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

Abstract

The application discloses an anti-aging recombinant humanized collagen composition and a preparation method and application thereof, and belongs to the technical field of biomedicine. The composition comprises a recombinant humanized collagen, a plant oil and a plant exosome freeze-dried powder, the recombinant humanized collagen and the plant oil are bonded into ionic liquids. The composition can improve the transdermal permeability of the recombinant humanized collagen and improve the in-vitro normal-temperature storage stability of the plant exosome. The application further provides a preparation method of the composition. The recombinant humanized collagen and the plant oil are atomized into positively-charged particles and negatively-charged particles respectively through an electrostatic atomization method, so that the activation energy of acid-base neutralization reaction of the two is reduced, and the two are bonded into ionic liquids through proton exchange.

Description

An anti-aging recombinant humanized collagen composition, its preparation method and application Technical Field

[0001] This invention relates to the field of cosmetic technology, and more specifically, to an anti-aging recombinant humanized collagen composition, its preparation method, and its application. Background Technology

[0002] Recombinant humanized collagen is collagen produced through genetic engineering technology. It possesses a structure and function highly similar to natural human collagen. Because its amino acid sequence is almost identical to that of human collagen, recombinant humanized collagen exhibits extremely low immune response in the skin, reducing the risk of allergies and rejection, thus demonstrating excellent biocompatibility. Furthermore, it avoids potential viral contamination and impurities that may arise during the extraction process, significantly improving product safety. 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 the moisture in the skin, keep the skin moisturized, and reduce dryness and 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 various scenarios such as postoperative care and sensitive skin repair; (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 from the external environment (such as ultraviolet rays, pollutants, etc.).

[0003] Despite the numerous advantages that recombinant humanized collagen has shown in cosmetics and skincare products, its relatively large molecular weight limits its ability to penetrate the skin barrier, resulting in poor transdermal absorption and hindering its effectiveness.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] This invention proposes an anti-aging recombinant humanized collagen composition, which includes recombinant humanized collagen and an ionic liquid prepared from plant oil. The transdermal efficiency of the ionicly liquefied recombinant humanized collagen is greatly improved; and it has a good stabilizing effect on plant exosomes as a dispersant.

[0006] This invention is implemented as follows:

[0007] In a first aspect, the present invention provides an anti-aging recombinant humanized collagen composition, comprising, by weight percentage: 53%–71% recombinant humanized collagen, 28%–45% vegetable oil, and 0.5%–2.5% lyophilized plant exosome powder. The recombinant humanized collagen and vegetable oil are bonded together to form an ionic liquid.

[0008] In some embodiments, 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.

[0009] In some implementations, the vegetable oil is selected from grape seed oil or meadowfoam seed oil.

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

[0011] In some embodiments, the ionic liquid is prepared as follows: an aqueous solution of recombinant humanized collagen is sprayed into a reaction vessel through a first electrostatic spray gun to form positively charged particles; an ethanol solution of vegetable oil is sprayed into the reaction vessel through a second electrostatic spray gun to form negatively charged particles; the positively charged particles and the negatively charged particles are attracted and combined by electrostatic attraction and collect at the bottom of the vessel; then stirring is started to homogenize, and the temperature of the reaction vessel is maintained at 40-60°C until the liquid phase is homogeneous; the solvent is removed by vacuum distillation to obtain the ionic liquid.

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

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

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

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

[0016] In some implementations, homogenization conditions include a stirring speed of 1000–3000 rpm.

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

[0018] In some implementations, plant exosome lyophilized powder is obtained by freeze-drying an exosome solution mixed with a freeze-drying protectant.

[0019] In some embodiments, the freeze-drying protectant includes mannitol and sugars selected from at least one of trehalose, glucose, sucrose, and pectin.

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

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

[0022] Fourthly, the present invention provides an anti-aging essence, comprising, by weight percentage, the following components: 3%–5% of the anti-aging recombinant humanized collagen composition of any of the above embodiments, 0.8%–2% of emulsifier, 0.2%–0.5% of thickener, 1%–3% of emollient, 5%–12% of moisturizer, 0.1%–0.3% of preservative, 0.03%–0.08% of pH adjuster, 0.02%–0.08% of disodium EDTA, and the balance being 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 phosphate, and cetearyl 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 caprylic / capric triglyceride, squalane, olive oil, polydimethylsiloxane, isononyl isononanoate, and neopentyl glycol diheptanoate.

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

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

[0028] In some implementations, 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 transdermal permeability of recombinant humanized collagen and improve the in vitro room temperature storage stability of plant exosomes.

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

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 shows the relationship between light transmittance of PO and temperature in Examples 1-3;

[0034] Figure 2 shows the physical images of the collagen-vegetable oil mixture obtained in Example 1 and Comparative Example 1.

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

[0036] Figure 4 shows the percutaneous permeation rate of PO in Example 1, the collagen-vegetable oil mixture obtained in Comparative Example 1, and the collagen aqueous solution over time.

[0037] Figure 5 shows the curves showing the change in the average diameter of exosomes over time in the compositions of Example 1, Comparative Example 4, and Comparative Example 5. Detailed Implementation

[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 clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] In the description of the embodiments of this application, the technical terms "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly indicating the number, specific order, or primary and secondary relationship of the indicated technical features.

[0040] The term "embodiment" in this document means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment that is mutually exclusive with other embodiments.

