity index of the liposome and the like caused by oxidation phenomenon in the preparation process are prevented. The micro-jet homogenizing technology used in the invention is a high-efficiency nano-scale emulsifying and dispersing treatment technology, and has the advantages of extremely high shearing force, and the materials are subjected to refining and uniform mixing treatment through dynamic high-pressure micro-jet, high-shearing, high-energy collision (turbulence collision) and cavitation effect, so that the particle size reduction and the particle size distribution are realized more rapidly. The micro-jet homogenizing technology can ensure that the pressure of the material is a constant peak value in the treatment process, so that the particle size of the material is reduced faster and the particle size distribution is narrower, the treatment effect is good in reproducibility, and the treatment effect can be kept consistent in pilot scale production and large-scale production.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a graph showing the detection result of the encapsulation efficiency of the product I provided in the example of the present invention for 30 days.

FIG. 2 is a graph showing the results of the measurement of the effect of products I-IX on fibroblast proliferation, wherein # P <0.01, # P <0.0001, compared with the blank.

FIG. 3 is a graph of a keratinocyte scratch test of products I-IX provided by an embodiment of the invention.

FIG. 4 is a graph showing the results of detection of the effect of products I-IX on keratinocyte scratch healing rate, wherein # # # P <0.0001 compared with the control group.

Fig. 5 is a graph of the clearance results of the positive control in ABTS radical clearance experiments provided by the examples of the present invention.

FIG. 6 is a graph of the detection results of the ABTS radical scavenging rate of the products I to IX provided by the embodiment of the invention.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

In order to solve the problem of low bioavailability of the longxuridine A in the prior art, the embodiment of the invention provides a preparation experiment of the longxuridine A nano liposome, and physical properties are measured, and the product prepared by the embodiment of the invention has smaller particle size, better physical stability and uniformity.

Further, the embodiment of the invention provides a test experiment of encapsulation efficiency and stability, and the experiment shows that the longxuelin A nano liposome provided by the embodiment of the invention has higher encapsulation efficiency and better stability. After the nano liposome of the longxuelin A is dispersed in water, a uniform colloid solution can be formed, which is beneficial to improving the solubility, the stability and the bioavailability of the nano liposome, and the defects of poor physical stability, low encapsulation efficiency and the like of the traditional liposome are overcome, so that the longxuelin A stays on the skin for a long time to achieve the optimal slow release effect.

Further, the embodiment of the invention provides a fibroblast proliferation assay, a cell repair assay and an ABTS free radical scavenging experiment, and the embodiment of the invention provides the longxuexin A nanoliposome which has obvious effect of promoting fibroblast proliferation, obvious capability of promoting wound repair and obvious capability of scavenging free radicals. The moisturizing and repairing functions of the liposome are synergistic with those of the longxuelin A, so that the skin repairing is further promoted, and the liposome has excellent nursing and repairing effects on skin wounds, damaged skin barriers and the like.

Further, the embodiment of the invention provides an application experiment of skin repair, and the embodiment of the invention provides the dracaena cochinchinensis A nanoliposome which has obvious effect of repairing damaged skin. After the nano-liposome of the longxuexin A is dispersed in water, a uniform colloid solution can be formed, the nano-liposome of the longxuexin A can effectively penetrate through the surface layer of the skin, is enriched in skin tissues at high concentration and stays for a long time, realizes the skin targeted delivery of the longxuexin A, and enables the longxuexin A to exert a powerful repairing effect.

The technical scheme of the invention will be further described in connection with specific embodiments.

Example 1

The embodiment provides a preparation experiment of a longxuelin A nano liposome.

10G of hydrogenated lecithin, 0.3g of PEG-7 glycerol cocoate, 3g of cholesterol and 15g of caprylic/capric triglyceride are mixed, and subjected to homogenizing and dispersing treatment at a speed of 14000rpm under the condition of heating in a water bath at 70 ℃ to obtain a mixed solution I;

Adding 6.7g of the longxuexin A into 40g of glycerin, stirring and dissolving at the speed of 1000rpm under the condition of heating in a water bath at 50 ℃ in an inert atmosphere until the longxuexin A is uniformly dissolved, and obtaining an alcohol solution I of the longxuexin A;

Mixing and homogenizing shearing the mixed solution I, the alcohol solution I of the longxuexin A and 25g of ultrapure water at the speed of 10000rpm for 7min to obtain mixed colostrum I;

The mixed colostrum I is subjected to micro-jet homogenization treatment at 25000psi for 5 times to obtain a product I.

