Rose extracellular matrix extract as well as preparation method and application thereof

Abstract

The invention relates to a rose extracellular matrix extract as well as a preparation method and application thereof, and belongs to the technical field of plant extract processing. The preparation method of the rose extracellular matrix extract comprises the following steps: (1) mixing withered rose petals with water, and extracting to obtain a rose extracting solution; (2) freeze-drying and crushing fresh rose petals to obtain crushed rose petals, mixing the crushed rose petals with water, and performing ultrasonic extraction to obtain a rose extract; and (3) mixing the rose extract with the rose extracting solution to react, separating, removing impurities, and sterilizing to obtain the rose extracellular matrix extract. The rose extracellular matrix extract with relatively high glycoprotein content, relatively high hydroxyproline content and better bioavailability can be prepared.

Description

Technical Field

[0001] The present application relates to the technical field of plant extract processing, and in particular to a rose extracellular matrix extract and a preparation method and application thereof. Background Art

[0002] ECM refers to the extracellular matrix, which is the microenvironment for normal cell growth; for human skin, the better the cell microenvironment, the younger the skin. For animal cells, the extracellular matrix is ​​synthesized by cells and secreted outside the cells. It is a macromolecule distributed on the cell surface or between cells. It is mainly composed of some polysaccharides, proteins, and proteoglycans, which regulate the physiological activities of tissues and cells. The cell wall of plant cells is equivalent to the extracellular matrix in plants. It mainly includes collagen, non-collagen, elastin, proteoglycans, and amino acid polysaccharides. The basement membrane is at the base of epithelial or endothelial cells, and the intercellular adhesion structure is the interstitial connective tissue. Glycoprotein is a protein containing oligosaccharide chains, which are connected by covalent bonds. The oligosaccharide chains are usually linked to proteins through glycosylation during co-translational modification or post-translational modification. They are generally soluble in water, dilute acid, and dilute alkaline solutions, but not easily soluble in ethanol. A suitable extraction process is more critical.

[0003] Plant ECM contains a variety of glycoproteins, with a protein content of 16.33%, among which the content of hydroxyproline is relatively high. Hydroxyproline is an amino acid with a high content in collagen and a signature component of collagen. The human body cannot synthesize it independently. Supplementing hydroxyproline can effectively help the body rebuild collagen and regenerate and renew elastin.

[0004] The existing methods for producing hydroxyproline mainly include protein hydrolysis, chemical synthesis, biological fermentation, plant extraction, etc. The hydrolysis method and chemical synthesis method have high purity, but strong acid and strong alkali are used in the preparation process, the cost of raw materials in the extraction process is high, the waste pollution is serious, the process is complicated, etc., and they are gradually eliminated by the market. The plant extraction method is to extract compounds from plants in nature, but due to the influence of seasons, environment, etc. on plant growth and development, the types and contents of the compounds produced are different. Summary of the invention

[0005] In view of the deficiencies of the prior art, the purpose of the embodiments of the present application includes providing a rose extracellular matrix extract and a preparation method and application thereof, which can prepare a rose extracellular matrix extract with a high glycoprotein content and a relatively high hydroxyproline content and better bioavailability.

[0006] In a first aspect, the present application provides a method for preparing a rose extracellular matrix extract, comprising:

[0007] (1) Take withered rose petals, mix them with water for extraction to obtain a rose extract.

[0008] (2) Take fresh rose petals, perform freeze-drying and pulverization to obtain a pulverized rose petal product, mix the pulverized rose petal product with water for ultrasonic extraction to obtain a rose extract.

[0009] (3) Mix and react the rose extract with the rose extract solution, and obtain an extracellular matrix extract of rose through separation, impurity removal, and sterilization.

