Loureirin A nano-liposome as well as preparation method and application thereof

Abstract

The invention discloses a loureirin A nano-liposome as well as a preparation method and application thereof, belongs to the technical field of nano-liposomes, and solves the problem of low bioavailability of loureirin A in the prior art. Every 100 parts by weight of the loureirin A nano-liposome comprises the following components: 1-10 parts by weight of loureirin A, 5-15 parts by weight of an emulsifier, 0-1 part by weight of a co-emulsifier, 1-3 parts by weight of cholesterol, 5-20 parts by weight of grease, 30-50 parts by weight of polyol and the balance of water. The loureirin A nano-liposome provided by the invention not only solves the problem that the loureirin A is difficult to add in pharmaceutical preparations and cosmetic preparations, but also improves the affinity of the loureirin A and keratinocytes, so that the loureirin A nano-liposome has better percutaneous infiltration capacity, and is safe, non-toxic and non-irritant.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoliposomes, and more specifically to a dragon's blood A nanoliposome and a preparation method and application thereof. Background Art

[0002] Dragon's Blood is a resin extracted from the oily wood of the Dracaena cochinchinensis plant of the Liliaceae family. It is mainly produced in southern Yunnan and southern Guangxi, my country, and is a traditional Chinese medicinal material and ethnic medicinal material. Loureirin A (chemical name 4'-hydroxy-2,4-dimethoxydihydrochalcone) is the main free dihydrochalcone component in the original plant of Dragon's Blood, and is one of the main active ingredients of Dragon's Blood. It is usually used in the pharmacological study of Dragon's Blood. In recent years, the active monomer Loureirin A has received increasing attention from domestic and foreign scholars in the research field.

[0003] The skin is composed of the epidermis, dermis, subcutaneous tissue and its appendages (sweat glands, sebaceous glands, blood vessels, etc.). It is the first line of defense for the human body against infection and injury. As an organ in direct contact with the external environment, it is vulnerable to damage from external factors, and the repair of skin damage is the result of the action of multiple cells and factors. Dragon's blood A has broad application prospects in wound healing and regulatory regeneration of skin damage. It can promote the proliferation and differentiation of hair follicle stem cells, provide more seed cells for skin wound repair, and effectively inhibit in vitro thrombin activity, promote blood circulation and remove blood stasis, improve wound microcirculation, and regulate cell differentiation and growth-related factor secretion. In the inflammatory response stage of skin wound repair, dragon's blood A can play an anti-inflammatory role and effectively prevent wound infection. Dragon's blood A also has extremely strong antioxidant properties, and its antioxidant capacity is stronger than N-acetyl-L-cysteine ​​(NAC). It can increase the activity of superoxide dismutase (SOD), enhance the efficiency of free radical scavenging, reduce wound oxidative stress, and help repair the epidermis and skin appendages. In addition, dragon's blood A regulates the balance between fibroblast proliferation and apoptosis, reduces extracellular matrix deposition, prevents the proliferation of connective tissue after skin injury, and inhibits the formation of pathological scars.

[0004] However, as a flavonoid compound, dragon's blood A has problems such as poor water solubility, poor permeability and low bioavailability. Summary of the invention

[0005] The invention provides a lordreyadin A nano liposome and a preparation method and application thereof, so as to solve the problem of low bioavailability of lordreyadin A in the prior art.

[0006] In the first aspect, the present invention provides a dragon's blood A nanoliposome, which comprises the following components per 100 parts by weight: 1-10 parts of dragon's blood 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.

[0007] As a possible implementation, 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 several of PEG-7 glyceryl cocoate, PEG-30 dipolyhydroxystearate, polyglyceryl-10 laurate, polyglyceryl-10 oleate and 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.

[0008] In a second aspect, the present invention provides a method for preparing the Loureirin A nanoliposomes described in any possible implementation of the first aspect, comprising the following steps: mixing the emulsifier, the co-emulsifier, the cholesterol and the oil, and performing a homogenous dispersion treatment under a closed and heated condition of 60 to 80° C. to obtain a mixed solution; adding the Loureirin A to the polyol, and stirring the mixture under an inert atmosphere and a heated condition of 40 to 60° C. to obtain an alcohol solution of Loureirin A; mixing the mixed solution, the alcohol solution of Loureirin A and the water, and performing a homogenous shear treatment to obtain a mixed colostrum; and performing a microfluidization homogenization treatment on the mixed colostrum under closed conditions to obtain the Loureirin A nanoliposomes.