Example 2

10G of egg yolk lecithin, 0.3g of polyglycerol-10 laurate, 3g of cholesterol and 15g of C12-15 alcohol benzoate are mixed, and subjected to homogenizing and dispersing treatment at a speed of 12000rpm under the condition of 80 ℃ water bath heating, and dispersed into a whole to obtain a mixed solution II;

Adding 6.7g of the longxuexin A into 40g of 1, 3-propanediol, stirring and dissolving at the speed of 1200rpm under the condition of heating in a water bath at the temperature of 40 ℃ under inert atmosphere until the longxuexin A is dissolved uniformly to obtain an alcohol solution II of the longxuexin A;

Mixing and homogenizing shearing the mixed solution II, the alcohol solution II of the longxuridine A and 25g of ultrapure water at the speed of 12000rpm for 5min to obtain mixed colostrum II;

the mixed colostrum II is subjected to micro-jet homogenization treatment at 20000psi pressure, and the circulation times are 6 times, so that the product II is obtained.

Example 3

10G of soybean lecithin, 0.3g of PEG-30 dimer hydroxystearate, 3g of cholesterin and 15g of squalane are mixed, and subjected to homogenizing and dispersing treatment at a speed of 16000rpm under the heating condition of a water bath at 60 ℃ to obtain a mixed solution III;

adding 6.7g of the longxuexin A into 40g of 1, 2-pentanediol, stirring and dissolving at the speed of 800rpm under the condition of heating in a water bath at the temperature of 60 ℃ in an inert atmosphere, and obtaining an alcohol solution III of the longxuexin A after uniform dissolution;

Mixing and homogenizing shearing the mixed solution III, the alcohol solution III of the longxuridine A and 25g of ultrapure water at a speed of 8000rpm for 10min to obtain mixed colostrum III;

The mixed colostrum III is subjected to micro-jet homogenization treatment at 30000psi for 3 times to obtain the product III.

Example 4

15G of hydrogenated lecithin, 0.1g of PEG-7 glycerol cocoate, 1.2g of cholesterol and 5g of caprylic/capric triglyceride are mixed, and subjected to homogenizing and dispersing treatment at a speed of 14000rpm under the condition of heating in a water bath at 70 ℃ to obtain a mixed solution IV;

adding 6.7g of the longxuexin A into 50g of glycerin, stirring and dissolving at the speed of 1000rpm under the condition of heating in a water bath at 50 ℃ in an inert atmosphere until the longxuexin A is uniformly dissolved, and obtaining an alcohol solution IV of the longxuexin A;

Mixing and homogenizing shearing the mixed solution IV, the alcohol solution IV of the longxuridine A and 22g of ultrapure water at the speed of 10000rpm for 7min to obtain mixed colostrum IV;

The mixed colostrum IV is subjected to micro-jet homogenization treatment at 25000psi for 5 times to obtain the product IV.

Example 5

Mixing 5g of hydrogenated lecithin, 1g of PEG-7 glycerol cocoate, 3g of cholesterol and 20g of caprylic/capric triglyceride, and carrying out homogenizing and dispersing treatment with the speed of 14000rpm under the condition of heating in a water bath at 70 ℃ to obtain a mixed solution V;

Adding 6.7g of the longxuexin A into 30g of glycerol, stirring and dissolving at the speed of 1000rpm under the condition of heating in a water bath at 50 ℃ in an inert atmosphere until the longxuexin A is dissolved uniformly, and obtaining an alcohol solution V of the longxuexin A;

Mixing and homogenizing shearing the mixed solution V, the alcohol solution V of the longxuexin A and 34.3g of ultrapure water at the speed of 10000rpm for 7min to obtain mixed colostrum V;

The mixed colostrum V is subjected to micro-jet homogenization treatment at 25000psi for 5 times to obtain a product V.

Comparative example 1

The embodiment provides a preparation experiment of nano liposome.