[0010] The preparation method of the extracellular matrix extract of rose provided by this application uses the water extract of withered rose petals to mix and react with the rose extract, and can prepare an extracellular matrix extract of rose with a relatively high glycoprotein content, a relatively high hydroxyproline content therein, and better bioavailability. On the one hand, during the withering process of rose petals, there are relatively many sugar-decomposing enzymes and protein-decomposing enzymes produced. The rose extract solution obtained by mixing withered rose petals with water is rich in natural sugar-decomposing enzymes and protein-decomposing enzymes. When mixed and reacted with the rose extract, the rich sugar-decomposing enzymes and protein-decomposing enzymes perform an efficient decomposition reaction on the rose extract. At the same time, the natural sugar-decomposing enzymes and protein-decomposing enzymes make the decomposition products natural and safe, with better bioavailability. On the other hand, the selected fresh rose petals have a relatively high glycoprotein content and a relatively high hydroxyproline content therein. Freeze-drying minimizes the loss of glycoprotein and hydroxyproline therein to the greatest extent. Then, through mixing the pulverized rose petal product with water for ultrasonic extraction, the sugar and protein in fresh rose petals can be efficiently extracted. When mixed and reacted with the rose extract solution, the sugar extracted from fresh rose petals can also improve the reaction activity of sugar-decomposing enzymes and protein-decomposing enzymes, further improving the extraction efficiency. Thus, an extracellular matrix extract of rose with a relatively high glycoprotein content, a relatively high hydroxyproline content therein, and better bioavailability is prepared.

[0011] In some embodiments of this application, the withered rose petals are rose petals that have withered for 1 - 10 days.

[0012] The withered rose petals for 1 - 10 days provided by this application are at a stage with a relatively high content and activity of sugar-decomposing enzymes and protein-decomposing enzymes, which is conducive to a more efficient reaction, and the prepared extracellular matrix extract of rose has a relatively high glycoprotein content, a relatively high hydroxyproline content therein, and better bioavailability.

[0013] In some embodiments of this application, the withered rose petals and water are mixed at a weight ratio of 1:5 - 15, and stirred and extracted at room temperature for 2 - 4 h.

[0014] The withered rose petals provided by the present application are mixed with water in a suitable weight ratio and extracted under suitable conditions, so that the carbohydrase and protease contained in the withered rose petals can be efficiently extracted, and a rose extract rich in natural carbohydrase and protease can be obtained.

[0015] In some embodiments of the present application, the fresh rose petals selected are the rose petals about to bloom.

[0016] The rose petals about to bloom provided by the present application contain higher glycoprotein and hydroxyproline therein, and a rose extracellular matrix extract with a higher content of glycoprotein and hydroxyproline therein can be extracted.

[0017] In some embodiments of the present application, the freeze-dried fresh rose petals in step (2) are pulverized at 4500 - 5000 rpm and screened through 50 - 80 meshes to obtain rose petal pulverizate.

[0018] The freeze-dried fresh rose petals provided by the present application are pulverized under suitable conditions and screened to a suitable particle size, which is more conducive to subsequent efficient extraction.

[0019] In some embodiments of the present application, the rose petal pulverizate is mixed with water in a weight ratio of 1:10 - 50, and ultrasonic extraction is carried out at a power of 200 - 300 W and a temperature of 30 - 50 °C for 20 - 50 min.

[0020] The rose petal pulverizate provided by the present application is mixed with water in a suitable weight ratio, and assisted by ultrasonic extraction under suitable conditions, so that the sugar and protein in the fresh rose petals can be extracted more efficiently.

[0021] In some embodiments of the present application, the rose extract and the rose extract solution are mixed in a weight ratio of 1:5 - 10 and reacted at 35 - 37 °C for 40 - 55 min.

[0022] The rose extract and the rose extract solution provided by the present application are mixed in a suitable weight ratio and reacted under suitable conditions, so that the rose extract can be decomposed more efficiently, and a rose extracellular matrix extract with a relatively high glycoprotein content, a relatively high hydroxyproline content and better bioavailability can be obtained.

[0023] In some embodiments of the present application, in step (3), microfiltration with 0.45 - 0.5 μm, microfiltration with 0.22 - 0.3 μm, ultrafiltration with 100 - 300 kDa and membrane filtration with 10 - 20 kDa are carried out in sequence for separation and impurity removal.