[0041] In the embodiments of this application, the term "or / and" is only a description of the relationship between related objects, indicating that there can be three kinds of relationships. For example, A or / and B can represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

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

[0043] In this application embodiment, "multiple" means two or more (including two), similarly, "multiple groups" means two or more (including two groups), and "multiple layers" means two or more (including two layers), unless otherwise explicitly specified and limited.

[0044] In the embodiments of this application, "at least one" means one or more.

[0045] In the embodiments of this application, the technical terms "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," and "horizontal," etc., indicating the orientation or positional relationship, are based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing the embodiments of this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation or be constructed in a specific orientation, etc., and should not be construed as a limitation on the embodiments of this application. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of this application according to the specific circumstances.

[0046] In a first aspect, the present invention provides an anti-aging recombinant humanized collagen composition, comprising, by weight percentage: 53%–71% recombinant humanized collagen, 28%–45% vegetable oil, and 0.5%–2.5% lyophilized plant exosome powder. The recombinant humanized collagen and vegetable oil are bonded together to form an ionic liquid.

[0047] Recombinant humanized collagen exhibits numerous advantages in cosmetics and skincare products, but its relatively large molecular weight limits its ability to penetrate the skin barrier, resulting in poor transdermal absorption and hindering its efficacy. This invention improves the transdermal permeability of recombinant humanized collagen by ionizing it with plant oils. This is because, firstly, after assembly into an ionic liquid, the plant oil enhances the hydrophobicity of the molecules, making the assembled molecules more lipid-soluble. The long-chain alkyl groups of the plant oil act as "switches," facilitating the introduction of the hydrophilic recombinant humanized collagen. Secondly, due to its hydrophilic nature, recombinant humanized collagen typically has a large hydration radius in aqueous solutions, but after assembly into an ionic liquid with plant oils, its molecular radius decreases, thus facilitating its passage through the skin barrier.

[0048] Plant exosomes are nanoscale vesicles secreted by plant cells. These vesicles contain various functional proteins, RNA, and metabolites, and have the potential to regulate intercellular communication, promote cell repair, and enhance skin barrier function. Derived from natural plants, plant exosomes exhibit good biocompatibility with human skin, reducing the risk of allergic reactions and other side effects. Studies have shown that plant exosomes can carry various bioactive molecules, including antioxidants, anti-inflammatory factors, and growth factors, which help improve skin health. However, exosomes are unstable and easily inactivated. To maintain the structural integrity of exosomes, these formulations must be maintained and transported at relatively low temperatures to preserve their biological activity. However, the loss of exosome biological activity often occurs during storage and preservation. Generally, liquid formulations are stored at ultra-low temperatures (e.g., not higher than -60°C) and transported under frozen conditions, requiring thawing before use. Furthermore, prolonged ultra-low temperature storage, repeated freeze-thaw cycles during use, or improper handling often lead to a rapid and significant decline in the biological activity of exosomes, thus limiting their normal use.

[0049] In the composition provided by this invention, the ionic liquid can improve the in vitro room temperature storage stability of plant exosomes, making it a promising candidate for use in skin care products stored at room temperature. This is because the ionic liquid contains a large number of hydrophobic alkyl groups (derived from plant oils), which have high compatibility with the bilayer phospholipid membrane of exosomes, enabling them to be well dispersed and without liquid exchange conditions, thereby preventing exosome aggregation or degradation.

[0050] In some embodiments, the molecular weight of the recombinant humanized collagen is 2kDa to 60kDa, for example, it can be 2kDa, 2.8kDa, 4kDa, 10kDa, 20kDa, 30kDa, 40kDa, 50kDa, 55kDa, 60kDa or any value within 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.

[0051] In some implementations, the vegetable oil is selected from grape seed oil or meadowfoam seed oil.

[0052] Grape seed oil and meadowfoam seed oil have high content of long-chain fatty acids, strong stability and skin affinity. As anions in ionic liquids, they can enhance the affinity of ionic liquids for the skin, thus facilitating the delivery of recombinant humanized collagen.

[0053] at the same time

[0054] Grape seed oil is a widely used active ingredient in skincare products, possessing antioxidant, moisturizing, and anti-inflammatory properties. It is rich in polyphenolic compounds, especially proanthocyanidins (OPC), a powerful natural antioxidant. Antioxidants neutralize free radicals, reducing oxidative stress damage to skin cells and thus slowing down the skin aging process. Meadowfoam seed oil helps prevent moisture loss and promotes skin regeneration, acting as a skin metabolism activator. It modifies keratinocyte structure, regulates the exudation of water and compounds, reabsorbs moisture, and forms hydrates in epidermal cells, thereby locking in moisture in the skin's epidermis.

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

[0056] Peony exosomes possess anti-inflammatory, antioxidant, and moisturizing effects, helping to enhance the skin barrier function, lock in moisture, and prevent dry skin. Ginseng is rich in active ingredients such as ginsenosides, and its exosomes can promote skin cell proliferation and repair, 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 exosomes also have a protective effect against skin damage caused by ultraviolet radiation and oxidative stress, and can inhibit the expression of pro-apoptotic genes. Pomegranate exosomes have antioxidant capabilities. Pomegranate is rich in polyphenolic compounds, especially ellagic acid, which have strong antioxidant properties, helping to resist environmental damage and protect the skin from free radical damage. Pomegranate exosomes help reduce skin inflammation and alleviate discomfort in sensitive skin. Pomegranate exosomes may also 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. They also possess significant anti-inflammatory properties, making them suitable for treating inflammatory skin conditions such as eczema and dermatitis. The active ingredients in Centella asiatica exosomes help stimulate collagen production, strengthen skin structure, and improve skin firmness.