10G of hydrogenated lecithin, 0.3g of PEG-7 glycerol cocoate, 3g of cholesterol and 15g of caprylic/capric triglyceride are mixed, and subjected to homogenizing and dispersing treatment at a speed of 14000rpm under the condition of heating in a water bath at 70 ℃ to obtain a mixed solution VI;

adding 6.7g of the longxuexin A into 40g of glycerin, stirring and dissolving at a speed of 1000rpm under the condition of heating in a 50 ℃ water bath under an inert atmosphere until the longxuexin A is uniformly dissolved, and obtaining an alcohol solution VI of the longxuexin A;

The alcoholic solution VI of the longxuexin A was added to 25g of ultrapure water, stirred at 60℃for 30min, and then added dropwise to the mixed solution VI under shearing conditions at a rotational speed of 15000rpm, the shearing temperature being 55℃and the time being 10min, to give the product VI.

Comparative example 2

The example provides an experiment for preparing a solution of longxuelin A.

6.7G of the longxuexin A is added into 40g of glycerin, dispersed by ultrasonic treatment at room temperature for 50min, 53.3g of ultrapure water is added, and the product VII is obtained by stirring uniformly.

Comparative example 3

The embodiment provides a preparation experiment of nano liposome.

6.7G of longxuein A, 10g of lecithin, 3g of cholesterol and 15g of Tween 80 are added into 30mL of absolute ethanol and heated at 65 ℃ to obtain a mixed solution VIII;

evaporating the mixed solution VIII on a rotary evaporator under reduced pressure (50 ℃, 45r/min and 0.10 MPa) to remove absolute ethyl alcohol to form a layer of uniform lipid film, and then adding phosphate buffer solution (the concentration is 0.05mol/L, pH and is 6.0) for eluting and dispersing to obtain lipid hydration suspension VIII;

homogenizing the lipid hydration suspension VIII at 600bar pressure for 5 times to obtain the product VIII.

Comparative example 4

The embodiment provides a preparation experiment of nano liposome.

10G of hydrogenated lecithin, 0.3g of PEG-7 glycerol cocoate, 3g of cholesterol and 15g of caprylic/capric triglyceride are mixed, and subjected to homogenizing and dispersing treatment at a speed of 14000rpm under the condition of heating in a water bath at 70 ℃ to obtain a mixed solution IX;

mixing and homogenizing shearing the mixed solution IX and 25g of ultrapure water at a speed of 10000rpm for 7min to obtain mixed colostrum IX;

the mixed colostrum IX was subjected to microfluidization at 25000psi for 5 cycles to give product IX.

Experimental example 1

This experimental example provides a physical property determination of the product.

The test material was diluted with deionized water, and the hydrodynamic particle size and Polydispersity (PDI) of the liposomes were measured using dynamic light scattering techniques and measured 3 times in parallel at 25 ℃.

In the experimental example, products I-IX prepared in examples 1-5 and comparative examples 1-4 are respectively used as objects to be tested, hydrodynamic particle sizes and PDI (polymer dispersed particles) of the objects to be tested are measured after four times of dilution, and the average value is obtained to obtain the results shown in Table 1.

TABLE 1 hydrodynamic particle size and PDI results

	Hydrodynamic particle size	PDI
			Product I	82.3	0.082
Product II	84.5	0.098
			Product III	88.7	0.127
Product IV	92.6	0.158
			Product V	89.8	0.146
Product VI	130.3	0.275
			Product VII	/	/
Product VIII	156.7	0.261
			Product IX	212.5	0.367

As can be seen from Table 1, the particle size of the products I-V is less than 100nm, the particle size of the product VI is 130.3nm, the product VII is a solution, no particle size data exists, the particle size of the product VIII is 156.7nm, the particle size of the product IX is 212.5nm, and the particle sizes of the products I-V are smaller, so that the bioavailability of the entrapped active ingredients is improved more favorably. The PDI of the products I-V is also obviously smaller than that of the products VI-IX, and the smaller the PDI value is, the more concentrated the particle size distribution of the liposome is, namely the more uniform the particle size is. Therefore, the products I-V prepared in the embodiment are considered to be better, and have smaller particle size, better physical stability and uniformity.

Experimental example 2

The present examples provide stability testing experiments for the products.

Placing the object to be tested in a closed container, respectively placing for 90d at room temperature, 4 ℃ -18 ℃ and 45 ℃, naturally recovering the object to be tested to room temperature, checking whether the property of the sample is agglomerated or layered after the initial state and 90 days, and testing the particle size of the sample.