[0024] The separation and impurity removal provided by the present application includes two microfiltrations, ultrafiltration and membrane filtration in sequence, which can effectively remove other impurities and play a purification role, so that the glycoprotein in the obtained rose extracellular matrix extract is relatively pure.

[0025] In a second aspect, an embodiment of the present application provides a rose extracellular matrix extract prepared by the preparation method of the first aspect.

[0026] The rose extracellular matrix extract provided by the present application is prepared according to the above preparation method, contains a high content of glycoprotein and hydroxyproline therein, and has good bioavailability.

[0027] In a third aspect, an embodiment of the present application provides an application of the rose extracellular matrix extract of the second aspect in the preparation of skin care products.

[0028] Since the rose extracellular matrix extract provided by the present application contains a high content of glycoprotein and hydroxyproline therein, and has good bioavailability, when it is applied to the preparation of skin care products, the skin care products have good moisturizing, antioxidant and anti-aging effects, and are safe, mild, and more easily metabolized and utilized by the human body. Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings.

[0030] Figure 1 It is a diagram of the corner of the eye of a subject before using the rose extracellular matrix extract provided in Example 1 in Test Example 6 of the present application.

[0031] Figure 2 It is a diagram of the corner of the eye of a subject 28 days after using the rose extracellular matrix extract provided in Example 1 in Test Example 6 of the present application. Detailed Embodiments

[0032] To make the objectives, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0033] An embodiment of the present application provides a preparation method of a rose extracellular matrix extract, including:

[0034] (1) Take rose petals that have withered for 1 - 10 days, mix and extract them with water at a weight ratio of 1:5 - 15 to obtain a rose extract;

[0035] (2) Take freshly blooming rose petals that are about to bloom, perform freeze-drying, crush them at 4500 - 5000 rpm, and screen them through a 50 - 80 mesh sieve to obtain crushed rose petals. Mix the crushed rose petals with water at a weight ratio of 1:10 - 50, and perform ultrasonic extraction at a power of 200 - 300 W and a temperature of 30 - 50 °C for 20 - 50 min to obtain a rose extract;

[0036] (3) Mix the rose extract and the rose extraction solution at a weight ratio of 1:5 - 10, react at 35 - 37 °C for 40 - 55 min, successively perform microfiltration through 0.45 - 0.5 μm, 0.22 - 0.3 μm, ultrafiltration through 100 - 300 kDa, membrane filtration through 10 - 20 kDa for separation and impurity removal, and sterilize at 100 - 120 °C for 10 - 15 min to obtain a rose extracellular matrix extract.

[0037] This application uses the water extract of rose petals that have withered for 1 - 10 days and mixes and reacts it with the rose extract. Rose petals that have withered for 1 - 10 days have a relatively high content and activity of sugar-decomposing enzymes and protein-decomposing enzymes. When mixed with water at an appropriate weight ratio and extracted under appropriate conditions, the sugar-decomposing enzymes and protein-decomposing enzymes contained in the withered rose petals can be efficiently extracted, obtaining a rose extraction solution rich in natural sugar-decomposing enzymes and protein-decomposing enzymes; freshly blooming rose petals contain a relatively high amount of glycoprotein and hydroxyproline therein. After freeze-drying and crushing, the loss of glycoprotein and hydroxyproline therein is minimized as much as possible, and then mixed with water at an appropriate weight ratio and assisted by ultrasonic extraction under appropriate conditions, the sugar and protein in the fresh rose petals can be extracted more efficiently; the rose extraction solution rich in natural sugar-decomposing enzymes and protein-decomposing enzymes is mixed with the rose extract rich in sugar and protein at an appropriate weight ratio and reacts under appropriate conditions, thereby efficiently preparing a rose extracellular matrix extract with a relatively high glycoprotein content and a relatively high hydroxyproline content and better bioavailability.

[0038] Optionally, the withered rose petals are rose petals that have withered for 3 - 8 days. Rose petals that have withered for 3 - 8 days have a higher content and activity of sugar-decomposing enzymes and protein-decomposing enzymes, which is beneficial for a more efficient reaction. As an example, the withering time of the withered rose petals can be, but is not limited to, any one value or the range value between any two of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days.

