Preparation method and application of bicontinuous phase gel based on water-soluble oligopeptide and grease

Abstract

The invention provides a preparation method and application of bicontinuous phase gel based on water-soluble oligopeptide and grease, the bicontinuous phase gel is prepared from water-soluble oligopeptide, non-volatile grease and an aqueous solution with the pH value in the range of 3-10, and a formed system is of a bicontinuous phase structure in which hydrogel and organogel coexist. And the water-soluble oligopeptide is used as an amphiphilic matrix in the system to play a role of a gel skeleton. According to the bicontinuous phase gel, the water-soluble oligopeptide, the non-volatile grease and the aqueous solution are used as core raw materials, no surfactant needs to be additionally added, the bicontinuous phase gel can be directly prepared in one step, and meanwhile the gel has the excellent functional component loading capacity and the oral bioavailability improving effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparation carriers and efficacy component delivery, and particularly relates to a preparation method and application of a double-continuous-phase gel based on water-soluble short peptides and oil. BACKGROUND

[0002] In the field of gel materials, hydrogels have a polar solvent (usually water) as a continuous phase, have good biocompatibility and water-soluble substance loading capacity; organic gels (or oil gels) have a non-polar liquid (such as an organic solvent, mineral oil, vegetable oil) as a continuous phase, and are suitable for loading lipophilic substances. Double-continuous-phase gels (Bijel) are a composite system composed of hydrogels and organic gels, and the core feature is that both liquid phases are in a continuous state without a distinct dispersed phase, and have the advantages of both hydrogels and organic gels, can simultaneously load lipophilic and hydrophilic efficacy components, and due to the stable three-dimensional network structure, are not prone to aggregation or flocculation, and have significantly better physical and chemical stability than traditional emulsions or single gels.

[0003] However, the traditional preparation method of the double-continuous-phase gel has many limitations, mainly including:

[0004] 1. Complex raw material composition: usually, polyols, cellulose, carbomer and a large amount of surfactants (such as sodium dodecyl sulfate) need to be added to stabilize the system, which not only increases the production cost, but also may introduce safety risks, such as skin irritation and oral toxicity of surfactants.

[0005] 2. Complicated process and high energy consumption: the preparation process needs to prepare hydrogels and oil gels separately, and needs to go through heating, cooling or multiple pH value adjustment steps during the process, which is complicated to operate, has high energy consumption, and is prone to cause inactivation of heat-sensitive efficacy components;

[0006] 3. Insufficient stability and applicability: the traditional system has a limited loading capacity for efficacy components (usually ≤5%), and is prone to phase separation during storage, especially in oral application scenarios, it is difficult to cope with the complex pH value and enzyme environment of the gastrointestinal tract, resulting in low drug bioavailability.

[0007] In existing related patents, for example, the Chinese patent with publication number CN113576930A “Double-continuous-phase makeup removing gel for washing and removing in one step and preparation method thereof”, the technical solution still needs to use carbomer as the hydrogel matrix, and the system needs to be heated to 70℃ during the preparation process to realize gel formation, which not only increases the complexity of raw materials due to the dependence on additional gel matrix, but also limits the applicability of heat-sensitive efficacy components due to the heating process, and cannot meet the application requirements of double-continuous-phase gels in terms of “low additive dependence and high stability”.

[0008] Therefore, developing a double-continuous-phase gel preparation method with simple raw materials, simple process, low energy consumption and wide applicability has become a key problem to be solved in the field. SUMMARY

[0009] In view of the problems of complex process, dependence on heating and a large number of additives, insufficient stability and bioavailability in the existing double-continuous-phase gel preparation technology, the purpose of the present application is to provide a double-continuous-phase gel preparation method and application based on water-soluble short peptides and oil, which uses water-soluble short peptides, non-volatile oil and aqueous solution as core raw materials, does not require additional surfactants, and can directly prepare double-continuous-phase gel in one step. Meanwhile, the gel has excellent loading capacity for functional ingredients and can improve oral bioavailability.

[0010] The purpose of the present application is achieved by the following technical solutions:

[0011] The first aspect of the present application provides a double-continuous-phase gel based on water-soluble short peptides and oil, wherein the double-continuous-phase gel is prepared from water-soluble short peptides, non-volatile oil and aqueous solution with a pH value in the range of 3-10, the formed system is a double-continuous-phase structure coexisting with hydrogel and organic gel, and the water-soluble short peptides act as an amphiphilic matrix to play a gel skeleton role in the system.