In the experimental example, products I-IX prepared in examples 1-5 and comparative examples 1-4 are respectively used as objects to be tested, and stability of the objects at different temperatures is measured to obtain results shown in a table 2, wherein the results are shown as whether agglomeration or layering phenomenon/particle size exists or not.

TABLE 2 stability test results

As shown in Table 2, the products I to V are not separated out and layered after being placed at-18 ℃,4 ℃ and room temperature for 90 days at 45 ℃, which indicates that the physical morphology and structure are kept well, the particle size is smaller, no obvious change is caused after being placed, the layering condition of the products VI, VII, VIII and IX is caused, the layering condition of the products VII and IX is caused, the particle sizes are larger, and the particle sizes are increased after being placed. Therefore, the products I-V are better and have better physical stability, wherein the particle size of the product I prepared in the embodiment 1 is minimum, the variation range of the particle size is minimum, and the effect is optimal.

Experimental example 3

This experimental example provides an encapsulation efficiency test of the product.

Taking 2mL of an object to be detected, passing through a 0.22 mu m organic filter membrane, taking 100 mu L of methanol for demulsification, measuring the content (w 1) of active ingredients of the object to be detected and the mass (w 0) of active ingredients of the input longxuexin A when the object to be detected is prepared by adopting an HPLC method, calculating the encapsulation efficiency according to a formula (w 1/w 0) multiplied by 100%, and carrying out long-term tracking on the encapsulation efficiency.

In this experimental example, the product I prepared in example 1 was used as a test substance, and the encapsulation efficiency was measured. The results shown in Table 3 were obtained.

TABLE 3 results of product I encapsulation efficiency test

Day	Day 0	For 7 days	14 Days	21 Days	For 30 days
						Encapsulation efficiency/%	98.6	98.3	98.3	98.3	98.2

As shown in Table 3, the encapsulation efficiency of the product I is 98.2+/-0.2%, and the encapsulation efficiency is maintained above 98% after one month, which indicates that the encapsulation efficiency stability of the product I is better.

Generally, higher encapsulation efficiency helps the formulation maintain long-term stability and consistency. As can be seen from the results in Table 3 and FIG. 1, the retention rate of the longxuelin A in the product I is 98.6% after the liposome preparation process, so that the preparation process provided by the invention can be considered to retain the activity of the longxuelin A to the greatest extent, avoid a large amount of loss and improve the utilization rate. The encapsulation efficiency of the product I is tracked for a long time, the encapsulation efficiency of the product I is not reduced basically within one month, and the encapsulation efficiency is still maintained to be more than 98% after one month, which indicates that the encapsulation efficiency stability of the product I prepared in the example 1 is very good, and the phenomena of leakage and the like are not easy to occur in the storage and transportation processes.

Experimental example 4

The experimental example provides an assay for the effect of the product on fibroblast proliferation.

Adding 1g of the to-be-detected substance into 100mL of DMEM culture medium to be fully dissolved to obtain a to-be-detected substance solution, inoculating the fibroblast in the logarithmic phase into a 96-well plate, adjusting the density of the fibroblast to 1X 10 ⁴ per hole, adaptively culturing for 1d, removing the old culture medium, adding 100 mu L of the to-be-detected substance solution, taking a blank DMEM culture medium as a control group, repeating 3 holes per group, continuously culturing for 48h, adding 10 mu L of CCK-8 solution per hole, incubating for 2h, measuring absorbance (A) at 450nm, and calculating the proliferation rate of the cells.

In the experimental example, products I-IX prepared in examples 1-5 and comparative examples 1-4 are respectively used as objects to be tested, and the respective cell proliferation rates are measured to obtain the results shown in FIG. 2.

Fibroblasts play an important role in maintaining tissue integrity and as a local precursor reservoir. After acute injury or burn to the skin, fibroblasts in the dermis begin to proliferate, migrate to the wound site, and activate at the wound site, form extracellular matrix, remodel the wound bed. As can be seen from FIG. 2, the results of the products I to V were better than those of the comparative examples. The product I has the most obvious effect of promoting the proliferation of the fibroblasts, and the proliferation rate of the cells is 29%. The method shows that the longxuelin A in the product prepared by the preparation process provided by the invention is easier to enter into fibroblasts to play a role, and has remarkable advantages in the aspect of repairing skin injury.

