Anti-aging recombinant humanized collagen composition as well as preparation method and application thereof

Abstract

The invention discloses an anti-aging recombinant humanized collagen composition as well as a preparation method and application thereof, the composition comprises recombinant humanized collagen, vegetable oil and plant exosome freeze-dried powder, and the recombinant humanized collagen and the vegetable oil are bonded into ionic liquid. According to the composition, the percutaneous permeability of the recombinant humanized collagen can be improved, and the in-vitro normal-temperature storage stability of the plant exosome is improved. The invention also provides a preparation method of the composition, the recombinant humanized collagen and the vegetable oil are respectively atomized into positively charged particles and negatively charged particles through an electrostatic atomization method, so that the acid-base neutralization reaction activation energy of the recombinant humanized collagen and the vegetable oil is reduced, and the recombinant humanized collagen and the vegetable oil are promoted to be bonded into ionic liquid through proton exchange.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to an anti-aging recombinant humanized collagen composition and a preparation method and application thereof. Background Art

[0002] Recombinant humanized collagen is produced through genetic engineering technology. It has a structure and function that is highly similar to natural human collagen. Because its amino acid sequence is almost completely consistent with the body's own collagen, recombinant humanized collagen has a very low immune response in the skin, reducing the risk of allergies and rejection, and thus has excellent biocompatibility. Recombinant humanized collagen avoids viral contamination and impurities that may be caused by the extraction process, greatly improving the safety of the product. The application of recombinant humanized collagen in cosmetics and skin care products brings many advantages: (1) Significant moisturizing effect: Collagen has good water absorption and water retention capacity, which can lock in moisture in the skin, keep the skin hydrated, and reduce dryness and the formation of fine lines; (2) Promote skin repair: Recombinant humanized collagen can stimulate the proliferation of fibroblasts and the synthesis of collagen, accelerate the repair process of damaged skin, and is suitable for postoperative care, sensitive skin repair and other scenarios; (3) Anti-aging effect: Collagen can enhance the elasticity and firmness of the skin, reduce wrinkles and sagging, delay the aging process of the skin, and make the skin smoother and more delicate; (4) Antioxidant properties: Recombinant humanized collagen has a certain antioxidant capacity, which can help neutralize free radicals and protect the skin from damage by the external environment (such as ultraviolet rays, pollutants, etc.).

[0003] Although recombinant humanized collagen shows many advantages in cosmetics and skin care products, its relatively large molecular weight limits its ability to penetrate the skin barrier, resulting in poor percutaneous absorption, making it difficult to exert its efficacy.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The present invention provides an anti-aging recombinant humanized collagen composition, which includes recombinant humanized collagen and ionic liquid prepared from plant oil. The ionic liquid recombinant humanized collagen has greatly improved skin permeation efficiency; and the ionic liquid has a good stabilizing effect on plant exosomes when used as a dispersant.

[0006] The present invention is achieved in that:

[0007] In the first aspect of the present invention, there is provided an anti-aging recombinant humanized collagen composition, which, calculated by mass percentage, comprises the following components: 53% - 71% of recombinant humanized collagen, 28% - 45% of vegetable oil, and 0.5% - 2.5% of freeze-dried plant exosomes. The recombinant humanized collagen and the vegetable oil are bonded to form an ionic liquid.

[0008] In some embodiments, the molecular weight of the recombinant humanized collagen is 2 kDa - 60 kDa; the recombinant humanized collagen is selected from at least one of recombinant type I humanized collagen, recombinant type III humanized collagen, and recombinant type XVII humanized collagen.

[0009] In some embodiments, the vegetable oil is selected from grape seed oil or Limnanthes alba seed oil.

[0010] In some embodiments, the plant exosomes are selected from at least one of paeonia exosomes, ginseng exosomes, pomegranate exosomes, and centella asiatica exosomes.

[0011] In some embodiments, the preparation method of the ionic liquid is as follows: The aqueous solution of recombinant humanized collagen is sprayed into the reaction kettle through a first electrostatic spraying gun to form positively charged particles, and the ethanol solution of vegetable oil is sprayed into the reaction kettle through a second electrostatic spraying gun to form negatively charged particles. The positively charged particles and the negatively charged particles are electrostatically attracted and combined and then gather at the bottom of the kettle. Then, stirring is started for homogenization, and the temperature of the reaction kettle is maintained at 40 - 60 °C until the liquid phase is uniform; the solvent is removed by vacuum distillation to obtain the ionic liquid.

[0012] In some embodiments, the process parameters when the first electrostatic spraying gun sprays the aqueous solution of recombinant humanized collagen include: voltage of 20 - 40 kV, current of 10 - 50 μA, and flow rate of 10 - 20 mL / min.

[0013] In some embodiments, the process parameters when the second electrostatic spraying gun sprays the ethanol solution of vegetable oil include: voltage of 20 - 40 kV, current of 10 - 50 μA, and flow rate of 4 - 8 mL / min.

[0014] In some embodiments, the percentage content of recombinant humanized collagen in the aqueous solution of recombinant humanized collagen is 1% - 3%.

[0015] In some embodiments, the percentage content of vegetable oil in the ethanol solution of vegetable oil is 1% - 5%.

[0016] In some embodiments, the conditions for homogenization include: stirring speed of 1000 - 3000 rpm.

[0017] Second aspect, the present invention provides a method for preparing the anti-aging recombinant humanized collagen composition in any of the above embodiments, comprising the following steps: dispersing the freeze-dried powder of plant exosomes in an ionic liquid to obtain a suspension, thereby preparing the anti-aging recombinant humanized collagen composition.

[0018] In some embodiments, the freeze-dried powder of plant exosomes is obtained by mixing an exosome solution with a lyoprotectant and freeze-drying.

[0019] In some embodiments, the lyoprotectant includes mannitol and sugars, and the sugars are selected from at least one of trehalose, glucose, sucrose, and pectin.

[0020] In some embodiments, the exosome solution is obtained by soaking the fragmented plant material in a buffer solution and separating it by differential centrifugation or density gradient centrifugation.

[0021] Third aspect, the present invention provides the application of the anti-aging recombinant humanized collagen composition in any of the above embodiments. The anti-aging recombinant humanized collagen composition can be applied to skin care products, such as creams, serums, lotions, etc.

[0022] Fourth aspect, the present invention provides an anti-aging essence, which, calculated by mass percentage, comprises the following components: 3% - 5% of the anti-aging recombinant humanized collagen composition in any of the above embodiments, 0.8% - 2% of an emulsifier, 0.2% - 0.5% of a thickener, 1% - 3% of an emollient, 5% - 12% of a humectant, 0.1% - 0.3% of a preservative, 0.03% - 0.08% of a pH regulator, 0.02% - 0.08% of disodium EDTA, and the balance is water.

[0023] In some embodiments, the emulsifier is selected from at least one of PEG hydrogenated castor oil, polysorbate, span, polyglycerol laurate, polyglycerol oleate, polyglycerol myristate, cetyl alcohol phosphate, and cetostearyl alcohol polyether-20.

[0024] In some embodiments, the thickener is selected from at least one of carbomer, xanthan gum, and hydroxyethyl cellulose.

[0025] In some embodiments, the emollient is selected from at least one of triglyceride caprylic / capric acid, squalane, olive oil, polydimethylsiloxane, isononyl isononanoate, and neopentyl glycol diheptanoate.