[0012] Further, the water-soluble short peptides are ultra-short peptides composed of 2-20 amino acids; preferably, they are mixtures of natural ultra-short peptides, such as animal and plant protein hydrolysates, and the molecular weight of the water-soluble short peptides is less than 1000 daltons.

[0013] Further, the non-volatile oil includes natural, synthetic or semi-synthetic fatty oil, fatty acid, and natural, synthetic or semi-synthetic monoglyceride, diglyceride or triglyceride; preferably, it is natural vegetable oil, particularly preferably refined soybean oil, refined olive oil or refined sesame oil.

[0014] Further, the aqueous solution includes pure water, organic acid or inorganic acid solution with a pH value of 3-10, and solution containing organic salt or inorganic salt with a pH value of 3-10; the organic acid or inorganic acid solution includes citric acid solution, acetic acid solution, the inorganic acid or inorganic base solution includes hydrochloric acid solution, sodium hydroxide solution, the organic salt includes sodium citrate, and the inorganic salt includes sodium chloride; particularly preferably, the acid solution is adjusted to a pH value of 4-5 with citric acid or acetic acid.

[0015] The second aspect of the present application provides a preparation method of the double-continuous-phase gel based on water-soluble short peptides and oil according to the first aspect, which comprises the following steps:

[0016] (1) Put the water-soluble short peptide solid powder into a mixing container, pre-mix, and ensure that the powder is uniformly dispersed without obvious clumping;

[0017] (2) Add the pre-mixed non-volatile oil to the pre-mixed water-soluble short peptide powder, continue to stir and soak, and form a uniform paste mixture or oil suspension;

[0018] (3) Keep the stirring state unchanged, slowly add the pre-mixed water solution with a pH value of 3-10 to the above paste mixture or oil suspension, continue to stir after the addition is completed, and until the system is naturally layered, the upper layer is a transparent oil phase, and the lower layer is an opaque gel phase;

[0019] (4) Separate and recover the excess oil in the upper layer, and the lower layer is the double-continuous phase gel.

[0020] Further, the mass ratio of the non-volatile oil to the water-soluble short peptide is ≥0.5; preferably, the mass ratio of the two is 2.0.

[0021] Further, the mass ratio of the water solution to the water-soluble short peptide is ≤1.0; preferably, the mass ratio of the two is 0.3.

[0022] Further, in the double-continuous phase gel system prepared, the mass ratio of the oil to the water-soluble short peptide is ≤0.5; preferably, the mass ratio of the residual oil / water-soluble short peptide in the system is 0.2. The residual oil amount in the system = initial oil amount - recovered oil amount, and the residual oil needs to be uniformly dispersed in the gel skeleton without obvious free oil droplets.

[0023] The third aspect of the application provides the use of the double-continuous phase gel of the first aspect or the double-continuous phase gel prepared by the method of the second aspect, wherein the double-continuous phase gel is used as a carrier for delivering the effective components including drugs, cosmetics, food, health products or agricultural chemicals.

[0024] Further, the effective components include but are not limited to active pharmaceutical ingredients API, peptides, polypeptides, proteins, nucleic acids, traditional Chinese medicine active ingredients, plant extract drugs, vaccines, cosmetic effective components, and pesticide components; the nucleic acids include DNA, RNA or fragments thereof, and the RNA is preferably RNAi.

[0025] Further, when the double-continuous phase gel is used for delivering the effective components, the following two addition methods are adopted:

[0026] (1) Pre-addition: mix the effective components with one or more of the water-soluble short peptide solid powder, the non-volatile oil, or the water solution with a pH value of 3-10 uniformly, and then prepare the double-continuous phase gel system according to the steps of the second aspect;

[0027] wherein the solid efficacy ingredient is mixed uniformly with the water-soluble short peptide solid powder; the liquid efficacy ingredient is pre-emulsified with the non-volatile oil or the water solution with pH value of 3-10;

[0028] (2) Post-adding: first prepare the bicontinuous phase gel system according to the steps of the second aspect, and then add the efficacy ingredient into the gel system by spraying or stirring mixing to ensure uniform dispersion of the efficacy ingredient.

[0029] Further, auxiliary ingredients for improving the performance of the bicontinuous phase gel system can be added during preparation, which include but are not limited to surfactants, emulsifiers, antioxidants, preservatives, flavors, and taste masking agents.

[0030] The present application has the following beneficial effects compared with the prior art:

[0031] 1. The present application first prepares a bicontinuous phase gel using three types of core raw materials: water-soluble short peptides, non-volatile oils, and water solutions, without the need for additional exogenous gel matrix (such as carbomer) and a large amount of surfactants, thereby reducing costs while avoiding irritation and toxicity risks; the short peptides are preferably natural animal and plant protein hydrolysates, and the oils are selected from food / pharmaceutical grade natural plant oils, which meet the safety standards of the pharmaceutical, cosmetic, and food industries, and have a wide range of applications.

