Holothurian peptide and magnolol liposome, preparation method and application thereof

Abstract

The application relates to the technical field of honokiol liposomes, and particularly discloses sea cucumber peptide-honokiol liposomes, a preparation method and application thereof. The preparation raw materials of the sea cucumber peptide-honokiol liposomes comprise the following components in parts by weight: phospholipid 0.5-4 parts, cholesterol 0.1-3 parts, a surfactant 0-4 parts, vitamin E 0-1 part, a buffer 1-10 parts, an aqueous solvent 60-100 parts, honokiol 0-1 part, sea cucumber peptide 0.1-5 parts and a preservative 0.1-5 parts. The obtained sea cucumber peptide-honokiol liposomes have excellent standing stability, centrifugal stability and dilution stability.

Description

Technical Field

[0001] This application relates to the technical field of magnolol liposomes, specifically to sea cucumber peptide-magnolol liposomes, their preparation methods, and applications. Background Technology

[0002] Currently, methods used in liposome preparation include thin-film dispersion, organic solvent injection, double emulsion, reverse evaporation, and chemical gradient methods. The wall material of liposomes is generally based on phospholipids and cholesterol. As a common loading material, liposomes have broad application value.

[0003] However, there is currently very little research on liposome loading or encapsulation of sea cucumber peptides. Nevertheless, sea cucumber peptides are considered promising and valuable peptides for research due to their various health benefits. For example:

[0004] ① Sea cucumber peptides and magnolol liposomes enhance the transdermal absorption of sea cucumber peptides;

[0005] In recent years, sea cucumber peptides have attracted increasing attention from researchers due to their bioactivity. Furthermore, sea cucumber peptides are abundant in my country, and their good solubility, stability, emulsification properties, and ease of digestion and absorption by the human body determine their potential for development into nutritional and functional foods, skincare products, and therapeutic drugs. Therefore, they have broad application prospects and market value.

[0006] Currently, sea cucumber peptide-based products on the market are mainly sold as nutritional supplements. Most mid-to-high-end sea cucumber peptide products are marketed based on their compound effects, while the development of skincare-related sea cucumber peptide products is relatively insufficient, due to the high value and weak absorption of sea cucumber peptides.

[0007] ② Sea cucumber peptides and magnolol liposomes enhance the anti-inflammatory effects of sea cucumber peptides;

[0008] Inflammation is a defensive response of the body to external stimuli. The body maintains its homeostasis by generating spontaneous responses, but inflammatory responses are also the basis of the pathogenesis of many diseases. It is speculated that its anti-inflammatory activity may be related to the composition, sequence and molecular weight of amino acids.

[0009] ③ Sea cucumber peptides and magnolol liposomes solve the problem of magnolol's insolubility in water;

[0010] Magnolol is one of the main active ingredients in Magnolia officinalis and is an important material basis for its medicinal effects. Modern pharmacological studies have confirmed that magnoolol possesses various pharmacological activities, including anti-inflammatory, antibacterial, antioxidant, calmodulin antagonism, and gastrointestinal function regulation. However, due to the presence of phenolic hydroxyl groups in its structure, it has poor stability and is easily oxidized. In particular, its poor water solubility greatly limits the formulation development and clinical application of magnoolol.

[0011] Liposomes, as a drug carrier, can encapsulate some unstable and easily oxidized drugs. The drugs are protected by the liposome bilayer membrane, which greatly improves the stability of the drugs and solves the solubility problem of poorly soluble drugs.

[0012] This application's research design encapsulates magnolol and sea cucumber peptides in liposomes to improve their stability and solubility. Summary of the Invention

[0013] This application provides sea cucumber peptide- and magnolol liposomes, their preparation methods, and applications. These liposomes are suitable for loading sea cucumber peptides and magnolol; they exhibit significantly superior stability, thus offering greater operability and wider application when used as raw materials for the further preparation of various skincare products.

[0014] This application involves the following:

[0015] 1. Sea cucumber peptide- and magnolol liposomes, wherein the raw materials for preparing the sea cucumber peptide- and magnolol liposomes comprise the following components in parts by weight:

[0016] Phospholipids 0.5-4 parts, cholesterol 0.1-3 parts, surfactants 0-4 parts, vitamin E 0-1 parts, buffers 1-10 parts, aqueous solvents 60-100 parts, magnolol 0-1 parts, sea cucumber peptides 0.1-5 parts, preservatives 0.1-5 parts.

[0017] 2. The sea cucumber peptide and magnolol liposomes according to claim 1, wherein the mass ratio of the sea cucumber peptide to the sum of the masses of the phospholipids, cholesterol, surfactant, magnolol, and vitamin E is 0.5-3:3; preferably 0.5-1.2:3 or 1.8-3:3.

[0018] 3. The sea cucumber peptide-honokiol liposome according to item 1 or 2, wherein the mass ratio of honokiol to vitamin E is 0.5-10:1; preferably 1-3:1; more preferably 0.5-1.2:1 or 1.8-3:1.

[0019] 4. The sea cucumber peptide-and magnolol liposomes according to claim 1, wherein the mass of vitamin E accounts for 0-6 wt.% of the sum of the mass of the magnolol, phospholipids, cholesterol, surfactant, and vitamin E.

[0020] 5. The sea cucumber peptide-and magnolol liposomes according to item 1 or 4, wherein the surfactant accounts for 0-60 wt.% of the sum of the masses of the magnolol, phospholipids, cholesterol, surfactant, and vitamin E; preferably 10-50 wt.%.

[0021] 6. The sea cucumber peptide and magnolol liposomes according to claim 1, wherein the preservative is present in an amount of 0.5-4 wt.% of the sum of the masses of the phospholipids, cholesterol, surfactant, vitamin E, buffer, aqueous solvent, magnolol, and sea cucumber peptide.

[0022] 7. The sea cucumber peptide and magnolol liposomes according to claim 1, wherein the preservative is selected from any one or more of 1,2-hexanediol, 1,2-pentanediol, phenoxyethanol and vinyl resins; preferably 1,2-hexanediol or 1,2-pentanediol or a combination thereof.

[0023] This application also provides a method for preparing the above-mentioned sea cucumber peptide and magnolol liposomes, comprising the following steps:

[0024] The phospholipids, cholesterol, surfactants, vitamin E, and magnolol were dissolved in a first organic solvent to obtain a lipid stock solution.

[0025] After the lipid stock solution is mixed evenly with an aqueous solvent and a buffer, a first hydration reaction is carried out to obtain a lipid solution.

[0026] After removing the first organic solvent from the lipid solution, dispersing and filtering, a blank liposome suspension is obtained.

[0027] After the blank liposome suspension and sea cucumber peptide were mixed evenly, a second hydration reaction was carried out, followed by dispersion, filtration, and the addition of preservatives to obtain a sea cucumber peptide- and magnolol liposome suspension.

[0028] 9. According to the preparation method described in item 8, the condition parameters of the first hydration reaction include:

[0029] The temperature of the first hydration reaction is 35-65℃; and / or,

[0030] The first hydration reaction takes 20-60 minutes; and / or,

[0031] The drop rate of the lipid stock solution is 0.5-5 mL / min;

[0032] And / or,

[0033] The conditions and parameters for the second hydration reaction include:

[0034] The temperature of the second hydration reaction is 20-55℃; and / or,

[0035] The second hydration reaction takes 20-60 minutes; and / or,

[0036] The lipid stock solution is added at a rate of 0.5-5 mL / min.

[0037] Optionally, the dropping rate of the lipid stock solution is 0.5-2 mL / min.

[0038] Optionally, the first hydration temperature is 40-45℃.

[0039] Optionally, the first hydration reaction time is 25-35 min.

[0040] Optionally, the drop rate of the lipid stock solution is 1-3 mL / min.

[0041] 10. Use of the sea cucumber peptide-and magnolol liposomes described in any one of items 1-7, and / or the sea cucumber peptide-and magnolol liposomes prepared by the preparation method described in item 8 or 9; for use in the preparation of skin care products.

[0042] 11. A skin care product comprising sea cucumber peptide-and magnolol liposomes as described in any one of items 1-7, and / or sea cucumber peptide-and magnolol liposomes prepared by the preparation method described in item 8 or 9.

[0043] 12. The skin care product according to item 11, wherein the content of sea cucumber peptide and magnolol liposomes in the skin care product is 0.05-0.5 wt%.

[0044] Invention Effects

[0045] 1. The liposomes prepared in this application are lipid-like in shape similar to sebum. The liposomes have high compatibility with the lipids of the stratum corneum of the skin, and the drug is easily absorbed by the stratum corneum, thereby playing a role in continuous drug release.

[0046] 2. The liposomes prepared in this application have magnolol added to their wall material. The anti-inflammatory mechanism of magnolol is to block the PI3K / Akt, ERK / MAPK and TLR / MAPK signaling pathways, inhibit the expression of inflammatory cytokines, and also to produce a broad-spectrum anti-inflammatory effect by directly inhibiting the enzyme activity of iNOS, COX-2 and 5-LO, blocking the synthesis and release of nitric oxide, TGs and LTs, and inhibiting histamine release.

[0047] 3. The liposomes prepared in this application exhibit significantly superior static stability, centrifugal stability, and dilution stability after being loaded with sea cucumber peptides. Therefore, they can be used in various processing techniques to prepare a variety of skin care products. Attached Figure Description

[0048] Figure 1 Centrifugal stability diagram of sea cucumber peptide- and magnolol liposomes from Example 1.

[0049] Figure 2 Transmission electron microscopy image of the sea cucumber peptide- and magnolol liposome suspension from Example 1.

[0050] Figure 3 Particle size distribution of sea cucumber peptide- and magnolol liposome suspensions from Example 1.

[0051] Figure 4 The effect of vitamin E content in blank liposomes on their static stability in Example 4.

