Composition for promoting generation of elastin as well as preparation method and application of composition

Abstract

The invention discloses a composition for promoting generation of elastin as well as a preparation method and application of the composition, and belongs to the field of medical, dental or cosmetic preparations. The composition is prepared from a plant extract, an exosome, carboxymethyl chitosan, acetylated sodium hyaluronate, soluble collagen, soluble proteoglycan, tremella polysaccharide and a plurality of deionized water according to a modern freeze-drying process, and the raw materials and the exosome mutually promote the function of promoting elastin generation. The production of elastin is remarkably promoted, the elasticity of aged or damaged skin is effectively improved, the raw materials are cheap, and the composition is suitable for industrial production.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical, dental or cosmetic preparations, and particularly relates to a composition for promoting elastin production, a preparation method thereof and an application thereof. Background Art

[0002] Large skin elastin is an important protein, mainly present in the skin, blood vessels, lungs and other connective tissues. Its main function is to endow tissues with elasticity and toughness, enabling them to return to their original state after being stretched or compressed. Studies have found that the precursor of elastin, tropoelastin, begins to be expressed in the late fetal life, reaches a relatively high level in the neonatal stage, and then gradually decreases. There is almost no new elastin production after adulthood. The half-life of elastin is about 70 years, so it needs to function throughout the year but is not renewed for decades. With age, or long-term ultraviolet exposure, or bad habits such as smoking, poor eating habits and lack of sleep, the elastin in the skin will be damaged, accelerating skin aging. Therefore, the production of elastin is crucial in the process of skin repair and anti-aging.

[0003] CN113453674A discloses a promoter for the production of type I collagen or elastin, which mainly uses natural mycosporine-like amino acids or salts as active ingredients. It is found that mycosporine-like amino acids or their salts have a strong promoting effect on the production of type I collagen and elastin, which is so strong that it cannot be imagined by the promoting effect of fibroblast growth. CN115515558A discloses an elastin production promoter and a skin cosmetic, which mainly uses neuraminidase as an active ingredient. Applying it to skin cosmetics can improve or prevent the reduction of dermal elastin with age.

[0004] Exosomes are extracellular matrix vesicles secreted by stem cells such as umbilical cord mesenchymal stem cells, umbilical cord stem cells, and placental stem cells, and contain various cytokines and protein components such as transforming growth factor B, vascular endothelial growth factor, hepatocyte growth factor, superoxide dismutase, interleukin 6, epidermal growth factor, collagen, fibronectin, platelet-derived growth factor, etc., and have the effects of preventing oxidative damage, inhibiting inflammatory reactions, endothelial repair, tissue and organ repair, metabolism regulation, immune system regulation, etc.

[0005] CN110279648A discloses a beauty preparation containing exosomes and its uses. It uses exosomes secreted by adipose-derived mesenchymal stem cells as the active ingredient, which has the activity of inhibiting elastase and collagenase, can effectively combat aging, improve skin elasticity, and improve skin wrinkles. When used in combination with other whitening compositions on the skin, it can achieve a better effect of whitening and firming the skin. CN117106704A discloses an exosome composition for mesoderm anti-aging, its preparation method and applications. It uses exosomes derived from fibroblasts and exosomes derived from mesenchymal stem cells prepared according to a volume ratio of 1:1, which can significantly promote collagen synthesis, the production of elastin and extracellular matrix remodeling, and is crucial for maintaining skin firmness and elasticity.

[0006] The above-mentioned prior art uses exosomes as the active ingredient in beauty preparations and other products, which can promote the production of elastin and effectively improve skin elasticity. However, there are still some drawbacks: First, exosomes are mainly derived from stem cells, and the preparation process is complex and expensive. The public needs a relatively inexpensive cosmetic. Second, no substances that promote each other have been found to synergistically promote the production of elastin with exosomes. Summary of the Invention

[0007] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a composition for promoting the production of elastin, its preparation method and applications to solve the problems mentioned in the background technology.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] The composition for promoting the production of elastin includes the following components in parts by mass:

[0010]

[0011] Preferably, it further includes at least one of the following plant extracts:

[0012] Bactris gasipaes fruit extract, Saussurea involucrata extract, Agave tequilana leaf extract;

[0013] The total addition amount of the plant extracts is 5 - 35.