[0026] In some embodiments, the humectant is at least one of allantoin, sodium hyaluronate, glycerol polyether-26, propylene glycol, dipropylene glycol, sorbitol, glycerol, butylene glycol, betaine, erythritol, and trehalose;

[0027] In some embodiments, the pH regulator is selected from at least one of triethanolamine and citric acid.

[0028] In some embodiments, the preservative is at least one of phenoxyethanol, ethylhexylglycerin, methylparaben, and p-hydroxyacetophenone.

[0029] The present invention has the following beneficial effects:

[0030] (1) The present invention provides an anti-aging recombinant humanized collagen composition, which can improve the percutaneous permeability of recombinant humanized collagen and enhance the in vitro room temperature storage stability of plant exosomes.

[0031] (2) The present invention provides a preparation method of an anti-aging recombinant humanized collagen composition. By the electrostatic atomization method, recombinant humanized collagen and vegetable oil are atomized into positively charged particles and negatively charged particles respectively, thereby reducing the activation energy of the acid-base neutralization reaction between the two and promoting the bonding of the two into an ionic liquid through proton exchange. Description of the Drawings

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and thus should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other relevant drawings can also be obtained based on these drawings.

[0033] Figure 1 It is the curve of the light transmittance of PO in Examples 1-3 changing with temperature;

[0034] Figure 2 It is the physical diagram of the collagen-vegetable oil mixture obtained from PO in Example 1 and Comparative Example 1;

[0035] Figure 3 It is the differential scanning calorimetry (DSC) test results of recombinant humanized collagen, vegetable oil, the ionic liquid PO in Example 1, and the mixture of collagen and vegetable oil in Comparative Example 1.

[0036] Figure 4 It is the curve of the percutaneous permeation amount of PO in Example 1, the collagen-vegetable oil mixture obtained from Comparative Example 1, and the collagen aqueous solution changing with time;

[0037] Figure 5 It is the curve of the average diameter of exosomes in the compositions of Example 1, Comparative Example 4, and Comparative Example 5 changing with time. Detailed Embodiments

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can all be obtained as conventional products through commercial purchase.

[0039] In the description of the embodiments of the present application, technical terms such as "first" and "second" are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the quantity, specific order, or primary and secondary relationship of the indicated technical features.

[0040] The mention of "embodiment" in this article means that the specific features, structures, or characteristics described in combination with the embodiment may be included in at least one embodiment of the present application. The occurrence of this phrase in various positions in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments.

[0041] In the embodiments of the present application, the term "or / and" is only a description of the association relationship of associated objects, indicating that there can be three relationships. For example, A or / and B can represent three situations: A exists alone, A and B exist simultaneously, and B exists alone.

[0042] In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0043] In the embodiments of the present application, the meaning of "a plurality of" refers to two or more (including two). Similarly, "a plurality of groups" refers to two or more groups (including two groups), and "a plurality of layers" refers to two or more layers (including two layers), unless otherwise clearly specified and limited.

[0044] In the embodiments of the present application, the meaning of "at least one" refers to one or more than one.

[0045] In the embodiments of the present application, the orientation or position relationship indicated by technical terms such as "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", etc. is based on the orientation or position relationship shown in the drawings. This is only for the convenience of describing the embodiments of the present application and simplifying the description, rather than indicating or implying that the indicated device or element must have a specific orientation, be constructed in a specific orientation, etc. It cannot be understood as a limitation on the embodiments of the present application. Those skilled in the art can understand the specific meanings of the above terms in the embodiments of the present application according to specific circumstances.

[0046] In a first aspect of the present invention, an anti-aging recombinant humanized collagen composition is provided, which comprises the following components by mass percentage: 53% to 71% recombinant humanized collagen, 28% to 45% vegetable oil, and 0.5% to 2.5% plant exosome freeze-dried powder. The recombinant humanized collagen and the vegetable oil are bonded to form an ionic liquid.

[0047] Recombinant humanized collagen shows many advantages in cosmetics and skin care products, but its relatively large molecular weight limits its ability to penetrate the skin barrier, and its percutaneous absorption is poor, which makes it difficult to exert its efficacy. The present invention improves its percutaneous permeability by ionizing recombinant humanized collagen and vegetable oil. The reason is that, on the one hand, after being assembled into an ionic liquid, the vegetable oil enhances the hydrophobicity of the molecule, making the assembled molecule more lipophilic, and the long-chain alkyl of the vegetable oil acts as a "switch", which is conducive to the introduction of hydrophilic recombinant humanized collagen; on the other hand, due to its hydrophilicity, recombinant humanized collagen usually has a large hydration radius in aqueous solution, but after being assembled into an ionic liquid with vegetable oil, its molecular radius is reduced, which is conducive to passing through the skin barrier.

[0048] Plant exosomes are nanoscale vesicles secreted by plant cells. These vesicles contain a variety of functional proteins, RNAs and metabolites, and have the potential to regulate intercellular communication, promote cell repair and enhance skin barrier function. Plant exosomes are derived from natural plants and have good biocompatibility with human skin, reducing the risk of allergic reactions and other side effects. Studies have shown that plant exosomes can carry a variety of bioactive molecules, including antioxidants, anti-inflammatory factors and growth factors, which help improve skin health. However, exosomes have low stability and are easily inactivated. In order to maintain the structural integrity of exosomes, such preparations must be maintained and transported at relatively low temperatures to maintain their biological activity. However, the loss of exosome biological activity often occurs during the storage and preservation stage. Generally, liquid preparations are made and stored at ultra-low temperatures (for example, not higher than -60°C), transported under low-temperature freezing conditions, and need to be thawed before use. In addition, if the ultra-low temperature storage time is too long, the exosomes are repeatedly frozen and thawed during use, or are used improperly, the biological activity of the exosomes will often drop rapidly and significantly, thereby limiting its normal use.

[0049] In the composition provided by the present invention, the ionic liquid can improve the in vitro storage stability of plant exosomes at room temperature, making it possible to be applied to skin care products stored at room temperature. The reason is that the ionic liquid contains a large amount of hydrophobic alkyl (derived from plant oil) and has high compatibility with the double-layer phospholipid membrane of the exosome, so that it can disperse the exosomes well, and there is no liquid exchange condition, thereby preventing the exosomes from aggregating or degrading.

[0050] In some embodiments, the molecular weight of the recombinant humanized collagen is 2 kDa to 60 kDa, for example, it can be 2 kDa, 2.8 kDa, 4 kDa, 10 kDa, 20 kDa, 30 kDa, 40 kDa, 50 kDa, 55 kDa, 60 kDa or any value within 2 kDa to 60 kDa; the recombinant humanized collagen is selected from at least one of recombinant type I humanized collagen, recombinant type III humanized collagen, and recombinant type XVII humanized collagen.

[0051] In some embodiments, the vegetable oil is selected from grape seed oil or Limnanthes alba seed oil.

[0052] Grape seed oil and Limnanthes alba seed oil have a high content of long-chain fatty acids, strong stability and skin-friendly properties. As the anion of the ionic liquid, they can improve the affinity of the ionic liquid for the skin, thus facilitating the introduction of recombinant humanized collagen.