[0052] Figure 5 The effect of Tween-80 content in blank liposomes on their static stability in Example 5.

[0053] Figure 6 The effect of the mass ratio of sea cucumber peptides to lipids on the static stability of sea cucumber peptide liposomes.

[0054] Figure 7 Effect of the mass ratio of vitamin E to honokiol on the static stability of honokiol liposomes.

[0055] Figure 8 The effect of the mass ratio of sea cucumber peptide to lipid on the static stability of honokiol liposomes when the mass ratio of honokiol to vitamin E is 1:1.

[0056] Figure 9 The effect of the mass ratio of sea cucumber peptide to lipid on the static stability of honokiol liposomes when the mass ratio of honokiol to vitamin E is 2:1.

[0057] Figure 10 The static stability of sea cucumber peptide- and magnolol liposomes after the addition of preservatives. Detailed Implementation

[0058] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in their functions.

[0059] As used throughout the specification and claims, the terms "comprising" or "including" are open-ended and should be interpreted as "comprising but not limited to". The subsequent descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0060] It should be understood that the embodiments of this application described herein include embodiments that are "composed of" and / or "substantially composed of". References to values ​​or parameters of "about" herein include (and describe) variations of that value or parameter itself. For example, a reference to "about X" includes a description of "X".

[0061] As used herein, references to “not” values ​​or parameters generally refer to and describe “except” values ​​or parameters. For example, “The method is not used to treat type X cancer” means that the method is used to treat cancers other than type X.

[0062] As used in this article, the term “approximately XY” has the same meaning as “approximately X to approximately Y”.

[0063] As used herein and in the appended claims, the singular forms “a / an” and “the” include the plural objects unless the context clearly indicates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. Therefore, this statement is intended as a preliminary basis for the use of exclusive terms such as “only” or “merely” in conjunction with the description of the elements of the claim, or for the use of the limitation of “no”.

[0064] As used herein, the term "and / or" in words such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, as used herein, the term "and / or" in words such as "A, B and / or C" is intended to include each of the following embodiments: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0065] As used herein, "sea cucumber peptide" is a small-molecule active substance extracted from sea cucumber, rich in collagen, amino acids, and polysaccharides, and possesses potential health benefits such as antioxidant and immune-enhancing effects. In some embodiments, the sea cucumber peptide is a polypeptide prepared from sea cucumber using an enzymatic hydrolysis method. In some embodiments, based on the mass of the sea cucumber peptide, the proportion of sea cucumber peptides with a relative molecular weight less than or equal to 2000 is ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or 100%; specifically, based on the mass of the sea cucumber peptide, the proportion of sea cucumber peptides with a relative molecular weight less than or equal to 2000 is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0066] This application provides a sea cucumber peptide-and magnolol liposome, wherein the raw materials for preparing the sea cucumber peptide-and magnolol liposome comprise the following components in parts by weight:

[0067] Phospholipids 0.5-4 parts, cholesterol 0.1-3 parts, surfactants 0-4 parts, vitamin E 0-1 parts, buffers 1-10 parts, aqueous solvents 60-100 parts, magnolol 0-1 parts, sea cucumber peptides 0.1-5 parts, preservatives 0.1-5 parts.

[0068] In some embodiments, the phospholipid is selected from any one or more of phosphatidylcholine (e.g., soybean phosphatidylcholine, egg yolk phosphatidylcholine, etc.), phosphatidylethanolamine (e.g., DOPF, DPPE, etc.), phosphatidylglycerol (e.g., DPPG, POPG, etc.), and phosphatidylinositol. In some embodiments, the phospholipid is a synthetic phospholipid, such as DSPC (distearate PC); in some embodiments, the phospholipid is a modified phospholipid, such as a PEG-modified phospholipid, such as DSPE-PEG. In some embodiments, the phospholipid is selected from plant-derived phospholipids or animal-derived phospholipids; the plant-derived phospholipid may be, for example, soybean lecithin, sunflower lecithin, rapeseed lecithin, etc.; the animal-derived lecithin may be, for example, egg yolk lecithin, bovine brain lecithin, etc. In some embodiments, the phospholipid is a saturated phospholipid; in some embodiments, the phospholipid is an unsaturated phospholipid, such as phosphatidylcholine, further for example, soybean lecithin, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC). In some embodiments, the phospholipids in the raw materials for preparing the sea cucumber peptides and magnolol liposomes are 0.5-4 parts by weight; for example, any range or any weight of 0.5 parts, 0.8 parts, 1.0 parts, 1.2 parts, 1.4 parts, 1.6 parts, 1.8 parts, 2.0 parts, 2.2 parts, 2.4 parts, 2.6 parts, 2.8 parts, 3.0 parts, 3.2 parts, 3.4 parts, 3.6 parts, 3.8 parts, 4.0 parts, and 0.5-4 parts.

[0069] In some embodiments, the cholesterol in the raw materials for preparing the sea cucumber peptide and magnolol liposomes comprises 0.1-3 parts by weight; for example, any range or any weight of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, and 0.1-3 parts.

[0070] In this application, the choice of surfactant is not limited, as long as it can achieve the functions of changing surface or interfacial tension, emulsification / demulsification; further, it may also have functions such as solubilization, wetting and penetration, dispersion and stabilization. In some embodiments, the surfactant is an ionic surfactant or a nonionic surfactant. In some embodiments, the ionic surfactant is an anionic surfactant, a cationic surfactant, or an amphoteric surfactant. In some embodiments, the nonionic surfactant is selected from any one or more combinations of polyoxyethylene ethers (e.g., fatty alcohol polyoxyethylene ether (AEO)), alkylphenol polyoxyethylene ethers, fatty acid esters (e.g., Tween, Span), glycosides, or block copolymers. In some embodiments, the surfactant is Tween, such as Tween-80, Tween-60, Tween-40, Tween-20, etc.

[0071] In some embodiments, the surfactant in the raw materials for preparing the sea cucumber peptide and magnolol liposomes comprises 0-4 parts by weight; for example, any range or any weight of 0 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, 1.0 parts, 1.1 parts, 1.2 parts, 1.3 parts, 1.4 parts, 1.5 parts, 1.6 parts, 1.7 parts, 1.8 parts, 1.9 parts, 2.0 parts, 2.1 parts, 2.2 parts, 2.3 parts, 2.4 parts, 2.5 parts, 2.6 parts, 2.7 parts, 2.8 parts, 2.9 parts, 3.0 parts, 3.2 parts, 3.4 parts, 3.6 parts, 3.8 parts, 4.0 parts, and 0-4 parts.

[0072] In some embodiments, the vitamin E in the raw materials for preparing the sea cucumber peptide and magnolol liposomes is 0-1 parts by weight; for example, any range or any weight of 0 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, 1.0 parts, and 0-1 parts.

[0073] In some embodiments, the buffer in the raw materials for preparing the sea cucumber peptide- and magnolol liposomes is 1-10 parts by weight; for example, 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, or any range or any weight of 1-10 parts. In some embodiments, the buffer is sodium dihydrogen phosphate / disodium hydrogen phosphate.

[0074] In some embodiments, the aqueous solvent in the raw materials for preparing the sea cucumber peptide and magnolol liposomes comprises 60-100 parts by weight; for example, any range or any weight of 60, 63, 66, 69, 73, 66, 79, 83, 86, 89, 93, 96, 100 parts, or 60-100 parts. In some embodiments, the aqueous solvent is water.

[0075] In some embodiments, the amount of magnolol in the raw materials for preparing the sea cucumber peptide-homologous liposomes is 0-1 parts by weight; for example, any range or any weight of 0 parts, 0.1 parts, 0.2 parts, 0.3 parts, 0.4 parts, 0.5 parts, 0.6 parts, 0.7 parts, 0.8 parts, 0.9 parts, 1.0 parts, and 0-1 parts.

[0076] In some embodiments, the sea cucumber peptide and magnolol liposome preparation raw materials contain sea cucumber peptide in the range of 0.1-5 parts by weight; for example, any range or any weight of 0.1, 0.5, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5, and 0.1-5 parts.

[0077] In some embodiments, the preservative is selected from one or more of 1,2-hexanediol, 1,2-pentanediol, PE9010 (i.e., phenoxyethanol / ethylhexylglycerol), or phenoxyethanol. In some embodiments, the preservative is selected from 1,2-hexanediol or 1,2-pentanediol. In this application, when the preservative is PE9010 (i.e., phenoxyethanol / ethylhexylglycerol) or phenoxyethanol, the resulting drug-loaded liposomes exhibit poor stability at 45°C and are prone to separation. In some embodiments, the preservative in the raw materials for preparing the sea cucumber peptide and magnolol liposomes is 0.1-5 parts by weight; for example, any range or any weight of 0.1, 0.5, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5, and 0.1-5 parts.

[0078] In some embodiments, the mass ratio of the sea cucumber peptide to the sum of the masses of the phospholipids, cholesterol, surfactant, magnolol, and vitamin E is 0.5-3:3; for example, 0.5-1.2:3 or 1.8-3:3; specifically, it can be any ratio within the range of 0.5:3, 0.6:3, 0.7:3, 0.8:3, 0.9:3, 1:3, 1.1:3, 1.2:3, 1.8:3, 1.9:3, 2:3, 2.1:3, 2.2:3, 2.3:3, 2.4:3, 2.5:3, 2.6:3, 2.7:3, 2.8:3, 2.9:3, 3:3, or 0.5-1.2:3 or 1.8-3:3. In one specific embodiment, this application tested the mass ratios of the sea cucumber peptide to the sum of the masses of the phospholipids, cholesterol, surfactant, magnolol, and vitamin E at 1:3, 1.5:3, and 2:3, respectively. The results showed that the centrifugal stability of all three groups of liposomes was excellent; however, the dilution stability of the liposomes in the 1.5:3 group was poor. Therefore, the liposomes in the 1.5:3 group can avoid dilution operations and maintain their operational stability for further use in subsequent processes.