[0009] As a possible implementation method, the conditions for the homogenous dispersion treatment are: a rate of 12000-16000rpm; and / or, the conditions for the stirring treatment are: a rate of 800-1200rpm; and / or, the conditions for the homogenous shear treatment are: a rate of 8000-12000rpm, a duration of 5-10min; and / or, the conditions for the microfluidic homogenization treatment are: a pressure of 20000-30000psi, and 3 to 6 cycles.

[0010] As a possible implementation method, the method comprises the following steps: mixing 10 g of hydrogenated lecithin, 0.3 g of PEG-7 glyceryl cocoate, 3 g of cholesterol and 15 g of caprylic / capric triglyceride, and performing a homogenizing dispersion treatment at a rate of 14000 rpm under a 70° C. water bath heating condition to disperse the mixture into a uniform whole to obtain a mixed solution; adding 6.7 g of dragon's blood A to 40 g of glycerol, and performing a stirring and dissolving treatment at a rate of 1000 rpm under an inert atmosphere and a 50° C. water bath heating condition to uniformly dissolve the mixture to obtain an alcohol solution of dragon's blood A; mixing and homogenizing and shearing the mixed solution, the alcohol solution of dragon's blood A and 25 g of ultrapure water at a rate of 10000 rpm for a shearing time of 7 min to obtain a mixed colostrum; and performing a microfluidization homogenization treatment on the mixed colostrum at a pressure of 25000 psi for 5 cycles to obtain the dragon's blood A nanoliposomes.

[0011] In a third aspect, the present invention provides a use of the Loreerain A nanoliposomes described in any possible implementation of the first aspect or the Loreerain A nanoliposomes prepared by the preparation method described in any possible implementation of the second aspect in preparing a skin repair preparation.

[0012] As a possible implementation, the preparation is used to promote fibroblast proliferation; and / or, the preparation is used to promote cell wound repair; and / or, the preparation is used to scavenge free radicals.

[0013] In a fourth aspect, the present invention provides a skin repair preparation, comprising the Loreerain A nanoliposomes described in any possible implementation of the first aspect or the Loreerain A nanoliposomes prepared by the preparation method described in any possible implementation of the second aspect and excipients.

[0014] As a possible implementation method, 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.

[0015] As a possible implementation, 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, integrators, 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.

[0016] Liposomes are ultra-microscopic spherical carriers formed by lipid bilayers. As a commonly used encapsulation and delivery system, they are widely used to protect, control and release bioactive substances. Liposomes contain skin-specific components such as phospholipids and cholesterol. They not only have good affinity with the skin and high safety, but also have a certain repair effect. Certain phospholipids in liposomes can enter the deep layers of the skin, enhance the fluidity and permeability of deep cells, thereby enhancing the metabolic activity of cells and activating cells. Liposome encapsulation can increase the penetration and retention effect of active ingredients, allowing them to overcome the barrier function of the stratum corneum, and be stored in the deep layers of the skin for slow release, sustained action, and improved efficacy.

[0017] The present invention provides a nanoliposome of lor's blood A, which not only solves the problem of difficulty in adding lor's blood A in pharmaceutical preparations and cosmetic preparations, but also improves the affinity of lor's blood A with keratinocytes, so that it has better transdermal penetration ability, and is safe, non-toxic and non-irritating. After being dispersed in water, the lor's blood A nanoliposome can form a uniform colloidal solution, which can effectively penetrate the skin surface, enrich in high concentration and stay for a long time in the skin tissue, realize the skin-targeted delivery of lor's blood A, and make lor's blood A play a powerful repair effect; on the other hand, it helps to improve its solubility, stability and bioavailability, and improves the shortcomings of poor physical stability and low encapsulation rate of traditional liposomes, so that lor's blood A can stay in the skin for a long time to achieve the best sustained release effect. In addition, the moisturizing and repairing function of the liposome itself synergizes with lor's blood A to further promote skin repair, and has excellent care and repair effects on skin trauma, damaged skin barrier, etc.