[0039] Optionally, the withered rose petals are mixed with water at a weight ratio of 1:5 - 10 for extraction. The withered rose petals are further mixed with water at an appropriate weight ratio and extracted under appropriate conditions, and the sugar-decomposing enzymes and protein-decomposing enzymes contained in the withered rose petals can be further efficiently extracted, obtaining a rose extraction solution rich in natural sugar-decomposing enzymes and protein-decomposing enzymes.

[0040] Optionally, the rose petal powder is mixed with water at a weight ratio of 1:10 - 40, and ultrasonic extraction is carried out at a power of 200 - 250 W and a temperature of 30 - 40 °C for 20 - 40 min. The rose petal powder and water are further mixed at a suitable weight ratio, and ultrasonic extraction under suitable conditions is further assisted, so that sugars and proteins in fresh rose petals can be extracted more efficiently.

[0041] Optionally, the rose extract and the rose extraction solution are mixed and reacted at a weight ratio of 1:5 - 8. The rose extract and the rose extraction solution are mixed at a suitable weight ratio and reacted under suitable conditions, so that the decomposition reaction of the rose extract can be carried out more efficiently, and a rose extracellular matrix extract with a relatively high content of glycoprotein, a relatively high content of hydroxyproline therein, and better bioavailability can be obtained.

[0042] In some embodiments of the present application, the rose extracellular matrix extract can also be concentrated to 2 - 3 times the mass of the rose petal powder and then freeze-dried. This is beneficial for the storage of the rose extracellular matrix extract and its application in the preparation of skin care products.

[0043] The embodiment of the present application provides a rose extracellular matrix extract prepared by the above preparation method.

[0044] The rose extracellular matrix extract of the present application is prepared according to the above preparation method, contains a relatively high content of glycoprotein and hydroxyproline therein, and has good bioavailability; thus, it has good moisturizing, antioxidant, and anti-aging effects, is safe and mild, and can be preferably applied to skin care products.

[0045] The embodiment of the present application provides an application of the rose extracellular matrix extract as described above in the preparation of skin care products.

[0046] The rose extracellular matrix extract of the present application contains a relatively high content of glycoprotein and hydroxyproline therein, has good bioavailability, and has good moisturizing, antioxidant, and anti-aging effects, and is safe and mild; when it is applied to the preparation of skin care products, the skin care products also have good moisturizing, antioxidant, and anti-aging effects, are safe and mild, and are more easily metabolized and utilized by the human body.

[0047] The features and properties of the present application are further described in detail below in conjunction with embodiments.

[0048] Example 1

[0049] This example provides a preparation method of a rose extracellular matrix extract, including:

[0050] (1) Take rose petals on the 6th day after withering, mix and extract them with water at a weight ratio of 1:5 to obtain a rose extraction solution;

[0051] (2) Take the fresh rose petals about to bloom, conduct freeze-drying, crush them at 4800 rpm, and screen them through 80 mesh to obtain rose petal powder. Mix the rose petal powder with water at a weight ratio of 1:10, and perform ultrasonic extraction at a power of 250 W and 40 °C for 30 min to obtain rose extract;

[0052] (3) Mix the rose extract and rose extraction solution at a weight ratio of 1:5, react at 37 °C for 40 min, successively conduct separation and impurity removal through 0.45 μm microfiltration, 0.22 μm microfiltration, 100 kDa ultrafiltration, and 20 kDa membrane filtration, and sterilize at 120 °C for 15 min to obtain rose extracellular matrix extract.