[0079] In some embodiments, the mass ratio of magnolol to vitamin E is any ratio within the range of 0.5-10:1, 1-8:1, 1-5:1, 1-3:1, or 0.5-10:1; specifically, for example, any ratio within the range of 0.5:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or 0.5-10:1. Furthermore, this application has experimentally found that liposomes prepared with different mass ratios of vitamin E to magnolol yield products with excellent static stability; that is, the mass ratio of vitamin E to magnolol has little effect on the stability of the resulting system.

[0080] In some embodiments, the mass percentage of vitamin E is 0-6 wt.% of the sum of the masses of the vitamin E, magnolol, phospholipids, cholesterol, surfactant, and vitamin E; for example, any mass percentage within the range of 0 wt.%, 0.5 wt.%, 1 wt.%, 1.5 wt.%, 2 wt.%, 2.5 wt.%, 3 wt.%, 3.5 wt.%, 4 wt.%, 4.5 wt.%, 5 wt.%, 5.5 wt.%, 6 wt.%, or 0-6 wt.%. Furthermore, this application has experimentally found that liposomes prepared with different mass percentages of vitamin E yield products with excellent static stability; that is, the mass percentage of vitamin E has little effect on the stability of the resulting system.

[0081] In some embodiments, the surfactant is any mass percentage within the range of 0-60 wt.%, 10-50 wt.%, 15-45 wt.%, or 0-60 wt.%, of the sum of the mass of the surfactant, honokiol, phospholipids, cholesterol, and vitamin E; specifically, it can be any mass percentage within the range of 0 wt.%, 5 wt.%, 10 wt.%, 15 wt.%, 20 wt.%, 25 wt.%, 30 wt.%, 35 wt.%, 40 wt.%, 45 wt.%, 50 wt.%, 55 wt.%, 60 wt.%, or 0-60 wt.%. Furthermore, this application has experimentally found that the static stability of liposomes prepared with different mass percentages of surfactant varies. Blank liposome suspensions with 15%, 30%, and 45% surfactant content are all stable; however, stratification occurs at 4°C when the surfactant content (wt.%) is 0% and 60%, respectively.

[0082] In some embodiments, the preservative is present at a mass of 0.5-4 wt.% of the sum of the masses of the phospholipids, cholesterol, surfactant, vitamin E, buffer, aqueous solvent, magnolol, and sea cucumber peptides; specifically, it may be 0.5 wt.%, 0.6 wt.%, 0.7 wt.%, 0.8 wt.%, 0.9 wt.%, 1.0 wt.%, 1.1 wt.%, 1.2 wt.%, 1.3 wt.%, 1.4 wt.%, 1.5 wt.%, 1.6 wt.%, 1.7 wt.%, 1.8 wt.%, 1.9 wt.%, etc. wt.%, 2.0wt.%, 2.1wt.%, 2.2wt.%, 2.3wt.%, 2.4wt.%, 2.5wt.%, 2.6wt.%, 2.7wt.%, 2.8wt.%, 2.9wt.%, 3.0wt.%, 3.1wt.%, 3.2wt.%, 3.3wt.%, 3.4wt.%, 3.5wt.%, 3.6wt.%, 3.7wt.%, 3.8wt.%, 3.9wt.%, 4.0wt.%, or any mass percentage in the range of 0.5-4wt.%. In some embodiments, the preservative is 0.5-2 wt.% of 1,2-hexanediol; in some embodiments, the preservative is 0.5-2 wt.% of 1,2-pentanediol and 0.5-3 wt.% of 1,2-hexanediol; for example, 0.5 wt.% of 1,2-pentanediol and 3 wt.% of 1,2-hexanediol, for example, 2 wt.% of 1,2-pentanediol and 0.5 wt.% of 1,2-hexanediol, for example, 1 wt.% of 1,2-pentanediol and 2 wt.% of 1,2-hexanediol.

[0083] This application also provides a method for preparing the above-mentioned sea cucumber peptide and magnolol liposomes, comprising the following steps:

[0084] The phospholipids, cholesterol, surfactants, vitamin E, and magnolol were dissolved in a first organic solvent to obtain a lipid stock solution.

[0085] After the lipid stock solution is mixed evenly with an aqueous solvent and a buffer, a first hydration reaction is carried out to obtain a lipid solution.

[0086] After removing the first organic solvent from the lipid solution and dispersing and filtering it, a blank liposome suspension is obtained.

[0087] After the blank liposome suspension and sea cucumber peptide were mixed evenly, a second hydration reaction was carried out, followed by dispersion, filtration, and the addition of preservatives to obtain a sea cucumber peptide- and magnolol liposome suspension.

[0088] In some embodiments, the temperature of the first hydration reaction is any temperature range within the range of 35-65°C, 45-55°C, or 35-65°C; specifically, it can be 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C, 49°C, 51°C, 53°C, 55°C, 57°C, 59°C, 61°C, 63°C, or 65°C. In this application, the blank liposome suspension obtained at hydration temperatures of 45°C and 55°C exhibits high stability; however, the blank liposome suspension obtained at hydration temperatures of 35°C and 65°C is unstable and struggles to maintain its uniform emulsion state at high temperatures.

[0089] In some embodiments, the time for the first hydration reaction is any time range within the range of 20-60 min, 25-50 min, 30-40 min, or 20-60 min; specifically, it can be any time range within the range of 20 min, 22 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 33 min, 35 min, 37 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 53 min, 55 min, 57 min, 60 min, or 20-60 min. In this application, when the hydration time is 30 min or 40 min, the resulting blank liposome suspension has high stability; however, when the hydration time is, for example, 60 min, the resulting blank liposome suspension is unstable and it is difficult to maintain its uniform emulsion state at high temperatures.

[0090] In some implementations, the lipid stock solution and buffer solution can be directly mixed; in others, the lipid stock solution is added dropwise to the buffer solution. However, direct mixing results in liposomes with low stability; for example, direct mixing may lead to layering (e.g., after direct mixing and standing, a uniformly dispersed emulsion is obtained, but layering occurs after centrifugation; or, after both direct mixing and centrifugation, a uniformly dispersed emulsion is obtained, but layering occurs after dilution and centrifugation). Therefore, dropwise addition is more advantageous for obtaining a uniform and stable emulsion. In some embodiments, the dropping rate of the lipid stock solution is any rate within the range of 0.5-5 mL / min, 0.5-4 mL / min, 0.5-3.5 mL / min, 0.5-3 mL / min, or 0.5-5 mL / min; for example, it can be any rate within the range of 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.7 mL / min, 1.8 mL / min, 1.9 mL / min, 2 mL / min, 2.2 mL / min, 2.5 mL / min, 2.7 mL / min, 2.9 mL / min, 3 mL / min, 4 mL / min, 5 mL / min, or 0.5-5 mL / min. Furthermore, experiments conducted in this application revealed that when mixing lipid stock solutions, the resulting blank liposome suspension system was unstable after the lipid stock solution was added dropwise at a rate of 3 ml / min and 4 ml / min. Therefore, choosing a lower dropwise rate was more conducive to obtaining a uniform and stable emulsion.

[0091] In some embodiments, the temperature of the second hydration reaction is 20-55°C; specifically, it can be any temperature within the range of 20°C, 21°C, 23°C, 25°C, 27°C, 29°C, 30°C, 32°C, 35°C, 37°C, 39°C, 41°C, 43°C, 45°C, 47°C, 49°C, 51°C, 53°C, 55°C, or 20-55°C. In some embodiments, the second hydration reaction takes 20-60 minutes; specifically, it can be any time range within the range of 20-60 minutes, such as 20 minutes, 22 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 33 minutes, 35 minutes, 37 minutes, 39 minutes, 40 minutes, 41 minutes, 42 minutes, 43 minutes, 44 minutes, 45 minutes, 46 minutes, 47 minutes, 48 ​​minutes, 49 minutes, 50 minutes, 51 minutes, 53 minutes, 55 minutes, 57 minutes, 60 minutes, or 20-60 minutes. In some embodiments, the drop rate of the lipid stock solution is 0.5-5 mL / min; for example, it can be any rate within the range of 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.7 mL / min, 1.8 mL / min, 1.9 mL / min, 2 mL / min, 2.2 mL / min, 2.5 mL / min, 2.7 mL / min, 2.9 mL / min, 3 mL / min, 4 mL / min, 5 mL / min, or 0.5-5 mL / min.

[0092] This application also provides the use of the above-mentioned sea cucumber peptide-and magnolol liposomes, and / or sea cucumber peptide-and magnolol liposomes prepared by the above-mentioned preparation method; which are used to prepare skin care products.

[0093] This application also provides a skin care product comprising the above-mentioned sea cucumber peptide-and magnolol liposomes, and / or sea cucumber peptide-and magnolol liposomes prepared by the above-mentioned preparation method.

[0094] In some embodiments, the content of sea cucumber peptides and magnolol liposomes in the skin care product is 0.05-0.5 wt%; for example, it can be any content in the range of 0.05 wt%, 0.06 wt%, 0.07 wt%, 0.08 wt%, 0.09 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, or 0.05-0.5 wt%.