[0018] In the preparation method of the nanoliposome of dragon's blood element A provided by the present invention, the stirring and dissolving process under inert atmosphere, and the homogeneous dispersion, mixed homogenization shearing, and microfluidization homogenization process carried out under sealing conditions are to prevent the problems such as the reduction of liposome membrane fluidity, the deterioration of encapsulation effect, the increase of liposome particle size and polydispersity index caused by the presence of oxidation phenomenon in the preparation process. The microfluidization homogenization technology used in the present invention is a kind of efficient nano-scale emulsification and dispersion processing technology, and its advantage lies in its extremely high shear force. Through dynamic high-pressure micro-jet, high shear, high-energy collision (turbulent collision) and cavitation effect, the material is refined and uniformly mixed, and a faster particle size reduction and a narrower particle size distribution are achieved. The microfluidization homogenization technology can ensure that the pressure of the material is a constant peak value during the processing, so as to achieve a faster material particle size reduction and a narrower particle size distribution, and the processing effect also has good reproducibility, and good reproducibility, and the processing effect can be kept consistent during pilot production and large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is a graph showing the encapsulation efficiency test results of Product I provided in an embodiment of the present invention for 30 days.

[0021] Figure 2 This is a graph showing the results of the determination of the effects of products I to IX provided in the examples of the present invention on the proliferation of fibroblasts; compared with the blank group, ##P<0.01, ####P<0.0001.

[0022] Figure 3 This is a diagram of a keratinocyte scratch test of products I to IX provided in the examples of the present invention.

[0023] Figure 4 This is a graph showing the test results of the effects of products I to IX provided in the examples of the present invention on the scratch healing rate of keratinocytes; compared with the control group, ####P<0.0001.

[0024] Figure 5 This is a diagram of the scavenging results of the positive control in the ABTS free radical scavenging experiment provided in an embodiment of the present invention.

[0025] Figure 6 This is a graph showing the test results of the ABTS free radical scavenging rates of products I to IX provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In order to solve the problem of low bioavailability of Loren'erhaerine A in the prior art, the embodiment of the present invention provides a preparation experiment of Loren'erhaerine A nanoliposomes and measures the physical properties. It can be seen that the product prepared in the embodiment of the present invention has a smaller particle size, better physical stability and uniformity.

[0028] Furthermore, the embodiments of the present invention provide test experiments for encapsulation efficiency and stability. It can be seen that the Lure's Blood A nanoliposomes provided by the embodiments of the present invention have a higher encapsulation efficiency and better stability. After being dispersed in water, the Lure's Blood A nanoliposomes can form a uniform colloidal solution, which helps to improve its solubility, stability and bioavailability, and improves the shortcomings of traditional liposomes such as poor physical stability and low encapsulation efficiency, so that Lure's Blood A can be retained in the skin for a long time to achieve the best sustained-release effect.

[0029] Furthermore, the embodiments of the present invention provide a fibroblast proliferation assay, a cell repair assay, and an ABTS free radical scavenging assay. It can be seen that the Lure's Blood A nanoliposomes provided by the embodiments of the present invention have a significant effect on promoting fibroblast proliferation, a significant ability to promote wound repair, and a significant ability to scavenge free radicals. The moisturizing and repairing function of the liposome itself works synergistically with Lure's Blood A to further promote skin repair, and has excellent care and repair effects on skin wounds, damaged skin barriers, etc.

[0030] Furthermore, the embodiments of the present invention provide an application experiment of skin repair, and it can be seen that the Lure's Blood A nanoliposomes provided by the embodiments of the present invention have a significant effect on repairing damaged skin. After being dispersed in water, the Lure's Blood A nanoliposomes can form a uniform colloidal solution, which can effectively penetrate the surface layer of the skin, accumulate in high concentrations and stay in the skin tissue for a long time, realize the skin-targeted delivery of Lure's Blood A, and enable Lure's Blood A to exert a powerful repair effect.

[0031] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0032] Example 1

[0033] This example provides an experiment for preparing Loreerain A nanoliposomes.

[0034] 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 I.

[0035] 6.7 g of Loreleirin A was added to 40 g of glycerol, and stirred and dissolved at a rate of 1000 rpm under an inert atmosphere and a 50° C. water bath heating condition until the mixture was uniformly dissolved, thereby obtaining an alcohol solution of Loreleirin A I;

[0036] The mixed solution I, the alcohol solution I of dragon's blood A and 25 g of ultrapure water were mixed and homogenized and sheared at a rate of 10000 rpm for 7 minutes to obtain mixed colostrum I;

[0037] The mixed colostrum I was subjected to microfluidization homogenization at a pressure of 25,000 psi for 5 cycles to obtain product I.