Experimental example 5

The experimental example provides an assay for the ability of the product to affect cell repair.

Adding the to-be-detected object into a DMEM culture medium to fully dissolve to prepare 20 mu L/mL to-be-detected object solution, taking keratinocytes (10 ⁶ cells/mL) in a logarithmic growth phase, inoculating 2mL of cell liquid into each hole in a 6-hole plate, culturing for 24 hours in an incubator, scratching the middle part of the back surface of the culture hole by using a pipette tip, washing by using PBS, adding 2mL of to-be-detected object solution into each hole, setting a negative control group, placing the cell plate in the incubator for culturing for 12 hours, taking out, observing under a lens, photographing to measure the scratch area, calculating the healing rate by the scratch area before and after culturing, and repeating the experiment for 3 times.

In the experimental example, products I-IX prepared in examples 1-5 and comparative examples 1-4 are respectively used as objects to be tested, and scratch healing rates are measured to obtain results shown in Table 4, FIG. 3 and FIG. 4.

TABLE 4 scratch healing Rate results statistics

Keratinocyte migration may reflect the process of wound repair, and keratinocyte (HaCaT) scratch healing rate is a common indicator of the ability of keratinocytes to migrate, which reflects the ability of cells to repair and regenerate after damage. As can be seen from Table 4, FIG. 3 and FIG. 4, the scratch healing rates between the products I-IX and the negative control group are all significantly different (p < 0.05), which indicates that the longxuexin A has an obvious promoting effect on the migration of HaCaT cells and has a strong wound repairing capability. The scratch healing rate of the products I-V is above 40%, particularly, the average scratch healing rate of the products I reaches 54.62%, the effect is optimal, the average scratch healing rate of the products VI is 22.49%, the average scratch healing rate of the products VII is 13.59%, the average scratch healing rate of the products VIII is 20.98%, and the average scratch healing rate of the products IX is 16.75%. Therefore, the preparation process provided by the invention can enable the longxuein A to more effectively permeate into the keratinocytes, thereby enhancing the repairing and regenerating capacity of the keratinocytes and showing remarkable superiority in promoting the repairing of skin injury.

Experimental example 6

The experimental example provides ABTS radical scavenging experiments.

And uniformly mixing 10 mu L of the object to be detected with 190 mu L of 0.25mmol/LABTS working solution/PBS, fully shaking to obtain an ABTS working solution of the object to be detected and a PBS solution of the object to be detected, incubating for 6 minutes at room temperature, and measuring the light absorption value at 405 nm. The absorbance of the ABTS working solution of the test object is designated as A1, the absorbance of the PBS solution of the test object is designated as A2, the absorbance of the blank ABTS working solution and the blank PBS is designated as A3, and the ABTS radical clearance is calculated according to the formula ABTS clearance= [1- (A1-A2)/A3 ] ×100%.

In the experimental example, products I-IX prepared in examples 1-5 and comparative examples 1-4 are respectively used as an object to be detected, and a positive control group is arranged, wherein the positive control group is formed by loading vitamin C at loading concentrations of 200, 150, 125, 100, 75, 50 and 25 mug/mL. The results shown in table 5, fig. 5 and fig. 6 were obtained.

TABTS radical scavenging results

When the IC50 of the positive control group was 40 to 70. Mu.g/mL, the system was considered to be effective, and as shown in FIG. 5, the IC50 of the positive control VC was 64.24. Mu.g/mL, and the system of this experimental example was effective. As can be seen from Table 5 and FIG. 6, the ABTS radical clearance of product I is highest, the average value is 62.78%, and the ABTS radical clearance of product II and product III are basically consistent. The ABTS free radical clearance of the products I-V is above 55%, and is obviously higher than that of the products VII, VIII and IX and higher than that of the products VI. The ability of the borneol A liposome prepared in the embodiments 1-5 to remove ABTS free radicals is obviously higher than that of other products, and the products I-V have excellent oxidation resistance. Therefore, the preparation process provided by the application can enable the longxuelin A to more effectively play an antioxidant effect, and has obvious superiority.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiment and all alterations and modifications that fall within the scope of the invention as described herein.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims

1. A nanoliposome of Loreerain A, characterized in that it comprises the following components per 100 parts by weight: 1-10 parts of Loreerain A, 5-15 parts of emulsifier, 0-1 parts of co-emulsifier, 1-3 parts of cholesterol, 5-20 parts of oil, 30-50 parts of polyol, and the balance is water.