[0053] Meanwhile

[0054] Grape seed oil is an active ingredient with antioxidant, moisturizing, and anti-inflammatory properties widely used in the skincare field. It is rich in polyphenolic compounds, especially proanthocyanidins (OPC), which are powerful natural antioxidants. Antioxidants can neutralize free radicals, reduce the damage of oxidative stress to skin cells, and thus delay the skin aging process; Limnanthes alba seed oil has the function of preventing water loss and regenerating the skin, and is a skin metabolism activator; it locks in moisture in the skin epidermis by changing the keratin tissue, regulating the exudation of water and compounds, reabsorbing water, and forming hydrates in the epidermal layer cells.

[0055] In some embodiments, the plant exosomes are selected from at least one of paeonia exosomes, ginseng exosomes, pomegranate exosomes, and centella asiatica exosomes.

[0056] Peony exosomes have anti-inflammatory, antioxidant, and moisturizing effects, which help enhance the skin barrier function, lock in moisture, and prevent skin dryness. Ginseng is rich in active ingredients such as ginsenosides, and its exosomes can promote the proliferation and repair of skin cells, helping to accelerate wound healing. The nutrients in ginseng exosomes help stimulate the production of collagen and elastin, making the skin firmer and more elastic. Ginseng exosomes contain powerful antioxidants that can combat free radicals, delay the skin aging process, and reduce the appearance of wrinkles and fine lines. Ginseng exosome-like has a protective effect on skin damage caused by ultraviolet rays and oxidative stress and can inhibit the expression of pro-apoptotic genes. Pomegranate exosomes have antioxidant capacity. Pomegranate is rich in polyphenolic compounds, especially ellagic acid, and these components have strong antioxidant properties, which help resist environmental damage and protect the skin from free radical damage; pomegranate exosomes help reduce skin inflammatory responses and relieve discomfort in sensitive skin; pomegranate exosomes may help inhibit melanin production, even out skin tone, and make the skin look brighter and healthier. Centella asiatica exosomes can repair damaged skin, accelerate wound healing, and improve scar tissue; Centella asiatica exosomes have obvious anti-inflammatory properties and are suitable for treating inflammatory skin diseases such as eczema and dermatitis. The active ingredients in Centella asiatica exosomes help stimulate collagen production, strengthen the skin structure, and enhance skin firmness.

[0057] In some embodiments, the preparation method of the ionic liquid is as follows: The aqueous solution of recombinant humanized collagen is sprayed into the reaction kettle through the first electrostatic spraying gun to form positively charged particles, and the ethanol solution of vegetable oil is sprayed into the reaction kettle through the second electrostatic spraying gun to form negatively charged particles. The positively charged particles and the negatively charged particles are electrostatically attracted and combined and then gather at the bottom of the kettle. Then, stirring is started for homogenization, and the temperature of the reaction kettle is maintained at 40 - 60 °C until the liquid phase is uniform; the solvent is removed by vacuum distillation to obtain the ionic liquid.

[0058] In the present invention, recombinant humanized collagen and vegetable oil are atomized into positively charged particles and negatively charged particles respectively by the electrostatic atomization method, thereby reducing the activation energy of the acid-base neutralization reaction between the two and promoting the bonding of the two into an ionic liquid through proton exchange.

[0059] In some embodiments, the process parameters when the first electrostatic spraying gun sprays the aqueous solution of recombinant humanized collagen include: voltage of 20 - 40 kV, current of 10 - 50 μA, and flow rate of 10 - 20 mL / min.

[0060] In some embodiments, the process parameters when the second electrostatic spraying gun sprays the ethanol solution of vegetable oil include: voltage of 20 - 40 kV, current of 10 - 50 μA, and flow rate of 4 - 8 mL / min.

[0061] The parameters of electrostatic spraying guns have an impact on the charge, particle size, flight speed, and spraying range of atomized droplets. Specifically, the greater the voltage, the greater the electric field strength, the faster the flight speed of charged particles, and the larger the spraying range. The greater the current, the greater the number of charged particles. The greater the flow rate, the larger the particle size of the atomized droplets.

[0062] In some embodiments, the percentage content of recombinant humanized collagen in the aqueous solution of recombinant humanized collagen is 1% - 3%.

[0063] In some embodiments, the percentage content of vegetable oil in the ethanol solution of vegetable oil is 1% - 5%.

[0064] In some embodiments, the conditions for homogenization include: the stirring speed is 1000 - 3000 rpm.

[0065] In a second aspect, the present invention provides a method for preparing the anti-aging recombinant humanized collagen composition in any of the above embodiments, including the following steps: dispersing the freeze-dried powder of plant exosomes in an ionic liquid to obtain a suspension, thereby preparing the anti-aging recombinant humanized collagen composition.

[0066] In some embodiments, the freeze-dried powder of plant exosomes is obtained by mixing an exosome solution with a lyoprotectant and freeze-drying.

[0067] In some embodiments, the lyoprotectant includes mannitol and sugars, and the sugars are selected from at least one of trehalose, glucose, sucrose, and pectin.

[0068] In some embodiments, the exosome solution is obtained by soaking the fragmented plant raw materials in a buffer solution and separating the exosome solution by differential centrifugation or density gradient centrifugation.

[0069] In a third aspect, the present invention provides the application of the anti-aging recombinant humanized collagen composition in any of the above embodiments. The anti-aging recombinant humanized collagen composition can be applied to skin care products, such as facial creams, essence, lotions, etc.

[0070] In a fourth aspect, the present invention provides an anti-aging essence, which, calculated by mass percentage, includes the following components: 3% - 5% of the anti-aging recombinant humanized collagen composition in any of the above embodiments, 0.8% - 2% of an emulsifier, 0.2% - 0.5% of a thickener, 1% - 3% of an emollient, 5% - 12% of a humectant, 0.1% - 0.3% of a preservative, 0.03% - 0.08% of a pH regulator, 0.02% - 0.08% of disodium EDTA, and the balance is water.

[0071] In some embodiments, the emulsifier is selected from at least one of PEG hydrogenated castor oil, polysorbate, span, polyglycerol laurate, polyglycerol oleate, polyglycerol myristate, cetyl alcohol phosphate, and cetostearyl alcohol polyether-20.

[0072] In some embodiments, the thickener is selected from at least one of carbomer, xanthan gum, and hydroxyethyl cellulose.

[0073] In some embodiments, the emollient is selected from at least one of triglyceride caprylic capric, squalane, olive oil, polydimethylsiloxane, isononyl isononanoate, and neopentyl glycol diheptanoate.

[0074] In some embodiments, the humectant is at least one of allantoin, sodium hyaluronate, glycerol polyether-26, propylene glycol, dipropylene glycol, sorbitol, glycerol, butylene glycol, betaine, erythritol, and trehalose;

[0075] In some embodiments, the pH regulator is selected from at least one of triethanolamine and citric acid.

[0076] In some embodiments, the preservative is at least one of phenoxyethanol, ethylhexylglycerin, methylparaben, and p-hydroxyacetophenone.

[0077] The features and properties of the present invention will be further described in detail below in conjunction with examples.

[0078] Example 1

[0079] An anti-aging recombinant humanized collagen composition, calculated by mass percentage, comprises the following components: 61.56% recombinant humanized collagen, 36.94% vegetable oil, and 1.5% plant exosome freeze-dried powder.

[0080] The recombinant humanized collagen is recombinant type I humanized collagen with a molecular weight of 47 ± 5 kDa, self-made CIS90 type.

