A lipopeptide-small molecule polypeptide supramolecular composition and a preparation method and application thereof

Abstract

The application discloses a kind of lipopeptide-small molecule polypeptide supramolecular composition and its preparation method and application, comprising, small molecule polypeptide active substance, amino acid and lipopeptide;With the total weight of composition, small molecule polypeptide active substance is 0.1%~5%, amino acid is 0.1%~10%, and lipopeptide is supplemented to 100%.The lipopeptide-small molecule polypeptide supramolecular composition prepared in the application has small particle size, good dispersibility, hydrophilicity and lipophilicity, can be self-assembled to form vesicle structure with similar structure of cell membrane, and can further carry other active components.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cosmetics and pharmaceutical technology, and particularly relates to a kind of lipopeptide-small molecule polypeptide supramolecular composition and its preparation method and application. BACKGROUND

[0002] Supramolecular chemistry is based on molecular assembly and intermolecular forces, and different types of intermolecular interactions can be distinguished according to their different degrees of strength, orientation and dependence on distance and angle. People can use intermolecular interaction forces as tools to assemble components or modules with specific structure and function into new supramolecular compositions according to the principle of supramolecular self-assembly.

[0003] Based on the designability of supramolecule, the regular structure of stable multi-functional composition is arranged by intermolecular hydrogen bonding, electrostatic attraction and van der Waals force and other interactions.

[0004] Supramolecular compositions can be used for precise delivery of active ingredients, and the flexibility of the active ingredients can be changed. After spontaneous deformation, the active ingredients can penetrate the skin, improve the solubility of poorly soluble active ingredients, promote the transdermal absorption and oral absorption rate of active ingredients, and significantly improve the bioavailability of active ingredients. It has low cost and high efficacy in the fields of cosmetics, functional foods and pharmaceuticals.

[0005] At present, there is no report on supramolecular composition composed of bioactive lipopeptide and small molecule polypeptide, and there is no report on its application to water to form vesicles for inclusion of active substances. SUMMARY

[0006] This part aims to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this part and the abstract and title of the specification to avoid obscuring the purpose of this part, the abstract and the title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0007] In view of the above and / or problems existing in the prior art, the present application is proposed.

[0008] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art, and to provide a kind of lipopeptide-small molecule polypeptide supramolecular composition.

[0009] To solve the above technical problems, the present application provides the following technical scheme: a kind of lipopeptide-small molecule polypeptide supramolecular composition, including, small molecule polypeptide active substance, amino acid and lipopeptide;

[0010] The small molecule polypeptide active is 0.1% to 5%, the amino acid is 0.1% to 10%, and the lipopeptide makes up to 100% based on the total weight of the composition.

[0011] As a preferred scheme of the composition of the present application, the lipopeptide comprises one or more of subtilin, fengycin, palmitoyl oligopeptide, di-lauroylamide glutamine lysine, iturin, bacillomycin, and polymyxin.

[0012] As a preferred scheme of the composition of the present application, the small molecule polypeptide active comprises one or more of ceramidin, acetyl octapeptide-3, and acetyl hexapeptide-8.

[0013] As a preferred scheme of the composition of the present application, the amino acid comprises one or more of arginine, lysine, histidine, arginine ethyl ester, lysine ethyl ester, and arginine monoglyceride.

[0014] As a preferred scheme of the composition of the present application, the lipopeptide and the amino acid are combined together through proton exchange, hydrogen bond and pi-pi interaction to form a supramolecular structure.

[0015] As a preferred scheme of the composition of the present application, the composition spontaneously forms a vesicle structure of 10 to 100 nm in water.

[0016] Still another object of the present application is to overcome the deficiencies in the prior art and provide a preparation method of the composition, comprising,

[0017] The lipopeptide, the small molecule polypeptide active, the amino acid and deionized water are uniformly mixed under an inert gas atmosphere, and then heated and stirred for reaction.

[0018] After cooling to room temperature, the mixture is subjected to high-pressure homogenization treatment for 2 to 8 times under a pressure of 400 to 800 bar, and then freeze-dried under vacuum to obtain the composition.