[0032] 2. In the preparation method of the bicontinuous phase gel, only four steps of "premixing - wetting - dropwise adding - separation" are required, without the need for heating and multiple pH adjustments, thereby avoiding the inactivation of heat-sensitive ingredients (such as polypeptides, vaccines, and curcumin) caused by high temperatures.

[0033] 3. When the bicontinuous phase gel system of the present application is used to deliver efficacy ingredients, the loaded efficacy ingredients are dissolved or encapsulated in the non-polar oil phase or the polar hydrogel layer, or attached in the form of free particles in the interlayer between the oil phase and the hydrogel phase. This bicontinuous phase gel self-emulsifies (SEDDS) to form microemulsion droplets in the gastrointestinal environment, inhibits the precipitation of drugs in the gastric solution, prolongs the gastrointestinal retention time, increases the stability of drugs in the gastrointestinal tract, and promotes lymphatic transport. Therefore, the bicontinuous phase gel of the present application can effectively improve the bioavailability of oral drugs due to its self-emulsifying properties. BRIEF DESCRIPTION OF DRAWINGS

[0034] The present application will be further described below in conjunction with the drawings and examples:

[0035] Figure 1 The figures are the appearance forms of different bicontinuous phase gels;

[0036] Figure 2 The figures are the microscope structure diagrams (40 times) of different bicontinuous phase gels;

[0037] Figure 3 Photos of different bicontinuous phase gels after self-emulsification;

[0038] In the above figures, A is Example 1 - blank bicontinuous phase gel, B is Example 2 - bicontinuous phase gel loaded with berberine, C is Example 3 - bicontinuous phase gel loaded with curcumin. DETAILED DESCRIPTION

[0039] The examples are provided to better illustrate the present application and should not be construed as limiting the present application to only the examples. Modifications and variations of the application that are obvious to those of skill in the art are to be considered within the scope of the present application.

[0040] The endpoints of the ranges and any values between the endpoints are included in the range. The endpoints of the ranges and the individual values are not included in the range unless the text specifically states otherwise. Ranges include the endpoints unless the context clearly dictates otherwise. The citation of any reference is not an admission that the reference is prior art, nor is it an admission of any description, claim, or drawings in the reference.

[0041] The application will be described in further detail by way of examples. It should be understood that the following examples are only used to more fully describe and illustrate the application and are not intended to limit the application.

[0042] In the following examples, the experimental instruments and raw materials involved are commercially available unless otherwise specified.

[0043] Raw materials, see Table 1.

[0044] Table 1

[0045]

[0046] Experimental instruments, see Table 2.

[0047] Table 2

[0048]

[0049] Example 1: Preparation of blank bicontinuous phase gel

[0050] This example provides a bicontinuous phase gel based on water-soluble short peptides and oil, and the preparation method comprises the following steps:

[0051] (1) Take 10 g of high content soybean peptide (molecular weight <1000 Dalton, purity 85%) and place it in a 500 mL beaker. Stir the mixture at a speed of 150 r / min for 8 minutes to ensure that the powder is evenly mixed without clumping.

[0052] (2) Continue to add 20g of refined olive oil to the beaker, maintain a stirring speed of 180r / min, and continue stirring and soaking at room temperature for 30 minutes to form a light yellow paste mixture.

[0053] (3) While stirring, slowly add 3g of purified water (pH 6.8) to the paste mixture at a rate of 1.5 drops / second. After the addition is complete, continue stirring for 12 minutes. The system will naturally separate into layers, with the upper layer being a transparent oil phase and the lower layer being a milky white gel phase.

[0054] (4) Centrifuge at 4000 r / min for 6 minutes to separate and recover 17.03 g of excess olive oil from the upper layer and obtain a bicontinuous phase gel (approximately 15.97 g) from the lower layer.

[0055] (5) Performance testing:

[0056] Bicontinuous phase gel morphology: such as Figure 1 As shown in Figure A, it is a milky white semi-solid with no obvious layering or free oil droplets and a uniform texture; the gel structure observed under a microscope is as follows. Figure 2 As shown in Figure A, a three-dimensional network gel framework structure is visible. The oil phase is uniformly distributed in the hydrogel phase in the form of fine channels, forming a bicontinuous structure with no obvious dispersed phase particles.