[0057] In some embodiments, the ionic liquid is prepared as follows: an aqueous solution of recombinant humanized collagen is sprayed into a reaction vessel through a first electrostatic spray gun to form positively charged particles; an ethanol solution of vegetable oil is sprayed into the reaction vessel through a second electrostatic spray gun to form negatively charged particles; the positively charged particles and the negatively charged particles are attracted and combined by electrostatic attraction and collect at the bottom of the vessel; then stirring is started to homogenize, and the temperature of the reaction vessel is maintained at 40-60°C until the liquid phase is homogeneous; the solvent is removed by vacuum distillation to obtain the ionic liquid.

[0058] This invention uses electrostatic atomization to atomize recombinant humanized collagen and vegetable oil into positively charged and negatively charged particles, respectively, thereby reducing the activation energy of their acid-base neutralization reaction and promoting their proton exchange bonding into an ionic liquid.

[0059] In some embodiments, the process parameters for spraying the aqueous solution of the recombinant humanized collagen with the first electrostatic spray gun 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 for spraying the ethanol solution of the vegetable oil with the second electrostatic spray gun 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 affect the charge, particle size, flight speed, and spray range of the atomized droplets. Specifically, the higher the voltage, the stronger the electric field, the faster the flight speed of the charged particles, and the larger the spray range; the higher the current, the more charged particles there are; and the higher the flow rate, the larger the atomized droplet size.

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

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

[0064] In some implementations, homogenization conditions include a stirring speed of 1000–3000 rpm.

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

[0066] In some implementations, plant exosome lyophilized powder is obtained by freeze-drying an exosome solution mixed with a freeze-drying protectant.

[0067] In some embodiments, the freeze-drying protectant includes mannitol and sugars selected from at least one of trehalose, glucose, sucrose, and pectin.

[0068] In some implementations, the exosome solution is obtained by immersing the fragmented plant material in a buffer solution and then separating it using differential centrifugation or density gradient centrifugation.

[0069] Thirdly, the present invention provides the application of the anti-aging recombinant humanized collagen composition in any of the above embodiments, which can be applied to skin care products such as face creams, serums, lotions, etc.

[0070] Fourthly, the present invention provides an anti-aging essence, comprising, by weight percentage, the following components: 3%–5% of the anti-aging recombinant humanized collagen composition of any of the above embodiments, 0.8%–2% of emulsifier, 0.2%–0.5% of thickener, 1%–3% of emollient, 5%–12% of moisturizer, 0.1%–0.3% of preservative, 0.03%–0.08% of pH adjuster, 0.02%–0.08% of disodium EDTA, and the balance being 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 phosphate, and cetearyl 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 caprylic / capric triglyceride, squalane, olive oil, polydimethylsiloxane, isononyl isononanoate, and neopentyl glycol diheptanoate.

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

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

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

[0077] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0078] Example 1

[0079] An anti-aging recombinant humanized collagen composition, comprising the following components by weight percentage: 61.56% recombinant humanized collagen, 36.94% vegetable oil, and 1.5% lyophilized plant exosome powder.

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

[0081] The vegetable oil in question is meadowfoam seed oil, purchased from Jiangxi Kangshengtang Pharmaceutical Co., Ltd.

[0082] The plant exosomes were pomegranate exosomes, prepared by differential centrifugation, with the specific steps as follows:

[0083] (1) Put the peeled pomegranate into a crusher, add an equal mass of 1× phosphate buffer (PBS), crush and extract juice, filter and collect the filtrate; centrifuge the filtrate at 4℃ and 800g for 30min and collect supernatant 1; centrifuge supernatant 1 at 4℃ and 2500g for 30min and collect supernatant 2; centrifuge supernatant 2 at 4℃ and 8000g for 75min and collect supernatant 3; centrifuge supernatant 3 at 4℃ and 14000g for 120min, discard the supernatant, and add 9 times the mass of 1× PBS buffer to obtain a 10% mass concentration pomegranate-derived exosome solution.

[0084] 100 μL of exosome solution was diluted in 2 mL of PBS, and the exosome concentration was calculated using a nanoparticle size analyzer. Each sample was repeated 3 times. The average particle size was 80.11 nm and the concentration was (5.22 ± 0.21) × 10⁸ / mL.

[0085] (2) Add trehalose and mannitol to the exosome solution from pomegranate, stir evenly, filter and obtain lyophilized solution; subject the lyophilized solution to vacuum freeze drying, first pre-freeze at -80℃ for 12h, then place it in a freeze dryer and freeze dry at -50℃ and 60Pa for 30h to obtain exosome lyophilized 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-mentioned anti-aging recombinant humanized collagen composition includes the following steps:

[0088] (1) A 3wt% recombinant humanized collagen aqueous solution is sprayed into the reactor through a first electrostatic spray gun to form positively charged particles. A 5wt% vegetable oil ethanol solution is sprayed into the reactor through a second electrostatic spray gun to form negatively charged particles. The positively charged particles and the negatively charged particles are attracted and combined by electrostatic attraction and then collect at the bottom of the reactor. Then, stirring is turned on to homogenize and the reactor temperature is kept at 50°C until the liquid phase is homogeneous.