[0014] Furthermore, the specific dosage of the plant extracts is:

[0015] Bactris gasipaes fruit extract 2 - 25

[0016] Saussurea involucrata extract 0.5 - 2

[0017] Agave tequilana leaf extract 2.5 - 10.

[0018] Preferably, the exosomes are extracted from human cell supernatant.

[0019] The preparation method of the aforementioned composition for promoting elastin production includes the following steps:

[0020] S1. Prepare component phases A and B; wherein phase A includes water, tremella polysaccharide, carboxymethyl chitosan, and sodium acetylated hyaluronate; phase B includes exosomes, soluble collagen, and soluble proteoglycan.

[0021] S2. Mix part of phase A and stir to at least 85°C until completely dissolved; seal and sterilize under high pressure and high temperature; then cool to ≤33°C;

[0022] S3. Mix and dissolve phases A and B and stir evenly; spread and freeze-dry.

[0023] Preferably, the phase B further includes plant extracts.

[0024] Preferably, the freeze-drying in step S3 adopts the following steps:

[0025] S1. Pre-freeze the product to -40±5°C and keep warm for at least 180 minutes;

[0026] S2. First temperature rise: -15°C for 60 minutes; keep warm at -15°C for at least 180 minutes;

[0027] S3. Second temperature rise: -5°C for 60 minutes; keep warm at 5°C for at least 180 minutes;

[0028] S4. Third temperature rise: 5°C for 60 minutes; keep warm at 5°C for at least 180 minutes;

[0029] S5. Fourth temperature rise: 30°C for 60 minutes; keep warm at 30°C for at least 180 minutes.

[0030] Preferably, during the freeze-drying process, the vacuum degree is maintained at 23±3 Pa.

[0031] The aforementioned composition for promoting elastin production is used in the preparation of skin care products, drugs, and medical beauty products.

[0032] It should be noted that carboxymethyl chitosan can promote wound healing in the field of medical health. By promoting the growth of fibroblasts and epidermal keratinocytes, improving the phagocytic ability of macrophages, it can accelerate the wound repair process.

[0033] Sodium acetylated hyaluronate is a chemical modification of natural sodium hyaluronate (HA) by introducing an acetyl group. Its bioavailability is 7 times that of ordinary HA, and its tolerance to hyaluronidase is better than that of ordinary HA. It can inhibit the generation of intracellular superoxide anion free radical ROS and improve the proliferation activity of fibroblasts after UVB irradiation.

[0034] Soluble collagen is obtained by using the human collagen gene as a template, optimizing and modifying it, and through fermentation induction, separation and purification. The obtained recombinant soluble collagen is highly consistent with human own collagen and has good skin affinity and tissue compatibility.

[0035] Soluble proteoglycan can promote the proliferation of keratinocytes and the synthesis of hyaluronic acid, promote the proliferation of fibroblasts, and promote fibroblasts to synthesize collagen and hyaluronic acid, thereby achieving the anti-aging effect.

[0036] The present invention specifically selects three plant extracts: Euterpe oleracea fruit extract, Saussurea involucrata extract, and Agave tequilana leaf extract:

[0037] Euterpe oleracea fruit extract (i.e., acai berry) is rich in various nutrients such as amino acids, oils, and anthocyanins, has a significant antioxidant effect, and can further promote the production of elastin in combination with other ingredients.

[0038] Saussurea involucrata extract is rich in antioxidant substances, can effectively scavenge free radicals in the body, and delay the aging process of the skin. It can also promote the production of collagen and elastin, make the skin more firm and elastic, and reduce the generation of wrinkles.

[0039] Agave tequilana leaf extract contains rich nutrients such as vitamin E and zinc, which are beneficial to the skin, can promote the synthesis of elastin, thereby enhancing skin elasticity; it also has the effects of moisturizing, soothing and anti-allergic, and active ingredients.

[0040] Compared with the prior art, the beneficial effects of the present invention are:

[0041] 1. The composition of the present invention promotes each other with exosomes in the function of promoting the production of elastin by adding substances such as plant extracts, polysaccharides, and sodium acetylated hyaluronate, greatly saving the production cost and enabling the composition to benefit the people.