[0038] Example 2

[0039] This example provides an experiment for preparing Loreerain A nanoliposomes.

[0040] 10 g of egg yolk lecithin, 0.3 g of polyglycerol-10 laurate, 3 g of cholesterol and 15 g of C12-15 alcohol benzoate were mixed, and the mixture was homogenized and dispersed at a rate of 12000 rpm under a heating condition of 80° C. in a water bath to obtain a mixed solution II.

[0041] 6.7 g of Loreleirin A was added to 40 g of 1,3-propylene glycol, and stirred and dissolved at a rate of 1200 rpm under an inert atmosphere and a 40° C. water bath heating condition until the mixture was uniformly dissolved, thereby obtaining an alcohol solution II of Loreleirin A;

[0042] The mixed solution II, the alcohol solution II of dragon's blood A and 25 g of ultrapure water were mixed and homogenized and sheared at a rate of 12000 rpm for 5 min to obtain mixed colostrum II;

[0043] The mixed colostrum II was subjected to microfluidization homogenization at a pressure of 20,000 psi for 6 cycles to obtain product II.

[0044] Example 3

[0045] This example provides an experiment for preparing Loreerain A nanoliposomes.

[0046] 10 g of soybean lecithin, 0.3 g of PEG-30 dipolyhydroxystearate, 3 g of cholesterol and 15 g of squalane were mixed, and the mixture was homogenized and dispersed at a rate of 16000 rpm under a heating condition of 60° C. water bath to obtain a mixed solution III.

[0047] 6.7 g of Loreleirin A was added to 40 g of 1,2-pentanediol, and the mixture was stirred and dissolved at a rate of 800 rpm under an inert atmosphere and a 60° C. water bath heating condition. After the mixture was uniformly dissolved, an alcohol solution of Loreleirin A III was obtained.

[0048] The mixed solution III, the alcohol solution III of dragon's blood A and 25 g of ultrapure water were mixed and homogenized and sheared at a rate of 8000 rpm for 10 min to obtain mixed colostrum III;

[0049] The mixed colostrum III was subjected to microfluidization homogenization at a pressure of 30,000 psi for 3 cycles to obtain product III.

[0050] Example 4

[0051] This example provides an experiment for preparing Loreerain A nanoliposomes.

[0052] 15 g of hydrogenated lecithin, 0.1 g of PEG-7 glyceryl cocoate, 1.2 g of cholesterol and 5 g of caprylic / capric triglyceride were mixed, and the mixture was homogenized and dispersed at a rate of 14000 rpm in a water bath at 70° C. to obtain a mixed solution IV.

[0053] 6.7 g of Loreleirin A was added to 50 g of glycerol, and stirred and dissolved at a rate of 1000 rpm under an inert atmosphere and a 50° C. water bath heating condition until the mixture was uniformly dissolved, thereby obtaining an alcohol solution IV of Loreleirin A;

[0054] The mixed solution IV, the alcohol solution IV of dragon's blood A and 22 g of ultrapure water were mixed and homogenized and sheared at a rate of 10000 rpm for 7 minutes to obtain mixed colostrum IV;

[0055] The mixed colostrum IV was subjected to microfluidization homogenization at a pressure of 25,000 psi for 5 cycles to obtain product IV.

[0056] Example 5

[0057] This example provides an experiment for preparing Loreerain A nanoliposomes.

[0058] 5 g of hydrogenated lecithin, 1 g of PEG-7 glyceryl cocoate, 3 g of cholesterol and 20 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 V.

[0059] 6.7 g of Loreleirin A was added to 30 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 V of Loreleirin A;

[0060] The mixed solution V, the alcohol solution V of dragon's blood A and 34.3 g of ultrapure water were mixed and homogenized and sheared at a rate of 10000 rpm for 7 minutes to obtain mixed colostrum V;

[0061] The mixed colostrum V was subjected to microfluidization homogenization at a pressure of 25,000 psi for 5 cycles to obtain product V.

[0062] Comparative Example 1

[0063] This example provides a preparation experiment of nanoliposomes.

[0064] 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 VI.