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

[0090] The mixing ratio of recombinant humanized collagen aqueous solution and vegetable oil ethanol solution is based on the formulation amount of the composition, 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 solvent in the solution obtained in step (1) is removed by vacuum distillation at 50°C to obtain the ionic liquid PO.

[0093] (3) Disperse the plant exosome freeze-dried powder in an ionic liquid to obtain a suspension, thus preparing an anti-aging recombinant humanized collagen composition.

[0094] Example 2

[0095] An anti-aging recombinant humanized collagen composition, comprising the following components by weight percentage: 71% recombinant humanized collagen, 28.5% vegetable oil, and 0.5% lyophilized plant exosome powder.

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

[0097] The vegetable oil in question is grape seed oil, purchased from Jiangxi Kang Shengtang Pharmaceutical Co., Ltd.

[0098] The plant exosomes were peony exosomes, prepared by differential centrifugation, with the specific steps as follows:

[0099] (3) Put the washed peony petals into a pulverizer, add an equal mass of 1× phosphate buffer (PBS), pulverize and extract juice, filter, and collect the filtrate; centrifuge the filtrate at 4℃ and 1000g for 30 min, and collect the supernatant 1; centrifuge the supernatant 1 at 4℃ and 5000g for 30 min, and collect the supernatant 2; centrifuge the supernatant 2 at 4℃ and 10000g for 60 min, and collect the supernatant 3; centrifuge the supernatant 3 at 4℃ and 20000g for 120 min, discard the supernatant, and add 9 times the mass of 1× PBS buffer to obtain a 10% mass concentration peony-derived exosome solution.

[0100] 100 μL of exosome solution was diluted in 2 mL of PBS, and the exosome concentration was calculated using a nanoparticle size analyzer. Each sample was repeated 3 times. The average particle size was 153.7 nm and the concentration was (7.4 ± 0.15) × 10⁷ / mL.

[0101] (4) Add sucrose and mannitol to the exosome solution from peony, stir evenly, filter and obtain lyophilized solution; subject the lyophilized solution to vacuum freeze drying, first pre-freeze at -80℃ for 12h, then place it in a freeze dryer and freeze dry at -50℃ and 60Pa for 30h to obtain exosome lyophilized powder.

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

[0104] (1) A 5 wt% recombinant humanized collagen aqueous solution is sprayed into the reactor through a first electrostatic spray gun to form positively charged particles. An 8 wt% vegetable oil ethanol solution is sprayed into the reactor through a second electrostatic spray gun to form negatively charged particles. The positively charged particles and the negatively charged particles are attracted and combined by electrostatic attraction and then collect at the bottom of the reactor. Then, stirring is turned on to homogenize and the reactor temperature is kept at 60°C until the liquid phase is homogeneous.

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

[0106] The mixing ratio of the recombinant humanized collagen aqueous solution and the vegetable oil ethanol solution is based on the formulation amount of the composition, that is, the mass ratio of recombinant humanized collagen to vegetable oil is 71:28.5.

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

[0108] (2) The solvent in the solution obtained in step (1) is removed by vacuum distillation at 50°C to obtain the ionic liquid PO.

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

[0110] Example 3

[0111] An anti-aging recombinant humanized collagen composition, comprising the following components by weight percentage: 54.17% recombinant humanized collagen, 43.33% vegetable oil, and 2.5% lyophilized plant exosome powder.

[0112] The recombinant humanized collagen has a molecular weight of 2.8 kDa and is a recombinant type III humanized collagen. Micro), purchased from Nanjing Dinowihua Biotechnology Co., Ltd.

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

[0114] The plant exosomes were Centella asiatica exosomes, prepared using a gradient density centrifugation method, with the specific steps as follows:

[0115] (1) Put the washed Centella asiatica into a pulverizer, add an equal mass of 1× phosphate buffer (PBS), pulverize and extract juice, filter, and collect the filtrate; centrifuge the filtrate at 4℃ and 2000g for 10 min, and collect the supernatant 1; centrifuge the supernatant 1 at 4℃ and 4000g for 30 min, and collect the supernatant 2; centrifuge the supernatant 2 at 4℃ and 10000g for 90 min, and collect the supernatant 3; centrifuge the supernatant 3 at 4℃ and 150000g for 120 min, discard the supernatant, and add 49 times the mass of 1× PBS buffer to obtain a 2% mass concentration refined extract 1;

[0116] (2) Prepare sucrose PBS solutions with mass fractions of 60%, 45%, 30%, and 15% respectively, and add them to centrifuge tubes in a volume ratio of 1:1:1:1. Then add the refined extract 1, place it in an ultra-high speed centrifuge, and centrifuge at 4℃ and 150,000g for 120 min. Take the solution in the 10%-30% sucrose solution layer as refined extract 2. Centrifuge refined extract 2 at 4℃ and 150,000g for 120 min to remove the sucrose solution, and add 9 times the mass of 1×PBS buffer to obtain a 10% mass concentration of Centella asiatica-derived exosome solution.

[0117] 100 μL of exosome solution was diluted in 2 mL of PBS, and the exosome concentration was calculated using a nanoparticle size analyzer. Each sample was repeated 3 times. The average particle size was 162.54 nm and the concentration was (5.3 ± 0.22) × 10⁷ / mL.

[0118] (3) Add trehalose and mannitol to the exosome solution from Centella asiatica, stir evenly, filter and obtain lyophilized solution; subject the lyophilized solution to vacuum freeze drying, first pre-freeze at -80℃ for 12h, then place it in a freeze dryer and freeze dry at -50℃ and 60Pa for 30h to obtain exosome lyophilized powder.