[0053] Example 2

[0054] (1) Take the rose petals on the 2nd day after withering, mix them with water at a weight ratio of 1:10 for extraction to obtain rose extraction solution;

[0055] (2) Take the fresh rose petals about to bloom, conduct freeze-drying, crush them at 5000 rpm, and screen them through 80 mesh to obtain rose petal powder. Mix the rose petal powder with water at a weight ratio of 1:50, and perform ultrasonic extraction at a power of 250 W and 40 °C for 30 min to obtain rose extract;

[0056] (3) Mix the rose extract and rose extraction solution at a weight ratio of 1:6, react at 37 °C for 50 min, successively conduct separation and impurity removal through 0.45 μm microfiltration, 0.22 μm microfiltration, 100 kDa ultrafiltration, and 10 kDa membrane filtration, and sterilize at 120 °C for 15 min to obtain rose extracellular matrix extract.

[0057] Example 3

[0058] (1) Take the rose petals on the 8th day after withering, mix them with water at a weight ratio of 1:15 for extraction to obtain rose extraction solution;

[0059] (2) Take the fresh rose petals about to bloom, conduct freeze-drying, crush them at 4500 rpm, and screen them through 50 mesh to obtain rose petal powder. Mix the rose petal powder with water at a weight ratio of 1:25, and perform ultrasonic extraction at a power of 250 W and 40 °C for 30 min to obtain rose extract;

[0060] (3) Mix the rose extract and rose extraction solution at a weight ratio of 1:8, react at 37 °C for 45 min, successively conduct separation and impurity removal through 0.45 μm microfiltration, 0.22 μm microfiltration, 100 kDa ultrafiltration, and 20 kDa membrane filtration, and sterilize at 120 °C for 15 min to obtain rose extracellular matrix extract.

[0061] Example 4

[0062] (1) Take the rose petals on the 20th day of withering, mix them with water at a weight ratio of 1:5 for extraction to obtain a rose extract;

[0063] (2) Take the fresh rose petals about to bloom, conduct freeze-drying, crush them at 4500 rpm, and sieve them through 50 meshes to obtain a crushed rose petal product. Mix the crushed rose petal product with water at a weight ratio of 1:10, and perform ultrasonic extraction at a power of 250 W and 40 °C for 30 min to obtain a rose extract;

[0064] (3) Mix the rose extract and the rose extract at a weight ratio of 1:5, react at 37 °C for 45 min, successively perform microfiltration through 0.45 μm, microfiltration through 0.22 μm, ultrafiltration through 100 kDa, and membrane filtration through 10 kDa for separation and impurity removal, and sterilize at 120 °C for 15 min to obtain a rose extracellular matrix extract.

[0065] Example 5

[0066] (1) Take the rose petals on the 6th day of withering, mix them with water at a weight ratio of 1:5 for extraction to obtain a rose extract;

[0067] (2) Take the fresh rose petals 24 h after blooming, conduct freeze-drying, crush them at 4800 rpm, and sieve them through 80 meshes to obtain a crushed rose petal product. Mix the crushed rose petal product with water at a weight ratio of 1:10, and perform ultrasonic extraction at a power of 250 W and 40 °C for 30 min to obtain a rose extract;

[0068] (3) Mix the rose extract and the rose extract at a weight ratio of 1:5, react at 37 °C for 40 min, successively perform microfiltration through 0.45 μm, microfiltration through 0.22 μm, ultrafiltration through 100 kDa, and membrane filtration through 10 - kDa for separation and impurity removal, and sterilize at 120 °C for 15 min to obtain a rose extracellular matrix extract.

[0069] Comparative Example 1

[0070] This comparative example provides a method for preparing a rose extracellular matrix extract, including:

[0071] (1) Take the fresh rose petals about to bloom, conduct freeze-drying, crush them at 4800 - rpm, and sieve them through 80 meshes to obtain a crushed rose petal product. Mix the crushed rose petal product with water at a weight ratio of 1:10, and perform ultrasonic extraction at a power of 250 W and 40 °C for 30 min to obtain a rose extract;

[0072] (2) Add 0.5% cellulase and 0.6% pectinase to the rose extract, react at 37 °C for 40 min, and successively perform microfiltration through 0.45 μm, 0.22 μm, ultrafiltration through 100 kDa, and membrane filtration through 20 kDa for separation and impurity removal, and sterilize at 120 °C for 15 min to obtain the rose extracellular matrix extract.