[0095] In some embodiments, the skincare product further includes excipients acceptable for use in skincare products; for example, these may be moisturizers (e.g., Bifida Ferment Lysate, Sodium Benzoate, 1,2-Pentanediol, 1,2-Hexanediol, Acetylated Sodium Hyaluronate, Sodium Hyaluronate, Sodium Hyaluronate Crosspolymer, Hydrolyzed Sodium Hyaluronate, Ethylhexylglycerin, Dipropylene Glycol, Betaine, Glycerin, Tocopheryl Acetate, Tranexamic Acid, Panthenol, Butylene Glycol, Biotin, Dipotassium Glycyrrhizate, Gynostemma Pentaphyllum Extract, Capryloyl Hydroxamic Acid, Allantoin, Oat Beta-Glucan, PEG / PPG / Polybutylene Glycol-8 / 5 / 3. Glycerin, etc.), solvents (e.g., polyethylene glycol-8), wetting agents (e.g., polyethylene glycol-8, isononyl isononanoate, etc.), plasticizers (e.g., sodium hyaluronate crosspolymer, polyacrylate crosspolymer-6, etc.), solubilizers (e.g., dipropylene glycol, etc.), emulsifiers (e.g., inulin lauryl carbamate, etc.), stabilizers (e.g., inulin lauryl carbamate, tocopheryl acetate, polyacrylate crosspolymer-6, etc.), thickeners (e.g., polyacrylate crosspolymer-6, xanthan gum, etc.), chelating agents (e.g., EDTA, etc.), and pH adjusters (e.g., citric acid, etc.). In some embodiments, the skincare product may further contain one or more active agents, such as skin repair agents (e.g., Bifida ferment lysate, sodium hyaluronate crosspolymer, hydrolyzed sodium hyaluronate, tocopheryl acetate, tranexamic acid, panthenol, dipotassium glycyrrhizate, oat beta-glucan, etc.), antioxidants (e.g., Bifida ferment lysate, betaine, tocopheryl acetate, tranexamic acid, panthenol, biotin, Gynostemma pentaphyllum extract, sodium citrate, ascorbate glucoside, citric acid, etc.), and anti-aging agents (e.g., Bifida ferment lysate, sodium hyaluronate crosspolymer, Gynostemma pentaphyllum extract, capryloyl hydroxyl group). Antimicrobial agents (e.g., oxime acid, ascorbate glucoside, etc.), antibacterial agents (e.g., phenoxyethanol, sodium benzoate, 1,2-pentanediol, ethylhexylglycerin, Gynostemma pentaphyllum extract, capryloyl hydroxamic acid, allantoin, etc.), anti-wrinkle agents (e.g., acetylated sodium hyaluronate, biotin, etc.), skin repair agents (e.g., sodium hyaluronate, etc.), anti-inflammatory agents (e.g., hydrolyzed sodium hyaluronate, betaine, tranexamic acid, panthenol, dipotassium glycyrrhizate, Gynostemma pentaphyllum extract, allantoin, etc.), whitening agents (e.g., niacinamide, tranexamic acid, biotin, capryloyl hydroxamic acid, ascorbate glucoside, etc.), and anti-allergy agents (e.g., panthenol, dipotassium glycyrrhizate, Gynostemma pentaphyllum extract, oat β-glucan, etc.).

[0096] In some embodiments, the skin care product comprises the following components by weight percentage: 5-15% antibacterial agent, polyethylene glycol-8 1-8%, moisturizer 1-8%, isononyl isononanoate 0.5-6%, dipropylene glycol 0.5-4%, 1,2-pentanediol 0.5-4%, betaine 0.1-2%, inulin lauryl carbamate 0.1-2%, tocopheryl acetate 0.05-1.5%, nicotinamide 0.05-1.2%, tranexamic acid 0.05-1%, panthenol 0.01-0.6%, polyacrylate crosspolymer 0.01-0.6%, biotin 0.01-0.4%, dipotassium glycyrrhizate 0.01-0.25%, xanthan gum 0.01-0.25%, sea cucumber peptide and magnolol liposomes 0.05-0.5%, sodium hyaluronate 0.01-0.1%, gynostemma pentaphyllum extract 0.005-0.02%, water as balance;

[0097] The antibacterial agents include Bifida ferment lysate, phenoxyethanol, and sodium benzoate.

[0098] The main moisturizing components in the moisturizer include 1,2-pentanediol, acetylated sodium hyaluronate, sodium hyaluronate, sodium hyaluronate crosspolymer, hydrolyzed sodium hyaluronate, and ethylhexylglycerin.

[0099] In some embodiments, the skin care product comprises the following components in weight percentages: 0.1-6% butylene glycol, 0.1-4% glycerin, 0.1-4% dipropylene glycol, 0.05-2% moisturizer, 0.05-0.3% panthenol, 0.05-0.3% sodium citrate, 0.05-0.3% allantoin, 0.05-0.3% antibacterial agent, 0.05-0.3% sea cucumber peptide and magnolol liposomes, 0.01-0.2% PEG / PPG / polybutylene glycol-8 / 5 / 3 glycerin, 0.01-0.1% sodium hyaluronate, 0.01-0.04% chelating agent, and 0.005-0.02% ascorbate glucoside;

[0100] The moisturizers include butylene glycol, 1,2-hexanediol, and capryloyl hydroxamic acid;

[0101] The main antibacterial components in antibacterial agents include phenoxyethanol and sodium benzoate.

[0102] Example

[0103] Specific embodiments of the present application will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0104] The material information involved in this application embodiment is as follows:

[0105] Soybean lecithin, CAS No. 8002-43-5, is typically added at a rate of 0.5-4 wt.% in this application.

[0106] Cholesterol, CAS No. 57-88-5, is typically added in the amount of 0.1-3 wt.% in this application;

[0107] Polysorbate-80 (also known as Tween-80), CAS No. 9005-65-6, is typically added in this application at a rate of 0.1-3 wt.%.

[0108] Tocopherol (i.e., vitamin E), CAS number 59-02-9 (or 119-13-1; 1046-18-4; 1406-66-2; 2074-53-5; 10191-41-0), is generally added in the present application at a rate of 0.01-1 wt.%.

[0109] Honokiol, CAS No. 33354-74-6, is typically added at a rate of 0.2 wt.% in this application.

[0110] Holothurin, a sea cucumber peptide, is typically added at a rate of 2.2 wt.% in this application.

[0111] Sodium dihydrogen phosphate, CAS No. 7558-80-7 (or 7632-85-6), is typically added in this application at a rate of 0.5-4 wt.%.

[0112] Disodium hydrogen phosphate, CAS No. 7558-79-4 (or 7782-18-5), is typically added in the present application at a rate of 1-5 wt.%; 1,2-hexanediol, CAS No. 107-41-5, is typically added in the present application at a rate of 0.1-3 wt.%.

[0113] Sea cucumber peptides:

[0114] ① Sea cucumber peptide is a type of marine bioactive peptide with various bioactive functions, such as soothing and moisturizing. Xi'an Huipu Biotechnology Co., Ltd. has developed sea cucumber peptide as a cosmetic ingredient.

[0115] ② Sea cucumber peptides were obtained by hydrolyzing sea cucumbers using an enzymatic process.

[0116] ③ The proportion of sea cucumber peptides with a relative molecular weight of less than or equal to 2000 is ≥90%; 17 amino acids in sea cucumber peptides were analyzed, and the highest content was arginine and glycine, followed by proline, glutamic acid, alanine and valine.

[0117] The sea cucumber peptides selected in this application are prepared according to CN 110613832 A. The specific process steps are as follows: Fresh sea cucumbers are cleaned by removing the mouth, viscera, and inner membrane, and then crushed. Six times the volume of ultrapure water is added to adjust the pH to 7.0. 1% neutral protease is added, and the solution is enzymatically hydrolyzed at 27°C for 8 hours. The hydrolysate is heated at 100°C for 40 minutes to inactivate the enzyme. The solution is then cooled to room temperature to obtain sea cucumber enzymatic hydrolysate. 95% ethanol is added to the sea cucumber enzymatic hydrolysate until the ethanol volume fraction reaches 80%. The solution is allowed to stand for 12 hours, centrifuged at 10,000 rpm for 30 minutes, and the ethanol in the supernatant is recovered. The mixture of sea cucumber peptides is passed through anion exchange resin and decolorized and deodorized with deionized water to obtain sea cucumber peptide extract. The sea cucumber peptide extract is then freeze-dried to obtain sea cucumber peptide extract.

[0118] Furthermore, the anion exchange resin is preferably a 717 type anion exchange resin.

[0119] The freeze-drying method is as follows: pre-freeze at -45℃ for 5 hours, vacuum dry at -30℃ (<10Pa) for 14 hours, then raise the temperature to 30℃ after another 10 hours and continue vacuum drying for 4 hours.

[0120] The sea cucumber peptides used in this application can also be commercially available. They were purchased from Xi'an Huipu Biotechnology Co., Ltd., with production batch number Y202502002 and specification of 100g.

[0121] Example 1. Preparation of sea cucumber peptide- and magnolol liposomes

[0122] Preparation of sea cucumber peptide- and magnolol liposomes:

[0123] 1) Preparation of blank liposomes:

[0124] Preparation of lipid stock solution: Prepare 1.8g of soybean lecithin (PC>70), 0.6g of cholesterol, 0.096g of vitamin E, 0.192g of magnolol, 1.082g of Tween-80, and 16mL of anhydrous ethanol. Mix soybean lecithin, cholesterol, vitamin E, magnolol, Tween-80, and anhydrous ethanol, stir, and dissolve in a water bath at 55℃ to obtain lipid stock solution.

[0125] Preparation of lipid solution: Take 83.3 ml of 0.2 mol / L (pH = 6.8) phosphate buffer solution (stored in a constant temperature water bath at 45℃ for later use), and in a constant temperature water bath at 45℃ with stirring at speed 2, add the lipid stock solution at a rate of 0.5 mL / min.