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

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

[0121] (1) A 1 wt% recombinant humanized collagen aqueous solution is sprayed into the reactor through a first electrostatic spray gun to form positively charged particles. A 2 wt% vegetable oil ethanol solution is sprayed into the reactor through a second electrostatic spray gun to form negatively charged particles. The positively charged particles and the negatively charged particles are attracted and combined by electrostatic attraction and then collect at the bottom of the reactor. Then, stirring is turned on to homogenize and the reactor temperature is kept at 40°C until the liquid phase is homogeneous.

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

[0123] The mixing ratio of the recombinant humanized collagen aqueous solution and the vegetable oil ethanol solution is based on the formulation 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 solvent in the solution obtained in step (1) is removed by vacuum distillation at 50°C to obtain the ionic liquid PO.

[0126] (3) Disperse the plant exosome freeze-dried powder in an ionic liquid to obtain a suspension, thus preparing an anti-aging recombinant humanized collagen composition.

[0127] Example 4

[0128] The difference from Example 1 is that the anti-aging recombinant humanized collagen composition comprises, by weight percentage, the following components: 70.35% recombinant humanized collagen, 28.15% vegetable oil, and 1.5% lyophilized plant exosome powder.

[0129] Example 5

[0130] The difference from Example 1 is that the anti-aging recombinant humanized collagen composition comprises, by weight percentage, the following components: 54.72% recombinant humanized collagen, 40.78% vegetable oil, and 1.5% lyophilized plant exosome powder.

[0131] Example 6

[0132] The difference from Example 1 is that the anti-aging recombinant humanized collagen composition comprises, by weight percentage, the following components: 62.19% recombinant humanized collagen, 37.31% vegetable oil, and 0.5% lyophilized plant exosome powder.

[0133] Example 7

[0134] The difference from Example 1 is that the anti-aging recombinant humanized collagen composition comprises, by weight percentage, the following components: 60.94% recombinant humanized collagen, 36.56% vegetable oil, and 2.5% lyophilized plant exosome powder.

[0135] Example 8

[0136] The difference from Example 1 is that the recombinant humanized collagen has a molecular weight of 2.8 kDa and is a recombinant type III humanized collagen (…). Micro), purchased from Nanjing Dinowihua 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 1 wt%, and the mass percentage concentration of the vegetable oil ethanol solution is 2 wt%.

[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 5 wt%, and the mass percentage concentration of the vegetable oil ethanol solution is 8 wt%.

[0141] Example 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 40kV, the current is 30μA, and the flow rate is 20mL / min; the voltage of the second electrostatic spray gun is 40kV, the current is 30μA, and the flow rate is 6.5mL / min.

[0143] Comparative Example 1

[0144] The difference from Example 1 is that the composition is prepared by the following method:

[0145] (1) Add a 3wt% aqueous solution of recombinant humanized collagen and a 5wt% ethanol solution of vegetable oil into a reaction vessel, then turn on the stirrer (2000 rpm) to homogenize, and keep the temperature of the reaction vessel at 50℃.

[0146] (2) The solvent in the solution obtained in step (1) is removed by vacuum distillation at 50°C to obtain a mixture of collagen and vegetable oil.

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

[0148] Comparative Example 2

[0149] The difference from Example 1 is that the composition is prepared by the following method:

[0150] (1) A 3 wt% aqueous solution of recombinant humanized collagen and a 5 wt% ethanol solution of vegetable oil were placed in the storage tank of the atomizing gun and injected into the reaction vessel cavity after atomization for mixing. After the liquid began to condense at the bottom of the reaction vessel, the stirring was turned on (speed was 2000 rpm) and the reaction was carried out at 50°C. After all the solutions were mixed, the stirring reaction was continued for 1 hour. The atomization flow rate was the same as in Example 1.

[0151] (2) The solvent in the solution obtained in step (1) is removed by vacuum distillation at 50°C to obtain a mixture of collagen and vegetable oil.

[0152] (3) Disperse the plant exosome freeze-dried powder in the above-mentioned collagen-plant 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, by weight percentage, comprises the following components: 62.5% recombinant humanized collagen and 37.5% vegetable oil; the preparation method is as follows:

[0155] (1) A 3wt% recombinant humanized collagen aqueous solution is sprayed into the reactor through a first electrostatic spray gun to form positively charged particles. A 5wt% vegetable oil ethanol solution is sprayed into the reactor through a second electrostatic spray gun to form negatively charged particles. The positively charged particles and the negatively charged particles are attracted and combined by electrostatic attraction and then collect at the bottom of the reactor. Then, stirring is turned on to homogenize and the reactor temperature is kept at 50°C until the liquid phase is homogeneous.

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

[0157] The mixing ratio of recombinant humanized collagen aqueous solution and vegetable oil ethanol solution is based on 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) The solvent in the solution obtained in step (1) is removed by vacuum distillation at 50°C to obtain the ionic liquid PO.

[0160] Comparative Example 4

[0161] A composition of recombinant humanized collagen and exosomes is provided, comprising the following components by mass percentage: 60% recombinant humanized collagen, 1.5% lyophilized plant exosome powder, and the balance being PBS buffer; the preparation method is as follows: the recombinant humanized collagen and lyophilized plant exosome powder are dispersed in PBS buffer and stirred to obtain the composition.