[0073] Comparative Example 2

[0074] This comparative example provides a method for preparing a rose extracellular matrix extract, which is different from Example 1 in that: in step (1), take unfaded rose petals and mix them with water at a weight ratio of 1:5 for extraction to obtain a rose extract.

[0075] Comparative Example 3

[0076] This comparative example provides a method for preparing a rose extracellular matrix extract, including:

[0077] (1) Take rose petals on the 6th day after withering, mix them with water at a weight ratio of 1:5 for extraction to obtain a rose extract;

[0078] (2) Take freshly picked rose petals about to bloom, perform freeze-drying, crush them at 4800 rpm, and screen them through 80 meshes to obtain a rose petal powder. Mix the rose petal powder with the rose extract at a weight ratio of 10:1, react at 37 °C for 55 min, and successively perform microfiltration through 0.45 μm, 0.22 μm, ultrafiltration through 100 kDa, and membrane filtration through 20 kDa for separation and impurity removal, and sterilize at 120 °C for 15 min to obtain the rose extracellular matrix extract.

[0079] The methods for preparing the rose extracellular matrix extract provided in Examples 1-5 and Comparative Examples 1-3 are shown in Table 1.

[0080] Table 1 Methods for Preparing Rose Extracellular Matrix Extract

[0081]

[0082] Note: " / " in Table 1 indicates that this step is not included.

[0083] Test Example 1

[0084] In this test example, the absorbance of the rose extracellular matrix extracts provided in Examples 1-5 and Comparative Examples 1-3 was measured at 595 nm using the Coomassie brilliant blue method, and the total glycoprotein content of each rose extracellular matrix extract was calculated. The specific method is as follows: (1) Reagent preparation: Coomassie brilliant blue G-250 staining solution: Dissolve 100 mg of Coomassie brilliant blue G-250 in 50 mL of 95% ethanol, add 100 mL of 85% phosphoric acid, make up the volume to 1 L with distilled water, and use after filtration. (2) Standard protein solution: Commonly used bovine serum albumin (BSA) is used as the standard protein, and a series of standard solutions with known concentrations (such as 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg / mL) are prepared. (3) Standard curve drawing: Take 1 mL of standard protein solutions with different concentrations, add 5 mL of Coomassie brilliant blue staining solution, mix well and let stand for 5 minutes. Measure the absorbance (A595) at a wavelength of 595 nm. Draw a standard curve with the protein concentration as the abscissa and the absorbance as the ordinate. (4) Sample determination: Take 1 mL of the sample to be tested, add 5 mL of Coomassie brilliant blue staining solution, mix well and let stand for 5 minutes. Measure the absorbance (A595) at a wavelength of 595 nm. (5) Result calculation: According to the standard curve, convert the absorbance value of the sample into the protein concentration.

[0085] The absorbance of each rose extracellular matrix extract was measured at 560 nm using the chloramine T method, and the hydroxyproline content was calculated. The specific method is as follows: (1) Reagent preparation: Chloramine T solution: Dissolve 1.41 g of chloramine T in 20 mL of distilled water, add 30 mL of methanol and 50 mL of citrate buffer (pH 6.0), and use after mixing. p-Dimethylaminobenzaldehyde (DMAB) solution: Dissolve 10 g of DMAB in 35 mL of 60% perchloric acid and 65 mL of isopropanol, and use after mixing. (2) Hydroxyproline standard solution: Prepare a series of hydroxyproline standard solutions with known concentrations (such as 1, 2, 4, 6, 8, 10 μg / mL). (3) Standard curve drawing: Take 1 mL of hydroxyproline standard solutions with different concentrations, add 1 mL of chloramine T solution, mix well and let stand at room temperature for 20 minutes. Add 1 mL of DMAB solution, mix well and heat in a water bath at 60 °C for 15 minutes. After cooling to room temperature, measure the absorbance (A558) at a wavelength of 558 nm. Draw a standard curve with the hydroxyproline concentration as the abscissa and the absorbance as the ordinate. (4) Sample determination: Take 1 mL of the sample to be tested, add 1 mL of chloramine T solution, mix well and let stand at room temperature for 20 minutes. Add 1 mL of DMAB solution, mix well and heat in a water bath at 60 °C for 15 minutes. After cooling to room temperature, measure the absorbance (A558) at a wavelength of 558 nm. (5) Result calculation: According to the standard curve, convert the absorbance value of the sample into the hydroxyproline concentration.