[0019] As a preferred scheme of the preparation method of the composition of the present application, the heating and stirring for reaction is performed at a heating temperature of 25 to 60°C and a stirring reaction time of 12 to 24 h.

[0020] Still another object of the present application is to overcome the deficiencies in the prior art and provide an application of the composition in cosmetic and drug delivery and sustained release.

[0021] The present application has the following beneficial effects:

[0022] (1) The lipopeptide-small molecule polypeptide supramolecular composition provided by the present application is green, non-toxic and non-irritating, because all the components are derived from biological fermentation.

[0023] (2) The lipopeptide-small molecule polypeptide supramolecular composition prepared by the present application is mainly composed of various amino acids and peptides, which are similar to the composition of skin cells and are easily absorbed by the skin.

[0024] (3) The lipopeptide-small molecule polypeptide supramolecular composition prepared by the present application has small particle size and good dispersibility, and has hydrophilicity and lipophilicity, and can self-assemble into a vesicle structure similar to the cell membrane, which can further carry other active components.

[0025] (4) The lipopeptide-small molecule polypeptide supramolecular composition prepared by the present application has excellent transdermal penetration performance, which not only protects the biological activity of the small molecule polypeptide active substance, but also delivers it to the effective site, fully exerting the biological efficacy of the small molecule polypeptide active substance; under the protection of the lipopeptide-small molecule polypeptide supramolecular structure, the active substance has rapid onset and is easily absorbed by the skin; the action is unidirectional, promoting the penetration of therapeutic drugs through the skin without affecting the in vivo process of endogenous substances; there is no interaction with excipients and active substances, which can effectively increase the transdermal absorption of drugs. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0027] Figure 1 The nuclear magnetic hydrogen spectrum of sample 1 prepared in Example 1 of the present application.

[0028] Figure 2 The DSC curve of sample 1 in Example 1 of the present application.

[0029] Figure 3 The TEM image of sample 1 in Example 1 of the present application.

[0030] Figure 4 The transdermal experiment result graph in Example 2 of the present application.

[0031] Figure 5 The transdermal experiment result graph in Example 3 of the present application.

[0032] Figure 6 The MMP-1 inhibition ability determination result graph in Example 4 of the present application. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below.

[0034] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without the specific details set forth in this description. In other instances, well-known methods have not been described in detail in order not to unnecessarily obscure aspects of the present application.

[0035] Second, the "one embodiment" or "an embodiment" appearing in the specification herein indicates that a specific feature, structure, or characteristic can be included in at least one implementation of the present application. The "in one embodiment" appearing in different places in the specification herein does not all refer to the same embodiment, nor is it an embodiment that is mutually exclusive with other embodiments.

[0036] The raw materials used in the present application are all commercially available products.

[0037] Example 1

[0038] The supermolecular composition was prepared according to the raw material ratio in Table 1:

[0039] (1) A certain amount of lipopeptide, small molecule polypeptide active substance, amino acid and deionized water were mixed well in a flask under an inert gas atmosphere, and then slowly heated to 50°C and stirred for 24h;

[0040] (2) After cooling to room temperature, the sample was treated by high pressure homogenization under 500 bar pressure for 5 times, and then freeze-dried under vacuum for 24h.

[0041] (3) DSC experiment: a certain amount of sample was weighed in a standard sealed aluminum pan, and an empty aluminum pan without sample was used as a blank sample; then the sample was heated from -40°C to 60°C at a rate of 2°C / min, and the heat change was recorded;

[0042] The entire experiment was carried out under a constant nitrogen flow of 30mL / min.

[0043] Table 1 Raw material ratio table (unit: g)

[0044] Sample 1 2 3 4 5 6 7 8 Subtilipetide 89 86 86 92 97 88 82 92 Acetyl hexapeptide-8 3 6 3 0 3 3 4 2 Arginine 8 8 11 8 0 9 8 6 Phase transition temperature / °C 10.2 - - - - 31.1 12 21

[0045] Note: "-" indicates that the phase transition temperature is not detected.