[0162] Comparative Example 5

[0163] The exosome solution derived from pomegranate in Example 1 was diluted with 1×PBS buffer to a mass concentration of 1.5% exosomes.

[0164] Test Example 1: Ultraviolet-Vis Absorption Spectroscopy (UV-vis)

[0165] The turbidity (light transmittance) of the test samples was studied using an Agilent Cary 100 Spectromete UV spectrophotometer. Quartz cuvettes were used for the measurements, and the wavelength was 600 nm. The transmittance of deionized water at 25℃ was used as the background zero. The light transmittance of different test samples is shown in Table 1. Furthermore, the stability of the test samples at different temperatures was investigated, and the light transmittance of the test samples at different temperatures is shown in Figure 1.

[0166] Table 1

[0167]

[0168] Table 1 shows that the ionic liquid provided by this invention has good light transmittance, indicating good phase homogeneity. Comparative Examples 1 and 2, obtained by simple mixing and homogenization or atomization mixing, showed severe phase separation in the recombinant humanized collagen and vegetable oil mixtures, indicating that neither the recombinant humanized collagen nor the vegetable oil was ionized. This demonstrates that the electrostatic atomization method provided by this invention effectively promotes charge exchange between the recombinant humanized collagen and vegetable oil to form salts. Furthermore, the feed ratio of recombinant humanized collagen and vegetable oil, the molecular weight of the recombinant humanized collagen, the concentration of the solution during preparation, and the electrostatic spraying parameters all affect the light transmittance of the prepared ionic liquid.

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

[0170] Figure 2 shows the physical images of 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 3 shows the differential scanning calorimetry (DSC) results for recombinant humanized collagen, vegetable oil, ionic liquid PO from Example 1, and a mixture of collagen and vegetable oil from Comparative Example 1. The results show that, compared to recombinant humanized collagen, ionic liquid PO did not exhibit a crystallization melting peak, indicating that the two phases fused without phase separation, suggesting that the recombinant humanized collagen and vegetable oil bonded to form a homogeneous phase. In contrast, the mixture from Comparative Example 1 still showed a crystallization melting peak for recombinant humanized collagen, indicating that Comparative Example 1 was merely a simple mixture of two phases without a phase transition. The results in Figures 1 and 2 demonstrate that recombinant humanized collagen and vegetable oil bonded to form an ionic liquid.

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

[0173] Test Example 2: Transdermal Absorption Test

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

[0175] The results show that the transdermal absorption was in the order of Example 1 > Comparative Example 1 ≈ pure collagen liquid, indicating that the recombinant humanized collagen and plant oil were assembled into an ionic liquid, which 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 plant oil enhanced the hydrophobicity of the molecules, making the assembled molecules more lipid-soluble. The long-chain alkyl group of the plant oil acts as a "switch", which is beneficial to introduce the hydrophilic recombinant humanized collagen; (2) Due to its hydrophilicity, the recombinant humanized collagen usually has a large hydration radius in aqueous solution, but after being assembled into an ionic liquid with plant oil, its molecular radius is reduced, which is beneficial to pass through the skin barrier.

[0176] Test Example 3: Exosome Storage Stability Test

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

[0178] The results show that the exosomes in the composition of Example 1 exhibited smaller particle size changes within 48 hours compared to the comparative example and the exosome solution, indicating that the exosomes can exist stably in the ionic liquid and that exosome aggregation can be avoided.

[0179] Test Example 4: In Vitro Anti-Aging Test

[0180] The free radical theory is currently the most widely accepted theory and the basis for published anti-aging function evaluation indicators. The free radical theory states that aging is caused by an excess of reactive oxygen species (ROS) in the body. Aging theories explain that age-related functional loss is due to the accumulation of ROS, leading to oxidative damage and tissue damage.

[0181] Superoxide dismutase (SOD) can scavenge free radicals accumulated in the body, preventing damage to cell structure. It converts harmful superoxide free radicals into H2O2, which is then broken down by catalase (CAT), thus protecting cells from damage. Glutathione peroxidase (GSH-Px) can scavenge extracellular H2O2, reducing the toxicity of reactive oxygen species in the body. It also protects the structural integrity and normal function of cells, slowing down the process of lipid peroxidation. Malondialdehyde (MDA) is a byproduct of lipid peroxidation damage and indirectly reflects the degree of cellular oxidative damage. Elevated MDA levels are a common marker of oxidative damage and aging.

[0182] This invention uses ELISA to detect the activity of antioxidant enzymes (SOD, GSH-Px, CAT) and the content of malondialdehyde (MDA) in cells to evaluate the anti-aging properties of the composition. Specifically, after HepG2 cells were cultured for 24 hours, they were seeded in 96-well plates at a density of 1×10⁵ cells / well. The cells were divided into a blank group, an H₂O₂ model group, and an experimental group. Each group was treated according to Table 2 to obtain test cells. The test cells of each group were collected, sonicated, centrifuged, and the supernatant was collected. The levels of SOD, CAT, GSH-Px and the content of MDA were determined according to the method in the ELISA kit instructions. The results are listed in Table 2.