[0081] The vegetable oil is Limnanthes Alba seed oil, purchased from Jiangxi Kangshengtang Pharmaceutical Co., Ltd.

[0082] The plant exosome is pomegranate exosome, prepared by differential centrifugation method, and the specific steps are as follows:

[0083] (1) Put the peeled pomegranates into a crushing device, add the same mass of 1× phosphate buffer solution (PBS), crush and juice, filter, and collect the filtrate; centrifuge the filtrate at 4°C and 800g for 30 min, and collect the supernatant 1; centrifuge the supernatant 1 at 4°C and 2500g for 30 min, and collect the supernatant 2; centrifuge the supernatant 2 at 4°C and 8000g for 75 min, and collect the supernatant 3; centrifuge the supernatant 3 at 4°C and 14000g for 120 min, discard the supernatant, and add 9 times the mass of 1× PBS buffer solution to obtain a 10% mass concentration exosome solution derived from pomegranates.

[0084] Take 100 μL of the exosome solution and dilute it in 2 mL of PBS. Use a nanoparticle size analyzer to measure the exosome concentration. Repeat each sample 3 times. The measured average particle size is 80.11 nm, and the concentration is (5.22 ± 0.21) × 108 / mL.

[0085] (2) Add trehalose and mannitol to the exosome solution derived from pomegranates, stir evenly, and filter to obtain a freeze-drying solution; perform vacuum freeze-drying on the freeze-drying solution. First, pre-freeze at -80°C for 12 h, then place it in a freeze-dryer and freeze-dry at -50°C and 60 Pa for 30 h to obtain exosome freeze-dried powder.

[0086] The concentration of trehalose in the freeze-drying solution is 8%, and the concentration of mannitol in the freeze-drying solution is 5%.

[0087] The preparation method of the above anti-aging recombinant humanized collagen composition includes the following steps:

[0088] (1) Spray the recombinant humanized collagen aqueous solution with a concentration of 3 wt% into the reaction kettle through the first electrostatic spraying gun to form positively charged particles, and spray the vegetable oil ethanol solution with a concentration of 5 wt% into the reaction kettle through the second electrostatic spraying gun to form negatively charged particles. The positively charged particles and the negatively charged particles are electrostatically attracted and combined and then gather at the bottom of the kettle. Then, start stirring for homogenization, and keep the temperature of the reaction kettle at 50°C until the liquid phase is homogeneous.

[0089] The voltage of the first electrostatic spraying gun is 20 kV, the current is 10 μA, and the flow rate is 15 mL / min; the voltage of the second electrostatic spraying gun is 30 kV, the current is 15 μA, and the flow rate is 4 mL / min.

[0090] The mixing ratio of the recombinant humanized collagen aqueous solution and the vegetable oil ethanol solution is carried out according to the composition formula amount, that is, the mass ratio of recombinant humanized collagen to vegetable oil is 61.56:36.94.

[0091] The rotation speed during homogenization is 2000 rpm.

[0092] (2) The solution obtained in step (1) is distilled under reduced pressure at 50 °C to remove the solvent, obtaining ionic liquid PO.

[0093] (3) The freeze-dried powder of plant exosomes is dispersed in the ionic liquid to obtain a suspension, thus preparing the anti-aging recombinant humanized collagen composition.

[0094] Example 2

[0095] An anti-aging recombinant humanized collagen composition, calculated by mass percentage, includes the following components: 71% recombinant humanized collagen, 28.5% vegetable oil, 0.5% freeze-dried powder of plant exosomes.

[0096] The recombinant humanized collagen is recombinant type III humanized collagen with a molecular weight of 55 ± 5 kDa, self-made CS90 type.

[0097] The vegetable oil is grape seed oil, purchased from Jiangxi Kangshengtang Pharmaceutical Co., Ltd.

[0098] The plant exosomes are paeonia lactiflora exosomes, prepared by differential centrifugation. The specific steps are as follows:

[0099] (3) The washed paeonia lactiflora petals are put into a crushing device, and an equal mass of 1× phosphate buffer solution (PBS) is added. Crush and juice, filter, and collect the filtrate; Centrifuge the filtrate at 4 °C and 1000g for 30 min, collect the supernatant 1; Centrifuge the supernatant 1 at 4 °C and 5000g for 30 min, collect the supernatant 2; Centrifuge the supernatant 2 at 4 °C and 10000g for 60 min, collect the supernatant 3; Centrifuge the supernatant 3 at 4 °C and 20000g for 120 min, discard the supernatant, and add 9 times the mass of 1× PBS buffer solution to obtain a 10% mass concentration of paeonia lactiflora-derived exosome solution.

[0100] Take 100 μL of the exosome solution and dilute it in 2 mL of PBS. Use a nanoparticle size analyzer to measure the exosome concentration. Each sample is repeated 3 times, and the measured average particle size is 153.7 nm, and the concentration is (7.4 ± 0.15) × 107 / mL.

[0101] (4) Add sucrose and mannitol to the paeonia lactiflora-derived exosome solution, stir evenly and filter to obtain a freeze-drying solution; Vacuum freeze-dry the freeze-drying solution. First, pre-freeze at -80 °C for 12 h, then place it in a freeze-dryer, and freeze-dry at -50 °C and 60 Pa for 30 h to obtain the freeze-dried powder of exosomes.

[0102] The concentration of sucrose in the freeze-drying solution is 5%, and the concentration of mannitol in the freeze-drying solution is 4%.

[0103] The method for preparing the above-mentioned anti-aging recombinant humanized collagen composition comprises the following steps:

[0104] (1) spraying a 5 wt % recombinant humanized collagen aqueous solution into a reactor through a first electrostatic spray gun to form positively charged particles, spraying a 8 wt % vegetable oil ethanol solution into a reactor through a second electrostatic spray gun to form negatively charged particles, the positively charged particles and the negatively charged particles are electrostatically attracted and combined and collected at the bottom of the reactor, and then stirring is started for homogenization, and the reactor temperature is maintained at 60° C., and homogenization is performed until the liquid phase is uniform;

[0105] The voltage of the first electrostatic spray gun is 30 kV, the current is 20 μA, and the flow rate is 10 mL / min; the voltage of the second electrostatic spray gun is 40 kV, the current is 30 μA, and the flow rate is 2 mL / min;

[0106] The mixing ratio of the recombinant humanized collagen aqueous solution and the vegetable oil ethanol solution is carried out according to the formula of the composition, that is, the mass ratio of the recombinant humanized collagen to the vegetable oil is 71:28.5.

[0107] The rotation speed during homogenization was 3000 rpm.

[0108] (2) The solution obtained in step (1) is distilled under reduced pressure at 50° C. to remove the solvent, thereby obtaining an ionic liquid PO.

[0109] (3) Dispersing the plant exosome freeze-dried powder in an ionic liquid to obtain a suspension, thereby preparing an anti-aging recombinant humanized collagen composition.

[0110] Example 3

[0111] An anti-aging recombinant humanized collagen composition comprises the following components, calculated by mass percentage: 54.17% recombinant humanized collagen, 43.33% vegetable oil, and 2.5% plant exosome freeze-dried powder.

[0112] The molecular weight of the recombinant humanized collagen is 2.8 kDa recombinant type III humanized collagen ( Micro) was purchased from Nanjing Dinovihua Biotechnology Co., Ltd.