[0046] The nuclear magnetic resonance hydrogen spectrum of the composition of sample 1 is shown in Figure 1 As can be seen from the nuclear magnetic resonance hydrogen spectrum of sample 1, the terminal methyl group on the sodium subtilipetrol, the hydrogen protons on the long aliphatic chain and the peptide bond α protons all appear in the composition of sample 1; at the same time, the hydrogen protons on the a position of arginine are displaced after combining with subtilipetrol, from 3.2ppm to 3.6ppm, indicating the appearance of a supermolecular structure.

[0047] The DSC curve of sample 1 composition, surfactin and arginine is shown in Figure 1. Figure 2 From the DSC graph, it can be found that single surfactin or arginine does not have phase transition behavior in the temperature range of -40 to 60°C, while sample 1 has a clear exothermic peak at 10.2°C, indicating that sample 1 is a new structure different from single surfactin or arginine.

[0048] Combining nuclear magnetic and DSC experiments can prove that samples 2-5 are not dissolved to form supramolecular structures, while samples 1, 6-8 have supramolecular structures different from single surfactin or arginine.

[0049] The TEM image of sample 1 is shown in Figure 2. Figure 3 The results show that the supramolecular composition has good water dispersibility and can self-assemble to form vesicle structures with a wall thickness of 2.7 nm. It can further carry other active components; the particle size is less than 100 nm and remains highly uniformly dispersed.

[0050] Example 2

[0051] Transdermal experiment 1:

[0052] Sample A Aqueous solution of acetyl hexapeptide-8 (concentration 300 ppm) Sample B 1% aqueous solution of sample 1 from Example 1

[0053] Experimental method:

[0054] The entire experiment requires selecting intact undamaged pigskin with equivalent thickness for transdermal experiment. After cutting the pigskin into about 2.5 cm x 2.5 cm, it is placed between the receiving pool and the supply pool of the Franz diffusion cell, with the stratum corneum facing the supply pool;

[0055] The receiving pool contains 5% Tween 80 in PBS buffer (pH = 7.4); 2 mL of 4-fold diluted sample is added to the supply pool; the assembled Franz diffusion cell is placed in the transdermal diffusion instrument, and the experimental conditions are set as follows: temperature is 37°C, stirring speed is 400 rpm, and transdermal time is 12 h.

[0056] After the transdermal experiment, the pigskin is taken out of the diffusion cell, and the surface of the pigskin is wiped clean with absorbent cotton to remove the remaining sample. Then, the tape stripping method is used to determine the content of dendrobium polysaccharide in the stratum corneum. 21 layers of skin are adhered to the surface of the pigskin with 3M tape, the first layer is discarded, and the remaining 20 layers are placed in a 15 mL centrifuge tube. 2 mL of isopropanol is added to the tube, and ultrasonic treatment is performed for 30 min. The supernatant is obtained by centrifugation at 5000 rpm for 30 min, and then filtered with a 0.22 μm organic filter membrane. The content of acetyl hexapeptide-8 is determined, which is the content of acetyl hexapeptide-8 in the stratum corneum.

[0057] The remaining skin surface was heated at 60°C for 15 s, and the viable epidermis layer was carefully cut off with a scalpel and placed in a 15 mL centrifuge tube. 2 mL of isopropanol was added, and the mixture was ultrasonicated for 30 min. The mixture was then centrifuged at 5000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. The content of acetyl hexapeptide-8 was determined, which was the content of acetyl hexapeptide-8 in the viable epidermis layer.

[0058] The remaining skin surface was heated at 60°C for 15 s, and the viable epidermis layer was carefully cut off with a scalpel and placed in a 15 mL centrifuge tube. 2 mL of isopropanol was added, and the mixture was ultrasonicated for 30 min. The mixture was then centrifuged at 5000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm organic filter membrane. The content of acetyl hexapeptide-8 was determined, which was the content of acetyl hexapeptide-8 in the viable epidermis layer.

[0059] The content of acetyl hexapeptide-8 was determined by high performance liquid chromatography.

[0060] The results of the transdermal experiment are shown in Table 1. Figure 4 As can be seen from the results, the transdermal effect of acetyl hexapeptide-8 in the supramolecular composition is four times that in an aqueous solution thereof, and the system effectively improves the transdermal absorption effect of acetyl hexapeptide-8.