[0086] The results are shown in Table 2.

[0087] Table 2 Glycoprotein Content in Rose Extracellular Matrix Extract

[0088]

[0089]

[0090] As can be seen from the results in Table 2, by comparing Examples 1-3 and Comparative Examples 1-3, the total glycoprotein content and hydroxyproline content in the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Examples 1-3 are both relatively high. This shows that by mixing the aqueous extract of withered rose petals with fresh rose extract in the present application, a rose extracellular matrix extract with a relatively high glycoprotein content, a relatively high hydroxyproline content, and better bioavailability can be prepared.

[0091] By comparing Example 1 with Examples 4-5, the total glycoprotein content and hydroxyproline content in the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Examples 1-3 are both relatively high. This shows that by mixing the aqueous extract of rose petals that have withered for 1-10 days with the rose extract of rose petals about to bloom in the present application, a rose extracellular matrix extract with a relatively high glycoprotein content and a relatively high hydroxyproline content can be prepared.

[0092] Moreover, by comparing Example 1 with Examples 2-3, the total glycoprotein content and hydroxyproline content in the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 are the highest.

[0093] Test Example 2

[0094] In this test example, the antioxidant effects of the rose extracellular matrix extracts provided in Examples 1-5 and Comparative Examples 1-3 were detected, and the specific steps are as follows:

[0095] Accurately weigh 11 mg of DPPH solid powder and mix it evenly with 11 mL of absolute ethanol. Wait until the solid is completely dissolved, then add 99 mL of absolute ethanol and mix evenly. Place it in a refrigerator at 4°C for later use. The DPPH concentration is 0.1 g / L. During the test, take 2 mL of the above DPPH solution + 1 mL of the sample to be tested as the test group, and the blank group is 1 mL of absolute ethanol. React at room temperature for 30 min, and measure the absorbance at 517 nm using an enzyme-linked immunosorbent assay reader. The DPPH scavenging rate = (A 0 -A) / A 0 ×100%, A 0 is the blank group, and A is the test group. The samples to be tested in the test group were diluted to 2% and 5% of the samples to be tested by the weighing method. The results are shown in Table 3.

[0096] Table 3 DPPH Scavenging Rate

[0097]

[0098] As can be seen from the results in Table 3, by comparing Example 1, Example 3 with Comparative Examples 1-3, the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 and Example 3 has a relatively high scavenging rate of DPPH. This shows that the rose extracellular matrix extract prepared by mixing the water extract of withered rose petals and fresh rose extract in this application has good antioxidant effects. Moreover, the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 has the highest DPPH scavenging rate and the best antioxidant effect.

[0099] Test Example 3

[0100] In this test example, the anti-aging effect of the rose extracellular matrix extract provided in Example 1 was detected, and the specific steps are as follows:

[0101] The rose extracellular matrix extract provided in Example 1 was diluted with serum-free medium containing 1% double antibiotics into test samples with concentrations of 0.01%, 0.2%, and 1% respectively;

[0102] HSF cells were selected, and the seeding density was 2×10 5 cells / mL. 100 μL was inoculated in a 96-well plate. After culturing in DMEM medium containing 15% fetal bovine serum and 1% double antibiotics for 24 h, the supernatant was aspirated and washed twice with sterile PBS. Then 100 μL of PBS was added for 10 MJ ultraviolet irradiation to form an ultraviolet aging model; after the irradiation, the PBS was discarded and 100 μL of the test sample was added to form the test group; the blank group was added with 100 μL of serum-free medium; the positive drug group was 12.5 μg / mL ascorbic acid; after continuing to culture for 24 h, MTT was measured for the lower-layer cells, and the cell survival rate was calculated to determine whether the concentration of the test sample was safe; the supernatant was measured for collagen content using Human COL-1 ELISA KIT, and the operation steps were carried out according to the kit instructions. The MTT results showed that each experimental concentration was non-toxic to the cells. The results are shown in Table 4.