[0042] 2. Through experimental determination, the composition of the present invention can significantly promote the production of elastin, thereby enhancing the elasticity of aging or damaged skin, having the effect of firming and anti-aging, and having important application value.

[0043] 3. The preparation process of the composition is simple. After the above freeze-drying process, different from the traditional powder form, it presents a novel flocculent structure appearance. The flocculent appearance completely retains the triple helix three-dimensional skeleton structure of macromolecular collagen and polysaccharides, without affecting the change of its molecular weight and molecular activity. After freeze-drying, there is no water residue in the composition, which is easy to store for a long time and is not easy to decompose and inactivate. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention will be further described with reference to the accompanying drawings. However, the embodiments shown in the drawings do not constitute any limitation to the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the following drawings without creative efforts.

[0045] Figure 1 It is the result of the expression of elastin gene by freeze-dried products 1-5 of the mixture. Detailed implementation manners

[0046] The embodiments of the present invention will be described in detail below. The examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention and should not be construed as a limitation to the present invention.

[0047] In the detailed implementation manners, unless otherwise specified, the experimental methods used are all conventional methods, and the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0048] The sources of some raw materials used in the present invention are as shown in Table 1 below:

[0049] Table 1

[0050] Name INCI Name Exosome / Carboxymethylchitosan CARBOXYMETHYLCHITOSAN Sodium Acetylated Hyaluronate SODIUMACETYLATEDHYALURONATE Soluble Collagen SOLUBLEELASTIN Soluble Proteoglycan SOLUBLEPROTEOGLYCAN Tremella Polysaccharide Tremella (Tremella fuciformis) Polysaccharide Euterpe oleracea Fruit Extract Euterpe oleracea Fruit Extract Saussurea involucrata Extract Saussurea involucrata Extract Agave tequilana Leaf Extract Agave tequilana Leaf Extract

[0051] Example 1 Preparation of a composition for promoting elastin production, including the following steps:

[0052] S1. Weigh the corresponding raw materials according to the following parts by mass

[0053] Phase A:

[0054]

[0055] Phase B:

[0056] Exosomes 5

[0057] Soluble collagen 0.15

[0058] Soluble proteoglycan 0.05

[0059] S2. Mix the components of Phase A and stir until at least 85 °C until completely dissolved; seal and sterilize under high pressure and high temperature; then cool to ≤ 33 °C;

[0060] S3. Mix and dissolve Phase A and Phase B and stir evenly; spread and freeze-dry. The freeze-drying adopts the following steps: during the freeze-drying process, the vacuum degree is maintained at 23 ± 3 Pa.

[0061] The product is pre-frozen to -40 ± 5 °C and kept warm for at least 180 minutes;

[0062] First temperature rise: -15°C for 60 minutes; keep at -15°C for at least 180 minutes;

[0063] Second temperature rise: -5°C for 60 minutes; keep at 5°C for at least 180 minutes;

[0064] Third temperature rise: 5°C for 60 minutes; keep at 5°C for at least 180 minutes;

[0065] Fourth temperature rise: 30°C for 60 minutes; keep at 30°C for at least 180 minutes.

[0066] Finally, turn off the freeze dryer. After adjusting the instrument to normal pressure and room temperature, take out the freeze-dried product.

[0067] Preparation of the composition for promoting elastin production in Example 2

[0068] The difference from Example 1 is that:

[0069] S1. Weigh the corresponding raw materials according to the following parts by mass

[0070] Phase A:

[0071]

[0072] Phase B:

[0073]

[0074] Preparation of the composition for promoting elastin production in Example 3

[0075] The difference from Example 1 is that:

[0076] S1. Weigh the corresponding raw materials according to the following parts by mass

[0077] Phase A:

[0078]

[0079] Phase B:

[0080]

[0081]

[0082] The difference between the preparation of the composition for promoting elastin production in Example 3 and Example 1 is that:

[0083] S1. Weigh the corresponding raw materials, Phase A:

[0084]

[0085] Phase B:

[0086]

[0087] Example 4 The difference in the preparation of the composition for promoting elastin production from Example 1 lies in:

[0088] S1. Weigh the corresponding raw material Phase A according to the following parts by mass:

[0089]

[0090] Phase B:

[0091]

[0092]

[0093] Example 5 The difference in the preparation of the composition for promoting elastin production from Example 1 lies in:

[0094] S1. Weigh the corresponding raw material Phase A according to the following parts by mass:

[0095]

[0096] Phase B:

[0097]

[0098] Example 6 The difference in the preparation of the composition for promoting elastin production from Example 1 lies in:

[0099] S1. Weigh the corresponding raw material Phase A according to the following parts by mass:

[0100]

[0101] Phase B:

[0102]

[0103]

[0104] Example 7 The difference in the preparation of the composition for promoting elastin production from Example 1 lies in:

[0105] S1. Weigh the corresponding raw material Phase A according to the following parts by mass:

[0106]

[0107] Phase B:

[0108] Exosomes 15 Soluble collagen 0.15 Soluble proteoglycan 0.05.

[0109] Example 8 The preparation of the composition for promoting elastin production is different from that of Example 1 in that:

[0110] S1. Weigh the corresponding raw materials of Phase A according to the following parts by mass:

[0111]

[0112] Phase B:

[0113] Exosomes 20 Soluble collagen 0.25 Soluble proteoglycan 0.1.

[0114] Example 9 The preparation of the composition for promoting elastin production is different from that of Example 1 in that:

[0115] S1. Weigh the corresponding raw materials of Phase A according to the following parts by mass:

[0116]

[0117] Phase B:

[0118] Exosomes 25

[0119] Soluble collagen 0.25

[0120] Soluble proteoglycan 0.1.

[0121] Example 10 The preparation of the composition for promoting elastin production

[0122] is different from that of Example 1 in that:

[0123] S1. Weigh the corresponding raw materials

[0124] Phase A:

[0125]

[0126] Phase B:

[0127] Exosomes 5

[0128] Soluble collagen 0.15

[0129] Soluble proteoglycan 0.05.

[0130] Test on the effect on elastin synthesis

[0131] Test the effect of the freeze-dried products prepared in the above partial examples on elastin synthesis by molecular biology methods.

[0132] Test principle: After adding raw materials or products to the cell culture system, it may cause changes in the gene transcription of molecules in the cells, resulting in changes in the amount of mRNA and corresponding changes in the expression level of proteins. By quantitatively analyzing the mRNA in the cells, the gene-level detection of the synthesis and expression of molecules can be carried out.

[0133] Evaluation index: The expression level of the elastin (ELN) gene.

[0134] Judgment criterion: Compared with the untreated control group, if the expression level of the ELN gene shows a significant increase after sample treatment, it indicates that the sample can promote the expression of the ELN gene and has the effects of firming, anti-wrinkle and reducing fine lines.

[0135] Main reagents:

[0136] 1. TRIzol reagent, brand: Thermo Fisher Scientific;

[0137] 2. One-step reverse transcription kit, brand: Novoprotein;

[0138] 3. Fluorescent quantitative kit, brand: Qiagen;

[0139] 4. DEPC-water, brand: Yuanye Bio-Technology;

[0140] 5. Chloroform, brand: Guangzhou Chemical Reagent Factory;

[0141] 6. Isopropanol, brand: Guangzhou Chemical Reagent Factory;

[0142] 7. FBS fetal bovine serum, brand:

[0143] Experimental method: After the human skin fibroblast BJ cell line in the 6-well plate grows to about 70% confluence, it is replaced with a medium containing 2% FBS. After starvation culture for 24 hours, the medium is replaced with a medium containing different concentrations of the test sample and treated for 24 hours. TRIzol reagent is used to extract RNA from the cells. After measuring the RNA concentration, the mRNA is reverse transcribed into cDNA, and fluorescence quantitative PCR is used as a template to detect the synthesis and expression of the test molecule at the gene level. Each sample is tested independently 3 times, and the results are shown in the form of mean ± standard deviation.

[0144] Test substances:

[0145] Test samples: Lyophilized products of mixtures T1-T5;

[0146] Test molecules: Elastin (ELN);

[0147] Reference calibration gene: β-actin (ACTB);

[0148] Blank control: Cells treated with medium containing 2% FBS for 24 hours.