[0057] Emulsification effect: To simulate the gastrointestinal environment, 2g of bicontinuous phase gel was dissolved in 40g of purified water and stirred until a light milky white homogeneous liquid was formed. Figure 3 (A), after standing for 24 hours, no stratification occurred.

[0058] Stability: When the bicontinuous phase gel was stored at 4℃, 25℃, and 40℃ for 30 days, no mold growth or phase separation was observed, and the hardness change rate was ≤5%.

[0059] Example 2: Preparation of Berberine Bicontinuous Phase Gel

[0060] This embodiment provides a bicontinuous phase gel based on water-soluble short peptides and lipids, which is used as a carrier for delivering berberine. The preparation method includes the following steps:

[0061] (1) Weigh 10g of sardine peptide composition (Valtyron, molecular weight <1000 Daltons, purity 85%) and 1g of berberine (pharmaceutical grade), place them in a mortar, grind and mix for 10 minutes to obtain a uniform mixed powder.

[0062] (2) Transfer the mixed powder to a 500mL beaker and stir at a rate of 160r / min for 6 minutes to premix.

[0063] (3) Continue to add 20 g of refined sesame oil into the beaker, keep the stirring rate at 200 r / min, and stir and soak at room temperature for 30 minutes to form a yellow-brown paste-like mixture,

[0064] (4) Keep stirring, slowly add 3 g of a citric acid solution (pH value 4.5, concentration 0.1 mol / L, conductivity 80 μS / cm) to the paste-like mixture at a rate of 1.2 drops per second, and continue stirring for 15 minutes after the addition is completed. The system is stratified, with a transparent oil phase on the top and a yellow-brown gel phase on the bottom.

[0065] (5) Separate 16.6 g of excess sesame oil from the top layer, and obtain a curcumin-loaded bicontinuous phase gel (about 16.4 g in mass) from the bottom layer.

[0066] (6) Performance detection:

[0067] The morphology of the curcumin-loaded bicontinuous phase gel is shown in FIG. 2B, which is a yellow-brown semi-solid with uniform color and no local dark clumps. The gel structure is observed under a microscope, as shown in FIG. 2B, and the yellow-brown curcumin particles (particle size ≤5 μm) are uniformly dispersed in the gel skeleton. The oil phase channels are tightly combined with the hydrogel phase, and there is no particle aggregation. Figure 1 Figure 2

[0068] The loading uniformity of the curcumin-loaded bicontinuous phase gel prepared in this example is detected by taking samples from different parts of the gel. The deviation of the curcumin content is ≤3%, indicating that the loading is uniform.

[0069] The emulsification effect is simulated in a gastrointestinal environment. 2 g of the curcumin-loaded bicontinuous phase gel prepared in this example is dissolved in 40 g of pure water, and a yellow-brown curcumin molecule (FIG. 2B) is observed in the microemulsion droplets obtained after uniform stirring. The droplets have good stability and do not settle after standing for 4 hours. Figure 3

[0070] The stability is tested by storing the gel at 4°C for 30 days. The curcumin content retention rate is ≥95%, and no degradation products are generated.

[0071] Example 3: Preparation of a curcumin bicontinuous phase gel

[0072] This example provides a bicontinuous phase gel based on a water-soluble short peptide and oil, which is used as a carrier for delivering curcumin. The preparation method comprises the following steps:

[0073] (1) Take 10 g of a high-content sardine peptide composition (Valtyron, molecular weight <1000 daltons, purity 85%) and 2 g of curcumin (food grade) and mix them in a marbled mortar for 1 minute to obtain a uniform powder.

[0074] ​​​(2) Weigh 0.04g of antioxidant-tocopherol acetate (food grade), add it to 20g of refined olive oil, stir at 180r / min for 5 minutes to ensure complete dissolution, and obtain olive oil-tocopherol acetate solution.

[0075] (3) Add the sardine peptide-curcumin mixed powder to the above olive oil-tocopherol acetate solution, maintain a stirring rate of 220 r / min, and stir and soak at room temperature for 30 minutes to form an orange-yellow paste mixture.

[0076] (4) While stirring, slowly add 2.5g of acetic acid solution with pH 5.0 (concentration 0.08mol / L) to the paste mixture at a rate of 1.8 drops / second. After the addition is complete, continue stirring for 13 minutes. The system will separate into layers, with the upper layer being a transparent oil phase and the lower layer being an orange-yellow gel phase.

[0077] (5) Centrifuge at 3500 r / min for 7 minutes to recover 17.5 g of excess olive oil from the upper layer and obtain a bicontinuous phase gel loaded with curcumin (approximately 16.94 g) from the lower layer.