[0126] Add the solution dropwise to obtain a lipid-like solution. It is important to note that during the dropwise addition process, the syringe needle should be tilted at a 40° angle to the container, and the direction of the liquid flowing out of the needle should be consistent with the rotation direction of the aqueous phase. After adding the solution, stir and hydrate for 30 minutes. The phosphate buffer solution is prepared as follows: 0.2 mol / L Na₂HPO₄: Weigh 71.6 g of Na₂HPO₄·12H₂O and dissolve it in 1000 mL of distilled water; 0.2 mol / L NaH₂PO₄: Weigh 31.2 g of NaH₂PO₄·2H₂O and dissolve it in 1000 mL of distilled water; Take 204 mL of 0.2 mol / L NaH₂PO₄ and 196 mL of 0.2 mol / L Na₂HPO₄ and mix well to obtain a 0.2 mol / L (pH = 6.8) phosphate buffer solution.

[0127] Preparation of blank liposome suspension: The lipid solution was transferred to a rotary evaporation flask and heated at a constant temperature of 55°C in a water bath, and then rotary evaporated at 0.08 MPa and 300 r / min. The resulting solution was ultrasonicated for 15 min in an ultrasonic cleaner at a water bath temperature of 35°C and 300 W. Then it was filtered through a 0.45 μm filter membrane to obtain the blank liposome suspension.

[0128] 2) Preparation of drug-loaded liposomes:

[0129] Under water bath conditions, 5 mL of a 0.86 g / mL sea cucumber peptide solution was added dropwise to 20 mL of a 0.043 g / mL blank liposome suspension at a rate of 2 mL / min. The solution was hydrated in a constant temperature water bath at 35°C for 40 min and then sonicated for 5 min. The resulting solution was further filtered through a 0.45 μm filter membrane to obtain a sea cucumber peptide- and magnolol liposome suspension. The final mass ratio of sea cucumber peptide to lipid was 1.5:3 when the sea cucumber peptide solution was added. The mass of lipid was calculated as: mass of soybean lecithin + mass of cholesterol + mass of vitamin E + mass of Tween-80 + mass of magnolol.

[0130] Example 2. Stability Test

[0131] Blank liposome suspensions and different sea cucumber peptide- and magnolol liposome suspensions (the mass ratios of sea cucumber peptide and lipid were 1:3, 1.5:3 and 2:3, respectively) were prepared according to the method in Example 1.

[0132] The stability of the different liposome suspensions obtained was tested. Specifically, it was divided into static stability test and centrifugal stability test.

[0133] (1) Static stability test: The presence or absence of precipitation or stratification of the liposome suspension can be preliminarily determined by visual observation. The appearance of different sample numbers is shown in Table 1. HK-Lips (blank liposome suspension) and HP-HK-Lips (sea cucumber peptide- and magnolol liposome suspension; the mass ratio of sea cucumber peptide to lipid is 1:3, 1.5:3 and 2:3 respectively) were observed by visual observation at 4±1℃ and 25±1℃ to see if they were stratified. After observation for ≥40 days, it was found that HK-Lips and HP-HK-Lips (the mass ratio of sea cucumber peptide to lipid is 1:3, 1.5:3 and 2:3 respectively) were stable.

[0134] Table 1

[0135]

[0136] (2) Centrifugal stability test: The liposome suspension was centrifuged at a certain speed (e.g., 10000 rpm) for a certain time (e.g., 10 min, 20 min, 30 min, 60 min), and the amount of precipitate and the clarity of the supernatant were observed. (Refer to Table 2 and...) Figure 1 The results showed that no obvious precipitation occurred after centrifugation of sea cucumber peptide- and magnolol liposomes, and the supernatant remained uniform, indicating that the samples had good dispersibility and high stability.

[0137] Table 2

[0138]

[0139]

[0140] Example 3. Particle size assay of sea cucumber peptide- and magnolol liposomes

[0141] (1) Transmission electron microscopy:

[0142] Sea cucumber peptide- and magnolol liposome suspensions were prepared using the method described in Example 1, and their suspensions were analyzed by transmission electron microscopy. (Refer to...) Figure 2 The sea cucumber peptide and magnolol liposomes prepared according to the method of Example 1 showed many spherical substances under a 2μm scale, and the spherical morphology of the liposomes and the uneven particle size distribution could be clearly observed.

[0143] (2) Particle size distribution

[0144] Liposomes were prepared using the method described in Example 1, and their particle size was determined using Dynamic Light Scattering (DLS). The testing principle is based on Brownian motion; when a laser beam irradiates liposomes in solution, scattered light is generated. By measuring the fluctuations in the intensity of the scattered light, the diffusion coefficient of the liposomes can be calculated, and then the particle size can be calculated using the Stokes-Einstein equation. The testing steps were as follows: 1. Sample preparation; 2. Instrument setup: Selecting a suitable DLS instrument; 3. Measurement: Placing the prepared liposome sample into the DLS instrument and measuring according to the instrument's operating instructions; 4. Data analysis. Specific results are as follows:

[0145] The particle size distribution of sea cucumber peptides and magnolol liposomes at 25°C is shown in the figure. Figure 3 As shown; specifically, its particle size information is as follows:

[0146] Mhp:M lipid = 1.5:3; Diam.10(nm): 78.32, Diam.50(nm): 134.51, Diam.90(nm): 231.04.

[0147] Example 4. Effect of Vitamin E Content on the Preparation of Blank Liposomes

[0148] A blank liposome suspension was prepared according to the method in Example 1, specifically as follows:

[0149] Preparation of lipid stock solution: Prepare 1.8g of soybean lecithin, 0.6g of cholesterol, 0.096g of vitamin E, 1.082g of Tween-80 and 16mL of anhydrous ethanol. Mix soybean lecithin, cholesterol, vitamin E, magnolol, Tween-80 and anhydrous ethanol, stir, and dissolve in a water bath at 55℃ to obtain lipid stock solution.

[0150] Preparation of the lipid solution: Take 83.3 ml of 0.2 mol / L (pH = 6.8) phosphate buffer solution (stored in a constant temperature water bath at 45°C for later use). In a constant temperature water bath at 45°C with stirring at speed 2, add the lipid stock solution dropwise at a rate of 0.5 mL / min to obtain the lipid solution. Note that during the dropwise addition process, the syringe needle should be tilted at a 40° angle to the container, and the direction of the liquid flowing from the needle should be consistent with the rotation direction of the aqueous phase. After adding the solution, stir and hydrate for 30 minutes. The preparation method of the phosphate buffer solution is the same as in Example 1.

[0151] Preparation of blank liposome suspension: The lipid solution was transferred to a rotary evaporation flask and heated at a constant temperature of 55°C in a water bath, and then rotary evaporated at 0.08 MPa and 300 r / min. The resulting solution was ultrasonicated for 15 min in an ultrasonic cleaner at a water bath temperature of 35°C and 300 W. Then it was filtered through a 0.45 μm filter membrane to obtain the blank liposome suspension.

[0152] Simultaneously, following the above method, blank liposome suspensions with vitamin E contents (wt.%) of 0%, 1.5%, 3%, 4.5%, and 6% were prepared. Wherein, the mass of lipids (g) = mass of soybean lecithin (g) + mass of cholesterol (g) + mass of vitamin E (g) + mass of Tween-80 (g). "Vitamin E content" means the percentage of vitamin E mass relative to the mass of lipids; for example, a vitamin E content (wt.%) of 1.5% means that the mass of vitamin E is 1.5% of the mass of lipids.

[0153] The static stability of each group of liposome suspensions was observed according to the method in Example 2. Figure 4 The results showed that blank liposome suspensions with vitamin E contents (wt.%) of 0%, 1.5%, 3%, 4.5%, and 6% were stable at 4℃; however, the subsequent optimization experiment selected a scheme with a vitamin E content of 3wt.%.

[0154] Example 5. Effect of Tween-80 content on the preparation of blank liposomes

[0155] A blank liposome suspension was prepared according to the method in Example 1, specifically as follows:

[0156] Preparation of lipid stock solution: Prepare 1.8g of soybean lecithin, 0.6g of cholesterol, 0.096g of vitamin E, 1.082g of Tween-80 and 16mL of anhydrous ethanol. Mix soybean lecithin, cholesterol, vitamin E, magnolol, Tween-80 and anhydrous ethanol, stir, and dissolve in a water bath at 55℃ to obtain lipid stock solution.

[0157] Preparation of the lipid solution: Take 83.3 ml of 0.2 mol / L (pH = 6.8) phosphate buffer solution (stored in a constant temperature water bath at 45°C for later use). In a constant temperature water bath at 45°C with stirring at speed 2, add the lipid stock solution dropwise at a rate of 0.5 mL / min to obtain the lipid solution. Note that during the dropwise addition process, the syringe needle should be tilted at a 40° angle to the container, and the direction of the liquid flowing from the needle should be consistent with the rotation direction of the aqueous phase. After adding the solution, stir and hydrate for 30 minutes. The preparation method of the phosphate buffer solution is the same as in Example 1.

[0158] Preparation of blank liposome suspension: The lipid solution was transferred to a rotary evaporation flask and heated at a constant temperature of 55°C in a water bath, and then rotary evaporated at 0.08 MPa and 300 r / min. The resulting solution was ultrasonicated for 15 min in an ultrasonic cleaner at a water bath temperature of 35°C and 300 W. Then it was filtered through a 0.45 μm filter membrane to obtain the blank liposome suspension.

[0159] Simultaneously, following the above method, blank liposome suspensions with Tween-80 contents (wt.%) of 0%, 15%, 30%, 45%, and 60% were prepared. Wherein, the mass of lipids (g) = the mass of soybean lecithin (g) + the mass of cholesterol (g) + the mass of vitamin E (g) + the mass of Tween-80 (g). "Tween-80 content" means the percentage of Tween-80 mass relative to the mass of lipids; for example, a Tween-80 content (wt.%) of 15% means that the mass of Tween-80 is 15% of the mass of lipids.