[0065] 6.7 g of Loreleirin A was added to 40 g of glycerol, and the mixture was stirred and dissolved at a rate of 1000 rpm under an inert atmosphere and a heating condition of a 50° C. water bath until the mixture was uniformly dissolved, thereby obtaining an alcohol solution VI of Loreleirin A;

[0066] Add the alcohol solution VI of dragon's blood A into 25g ultrapure water, stir at 60°C for 30min, then add dropwise into the mixed solution VI under the shearing condition of 15000rpm, the shearing temperature is 55°C, and the time is 10min to obtain product VI.

[0067] Comparative Example 2

[0068] This example provides an experiment for preparing a solution of Loren's blood A.

[0069] 6.7 g of dragon's blood A was added to 40 g of glycerol, and dispersed by ultrasonication at room temperature for 50 min. Then 53.3 g of ultrapure water was added and stirred evenly to obtain product VII.

[0070] Comparative Example 3

[0071] This example provides a preparation experiment of nanoliposomes.

[0072] 6.7 g of dragon's blood A, 10 g of lecithin, 3 g of cholesterol and 15 g of Tween 80 were added to 30 mL of anhydrous ethanol and heated at 65°C to obtain a mixed solution VIII;

[0073] The mixed solution VIII was evaporated under reduced pressure on a rotary evaporator (50°C, 45r / min, 0.10MPa) to remove anhydrous ethanol to form a uniform lipid film; then a phosphate buffer solution (concentration of 0.05mol / L, pH 6.0) was added for elution and dispersion to obtain a lipid hydrate suspension VIII;

[0074] The lipid hydrated suspension VIII was homogenized at a pressure of 600 bar for 5 cycles to obtain product VIII.

[0075] Comparative Example 4

[0076] This example provides a preparation experiment of nanoliposomes.

[0077] 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 in a water bath at 70° C. to obtain a mixed solution IX.

[0078] The mixed solution IX and 25 g of ultrapure water were mixed and homogenized at a rate of 10,000 rpm for 7 minutes to obtain mixed colostrum IX;

[0079] The mixed colostrum IX was subjected to microfluidization homogenization at a pressure of 25,000 psi for 5 cycles to obtain product IX.

[0080] Experimental Example 1

[0081] This experimental example provides the physical property measurement of the product.

[0082] The test substance was diluted with deionized water, and the hydrodynamic particle size and polydispersity index (PDI) of the liposomes were measured by dynamic light scattering technology at 25°C for 3 times in parallel.

[0083] In this experimental example, products I to IX prepared in Examples 1 to 5 and Comparative Examples 1 to 4 were used as test objects, respectively. After diluting four times, their hydrodynamic particle sizes and PDI were measured, and the average values ​​were taken to obtain the results shown in Table 1.

[0084] Table 1 Hydrodynamic particle size and PDI results

[0085] 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

[0086] As can be seen from Table 1, the particle size of product I to product V is less than 100nm, the particle size of product VI is 130.3nm, product VII is a solution with no particle size data, the particle size of product VIII is 156.7nm, and the particle size of product IX is 212.5nm; it can be seen that the particle size of product I to product V is smaller, which is more beneficial to improve the bioavailability of the encapsulated active ingredient. The PDI of product I to product V is also significantly smaller than that of product VI to product IX. The smaller the PDI value, the more concentrated the particle size distribution of the liposome, that is, the more uniform the particle size. Therefore, it is believed that product I to product V prepared in the embodiment is better, with a smaller particle size, better physical stability and uniformity.

[0087] Experimental Example 2

[0088] This experimental example provides a stability test experiment of the product.

[0089] Place the test object in a sealed container and place it at room temperature, 4°C, -18°C and 45°C for 90 days, respectively. Allow it to return to room temperature naturally. Check the sample properties for agglomeration or stratification in the initial state and after 90 days. The inspection results can be expressed as yes / no, and the particle size of the sample is tested.

[0090] In this experimental example, products I to IX prepared in Examples 1 to 5 and Comparative Examples 1 to 4 were used as test objects, respectively, and their stability at different temperatures was measured to obtain the results shown in Table 2, where the results in the table are "whether there is agglomeration or stratification phenomenon / particle size".