[0078] (6) Performance testing:

[0079] Bicontinuous phase gel morphology loaded with curcumin: such as Figure 1 As shown in Figure C, it presents as an orange-yellow semi-solid with a bright color and no layering. Under a microscope, the gel structure is observed as follows... Figure 2 As shown in Figure C, orange-yellow curcumin particles are uniformly attached to the oil-water interface. The oil phase channel diameter is about 1-5 μm, and the hydrogel phase forms a dense network structure with good stability.

[0080] Antioxidant effect: After storage at 25℃ for 30 days, the peroxide value of the oil phase in the gel was 6 meq / kg, which was significantly lower than that of the control group without added antioxidants (15 meq / kg).

[0081] Self-emulsification effect: Simulating the gastrointestinal environment, 2g of the bicontinuous phase gel loaded with berberine prepared in this example was dissolved in 40g of purified water. Figure 3 As shown in Figure C, the orange-yellow curcumin obtained after stirring is uniformly dispersed in the microemulsion droplets, with uniform droplet size and no fusion phenomenon.

[0082] Finally, it should be noted that the above description is only used to illustrate the technical solutions of the present invention and is not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention.

Claims

1. A bicontinuous phase gel based on water-soluble short peptides and oil, characterized in that, The bicontinuous phase gel is prepared from a water-soluble short peptide, a non-volatile oil, and an aqueous solution with a pH value in the range of 3-10, and forms a bicontinuous phase structure in which the water-soluble short peptide acts as an amphiphilic matrix to form a gel skeleton.

2. The bicontinuous phase gel of claim 1, wherein, The water-soluble short peptide is an ultrashort peptide composed of 2-20 amino acids.

3. The bicontinuous phase gel of claim 1, wherein, The non-volatile oil includes natural, synthetic or semi-synthetic fatty oil, fatty acid, and natural, synthetic or semi-synthetic monoglyceride, diglyceride or triglyceride.

4. The bicontinuous phase gel of claim 1, wherein, The aqueous solution includes pure water, organic acid or inorganic acid solution with a pH value of 3-10, and organic salt or inorganic salt solution with a pH value of 3-10.

5. The method for preparing a bicontinuous phase gel based on water-soluble short peptides and oil according to any one of claims 1 to 4, characterized in that, The preparation method comprises the following steps: (1) placing the water-soluble short peptide solid powder in a mixing container, pre-mixing to ensure uniform dispersion of the powder without obvious clumping; (2) adding the pre-mixed non-volatile oil to the pre-mixed water-soluble short peptide powder, continuously stirring to immerse, and forming a uniform paste mixture or oil suspension; (3) keeping the stirring state unchanged, slowly adding the pre-mixed aqueous solution with a pH value of 3-10 to the above paste mixture or oil suspension, continuing to stir after the addition is completed, and then allowing the system to naturally separate into an upper transparent oil phase and a lower opaque gel phase; (4) separating and recovering the excess oil in the upper layer, and the lower layer is the bicontinuous phase gel.

6. The production method according to claim 5, wherein The mass ratio of the non-volatile oil to the water-soluble short peptide is ≥0.

5.

7. The preparation method according to claim 5, characterized in that, The mass ratio of the aqueous solution to the water-soluble short peptide is ≤1.

0.

8. The preparation method according to claim 5, characterized in that, In the prepared bicontinuous phase gel system, the mass ratio of the oil to the water-soluble short peptide is ≤0.

5.

9. Use of a bicontinuous phase gel according to any one of claims 1 to 4, or of a bicontinuous phase gel prepared according to any one of claims 5 to 8, characterized in that, The bicontinuous phase gel is used as a carrier to deliver active ingredients including drugs, cosmetics, food, health products or agricultural chemicals.

10. Use according to claim 9, wherein When the bicontinuous phase gel is used to deliver active ingredients, the following two addition methods are used: (1) pre-addition: uniformly mixing the active ingredients with one or more of the water-soluble short peptide solid powder, non-volatile oil or aqueous solution with a pH value of 3-10, and then preparing the bicontinuous phase gel system according to the steps of claim 5; wherein the solid active ingredients are uniformly mixed with the water-soluble short peptide solid powder, and the liquid active ingredients are pre-emulsified with the non-volatile oil or the aqueous solution with a pH value of 3-10; (2) post-addition: first preparing the bicontinuous phase gel system according to the steps of claim 5, and then adding the active ingredients to the gel system by spraying or stirring to ensure uniform dispersion of the active ingredients.

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