[0061] Example 3

[0062] Transdermal experiment 2:

[0063] Sample A: The sample No. 1 in Example 1 was diluted with pure water to a mass content of 1%, and hydroxyl asperosaponin was added and stirred until it was completely dissolved, and the content of hydroxyl asperosaponin was 0.5%.

[0064] Sample B: An aqueous solution of hydroxyl asperosaponin (mass concentration of 0.5%)

[0065] Experimental method:

[0066] During the experiment, intact and undamaged pigskin with a thickness of about 2.5 cm x 2.5 cm was selected for the transdermal experiment. The pigskin was placed between the receiving pool and the supply pool of the Franz diffusion cell, with the stratum corneum facing the supply pool.

[0067] The receiving pool contained PBS buffer containing 5% Tween 80 (pH = 7.4); 2 mL of the sample diluted 4 times was added to the supply pool; the assembled Franz diffusion cell was placed in the transdermal diffusion instrument, and the experimental conditions were set as follows: temperature was 37°C, stirring speed was 400 rpm, and transdermal time was 12 h.

[0068] After the end of transdermal, the pigskin was taken out from the diffusion cell, the sample left on the surface of the pigskin was wiped clean with absorbent cotton, then the content of Dendrobium polysaccharide in the stratum corneum was determined by tape stripping method, 21 layers of skin on the surface of the pigskin were adhered by 3M tape, the first layer was discarded, and the remaining 20 layers were placed in a 15 mL centrifuge tube, 2 mL of isopropyl alcohol was added to the tube, ultrasonic treatment was performed for 30 min, centrifugation was performed at 5000 rpm for 30 min, the supernatant was filtered by 0.22 μm organic filter membrane, and then the content of madecassoside was determined, which was the content of madecassoside in the stratum corneum.

[0069] The remaining surface of the skin was heated at 60°C for 15 s, and the viable epidermis layer was carefully cut off with a scalpel, cut into pieces and placed in a 15 mL centrifuge tube, 2 mL of isopropyl alcohol was added, ultrasonic treatment was performed for 30 min, centrifugation was performed at 5000 rpm for 30 min, the supernatant was filtered by 0.22 μm organic filter membrane, and then the content of madecassoside was determined, which was the content of madecassoside in the viable epidermis layer.

[0070] The last remaining skin was cut into pieces and placed in a 15 mL centrifuge tube, 2 mL of isopropyl alcohol was added, ultrasonic treatment was performed for 30 min, and then centrifugation was performed at 5000 rpm for 30 min, the supernatant was filtered by 0.22 μm organic filter membrane, and then the content of madecassoside was determined, which was the content of madecassoside in the dermis layer.

[0071] The content of madecassoside was determined by high performance liquid chromatography.

[0072] The transdermal experiment results are as follows Figure 5 From the results, it can be seen that the lipopeptide-small molecule polypeptide supramolecular composition can self-assemble into a vesicular structure in water, which can promote the dissolution and transdermal absorption of water-soluble active substances; compared with the aqueous solution, the system effectively improves the transdermal absorption effect of madecassoside.

[0073] Example 4

[0074] MMP-1 inhibitory activity detection: (using Sigma kit);

[0075] Sample A: aqueous solution of acetyl hexapeptide-8 (consistent with the mass concentration of acetyl hexapeptide-8 in sample C, 300 ppm);

[0076] Sample B: aqueous solution of nicotinamide mononucleotide (consistent with the mass concentration of nicotinamide mononucleotide in sample C, 0.5% mass fraction);

[0077] Sample C: No. 1 sample in Example 1 is diluted with pure water, the mass content is 1%, and then nicotinamide mononucleotide is added and stirred until it is completely dissolved, and the mass content of nicotinamide mononucleotide is 0.5%.

[0078] Experimental process

[0079]

[0080] Data processing:

[0081] Slope = (Fluorescence value 2- Fluorescence value 1) / (Time 2- Time 1)

[0082] Relative inhibition rate % = (Slope 酶对照组 - Slope 样品组 ) / Slope 酶对照组 * 100

[0083] Time 1 and Time 2 represent two time points within 30 minutes.