[0183] Table 2

[0184]

[0185]

[0186] As shown in Table 2, compared with the blank group, the activities of SOD, CAT, and GSH-Px in the cells of the H2O2 model group were significantly reduced, while the content of MDA was significantly increased, indicating that H2O2 caused oxidative damage to the cells. However, when the cells were pretreated with the compositions of the examples or comparative examples in the experimental groups, the activities of SOD, CAT, and GSH-Px were significantly increased. These results indicate that the compositions can improve the activity of antioxidant enzymes and inhibit lipid peroxidation, thereby protecting cells from H2O2-induced oxidative damage. Furthermore, compared with Examples 1, 6, and 7 and Comparative Example 3, the percentage of exosomes in the composition affected its antioxidant (anti-aging) performance. As the content of exosomes increased, the antioxidant performance of the composition improved, but further increases did not significantly change the antioxidant performance. Comparative Example 4 is a mixture of recombinant humanized collagen and exosomes. The antioxidant properties of this composition are not much different from those of Example 1, indicating that recombinant humanized collagen and exosomes are antioxidant active ingredients. However, Comparative Example 5 only contains exosomes and exhibits poor antioxidant properties. In Comparative Example 1, since the recombinant humanized collagen and vegetable oil are simply mixed, the liquid is in the form of an emulsion, which is not conducive to the stable dispersion of exosomes, resulting in slightly worse antioxidant properties than Comparative Example 4.

[0187] Test Example 5: Skin Irritation Test

[0188] Skin irritation tests were conducted on the compositions prepared in Examples 1-8. Human trials were conducted on adults aged 25-50 years, with 10 subjects per sample. After the subjects' foreheads were cleaned, they were divided into left and right sections as the test area and the blank area. 300 μL of the sample (prepared with water to a 5 wt% aqueous solution) was applied to the test area of ​​the subjects. After 24 hours, the skin condition was evaluated according to the following criteria. The results are shown in Table 3.

[0189] Table 3

[0190]

[0191] The results show that the composition provided by this invention passed the skin irritation test.

[0192] Application Example 1

[0193] An anti-aging serum, by weight percentage, comprises the following components: 4% of the composition of Example 1, 1% of the emulsifier, 0.3% of the thickener, 1.5% of the emollient, 8% of the moisturizer, 0.2% of the preservative, 0.03% of the pH adjuster, 0.05% of the disodium EDTA, and the balance being water.

[0194] The emulsifier is a mixture of polyglycerol-10 dilaurate, cetearyl alcohol polyether-20, and PEG-60 hydrogenated castor oil in a mass ratio of 4:1:0.2; the thickener is carbomer; the emollient is a mixture of caprylic / capric triglyceride, squalane, and polydimethylsiloxane in a weight ratio of 1:1:2; the humectant is a mixture of succinate, sodium hyaluronate, 1,3-butanediol, and glycerin in a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; and the pH adjuster is citric acid.

[0195] Application Example 2

[0196] An anti-aging serum, by weight percentage, comprises the following components: 3% of the composition of Example 6, 0.8% of the emulsifier, 0.5% of the thickener, 2% of the emollient, 10% of the moisturizer, 0.1% of the preservative, 0.04% of the pH adjuster, 0.02% of the disodium EDTA, and the balance being water.

[0197] The emulsifier is a mixture of polyglycerol-10 dilaurate, cetearyl alcohol polyether-20, and PEG-60 hydrogenated castor oil in 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 in a weight ratio of 1:0.5:1; the humectant is a mixture of succinate, sodium hyaluronate, 1,3-butanediol, and glycerin in a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; and the pH adjuster is citric acid.

[0198] Application Example 3

[0199] An anti-aging serum, by weight percentage, comprises the following components: 5% of the composition of Example 7, 2% of the emulsifier, 0.2% of the thickener, 3% of the emollient, 5% of the moisturizer, 0.3% of the preservative, 0.06% of the pH adjuster, 0.08% of the disodium EDTA, and the balance being water.

[0200] The emulsifier is a mixture of polyglycerol oleate, cetyl phosphate, and cetearyl alcohol polyether-20 in a mass ratio of 4:1:0.8; the thickener is hydroxyethyl cellulose; the emollient is a mixture of caprylic / capric triglyceride, squalane, and polydimethylsiloxane in a weight ratio of 1:1:2; the humectant is a mixture of styracin, sodium hyaluronate, 1,3-butanediol, and glycerin in a weight ratio of 1:2:7:10; the preservative is hydroxyacetophenone; and the pH adjuster is citric acid.

[0201] Application Example 4

[0202] An anti-aging serum, by weight percentage, comprises the following components: 4% of the composition of Example 2, 1% of the emulsifier, 0.3% of the thickener, 1.5% of the emollient, 8% of the moisturizer, 0.2% of the preservative, 0.03% of the pH adjuster, 0.05% of the disodium EDTA, and the balance being water.

[0203] The emulsifier is a mixture of polyglycerol-10 dilaurate, cetearyl alcohol polyether-20, and PEG-60 hydrogenated castor oil in a mass ratio of 4:1:0.2; the thickener is carbomer; the emollient is a mixture of caprylic / capric triglyceride, squalane, and polydimethylsiloxane in a weight ratio of 1:1:2; the humectant is a mixture of succinate, sodium hyaluronate, 1,3-butanediol, and glycerin in a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; and the pH adjuster is citric acid.

[0204] Application Example 5

[0205] An anti-aging serum, by weight percentage, comprises the following components: 4% of the composition of Example 3, 1% of the emulsifier, 0.3% of the thickener, 1.5% of the emollient, 8% of the moisturizer, 0.2% of the preservative, 0.03% of the pH adjuster, 0.05% of the disodium EDTA, and the balance being water.