[0113] The vegetable oil is meadowfoam seed oil, which was purchased from Jiangxi Kangshengtang Pharmaceutical Co., Ltd.

[0114] The plant exosomes are Centella asiatica exosomes, which are prepared by gradient density centrifugation, and the specific steps are as follows:

[0115] (1) Put the washed Centella asiatica into a crushing device, add 1× phosphate buffer solution (PBS) of the same mass, crush and juice, filter, and collect the filtrate; centrifuge the filtrate at 4°C and 2000g for 10 min, and collect the supernatant 1; centrifuge the supernatant 1 at 4°C and 4000g for 30 min, and collect the supernatant 2; centrifuge the supernatant 2 at 4°C and 10000g for 90 min, and collect the supernatant 3; centrifuge the supernatant 3 at 4°C and 150000g for 120 min, discard the supernatant, and add 49 times the mass of 1× PBS buffer solution to obtain the refined extract 1 with a mass concentration of 2%.

[0116] (2) Prepare sucrose-PBS solutions with mass fractions of 60%, 45%, 30%, and 15% respectively, and add them to the centrifuge tube in a volume ratio of 1:1:1:1 in sequence; then add the refined extract 1, put it into an ultra-high-speed centrifuge, centrifuge at 4°C and 150000g for 120 min, and take the solution in the 10%-30% sucrose solution layer as the refined extract 2; centrifuge the refined extract 2 at 4°C and 150000g for 120 min to remove the sucrose solution, and add 9 times the mass of 1× PBS buffer solution to obtain the exosome solution derived from Centella asiatica with a mass concentration of 10%.

[0117] Take 100 μL of the exosome solution and dilute it in 2 mL of PBS. Use a nanoparticle size analyzer to measure the exosome concentration. Repeat each sample 3 times. The measured average particle size is 162.54 nm, and the concentration is (5.3 ± 0.22) × 107 / mL.

[0118] (3) Add trehalose and mannitol to the exosome solution derived from Centella asiatica, stir evenly and filter to obtain the freeze-drying solution; carry out vacuum freeze-drying on the freeze-drying solution. First, pre-freeze at -80°C for 12 h, then place it in a freeze-dryer, and freeze-dry at -50°C and 60 Pa for 30 h to obtain the exosome freeze-dried powder.

[0119] The concentration of the trehalose in the freeze-drying solution is 3%, and the concentration of the mannitol in the freeze-drying solution is 2%.

[0120] The preparation method of the above anti-aging recombinant humanized collagen composition includes the following steps:

[0121] (1) Spray the recombinant humanized collagen aqueous solution with a concentration of 1 wt% into the reaction kettle through the first electrostatic spraying gun to form positively charged particles, spray the vegetable oil ethanol solution with a concentration of 2 wt% into the reaction kettle through the second electrostatic spraying gun to form negatively charged particles. The positively charged particles and the negatively charged particles are electrostatically attracted and combined and then gather at the bottom of the kettle. Then start stirring for homogenization, and keep the temperature of the reaction kettle at 40°C until the liquid phase is uniform.

[0122] The voltage of the first electrostatic spraying gun is 40 kV, the current is 30 μA, and the flow rate is 20 mL / min; the voltage of the second electrostatic spraying gun is 40 kV, the current is 30 μA, and the flow rate is 6.5 mL / min;

[0123] The mixing ratio of the recombinant humanized collagen aqueous solution and the vegetable oil ethanol solution is carried out according to the formula amount of the composition, that is, the mass ratio of recombinant humanized collagen to vegetable oil is 54.17:43.33.

[0124] The rotation speed during homogenization is 1000 rpm.

[0125] (2) The solution obtained in step (1) is distilled under reduced pressure at 50 °C to remove the solvent, obtaining ionic liquid PO.

[0126] (3) The freeze-dried plant exosomes are dispersed in the ionic liquid to obtain a suspension, that is, the anti-aging recombinant humanized collagen composition is prepared.

[0127] Example 4

[0128] The difference from Example 1 is that the anti-aging recombinant humanized collagen composition, calculated by mass percentage, includes the following components: 70.35% recombinant humanized collagen, 28.15% vegetable oil, 1.5% freeze-dried plant exosomes.

[0129] Example 5

[0130] The difference from Example 1 is that the anti-aging recombinant humanized collagen composition, calculated by mass percentage, includes the following components: 54.72% recombinant humanized collagen, 40.78% vegetable oil, 1.5% freeze-dried plant exosomes.

[0131] Example 6

[0132] The difference from Example 1 is that the anti-aging recombinant humanized collagen composition, calculated by mass percentage, includes the following components: 62.19% recombinant humanized collagen, 37.31% vegetable oil, 0.5% freeze-dried plant exosomes.

[0133] Example 7

[0134] The difference from Example 1 is that the anti-aging recombinant humanized collagen composition, calculated by mass percentage, includes the following components: 60.94% recombinant humanized collagen, 36.56% vegetable oil, 2.5% freeze-dried plant exosomes.

[0135] Example 8

[0136] The difference from Example 1 is that the molecular weight of the recombinant humanized collagen is 2.8 kDa recombinant humanized type III collagen ( Micro) was purchased from Nanjing Dinovihua Biotechnology Co., Ltd.

[0137] Example 9

[0138] The difference from Example 1 is that in step (1) of the method for preparing the anti-aging recombinant humanized collagen composition, the mass percentage concentration of the recombinant humanized collagen aqueous solution is 1wt%, and the mass percentage concentration of the vegetable oil ethanol solution is 2wt%.

[0139] Example 10

[0140] The difference from Example 1 is that in step (1) of the method for preparing the anti-aging recombinant humanized collagen composition, the mass percentage concentration of the recombinant humanized collagen aqueous solution is 5wt%, and the mass percentage concentration of the vegetable oil ethanol solution is 8wt%.

[0141] Embodiment 11

[0142] The difference from Example 1 is that in step (1) of the method for preparing the anti-aging recombinant humanized collagen composition, the voltage of the first electrostatic spray gun is 40 kV, the current is 30 μA, and the flow rate is 20 mL / min; the voltage of the second electrostatic spray gun is 40 kV, the current is 30 μA, and the flow rate is 6.5 mL / min.

[0143] Comparative Example 1

[0144] The difference from Example 1 is that the preparation method of the composition is as follows:

[0145] (1) A 3 wt % recombinant humanized collagen aqueous solution and a 5 wt % vegetable oil ethanol solution were added into a reactor, and then stirring (at a speed of 2000 rpm) was started for homogenization, and the reactor temperature was maintained at 50° C.;

[0146] (2) The solution obtained in step (1) is subjected to reduced pressure distillation at 50° C. to remove the solvent, thereby obtaining a collagen-vegetable oil mixture.

[0147] (3) Dispersing the plant exosome freeze-dried powder in the above-mentioned collagen-vegetable oil mixture to obtain a suspension, thereby obtaining a composition.

[0148] Comparative Example 2

[0149] The difference from Example 1 is that the preparation method of the composition is as follows:

[0150] (1) Place the aqueous solution of recombinant humanized collagen with a concentration of 3 wt% and the ethanol solution of vegetable oil with a concentration of 5 wt% in the liquid storage tanks of the atomizing guns respectively. After atomization, inject them into the reaction kettle cavity for mixing. After the condensed liquid starts to appear at the bottom of the reaction kettle, start stirring (rotation speed is 2000 rpm) and react at 50 °C. After all the solutions are mixed, continue stirring and reacting for 1 h; the atomization flow rate is the same as that in Example 1.