[0091] Table 2 Stability test results

[0092]

[0093] As shown in Table 2, after being placed at -18°C, 4°C, room temperature and 45°C for 90 days, products I to V had no precipitation and stratification, indicating that their physical morphology and structure were well maintained, and the particle size was small and there was no obvious change after placement; while products VI, VII, VIII and IX showed stratification, and products VII and IX showed obvious stratification, and the particle size was large, and the particle size increased after placement. Therefore, products I to V are better and have better physical stability, among which product I prepared in Example 1 has the smallest particle size, the smallest particle size change, and the best effect.

[0094] Experimental Example 3

[0095] This experimental example provides an encapsulation efficiency test experiment of the product.

[0096] Take 2 mL of the test substance, pass it through a 0.22 μm organic filter membrane, take 100 μL and use methanol to break the emulsion, and use HPLC to measure the effective ingredient content (w1) of the test substance and the mass of the active ingredient of dragon's blood A added when preparing the test substance (w0), calculate the encapsulation efficiency according to the formula (w1 / w0)×100%, and track the encapsulation efficiency over a long period of time.

[0097] In this experimental example, the product I prepared in Example 1 was used as the test object to measure the encapsulation efficiency. The results shown in Table 3 were obtained.

[0098] Table 3 Product I encapsulation efficiency test results

[0099] Day Day 0 7 days 14 days 21 days 30 days Encapsulation rate / % 98.6 98.3 98.3 98.3 98.2

[0100] As shown in Table 3, the encapsulation efficiency of product I is 98.2±0.2%, and the encapsulation efficiency remains above 98% after one month, indicating that the encapsulation efficiency of product I is relatively stable.

[0101] Generally speaking, a higher encapsulation efficiency helps the formulation maintain long-term stability and consistency. Figure 1The results show that after the liposome preparation process, the retention rate of dragon's blood A in product I is 98.6%. Therefore, it is believed that the preparation process provided by the present invention can retain the activity of dragon's blood A to the greatest extent, avoid a large amount of loss, and improve the utilization rate. The encapsulation rate of product I was tracked for a long time. The encapsulation rate of product I did not decrease substantially within one month, and the encapsulation rate remained above 98% after one month, indicating that the encapsulation rate stability of product I prepared in Example 1 is very good, and it is not easy to leak during storage and transportation.

[0102] Experimental Example 4

[0103] This experimental example provides an experiment to measure the effect of the product on the proliferation of fibroblasts.

[0104] 1 g of the test substance was added to 100 mL of DMEM medium and fully dissolved to obtain the test substance solution; fibroblasts in the logarithmic growth phase were seeded in a 96-well plate and the fibroblast cell density was adjusted to 1×10 4 After 1 day of adaptive culture, the old culture medium was removed and 100 μL of the test solution was added. The blank DMEM culture medium was used as the control group, and each group had 3 replicate wells. After 48 hours of continuous culture, 10 μL of CCK-8 solution was added to each well and incubated for 2 hours. The absorbance (A) was measured at 450 nm to calculate the cell proliferation rate.

[0105] In this experimental example, the products I to IX prepared in Examples 1 to 5 and Comparative Examples 1 to 4 were used as test objects, and their respective cell proliferation rates were measured to obtain the following results: Figure 2 Results shown.

[0106] Fibroblasts play an important role in maintaining tissue integrity and serving as a local precursor reserve. After acute injury or burn to the skin, fibroblasts in the dermis begin to proliferate, migrate to the wound, and activate at the wound to form extracellular matrix and reshape the wound bed. Figure 2 It can be seen that the effects of promoting fibroblast proliferation of products I to V are better than those of the comparative example. Product I has the most significant effect on promoting fibroblast proliferation, with a cell proliferation rate of 29%. This shows that the dragon's blood A in the product prepared by the preparation process provided by the present invention is easier to enter fibroblasts to exert its effect, and has significant advantages in repairing skin damage.

[0107] Experimental Example 5

[0108] This experimental example provides an experiment to measure the effect of the product on the cell repair ability.

[0109] The test substance was added to DMEM medium and fully dissolved to prepare a 20 μL / mL test substance solution; keratinocytes (10 6cell / mL), 2mL of cell solution was inoculated into each well of a 6-well plate, and the plate was cultured in an incubator for 24h. The middle of the back of the culture well was scratched with a pipette tip and washed with PBS; 2mL of the test solution was added to each well, and a negative control group was set up at the same time; the cell plate was placed in an incubator for culture for 12h and then taken out, and the scratch area was observed and photographed under a microscope to measure the scratch area. The healing rate was calculated based on the scratch area before and after culture, and the experiment was repeated 3 times for each group.