[0206] The emulsifier is a mixture of polyglycerol-10 dilaurate, cetearyl alcohol polyether-20, and PEG-60 hydrogenated castor oil in a mass ratio of 4:1:0.2; the thickener is carbomer; the emollient is a mixture of caprylic / capric triglyceride, squalane, and polydimethylsiloxane in a weight ratio of 1:1:2; the humectant is a mixture of succinate, sodium hyaluronate, 1,3-butanediol, and glycerin in a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; and the pH adjuster is citric acid.

[0207] Application Comparative Example 1

[0208] An anti-aging serum, by weight percentage, comprises the following components: 4% of the composition of Comparative Example 4, 1% of the emulsifier, 0.3% of the thickener, 1.5% of the emollient, 8% of the moisturizer, 0.2% of the preservative, 0.03% of the pH adjuster, 0.05% of the disodium EDTA, and the balance being water.

[0209] The emulsifier is a mixture of polyglycerol-10 dilaurate, cetearyl alcohol polyether-20, and PEG-60 hydrogenated castor oil in a mass ratio of 4:1:0.2; the thickener is carbomer; the emollient is a mixture of caprylic / capric triglyceride, squalane, and polydimethylsiloxane in a weight ratio of 1:1:2; the humectant is a mixture of succinate, sodium hyaluronate, 1,3-butanediol, and glycerin in a weight ratio of 1:2:7:10; the preservative is phenoxyethanol; and the pH adjuster is citric acid.

[0210] Test 6: Human Skin Elasticity Test

[0211] The serum prepared for the corresponding application cases was used to conduct skin elasticity tests on human skin. The subjects were women aged 35-55, randomly divided into 5 groups corresponding to 5 application cases, with 15 people in each group. A Cotometer MPA580 skin elasticity testing instrument (using the Reviscometer RV600 elastic fiber tissue testing probe) manufactured by CK (Germany) was used to measure the elasticity index of the skin in the forehead area 2cm above the center of the eyebrows, obtaining the initial elasticity index. Subjects used the serum (0.5±0.1g) morning and evening after cleansing their skin. The skin elasticity index of the test area was tested after 4 weeks of use. Each test was performed 5 times, and the average value was taken. The elasticity index was calculated using the following formula:

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

[0213] Uf—Maximum skin stretch; Ue—Skin elastic stretch after constant negative pressure is applied for 0.1 seconds; Ur—Skin elastic recovery value after negative pressure is removed for 0.1 seconds; Ua—Skin recovery value from the removal of negative pressure to the next continuous test when negative pressure is applied again; the negative pressure is kept constant at 450 mbar during the test. The closer the skin elasticity index is to 1, the faster and more the skin recovers after stretching, and the better the skin elasticity.

[0214] Table 4

[0215]

[0216]

[0217] The results show that the essence provided in the application example of this invention increases skin elasticity. Although Comparative Example 1 also improved skin elasticity, its effect was not as good as that of the application example. This is because the recombinant humanized collagen has enhanced permeability after ionization and its stability to exosomes is greatly improved, resulting in a significant increase in the bioavailability of the raw materials.

[0218] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An anti-aging recombinant humanized collagen composition, characterized in that, The product comprises, by mass percentage, the following components: 53%–71% recombinant humanized collagen, 28%–45% vegetable oil, and 0.5%–2.5% lyophilized plant exosome powder; the recombinant humanized collagen and the vegetable oil are bonded together to form an ionic liquid; the ionic liquid is prepared as follows: an aqueous solution of recombinant humanized collagen is sprayed into a reaction vessel through a first electrostatic spray gun to form positively charged particles; an ethanol solution of vegetable oil is sprayed into the reaction vessel through a second electrostatic spray gun to form negatively charged particles; the positively and negatively charged particles are electrostatically attracted and combine, then collect at the bottom of the vessel; stirring is then started for homogenization, and the temperature of the reaction vessel is maintained at 40–60°C until the liquid phase is homogeneous; the solvent is removed by vacuum distillation to obtain the ionic liquid; the vegetable oil is selected from grape seed oil or meadowfoam seed oil.

2. The anti-aging recombinant humanized collagen composition according to claim 1, characterized in that, The recombinant humanized collagen has a molecular weight of 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.

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

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

5. The method for preparing the anti-aging recombinant humanized collagen composition according to any one of claims 1 to 4, characterized in that, Includes the following steps: Plant exosome freeze-dried powder is dispersed in an ionic liquid to obtain a suspension, thus preparing an anti-aging recombinant humanized collagen composition.

6. The method for preparing the anti-aging recombinant humanized collagen composition according to claim 5, characterized in that, The plant exosome freeze-dried powder is obtained by freeze-drying an exosome solution mixed with a freeze-drying protectant; the freeze-drying protectant includes mannitol and sugars, the sugars being selected from at least one of trehalose, glucose, sucrose and pectin; the exosome solution is obtained by immersing the fragmented plant material in a buffer solution and then separating it using differential centrifugation or density gradient centrifugation.

7. An anti-aging essence, characterized in that, The composition comprises, by mass percentage, the following components: 3% to 5% of the anti-aging recombinant humanized collagen composition according to any one of claims 1 to 4, 0.8% to 2% of emulsifier, 0.2% to 0.5% of thickener, 1% to 3% of emollient, 5% to 12% of moisturizer, 0.1% to 0.3% of preservative, 0.03% to 0.08% of pH adjuster, 0.02% to 0.08% of disodium EDTA, and the balance being water.

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