[0160] The static stability of each group of liposome suspensions was observed according to the method in Example 2. Figure 5 The results showed that at 4℃, stratification occurred when the Tween-80 content (wt.%) was 0% and 60%, respectively; while the blank liposome suspensions with 15%, 30% and 45% were stable; however, the subsequent optimization experiment selected a scheme with a Tween-80 content of 30wt.%.

[0161] Example 6. Effect of parameters on the mixing of lipid stock solution and buffer solution on the preparation of blank liposomes

[0162] A blank liposome suspension was prepared according to the method in Example 1, specifically as follows:

[0163] Preparation of lipid stock solution: Prepare 1.8g of soybean lecithin, 0.6g of cholesterol, 0.096g of vitamin E, 1.082g of Tween-80 and 16mL of anhydrous ethanol. Mix soybean lecithin, cholesterol, vitamin E, magnolol, Tween-80 and anhydrous ethanol, stir, and dissolve in a water bath at 55℃ to obtain lipid stock solution.

[0164] Preparation of the lipid solution: Take 83.3 ml of 0.2 mol / L (pH = 6.8) phosphate buffer solution (stored in a constant temperature water bath at 45°C for later use). In a constant temperature water bath at 45°C with stirring at speed 2, add the lipid stock solution dropwise at a rate of 0.5 mL / min to obtain the lipid solution. Note that during the dropwise addition process, the syringe needle should be tilted at a 40° angle to the container, and the direction of the liquid flowing from the needle should be consistent with the rotation direction of the aqueous phase. After adding the solution, stir and hydrate for 30 minutes. The preparation method of the phosphate buffer solution is the same as in Example 1.

[0165] Preparation of blank liposome suspension: The lipid solution was transferred to a rotary evaporation flask and heated at a constant temperature of 55°C in a water bath, and then rotary evaporated at 0.08 MPa and 300 r / min. The resulting solution was ultrasonicated for 15 min in an ultrasonic cleaner at a water bath temperature of 35°C and 300 W. Then it was filtered through a 0.45 μm filter membrane to obtain the blank liposome suspension.

[0166] Meanwhile, referring to the above method, the following process parameters for preparing lipid solutions were optimized:

[0167] (1) Investigate the effect of hydration temperatures of 35℃, 45℃, 55℃ and 65℃;

[0168] (2) Investigate the effect of the drop rate of the lipid stock solution being 0.5 mL / min, 1 mL / min, 2 mL / min, 3 mL / min and 4 mL / min; and investigate the stability of directly mixing the lipid stock solution and phosphate buffer.

[0169] (3) The effect of hydration time of 20 min, 30 min, 40 min, 50 min and 60 min after mixing lipid stock solution and buffer solution was investigated.

[0170] The results showed that the blank liposome suspensions obtained at hydration temperatures of 45℃ and 55℃ exhibited high stability; however, the suspensions obtained at hydration temperatures of 35℃ and 65℃ were unstable, failing to maintain a uniform emulsion state at high temperatures. When mixing the lipid stock solution, adding it directly or adding phosphate buffer at rates of 3 ml / min and 4 ml / min resulted in unstable blank liposome suspensions; a rate of 0.5 ml / min was ultimately chosen. Regarding hydration time, the blank liposome suspensions obtained at hydration times of 30 min and 40 min were stable; however, excessively long hydration times, such as 60 min, resulted in blank liposome suspensions (or liposomes loaded with sea cucumber peptides) that remained unstable at high temperatures (e.g., 45℃).

[0171] Finally, the process parameters of hydration temperature of 45℃, drop rate of lipid stock solution of 0.5mL / min and hydration time of 30min were selected for subsequent experiments.

[0172] A blank liposome suspension was prepared using the optimized process parameters, and the static stability of the obtained liposome suspension was then observed according to the method in Example 2. The results are shown in Tables 4 and 5.

[0173] Table 4. Appearance observation of liposomes at 4℃

[0174] 1D √ √ √ 2D √ √ √ 3D √ √ √ 4D √ √ √ 5D √ √ √ 6D √ √ √ 7D √ √ √ 12D √ √ √ 15D √ √ √ 20D √ √ √ 25D √ √ √ 30D √ √ √

[0175] Table 5. Appearance observation of liposomes at 25℃

[0176] 1D √ √ √ 2D √ √ √ 3D √ √ √ 4D √ √ √ 5D √ √ √ 6D √ √ √ 7D √ √ √ 12D √ √ √ 15D √ √ √ 20D √ √ √ 25D √ √ √ 30D √ √ √

[0177] Example 7. Effect of the mass ratio of sea cucumber peptides to lipids on the preparation of sea cucumber peptide liposomes

[0178] Preparation of sea cucumber peptide liposomes:

[0179] 1) Preparation of blank liposomes:

[0180] Preparation of lipid stock solution: Prepare 1.8g of soybean lecithin, 0.6g of cholesterol, 0.096g of vitamin E, 1.082g of Tween-80 and 16mL of anhydrous ethanol. Mix soybean lecithin, cholesterol, vitamin E, magnolol, Tween-80 and anhydrous ethanol, stir, and dissolve in a water bath at 55℃ to obtain lipid stock solution.

[0181] Preparation of the lipid solution: Take 83.3 ml of 0.2 mol / L (pH = 6.8) phosphate buffer solution (stored in a constant temperature water bath at 45°C for later use). In a constant temperature water bath at 45°C with stirring at speed 2, add the lipid stock solution dropwise at a rate of 2 mL / min to obtain the lipid solution. Note that during the dropwise addition, the syringe needle should be tilted at a 40° angle to the container, and the direction of the liquid flowing from the needle should be consistent with the rotation direction of the aqueous phase. After adding the solution, stir and hydrate for 30 minutes. The preparation method of the phosphate buffer solution is the same as in Example 1.

[0182] Preparation of blank liposome suspension: The lipid solution was transferred to a rotary evaporation flask and heated at a constant temperature of 55°C in a water bath, and then rotary evaporated at 0.08 MPa and 300 r / min. The resulting solution was ultrasonicated for 15 min in an ultrasonic cleaner at a water bath temperature of 35°C and 300 W. Then it was filtered through a 0.45 μm filter membrane to obtain the blank liposome suspension.

[0183] 2) Preparation of drug-loaded liposomes:

[0184] Under water bath conditions, 5 mL of sea cucumber peptide solution was added dropwise to 20 mL of blank liposome suspension with a lipid concentration of 0.043 g / mL at a rate of 2 mL / min. The solution was hydrated in a constant temperature water bath at 35 °C for 40 min and then sonicated for 5 min. The resulting solution was further filtered through a 0.45 μm filter membrane to obtain a sea cucumber peptide- and magnolol liposome suspension. The final mass ratio of sea cucumber peptide to lipid was 1.5:3 when the sea cucumber peptide solution was added. The mass of lipid was calculated as: mass of soybean lecithin + mass of cholesterol + mass of vitamin E + mass of Tween-80.

[0185] Following the above method, sea cucumber peptide liposomes were prepared with mass ratios of sea cucumber peptides to lipids of 1:3, 1.5:3, 2:3, 2.5:3, and 3:3. Specifically, the masses of sea cucumber peptides and liposomes in each scheme are shown in Table 6.

[0186] Table 6

[0187] Subsequently, the static stability of each group of liposome suspensions was observed according to the method in Example 2, combined with... Figure 6The results showed that the sea cucumber peptide liposomes (HP-Lips; 1:3, 1.5:3, 2:3) were stable after ≥40 days of observation.

[0188] Example 8. Effect of the mass ratio of vitamin E to honokiol on honokiol liposomes

[0189] Preparation and magnolol liposomes:

[0190] Preparation of lipid stock solution: Prepare 1.8g of soybean lecithin, 0.6g of cholesterol, 0.096g of vitamin E, 0.192g of magnolol, 1.082g of Tween-80 and 16mL of anhydrous ethanol. Mix soybean lecithin, cholesterol, vitamin E, magnolol, Tween-80 and anhydrous ethanol, stir, and dissolve in a water bath at 55℃ to obtain lipid stock solution.

[0191] Preparation of the lipid solution: Take 83.3 ml of 0.2 mol / L (pH = 6.8) phosphate buffer solution (stored in a constant temperature water bath at 45°C for later use). In a constant temperature water bath at 45°C with stirring at speed 2, add the lipid stock solution dropwise at a rate of 0.5 mL / min to obtain the lipid solution. Note that during the dropwise addition process, the syringe needle should be tilted at a 40° angle to the container, and the direction of the liquid flowing from the needle should be consistent with the rotation direction of the aqueous phase. After adding the solution, stir and hydrate for 30 minutes. The preparation method of the phosphate buffer solution is the same as in Example 1.

[0192] Preparation of magnolol liposome suspension: The lipid solution was transferred to a rotary evaporation flask and heated at a constant temperature of 55°C in a water bath, and then rotary evaporated at 0.08 MPa and 300 r / min. The resulting solution was ultrasonicated for 15 min in an ultrasonic cleaner at a water bath temperature of 35°C and 300 W. Then it was filtered through a 0.45 μm filter membrane to obtain magnolol liposome suspension.

[0193] Following the above method, honokiol liposomes were prepared when the mass ratio of vitamin E to honokiol was 1:1 and 1:2, respectively.

[0194] Subsequently, the static stability of each group of liposome suspensions was observed according to the method in Example 2, combined with... Figure 7 The results showed that both magnolol liposomes and magnolol liposomes were stable after ≥40 days of observation.

[0195] Example 9. The effect of the mass ratio of magnolol to vitamin E and the mass ratio of sea cucumber peptide to lipid on the sea cucumber peptide-magnolol ratio. Effects of phenol liposomes

[0196] Preparation of sea cucumber peptide- and magnolol liposomes:

[0197] 1) Preparation of blank liposomes:

[0198] Preparation of lipid stock solution: Prepare 1.8g of soybean lecithin, 0.6g of cholesterol, 0.096g of vitamin E, 0.192g of magnolol, 1.082g of Tween-80 and 16mL of anhydrous ethanol. Mix soybean lecithin, cholesterol, vitamin E, magnolol, Tween-80 and anhydrous ethanol, stir, and dissolve in a water bath at 55℃ to obtain lipid stock solution.