[0151] (2) Distill off the solvent from the solution obtained in step (1) under reduced pressure at 50 °C to obtain a collagen-vegetable oil mixture.

[0152] (3) Disperse the freeze-dried plant exosomes in the above-mentioned collagen-vegetable oil mixture to obtain a suspension, and obtain the composition.

[0153] Comparative Example 3

[0154] The difference from Example 1 is that the anti-aging recombinant humanized collagen composition, calculated by mass percentage, includes the following components: 62.5% recombinant humanized collagen, 37.5% vegetable oil; the preparation method is as follows:

[0155] (1) Spray the aqueous solution of recombinant humanized collagen with a concentration of 3 wt% into the reaction kettle through the first electrostatic spraying gun to form positively charged particles, and spray the ethanol solution of vegetable oil with a concentration of 5 wt% into the reaction kettle through the second electrostatic spraying gun to form negatively charged particles. The positively charged particles and the negatively charged particles are electrostatically attracted and combined and then gather at the bottom of the kettle. Then start stirring for homogenization, and keep the temperature of the reaction kettle at 50 °C until the liquid phase is uniform;

[0156] The voltage of the first electrostatic spraying gun is 20 kV, the current is 10 μA, and the flow rate is 15 mL / min; the voltage of the second electrostatic spraying gun is 30 kV, the current is 15 μA, and the flow rate is 4 mL / min;

[0157] The mixing ratio of the aqueous solution of recombinant humanized collagen and the ethanol solution of vegetable oil is carried out according to the formulation amount of the composition, that is, the mass ratio of recombinant humanized collagen to vegetable oil is 61.56:36.94.

[0158] The rotation speed during homogenization is 2000 rpm.

[0159] (2) Distill off the solvent from the solution obtained in step (1) under reduced pressure at 50 °C to obtain the ionic liquid PO.

[0160] Comparative Example 4

[0161] Provided is a composition of recombinant humanized collagen and exosomes, which comprises the following components by mass percentage: 60% recombinant humanized collagen, 1.5% freeze-dried plant exosomes, and the balance is PBS buffer; the preparation method is as follows: the recombinant humanized collagen and the freeze-dried plant exosomes are dispersed in the PBS buffer and stirred to obtain the composition.

[0162] Comparative Example 5

[0163] Add 1×PBS buffer to the exosome solution derived from pomegranate in Example 1 and dilute it to a mass concentration of 1.5% of the exosomes.

[0164] Test Example 1: Ultraviolet-visible absorption spectrum (UV-vis)

[0165] Use an Agilent Cary 100 spectrometer ultraviolet spectrophotometer to study the turbidity (light transmittance) of the sample to be tested. A quartz cuvette is used for the test, and the collection wavelength is 600 nm. The transmittance of deionized water at 25°C is used as the background zero. The light transmittances of different samples to be tested are shown in Table 1. Further, study the stability of the samples to be tested at different temperatures. The light transmittances of the samples to be tested at different temperatures are as Figure 1 shown.

[0166] Table 1

[0167]

[0168] As can be seen from Table 1, the ionic liquid provided by the present invention has good light transmittance, indicating good homogeneity of each phase. The mixtures of recombinant humanized collagen and vegetable oil obtained by simple mixing and homogenization, or atomization mixing in Comparative Examples 1 and 2 have serious phase separation, indicating that neither recombinant humanized collagen nor vegetable oil is ionized, indicating that the electrostatic atomization method provided by the present invention effectively promotes the charge exchange and salt formation between recombinant humanized collagen and vegetable oil. Further, the feeding ratio of recombinant humanized collagen and vegetable oil, the molecular weight of recombinant humanized collagen, the concentration of the solution during the preparation process, and the electrostatic spraying parameters all have an impact on the light transmittance of the obtained ionic liquid.

[0169] Figure 1 Shows the light transmittances of Examples 1 to 3 at different temperatures, indicating that the PO provided by the present invention has good stability at 10-40°C.

[0170] Figure 2 Shows the physical pictures of the PO obtained in Example 1 and Comparative Example 1. It can be seen that Comparative Example 1 is more turbid than Example 1.

[0171] Figure 3Differential scanning calorimetry (DSC) test results of recombinant humanized collagen, vegetable oil, ionic liquid PO in Example 1, and the mixture of collagen and vegetable oil in Comparative Example 1; it can be seen from the results that compared with recombinant humanized collagen, no crystallization melting peak appears for ionic liquid PO, indicating that the two phases are fused and no phase separation occurs, suggesting that recombinant humanized collagen and vegetable oil are bonded to form a homogeneous phase; while the mixture in Comparative Example 1 still has the crystallization melting peak of recombinant humanized collagen, indicating that Comparative Example 1 is merely a simple mixture of two phases and no phase change occurs in the substance. From Figure 1 and Figure 2 The results show that recombinant humanized collagen and vegetable oil are bonded into an ionic liquid.

[0172] DSC test conditions: nitrogen atmosphere, heating rate of 10 °C / min, scanning range of 0 - 300 °C, using Hitachi DSC600 differential scanning calorimeter.

[0173] Test Example 2: Transdermal absorption test

[0174] The PO in Example 1, the PO in Comparative Example 1, and pure collagen solution (5% type I recombinant humanized collagen CIS90 type) were subjected to transdermal absorption test with reference to the Franz diffusion method in T / ZGKSL 010 - 2023 "Determination Method for Transdermal Absorption of Recombinant Collagen". The concentration of protein in the subcutaneous receiving fluid at different times was measured by HPLC, and the cumulative permeation amount at different times was calculated. The results are as Figure 4 shown.

[0175] It can be seen from the results that the transdermal absorption amounts are in the order of Example 1 > Comparative Example 1 ≈ pure collagen solution, indicating that after recombinant humanized collagen and vegetable oil are assembled into an ionic liquid, it is beneficial to improve its in vitro transdermal absorption performance. The possible reasons include the following: (1) After being assembled into an ionic liquid, the vegetable oil enhances the hydrophobicity of the molecule, making the assembled molecule more lipophilic. The long-chain alkyl group of the vegetable oil acts as a "switch", which is beneficial to introducing hydrophilic recombinant humanized collagen; (2) Due to its hydrophilicity, recombinant humanized collagen usually has a large hydration radius in aqueous solution, but after being assembled with vegetable oil into an ionic liquid, its molecular radius decreases, thus being beneficial to passing through the skin barrier.

[0176] Test Example 3: Exosome storage stability test

[0177] The average diameter of exosomes after standing for different times in Example 1, Comparative Example 4, and Comparative Example 5 was measured using a NanoSight NS 3000 nanoparticle size analyzer. The results are as Figure 5 shown.

[0178] As can be seen from the results, the particle size of exosomes in the composition of Example 1 changed less within 48 h than that of the comparative example and the exosome solution, indicating that exosomes can stably exist in ionic liquids and aggregation of exosomes can be avoided.

[0179] Test Example 4: Anti-aging test in vitro

[0180] The free radical theory is currently the most recognized and is the basis for the published anti-aging function evaluation indicators. The free radical theory states that aging is caused by an excessive amount of reactive oxygen species (ROS) in the body. The aging theory explains that age-related loss of function is due to the accumulation of ROS, which leads to oxidative damage and tissue damage.