[0149] The RNA concentrations in cells after the action of the freeze-dried products of mixtures T1 - T5 for 24 hours are shown in Table 2:

[0150] Table 2 RNA concentrations in cells after the action of the freeze-dried products of mixtures T1 - T5 for 24 hours

[0151]

[0152] Note: OD260 represents the absorbance of the sample at 260 nm; OD280 represents the absorbance of the sample at 280 nm; the RNA concentration is calculated according to the formula: RNA concentration = OD260 × 0.04 × dilution factor.

[0153] The results of fluorescence quantitative PCR are shown in Table 3:

[0154] Table 3 Gene expression results in cells after the action of the freeze-dried products of mixtures T1 - T5 for 24 hours

[0155]

[0156] Using the specific primers of the molecule to be detected, and taking the cDNA obtained after treating cells with different samples as the template for fluorescence quantitative PCR, by comparing the difference in the C T value between the target gene and the reference gene (ACTB), the relative quantification of the target gene can be achieved, that is, the 2 -ΔΔCT method, where ΔC T is the difference between the C T value of the target gene and the C T value of the reference gene, and ΔΔC T is the difference in ΔC T between different samples. Using GraphPad Prism 8 software to plot the 2 -ΔΔCT values Figure 1 to compare the differences between each gene sample and the blank control group.

[0157] As Figure 1 shown, after treating cells with the freeze-dried products of Examples 1 - 5, the synthesis level of elastin can be significantly increased. Compared with the blank control, after treating with the freeze-dried products 1, 2, 3, 4, 5 of Examples 1 - 5 respectively, the synthesis of elastin gene increased significantly to 2.34, 11.54, 14.32, 19.63, 12.15 times respectively, which can significantly promote the synthesis of elastin in the extracellular matrix of the skin dermis.

[0158] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. Any equivalent structure or equivalent process transformation made by using the content of the specification and drawings of the present invention, or directly or indirectly applied in other related technical fields, shall be similarly included in the patent protection scope of the present invention.

Claims

1. A composition for promoting elastin production, characterized in that: The following components are included in parts by mass:

2. The composition for promoting elastin production according to claim 1, characterized in that: Also includes at least one of the following plant extracts: Vegetarian Euterpe palm fruit extract, snow lotus extract, Agave tatarinowii leaf extract; The total added amount of the plant extract is 5-35.

3. The composition for promoting elastin production according to claim 2, characterized in that: Also includes the following components: Vegetarian Euterpe palm fruit extract 2-25 Snow lotus extract 0.5-2 Agave tataricus leaf extract 2.5-10.

4. The composition for promoting elastin production according to any one of claims 1 to 3, characterized in that: The exosomes are extracted from the supernatant of human cells.

5. The method for preparing the composition for promoting elastin production according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1, combine the components phase A and phase B; Phase A includes water, Tremella polysaccharide, carboxymethyl deacetylated chitosan, and acetylated sodium hyaluronate; Phase B includes exosomes, soluble collagen, and soluble proteoglycan. S2. Mix and stir the A phase to at least 85°C until completely dissolved; seal and sterilize under high pressure; then cool to ≤33°C; S3, mix and dissolve phase A and phase B and stir evenly; spread and freeze-dry.

6. The method for preparing the composition for promoting elastin production according to claim 5, characterized in that: The phase B also includes plant extracts.

7. The method for preparing the composition for promoting elastin production according to claim 5, characterized in that: The freeze-drying in step S3 adopts the following steps: S1. Pre-freeze the product to -40±5℃ and keep warm for at least 180 minutes; S2, first temperature rise: -15℃ for 60 minutes: -15℃ for at least 180 minutes; S3, second temperature rise: -5℃ for 60 minutes: 5℃ for at least 180 minutes; S4, the third temperature rise: 60 minutes at 5℃ and at least 180 minutes at 5℃; S5. Fourth heating: 60 minutes at 30℃; keep warm at 30℃ for at least 180 minutes.

8. The method for preparing the composition for promoting elastin production according to claim 7, characterized in that: During the freeze-drying process, the vacuum degree was maintained at 23 ± 3 Pa.

9. Use of the composition for promoting elastin production according to any one of claims 1 to 4 in the preparation of skin care products, medicines, and medical beauty products.

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