[0084] Fluorescence value 1 and 2 represent the fluorescence value at the corresponding time point.

[0085] The experimental results are shown in Figure 6 The lipopeptide-small molecule polypeptide supramolecular composition can self-assemble into a vesicular structure in water, which is beneficial to the dissolution and penetration of active substances, and the MMP-1 inhibition activity detection of sample C is obviously better than that of samples AB, and the structure can promote the improvement of the bioavailability of water-soluble active substances.

[0086] Example 5

[0087] Serum formula:

[0088]

[0089]

[0090] The serum is divided into normal temperature sample, heat-resistant sample, cold-resistant sample and light sample, respectively, the normal temperature sample is placed in the dark, the heat-resistant sample is placed in a constant temperature oven at (50±1℃), the cold-resistant sample is placed in a-18℃ refrigerator, and the light sample is placed in an artificial intelligent climate box. After 3 months, the sample appearance, color and odor are observed, and no oil-water separation phenomenon is observed, and the results are shown in the following table.

[0091] Table 9 Formula stability test

[0092]

[0093] The experimental results show that the sample provided by the present application is not obviously discolored and stratified after being placed at room temperature, high temperature, low temperature and light for 3 months after being compounded with the matrix, which can meet the actual application requirements.

[0094] In view of the low utilization rate of active substances, the lipopeptide-small molecule polypeptide-amino acid is effectively combined to form a supramolecular composition, which is green, non-toxic and non-irritating; has small particle size and good dispersibility, and has hydrophilicity and lipophilicity; can be sub-assembled to form a vesicle structure similar to the structure of a cell membrane, and can further carry other active components; has excellent transdermal penetration performance, can not only protect the biological activity of the small molecule polypeptide active substance, but also deliver it to the effective site, and fully exert the biological activity of the small molecule polypeptide active substance; the active substance has fast effect and is easy to be absorbed by the skin; has a single effect, promotes the penetration of therapeutic drugs through the skin, and does not affect the in vivo process of endogenous substances; has no interaction with excipients and active substances, and can effectively increase the transdermal absorption of drugs.

[0095] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and they should be included in the scope of the present application.

Claims

1. A lipopeptide-small molecule polypeptide supramolecular composition, characterized in that: Comprising, small molecule polypeptide active, amino acid and lipopeptide; The small molecule polypeptide active is 0.1% to 5%, the amino acid is 0.1% to 10%, and the lipopeptide makes up to 100% based on the total weight of the composition. The lipopeptide is one or more of surfactin, fengycin, palmitoyl oligopeptide, di(lauroylamide glutamine) lysine, iturin, bacillomycin and polymyxin. The small molecule polypeptide active is one or more of blue copper peptide, acetyl octapeptide-3 and acetyl hexapeptide-8. The amino acid is one or more of arginine, lysine, histidine, arginine ethyl ester, lysine ethyl ester and arginine monoglyceride.

2. The composition of claim 1, wherein: The lipopeptide and the amino acid are combined together through proton exchange, hydrogen bond and π-π interaction to form a supramolecular structure.

3. The composition of claim 1 or 2, wherein: The composition spontaneously forms a vesicle structure of 10 to 100 nm in water.

4. Process for the preparation of a composition according to any one of claims 1 to 3, characterized in that: Comprising, The lipopeptide, the small molecule polypeptide active, the amino acid and deionized water are uniformly mixed under an inert gas atmosphere, and then heated and stirred to react; After cooling to room temperature, the mixture is subjected to high-pressure homogenization under a pressure of 400 to 800 bar, and then freeze-dried under vacuum to obtain the composition.

5. The production method according to claim 4, characterized by: The homogenization is performed 2 to 8 times.

6. The production method according to claim 4, characterized by: The heating and stirring reaction, wherein, The heating temperature is 25 to 60℃, and the stirring reaction time is 12 to 24 h.

7. Use of the composition of any one of claims 1 to 3 in the preparation of a medicament having efficacy in delivery and sustained release.

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