[0181] Superoxide dismutase (SOD) can scavenge the free radicals accumulated in the body, prevent the damage caused by free radicals to the cell structure, and convert harmful superoxide free radicals into H 2 O 2 , and then it is decomposed by catalase (CAT), thereby protecting cells from its damage. Glutathione peroxidase (GSH-Px) can scavenge H 2 O 2 outside the cell, reduce the toxicity of reactive oxygen species in the body, and at the same time can also protect the structural integrity and normal function of cells and slow down the process of lipid peroxidation. Malondialdehyde (MDA) is a by-product of lipid peroxidation damage and indirectly reflects the degree of cell oxidative damage. An increase in the level of MDA is a common sign of oxidative damage and aging.

[0182] In the present invention, the anti-aging performance of the composition was evaluated by detecting the activities of antioxidant enzymes (SOD, GSH-Px, CAT) and the content of malondialdehyde (MDA) in cells by ELISA method. Specifically: after HepG2 cells were cultured for 24 h, they were inoculated into 96-well plates at a density of 1×105 cells / well. The cells were divided into a blank group, an H 2 O 2 model group and an experimental group. After the cells in each group were treated according to Table 2 to obtain the cells to be tested, the cells to be tested in each group were collected, ultrasonically disrupted, and the supernatant was taken by centrifugation. The levels of SOD, CAT, GSH-Px and the content of MDA were measured according to the method in the ELISA kit instructions, and the results are listed in Table 2.

[0183] Table 2

[0184]

[0185]

[0186] As can be seen from Table 2, H 2 O 2Compared with the blank group, the activities of SOD, CAT, and GSH-Px in the cells of the model group were significantly decreased, and the content of MDA was significantly increased, indicating that H 2 O 2 caused oxidative damage to the cells. However, when the composition of the example or comparative example was added to the experimental group for pre-treatment of the cells, the activities of SOD, CAT, and GSH-Px were significantly increased. The above results indicate that the composition can increase the activity of antioxidant enzymes and inhibit lipid peroxidation, thereby protecting the cells from oxidative damage induced by H 2 O 2 Furthermore, when comparing Example 1, 6, 7 with Comparative Example 3, the percentage content of exosomes in the composition affects its antioxidant (anti-aging) performance. As the content of exosomes increases, the antioxidant performance of the composition becomes better, but further increase leads to little change in antioxidant performance. Comparative Example 4 is a mixture of recombinant humanized collagen and exosomes, and the antioxidant performance of this composition is not much different from that of Example 1, indicating that recombinant humanized collagen and exosomes are antioxidant active ingredients; while Comparative Example 5 only has exosomes and shows poor antioxidant performance; in Comparative Example 1, since recombinant humanized collagen and vegetable oil are simply mixed, the liquid is in an emulsion state, which is not conducive to the stable dispersion of exosomes, resulting in slightly worse antioxidant performance than Comparative Example 4.

[0187] Test Example 5: Skin irritation test

[0188] The compositions prepared in Examples 1 to 8 were subjected to a skin irritation test using a human test. The test subjects were adults aged 25 to 50, with 10 people for each sample. After cleaning the foreheads of the subjects, the left and right parts were marked as the test area and the blank area; 24 hours after applying 300 μL of the sample (prepared as a 5 wt% aqueous solution by adding water) to the experimental sites of the subjects, the skin conditions were evaluated according to the following criteria, and the results are shown in Table 3.

[0189] Table 3

[0190]

[0191] It can be seen from the results that the composition provided by the present invention passed the skin irritation test.

[0192] Application Example 1

[0193] An anti-aging essence, calculated by mass percentage, includes the following components: 4% of the composition of Example 1, 1% of an emulsifier, 0.3% of a thickener, 1.5% of an emollient, 8% of a humectant, 0.2% of a preservative, 0.03% of a pH regulator, 0.05% of disodium EDTA, and the balance is water.

[0194] Among them, the emulsifier is a mixture of polyglyceryl-10 dilaurate, ceteth-20, and PEG-60 hydrogenated castor oil with a mass ratio of 4:1:0.2; the thickener is carbomer; the emollient is a mixture of triglyceride caprylic / capric acid, squalane, and polydimethylsiloxane with a weight ratio of 1:1:2; the humectant is a mixture of allantoin, sodium hyaluronate, 1,3-butanediol, and glycerin with a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; the pH regulator is citric acid.

[0195] Application Example 2

[0196] An anti-aging essence, calculated by mass percentage, includes the following components: 3% of the composition of Example 6, 0.8% of emulsifier, 0.5% of thickener, 2% of emollient, 10% of humectant, 0.1% of preservative, 0.04% of pH regulator, 0.02% of disodium EDTA, and the balance is water.

[0197] Among them, the emulsifier is a mixture of polyglyceryl-10 dilaurate, ceteth-20, and PEG-60 hydrogenated castor oil with a mass ratio of 4:1:0.2; the thickener is xanthan gum; the emollient is a mixture of isononyl isononanoate, squalane, and neopentyl glycol diheptanoate with a weight ratio of 1:0.5:1; the humectant is a mixture of allantoin, sodium hyaluronate, 1,3-butanediol, and glycerin with a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; the pH regulator is citric acid.

[0198] Application Example 3

[0199] An anti-aging essence, calculated by mass percentage, includes the following components: 5% of the composition of Example 7, 2% of emulsifier, 0.2% of thickener, 3% of emollient, 5% of humectant, 0.3% of preservative, 0.06% of pH regulator, 0.08% of disodium EDTA, and the balance is water.

[0200] Among them, the emulsifier is a mixture of polyglyceryl oleate, cetyl phosphate, and ceteth-20 with a mass ratio of 4:1:0.8; the thickener is hydroxyethyl cellulose; the emollient is a mixture of triglyceride caprylic / capric acid, squalane, and polydimethylsiloxane with a weight ratio of 1:1:2; the humectant is a mixture of allantoin, sodium hyaluronate, 1,3-butanediol, and glycerin with a weight ratio of 1:2:7:10; the preservative is hydroxyacetophenone; the pH regulator is citric acid.

[0201] Application Example 4

[0202] An anti-aging essence, calculated by mass percentage, includes the following components: 4% of the composition of Example 2, 1% of an emulsifier, 0.3% of a thickener, 1.5% of an emollient, 8% of a humectant, 0.2% of a preservative, 0.03% of a pH regulator, 0.05% of disodium EDTA, and the balance is water.

[0203] Among them, the emulsifier is a mixture of polyglyceryl-10 dilaurate, ceteth-20, and PEG-60 hydrogenated castor oil with a mass ratio of 4:1:0.2; the thickener is carbomer; the emollient is a mixture of triglyceride caprylic / capric acid, squalane, and polydimethylsiloxane with a weight ratio of 1:1:2; the humectant is a mixture of allantoin, sodium hyaluronate, 1,3-butanediol, and glycerol with a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; the pH regulator is citric acid.

[0204] Application Example 5

[0205] An anti-aging essence, calculated by mass percentage, includes the following components: 4% of the composition of Example 3, 1% of an emulsifier, 0.3% of a thickener, 1.5% of an emollient, 8% of a humectant, 0.2% of a preservative, 0.03% of a pH regulator, 0.05% of disodium EDTA, and the balance is water.