[0199] Preparation of the lipid solution: Take 83.3 ml of 0.2 mol / L (pH = 6.8) phosphate buffer solution (stored in a constant temperature water bath at 45°C for later use). In a constant temperature water bath at 45°C with stirring at speed 2, add the lipid stock solution dropwise at a rate of 0.5 mL / min to obtain the lipid solution. Note that during the dropwise addition process, the syringe needle should be tilted at a 40° angle to the container, and the direction of the liquid flowing from the needle should be consistent with the rotation direction of the aqueous phase. After adding the solution, stir and hydrate for 30 minutes. The preparation method of the phosphate buffer solution is the same as in Example 1.

[0200] Preparation of blank liposome suspension: The lipid solution was transferred to a rotary evaporation flask and heated at a constant temperature of 55°C in a water bath, and then rotary evaporated at 0.08 MPa and 300 r / min. The resulting solution was ultrasonicated for 15 min in an ultrasonic cleaner at a water bath temperature of 35°C and 300 W. Then it was filtered through a 0.45 μm filter membrane to obtain the blank liposome suspension.

[0201] 2) Preparation of drug-loaded liposomes:

[0202] Under water bath conditions, 5 mL of sea cucumber peptide solution was added dropwise to 20 mL of blank liposome suspension with a lipid concentration of 0.043 g / mL at a rate of 2 mL / min. The solution was hydrated in a constant temperature water bath at 35 °C for 40 min and then sonicated for 5 min. The resulting solution was further filtered through a 0.45 μm filter membrane to obtain a sea cucumber peptide- and magnolol liposome suspension. The final mass ratio of sea cucumber peptide to lipid was 1.5:3 when the sea cucumber peptide solution was added. The mass of lipid was calculated as: mass of soybean lecithin + mass of cholesterol + mass of vitamin E + mass of Tween-80 + mass of magnolol.

[0203] I. The static stability test of different liposomes was performed following the method in Example 2. Specifically, it included the following two groups:

[0204] (1) When the mass ratio of honokiol to vitamin E was 1:1, sea cucumber peptide-honokiol liposomes (HP-HK-LIPS) were prepared. The static stability of the liposomes was studied when the mass ratio of sea cucumber peptide to lipid was 1.5:3 and 2:3, respectively. See the results below. Figure 8 .

[0205] (2) When the mass ratio of honokiol to vitamin E was 2:1, sea cucumber peptide-honokiol liposomes (HP-HK-LIPS) were prepared. The static stability of the liposomes was studied when the mass ratio of sea cucumber peptide to lipid was 1.5:3 and 2:3, respectively. See the detailed results below. Figure 9 .

[0206] II. The centrifugal stability tests were performed on different liposomes following the method described in Example 2. Specifically, five groups were included:

[0207] (1) After centrifugation at 10000rpm for 10min, the centrifugal stability of sea cucumber peptide- and magnolol liposomes (HP-HK-LIPS, sea cucumber peptide:liposome (mass ratio) were 1:3, 1.5:3, and 2:3, respectively) was tested.

[0208] (2) After centrifugation at 10,000 rpm for 20 min, the centrifugal stability of sea cucumber peptide- and magnolol liposomes (HP-HK-LIPS, sea cucumber peptide: lipid (mass ratio) were 1:3, 1.5:3, and 2:3, respectively) was tested.

[0209] (3) After centrifugation at 10,000 rpm for 30 min, the centrifugation stability of sea cucumber peptide- and magnolol liposomes (HP-HK-LIPS, sea cucumber peptide: lipid (mass ratio) were 1:3, 1.5:3, and 2:3, respectively) was tested.

[0210] (4) After centrifugation at 10,000 rpm for 60 min, the centrifugal stability of sea cucumber peptide- and magnolol liposomes (HP-HK-LIPS, sea cucumber peptide:liposome (mass ratio) were 1:3, 1.5:3, and 2:3, respectively) was tested.

[0211] (5) After centrifugation at 20,000 rpm for 30 min, the centrifugation stability of sea cucumber peptide- and magnolol liposomes (HP-HK-LIPS, sea cucumber peptide:liposome (mass ratio) were 1:3, 1.5:3, and 2:3, respectively) was tested.

[0212] The liposomes obtained under the above five conditions were all stable.

[0213] III. The sea cucumber peptide- and magnolol liposomes (HP-HK-LIPS, sea cucumber peptide:liposome mass ratio of 1:3) used in this embodiment were subjected to dilution and centrifugation stability tests, specifically including the following three groups:

[0214] (1) The dilution and centrifugation stability of sea cucumber peptide- and magnolol liposomes (HP-HK-LIPS, sea cucumber peptide:liposome mass ratio of 1:3) was tested after dilution by 3, 6, and 9 times and centrifugation at 10,000 rpm for 30 min. All groups were stable.

[0215] (2) The dilution and centrifugation stability of sea cucumber peptide- and magnolol liposomes (HP-HK-LIPS, sea cucumber peptide:liposome mass ratio of 1.5:3) was tested after dilution by 3, 6, and 9 times and centrifugation at 10,000 rpm for 30 min. All groups were stratified and unstable.

[0216] (3) The centrifugal stability of sea cucumber peptide- and magnolol liposomes (HP-HK-LIPS, sea cucumber peptide:liposome mass ratio of 2:3) was tested after dilution by 3, 6, and 9 times and centrifugation at 10,000 rpm for 30 min. All groups were stable.

[0217] Table 7

[0218]

[0219] In summary, the results of Example 9 are as follows:

[0220] 1) HP-HK-Lips (1:3, 1.5:3, 2:3) showed visual stability ≥40 days at 4±1℃ and 25±1℃;

[0221] 2) HP-HK-Lips (1:3) was stable and did not separate into layers after high-speed centrifugation at 10000 r / min, 10 min, 20 min, 30 min and 60 min. It was stable at 10000 r / min and 30 min after dilution by 3 times, 6 times and 9 times.

[0222] 3) HP-HK-Lips (1.5:3) was stable and did not separate into layers after high-speed centrifugation at 10000 r / min, 10 min, 20 min, 30 min and 60 min. After dilution by 3 times, 6 times and 9 times, it separated into layers at 10000 r / min and 30 min.

[0223] 4) HP-HK-Lips (2:3) was stable and did not separate into layers after high-speed centrifugation at 10000 r / min, 10 min, 20 min, 30 min and 60 min. It was stable at 10000 r / min and 30 min after dilution by 3 times, 6 times and 9 times.

[0224] By comparing HP-HK-Lips (1:3, 1.5:3, 2:3), HP-HK-Lips (1:3, 2:3) currently has the best process and drug loading stability.

[0225] Example 10. Effects of different preservatives on sea cucumber peptide- and magnolol liposomes

[0226] Preparation of sea cucumber peptide- and magnolol liposomes:

[0227] 1) Preparation of blank liposomes:

[0228] Preparation of lipid stock solution: Prepare 1.8g of soybean lecithin, 0.6g of cholesterol, 0.096g of vitamin E, 0.192g of magnolol, 1.082g of Tween-80 and 16mL of anhydrous ethanol. Mix soybean lecithin, cholesterol, vitamin E, magnolol, Tween-80 and anhydrous ethanol, stir, and dissolve in a water bath at 55℃ to obtain lipid stock solution.

[0229] Preparation of the lipid solution: Take 83.3 ml of 0.2 mol / L (pH = 6.8) phosphate buffer solution (stored in a constant temperature water bath at 45°C for later use). In a constant temperature water bath at 45°C with stirring at speed 2, add the lipid stock solution dropwise at a rate of 0.5 mL / min to obtain the lipid solution. Note that during the dropwise addition process, the syringe needle should be tilted at a 40° angle to the container, and the direction of the liquid flowing from the needle should be consistent with the rotation direction of the aqueous phase. After adding the solution, stir and hydrate for 30 minutes. The preparation method of the phosphate buffer solution is the same as in Example 1.

[0230] Preparation of blank liposome suspension: The lipid solution was transferred to a rotary evaporation flask and heated at a constant temperature of 55°C in a water bath, and then rotary evaporated at 0.08 MPa and 300 r / min. The resulting solution was ultrasonicated for 15 min in an ultrasonic cleaner at a water bath temperature of 35°C and 300 W. Then it was filtered through a 0.45 μm filter membrane to obtain the blank liposome suspension.

[0231] 2) Preparation of drug-loaded liposomes:

[0232] Under water bath conditions, 5 mL of sea cucumber peptide solution was added dropwise to 20 mL of blank liposome suspension with a lipid concentration of 0.043 g / mL at a rate of 2 mL / min. The solution was hydrated in a constant temperature water bath at 35 °C for 40 min and then sonicated for 5 min. The resulting solution was further filtered through a 0.45 μm filter membrane to obtain a sea cucumber peptide- and magnolol liposome suspension. The final mass ratio of sea cucumber peptide to lipid was 1.5:3 when the sea cucumber peptide solution was added. The mass of lipid was calculated as: mass of soybean lecithin + mass of cholesterol + mass of vitamin E + mass of Tween-80 + mass of magnolol.

[0233] Preservatives were added to the obtained sea cucumber peptide- and magnolol liposome suspension. After adding the preservatives, the mass fraction of 1,2-hexanediol in the suspension was 1%; that is, for example, the added 1% 1,2-hexanediol means that 1,2-hexanediol accounts for 1% of the final suspension mass. The final suspension mass = mass of soybean lecithin + mass of cholesterol + mass of vitamin E + mass of Tween-80 + mass of magnolol + mass of sea cucumber peptide + mass of buffer solution (disodium hydrogen phosphate, sodium dihydrogen phosphate, water) + mass of hexanediol.