[0110] In this experimental example, the products I to IX prepared in Examples 1 to 5 and Comparative Examples 1 to 4 were used as test objects, and the scratch healing rates were measured, and the results were as shown in Table 4. Figure 3 and Figure 4 Results shown.

[0111] Table 4 Scratch healing rate results statistics

[0112]

[0113] The migration of keratinocytes can reflect the process of wound repair. The scratch healing rate of keratinocytes (HaCaT) is a common indicator to measure the migration ability of keratinocytes, which reflects the ability of cells to repair and regenerate after damage. Figure 3 and Figure 4 It can be seen that there are significant differences in the scratch healing rates between product groups Ⅰ to IX and between product groups Ⅰ to IX and the negative control group (p<0.05), indicating that dragon's blood A has a significant promoting effect on the migration of HaCaT cells and has a strong wound repair ability. The scratch healing rates of products Ⅰ to Ⅴ are all above 40%, especially the average scratch healing rate of product Ⅰ reaches 54.62%, with the best effect; the average scratch healing rate of product VI is 22.49%, the average scratch healing rate of product VII is 13.59%, the average scratch healing rate of product VIII is 20.98%, and the average scratch healing rate of product IX is 16.75%. Therefore, the preparation process provided by the present invention enables dragon's blood A to penetrate into keratinocytes more effectively, thereby enhancing the repair and regeneration ability of keratinocytes, and showing significant superiority in promoting skin damage repair.

[0114] Experimental Example 6

[0115] This experimental example provides an ABTS free radical scavenging experiment.

[0116] 10 μL of the test object was mixed with 190 μL of 0.25 mmol / LABTS working solution / PBS, and shaken thoroughly to obtain the ABTS working solution of the test object and the PBS solution of the test object, incubated at room temperature for 6 minutes, and the absorbance was measured at 405 nm. The absorbance of the ABTS working solution of the test object was recorded as A1, the absorbance of the PBS solution of the test object was recorded as A2, and the absorbance of the blank ABTS working solution and blank PBS was recorded as A3. The ABTS free radical scavenging rate was calculated according to the formula ABTS scavenging rate = [1-(A1-A2) / A3]×100%.

[0117] In this experimental example, the products I to IX prepared in Examples 1 to 5 and Comparative Examples 1 to 4 were used as test objects, and a positive control group was set up, wherein the positive control group was loaded with vitamin C at a loading concentration of 200, 150, 125, 100, 75, 50, and 25 μg / mL. The results are shown in Table 5. Figure 5 and Figure 6 Results shown.

[0118] Table 5 ABTS free radical scavenging rate results

[0119]

[0120] When the IC50 of the positive control group is between 40 and 70 μg / mL, the system is considered effective. Figure 5 It can be seen that the IC50 of the positive control VC is 64.24μg / mL, and this experimental example system is effective. Figure 6 It can be seen that product I has the highest ABTS free radical scavenging rate, with an average of 62.78%, and the ABTS free radical scavenging rates of products II and III are basically the same. The ABTS free radical scavenging rates of products I to V are all above 55%, significantly higher than those of products VII, VIII, and IX, and higher than product VI. The ability of the dragon's blood A liposomes prepared in Examples 1 to 5 of the present application to scavenge ABTS free radicals is significantly higher than that of other products, and products I to V have excellent antioxidant properties. Therefore, the preparation process provided by the present invention enables dragon's blood A to more effectively exert its antioxidant effect, showing obvious superiority.

[0121] Although the preferred embodiments of the present invention have been described, other changes and modifications may be made to these embodiments once those skilled in the art are aware of the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the preferred embodiments of the present invention and the scope of the present invention.

[0122] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A Loreerain A nanoliposome, characterized in that: The invention comprises the following components per 100 parts by weight: 1-10 parts of dragon's blood A, 5-15 parts of emulsifier, 0-1 parts of auxiliary 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: The following steps are involved: 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: the speed is 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: The following steps are involved: 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: The invention comprises the Loreerain A nanoliposomes as described in any one of claims 1 to 2 or the Loreerain A nanoliposomes prepared by the preparation method as described in any one of claims 3 to 5 and auxiliary materials.

9. The preparation according to claim 8, 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 auxiliary materials are 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, integrators, 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.

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