2. The nanoliposome according to claim 1, characterized in that the emulsifier is any one of hydrogenated lecithin, egg yolk lecithin, soybean lecithin, sunflower lecithin, distearoylphosphatidylcholine, and dipalmitoylphosphatidylcholine;

And/or, the co-emulsifier is one or a combination of PEG-7 glyceryl cocoate, PEG-30 dipolyhydroxystearate, polyglyceryl-10 laurate, polyglyceryl-10 oleate, polyglyceryl-10 myristate, and polyethylene glycol fatty acid glycerides;

And/or, the oil is any one of caprylic/capric triglyceride, C12-15 alcohol benzoate, glyceryl monostearate, squalane and acetylated monoglyceride;

And/or, the polyol is any one of glycerol, 1,3-propylene glycol, butylene glycol, 1,2-pentanediol, and dipropylene glycol.

3. A method for preparing the Loreerain A nanoliposome according to any one of claims 1 to 2, characterized in that it comprises the following steps:

The emulsifier, the co-emulsifier, the cholesterol and the oil are mixed, and homogenized and dispersed under a closed and heated condition of 60 to 80° C. to obtain a mixed solution;

Adding the Loureirin A to the polyol, stirring and treating under an inert atmosphere and heating conditions of 40 to 60° C., to obtain an alcohol solution of Loureirin A;

The mixed solution, the alcohol solution of Loreerain A and the water are mixed and sheared to obtain a mixed colostrum; the mixed colostrum is subjected to a microfluidization homogenization treatment under closed conditions to obtain the Loreerain A nanoliposomes.

4. The preparation method according to claim 3, characterized in that the conditions of the homogenization and dispersion treatment are: a speed of 12000-16000 rpm;

And/or, the stirring treatment condition is: the speed is 800-1200 rpm;

And/or, the conditions of the homogenization shearing treatment are: a rate of 8000-12000 rpm and a duration of 5-10 min;

And/or, the conditions of the microfluidization homogenization treatment are: pressure 20000-30000 psi, and number of cycles 3-6 times.

5. The preparation method according to claim 3, characterized in that it comprises the following steps:

10 g of hydrogenated lecithin, 0.3 g of PEG-7 glyceryl cocoate, 3 g of cholesterol and 15 g of caprylic/capric triglyceride were mixed, and the mixture was homogenized and dispersed at a rate of 14000 rpm under a heating condition of 70° C. in a water bath to obtain a mixed solution.

6.7 g of Loreleirin A was added to 40 g of glycerol, and stirred and dissolved at a rate of 1000 rpm in an inert atmosphere and a 50° C. water bath until the mixture was uniformly dissolved, thereby obtaining an alcohol solution of Loreleirin A;

The mixed solution, the alcohol solution of Loreleirin A and 25 g of ultrapure water were mixed, homogenized and sheared at a rate of 10,000 rpm for 7 minutes to obtain a mixed colostrum; the mixed colostrum was subjected to microfluidization homogenization at a pressure of 25,000 psi for 5 cycles to obtain the Loreleirin A nanoliposomes.

6. Use of the Loreerain A nanoliposomes according to any one of claims 1 to 2 or the Loreerain A nanoliposomes prepared by the preparation method according to any one of claims 3 to 5 in the preparation of skin repair preparations.

7. The use according to claim 6, characterized in that the preparation is used to promote the proliferation of fibroblasts;

And/or, the preparation is used to promote cell wound repair;

And/or, the formulation is used to scavenge free radicals.

8. A skin repair preparation, characterized in that it comprises the Loreerain A nanoliposomes according to any one of claims 1 to 2 or the Loreerain A nanoliposomes prepared by the preparation method according to any one of claims 3 to 5 and excipients.

9. The preparation according to claim 8 is characterized in that the dosage form of the preparation is any one of creams, emulsions, aqueous solutions, gels, oils, powders, block powders or solids, muds, aerosols, aerosols, organic solvents, wax-based, patches, films, and freeze-dried types.

10. The preparation according to claim 8, characterized in that the excipient is one or a combination of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, and release retardants.