[0234] In this embodiment, the preservatives added in different schemes are different, specifically (wt.%): (1) 1% 1,2-hexanediol; (2) 1% pentanediol + 2% 1,2-hexanediol; (3) 1% PE9010 (i.e., phenoxyethanol / ethylhexylglycerin); (4) 1% phenoxyethanol. The preservatives are added with reference to the mass of lipids, and the method for calculating the mass of lipids is as described above.

[0235] 1) The pH values ​​of the sea cucumber peptide- and magnolol liposome suspensions with added preservative (1% 1,2-hexanediol) were tested after being placed in an environment of 4℃ for 10, 17, 24 and 31 days. The specific results are shown in Table 8. The results show that the pH value is relatively stable.

[0236] Table 8

[0237]

[0238] Where “X” indicates that the system is separated from oil and water; “ / ” indicates that there are no test results, mainly because the sample is not stable at 45℃, so its pH value is not measured.

[0239] 2) The static stability of the liposome suspension was tested according to the method in Example 2. Specific results are shown in [link to example]. Figure 10 The results showed that the 1% hexanediol group and the 1% 1,2-pentanediol + 2% hexanediol group were stable after standing at 4℃, 25℃ and 45℃; while the 1% PE9010 group and the 1% phenoxyethanol group separated into layers after standing at 45℃, showing poor stability.

[0240] Example 11. Particle size analysis of different liposomes

[0241] Three types of sea cucumber peptide- and magnolol liposomes were prepared according to the method in Example 10 (the mass ratios of sea cucumber peptide and lipid were 1.5:3, 1:3 and 2:3, respectively). The particle size of the three types of liposomes was tested at 25°C. The specific results are shown in Table 9.

[0242] Table 9

[0243] Diam. 10 (nm) 78.32 104.65 122.37 Diam. 50 (nm) 134.51 193.45 210.38 Diam. 90 (nm) 231.04 357.58 361.69

[0244] Example 12. Encapsulation efficiency test

[0245] The encapsulation efficiency test method is as follows: the Kjeldahl nitrogen determination method is used to calculate the sea cucumber peptide content. The formula for calculating the encapsulation efficiency is: Encapsulation efficiency (%) = [(Total sea cucumber peptide content - Free sea cucumber peptide content) / Total sea cucumber peptide content] × 100%.

[0246] Following the above method, the encapsulation efficiency of the sea cucumber peptide- and magnolol liposomes obtained in Example 1 was tested, and the encapsulation efficiency was 54.5%.

[0247] Comparative Example 1.

[0248] Liposomes were prepared using a thin-film dispersion method. The specific steps are as follows:

[0249] 1) Preparation of blank liposomes:

[0250] Preparation of lipid stock solution: Prepare 1.8g soybean lecithin, 0.6g cholesterol, 0.096g vitamin E, 0.192g honokiol, 1.082g Tween-80 and 16mL anhydrous ethanol. Dissolve soybean lecithin, cholesterol, vitamin E, honokiol and Tween-80 (2) in ethanol solution, dissolve in a 55℃ water bath, and then evaporate by rotary evaporation at 0.008MPA in a 40℃ water bath at speed 2 to form a uniform lipid film.

[0251] Preparation of lipid solution: Add 83.3 ml of 0.2 mol / L (pH = 6.8) phosphate buffer solution (stored at 45°C in a constant temperature water bath for later use) to the homogenized membrane. After washing the membrane, transfer the suspension to the inlet, add a magnetic stir bar, and stir in a 45°C water bath for 40 min to obtain the lipid solution. The preparation method of the phosphate buffer solution is the same as in Example 1.

[0252] Preparation of blank liposome suspension: The lipid solution was sonicated in an ultrasonic cleaner with a water bath temperature of 35℃ and 300W for 15 minutes, and then passed through a 0.45um membrane to obtain a blank liposome suspension.

[0253] 2) Preparation of drug-loaded liposomes:

[0254] Under water bath conditions, 5 ml of a 0.86 g / mL sea cucumber peptide solution was slowly added dropwise to 20 ml of blank liposome suspension. The solution was hydrated in a constant temperature water bath at 35°C for 40 min, followed by sonication for 5 min. The resulting solution was then filtered through a 0.45 μm filter membrane to obtain a sea cucumber peptide liposome suspension. The final mass ratio of sea cucumber peptide to lipids was 1.5:3, where the mass of lipids equals the mass of soybean lecithin + the mass of cholesterol + the mass of vitamin E + the mass of Tween-80.

[0255] The resulting sea cucumber peptide liposome suspension tends to separate into layers after standing, making it difficult to obtain a stable product.

[0256] Example 13. Skincare Product 1

[0257] A skin care product was prepared using the sea cucumber peptide and magnolol liposomes obtained in Example 1. The components of the skin care product are shown in Table 10.

[0258] Table 10

[0259]

[0260]

[0261] Example 14. Skincare Product 2

[0262] A skin care product was prepared using the sea cucumber peptide and magnolol liposomes obtained in Example 1. The components of the skin care product are shown in Table 11.

[0263] Table 11

[0264]

[0265] The description in this disclosure is provided for illustrative and descriptive purposes only and is not intended to be exhaustive or to limit the disclosure to its forms. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described in order to better illustrate the principles and practical application of this disclosure and to enable those skilled in the art to understand this disclosure and to design various embodiments with various modifications suitable for a particular purpose.

Claims

1. Sea cucumber peptide- and magnolol liposomes, wherein the raw materials for preparing the sea cucumber peptide- and magnolol liposomes comprise the following components in parts by weight: Phospholipids 0.5-4 parts, cholesterol 0.1-3 parts, surfactants 0.1-4 parts, vitamin E 0-1 part, phosphate buffer 1-10 parts, water 60-100 parts, magnolol 0.1-1 part, sea cucumber peptides 0.1-5 parts, preservatives 0.1-5 parts; The preparation method of the sea cucumber peptide and magnolol liposomes includes the following steps: The phospholipids, cholesterol, surfactants, vitamin E, and magnolol were dissolved in a first organic solvent to obtain a lipid stock solution. After the lipid stock solution is mixed evenly with water and phosphate buffer, a first hydration reaction is carried out to obtain a lipid solution. After removing the first organic solvent from the lipid solution and dispersing and filtering it, a blank liposome suspension is obtained. After the blank liposome suspension and sea cucumber peptide were mixed evenly, a second hydration reaction was carried out, followed by dispersion and filtration, and then a preservative was added to obtain a sea cucumber peptide- and magnolol liposome suspension. The temperature of the first hydration reaction is 45-55℃; the time of the first hydration reaction is 30-40 min; and the dropping rate of the lipid stock solution is 0.5-2 mL / min. in, The surfactant comprises 15-45 wt.% of the sum of the masses of the surfactant, honokiol, phospholipids, cholesterol, and vitamin E. The mass ratio of the sea cucumber peptide to the sum of the masses of the phospholipids, cholesterol, surfactant, magnolol, and vitamin E is 0.5-1.2:3 or 1.8-3:

3. The preservative is present in an amount of 0.5-4 wt.% of the sum of the masses of the phospholipids, cholesterol, surfactants, vitamin E, phosphate buffer, water, magnolol, and sea cucumber peptides. The preservative is 1,2-hexanediol or a combination of 1,2-pentanediol and 1,2-hexanediol; The surfactant is Tween; The first organic solvent is anhydrous ethanol.

2. The sea cucumber peptide-honokiol liposome according to claim 1, wherein the mass ratio of honokiol to vitamin E is 0.5-10:

1.

3. The sea cucumber peptide-honokiol liposome according to claim 1, wherein the mass ratio of honokiol to vitamin E is 1-3:

1.

4. The sea cucumber peptide-and magnolol liposome according to claim 1, wherein the mass of vitamin E accounts for 0-6 wt.% of the sum of the mass of magnolol, phospholipids, cholesterol, surfactant and vitamin E.

5. A method for preparing sea cucumber peptide and magnolol liposomes according to any one of claims 1-4, comprising the following steps: The phospholipids, cholesterol, surfactants, vitamin E, and magnolol were dissolved in a first organic solvent to obtain a lipid stock solution. After the lipid stock solution is mixed evenly with water and phosphate buffer, a first hydration reaction is carried out to obtain a lipid solution. After removing the first organic solvent from the lipid solution and dispersing and filtering it, a blank liposome suspension is obtained. After the blank liposome suspension and sea cucumber peptide were mixed evenly, a second hydration reaction was carried out, followed by dispersion and filtration, and then a preservative was added to obtain a sea cucumber peptide- and magnolol liposome suspension. in, The conditional parameters for the first hydration reaction include: The temperature of the first hydration reaction is 45-55℃; the time of the first hydration reaction is 30-40 min; and the dropping rate of the lipid stock solution is 0.5-2 mL / min.

6. The preparation method according to claim 5, The conditions and parameters for the second hydration reaction include: The temperature for the second hydration reaction is 20-55℃; And / or, The second hydration reaction takes 20-60 minutes; and / or, The lipid stock solution is added at a rate of 0.5-5 mL / min.

7. Use of the sea cucumber peptide-and magnolol liposomes according to any one of claims 1-4, and / or the sea cucumber peptide-and magnolol liposomes prepared by the preparation method according to claim 5 or 6; for use in the preparation of skin care products.

8. A skin care product comprising sea cucumber peptide-and magnolol liposomes as described in any one of claims 1-4, and / or sea cucumber peptide-and magnolol liposomes prepared by the preparation method described in claim 5 or 6.

9. The skin care product according to claim 8, wherein the content of sea cucumber peptide and magnolol liposomes in the skin care product is 0.05-0.5 wt%.

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