[0206] Among them, the emulsifier is a mixture of polyglyceryl-10 dilaurate, ceteth-20, and PEG-60 hydrogenated castor oil with a mass ratio of 4:1:0.2; the thickener is carbomer; the emollient is a mixture of triglyceride caprylic / capric acid, squalane, and polydimethylsiloxane with a weight ratio of 1:1:2; the humectant is a mixture of allantoin, sodium hyaluronate, 1,3-butanediol, and glycerol with a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; the pH regulator is citric acid.

[0207] Application Comparative Example 1

[0208] An anti-aging essence, calculated by mass percentage, includes the following components: 4% of the composition of Comparative Example 4, 1% of an emulsifier, 0.3% of a thickener, 1.5% of an emollient, 8% of a humectant, 0.2% of a preservative, 0.03% of a pH regulator, 0.05% of disodium EDTA, and the balance is water.

[0209] Among them, the emulsifier is a mixture of polyglyceryl-10 dilaurate, ceteth-20, and PEG-60 hydrogenated castor oil with a mass ratio of 4:1:0.2; the thickener is carbomer; the emollient is a mixture of triglyceride caprylic / capric acid, squalane, and polydimethylsiloxane with a weight ratio of 1:1:2; the humectant is a mixture of allantoin, sodium hyaluronate, 1,3-butanediol, and glycerol with a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; the pH regulator is citric acid.

[0210] Test 6: Human Skin Elasticity Test

[0211] The essence prepared from the application examples was subjected to a human skin elasticity test. The test subjects were women aged between 35 and 55 years old, randomly divided into 5 groups corresponding to 5 application examples, with 15 people in each group. The skin elasticity test instrument Cotometer MPA580 manufactured by CK Company in Germany (the probe is the Reviscometer RV600 elastic fiber tissue test probe) was used to measure the elasticity index of the forehead area 2 cm above the center of the glabella of the subjects' skin, and the initial elasticity index was obtained. After cleaning their skin in the morning and evening, the subjects used this essence (the dosage was 0.5 ± 0.1 g), and the skin elasticity index of the test area was measured 4 weeks after using the cream. Each test was measured 5 times and the average value was taken. The elasticity indices R1, R2, and R3 were calculated by the following formulas:

[0212] R1 1 = Ua / Uf, R1 2 = Ur / Ue, R2 3 = Ur / Uf, R3

[0213] Uf - the maximum skin stretch; Ue - the elastic stretch of the skin at 0.1 s after applying a constant negative pressure to the skin; Ur - the elastic recovery value of the skin 0.1 s after canceling the negative pressure; Ua - the recovery value of the skin from canceling the negative pressure to the next continuous test when adding negative pressure to the skin surface again; during the test, the negative pressure was kept constant at 450 mbar. The closer the skin elasticity index is to 1, that is, the faster and more the skin recovers after stretching, the better the skin elasticity.

[0214] Table 4

[0215]

[0216]

[0217] It can be seen from the results that the essence provided by the application examples of the present invention has the effect of increasing skin elasticity. Although Comparative Example 1 has an improvement effect on skin elasticity, its effect is inferior to that of the application examples. This is because the permeability of recombinant humanized collagen is enhanced after ionization, and the stability effect on exosomes is greatly improved, resulting in a greatly improved biological utilization rate of the raw materials.

[0218] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An anti-aging recombinant humanized collagen composition, characterized in that: Calculated by mass percentage, it includes the following components: 53% to 71% recombinant humanized collagen, 28% to 45% vegetable oil, and 0.5% to 2.5% plant exosome freeze-dried powder.

2. The anti-aging recombinant humanized collagen composition according to claim 1, characterized in that: The recombinant humanized collagen and the vegetable oil are bonded to form an ionic liquid.

3. The anti-aging recombinant humanized collagen composition according to claim 1 or 2, characterized in that: The molecular weight of the recombinant humanized collagen is 2kDa to 60kDa; the recombinant humanized collagen is selected from at least one of recombinant type I humanized collagen, recombinant type III humanized collagen and recombinant type XVII humanized collagen.

4. The anti-aging recombinant humanized collagen composition according to claim 1 or 2, characterized in that: The vegetable oil is selected from grape seed oil or meadowfoam seed oil.

5. The anti-aging recombinant humanized collagen composition according to claim 1 or 2, characterized in that: The plant exosomes are selected from at least one of peony exosomes, ginseng exosomes, pomegranate exosomes and Centella asiatica exosomes.

6. The anti-aging recombinant humanized collagen composition according to any one of claims 2 to 5, characterized in that: The preparation method of the ionic liquid is as follows: an aqueous solution of recombinant humanized collagen is sprayed into a reactor through a first electrostatic spray gun to form positively charged particles, and an ethanol solution of vegetable oil is sprayed into a reactor through a second electrostatic spray gun to form negatively charged particles, the positively charged particles and the negatively charged particles are electrostatically attracted and combined and gathered at the bottom of the reactor, and then stirring is started for homogenization, and the temperature of the reactor is maintained at 40-60° C., and homogenization is performed until the liquid phase is uniform; and the solvent is removed by reduced pressure distillation to obtain the ionic liquid.

7. The anti-aging recombinant humanized collagen composition according to claim 6, characterized in that: The process parameters when the first electrostatic spray gun sprays the aqueous solution of the recombinant humanized collagen include: voltage of 20 to 40 kV, current of 10 to 50 μA, and flow rate of 10 to 20 mL / min; And / or, the process parameters of the second electrostatic spray gun when spraying the ethanol solution of the vegetable oil include: voltage of 20 to 40 kV, current of 10 to 50 μA, and flow rate of 4 to 8 mL / min; And / or, the percentage content of recombinant humanized collagen in the aqueous solution of recombinant humanized collagen is 1% to 3%; And / or, the percentage content of vegetable oil in the vegetable oil ethanol solution is 1% to 5%; And / or, the homogenizing conditions include: a stirring speed of 1000 to 3000 rpm.

8. The method for preparing the anti-aging recombinant humanized collagen composition according to any one of claims 1 to 7, characterized in that: The following steps are involved: The plant exosome freeze-dried powder is dispersed in an ionic liquid to obtain a suspension, thereby preparing an anti-aging recombinant humanized collagen composition.

9. The method for preparing the anti-aging recombinant humanized collagen composition according to claim 8, characterized in that: The plant exosome freeze-dried powder is obtained by freeze-drying an exosome solution mixed with a freeze-drying protective agent; The freeze-drying protective agent includes mannitol and sugar, and the sugar is selected from at least one of trehalose, glucose, sucrose and pectin; The exosome solution is obtained by soaking the crushed plant material in a buffer solution and separating the plant material by differential centrifugation or density gradient centrifugation.

10. An anti-aging essence, characterized in that: Calculated by mass percentage, the composition comprises the following components: 3% to 5% of the anti-aging recombinant humanized collagen composition according to any one of claims 1 to 7, 0.8% to 2% of an emulsifier, 0.2% to 0.5% of a thickener, 1% to 3% of an emollient, 5% to 12% of a moisturizer, 0.1% to 0.3% of a preservative, 0.03% to 0.08% of a pH regulator, 0.02% to 0.08% of disodium EDTA, and the remainder being water.

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