[0103] Table 4 COL-1 content

[0104] Sample COL-1 content (ng / mL) Blank group 28.29±0.26 Model group <![CDATA[4.86±0.05 ### > Positive drug group <![CDATA[16.75±0.36 *** > 0.01% Example 1 <![CDATA[7.68±0.62 * > 0.2% Example 1 <![CDATA[13.10±0.24 *** > 1% Example 1 <![CDATA[20.65±0.33 *** >

[0105] Note: In Table 4 ### represents vs the blank group, p < 0.001; * represents vs the model group, 0.01 < p < 0.05, *** represents vs the model group, p < 0.001.

[0106] As can be seen from the results in Table 4, the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 at a concentration of 1% can significantly increase the content of COL-1 in HSF cells in the model group, and is higher than the content of COL-1 achieved in HSF cells with ascorbic acid at 12.5 μg / mL. This indicates that the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 at a concentration of 1% can resist ultraviolet-induced aging and promote collagen production.

[0107] Test Example 4

[0108] In this test example, the cell migration experiment was conducted on the rose extracellular matrix extract provided in Example 1, and the specific steps are as follows:

[0109] The rose extracellular matrix extract provided in Example 1 was diluted into test samples with concentrations of 0.01%, 0.2%, and 1% respectively using serum-free medium containing 1% double antibody;

[0110] Hacat cells were selected, and the seeding density was 4×10 5 cells / mL. 100 uL was inoculated in a 24-well plate and cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody in a 5% CO 2 After culturing in the cell incubator for 24 h, the cell density was approximately 70%-80%. Use a 100 μL pipette tip to draw a line in the 24-well plate, discard the supernatant, wash twice with serum-free medium, remove the medium, and add 500 μL of the test sample as the test group; the blank group was added with 500 μL of serum-free medium; the positive drug group was 100 μg / mL of EGF epidermal growth factor. Continue to culture and discard the supernatant again at 24 h, 48 h, and 72 h respectively, wash twice with serum-free medium, remove the medium, and repeat the above operations. Take pictures under the microscope at 0 h, 24 h, 48 h, and 72 h, and use Image J software to calculate the scratch area. The scratch healing rate = (initial scratch area - scratch area at time t) / initial scratch area. The results are shown in Table 5.

[0111] Table 5 Scratch healing rate

[0112]

[0113]

[0114] Note: * represents vs the blank group, 0.01 < p < 0.05, ** represents vs the blank group, 0.001 < p < 0.01.

[0115] As can be seen from the results in Table 5, after 24 hours of the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 with a concentration of 1%, the cell scratch healing rate can be effectively increased. It shows that the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 with a concentration of 1% can significantly promote cell migration and has a repair effect on the skin.

[0116] Test Example 5

[0117] In this test example, the moisturizing effect of the rose extracellular matrix extract provided in Example 1 was detected with reference to the standards of "QB / T 4256-2011 Evaluation Guidelines for the Moisturizing Efficacy of Cosmetics" and "Technical Specifications for Cosmetics Safety". The specific steps are as follows:

[0118] 1. Clinical laboratory environment requirements: The temperature is 20°C - 22°C, and the humidity is 40% - 60%, and real-time dynamic monitoring is carried out.

[0119] 2. Number of subjects: 30 people, and 24 - 30 effective volunteers.

[0120] 3. Subject conditions:

[0121] (1) Aged between 16 - 65 years old (excluding pregnant or lactating women);

[0122] (2) The basic value of the capacitance method skin moisture meter for the forearm test area is between 15 - 45, and other capacitance method measuring instruments can be calibrated with Cornemeter;

[0123] (3) Without allergic diseases and no history of allergies to cosmetics and other topical preparations;

[0124] (4) Skilled in reading Chinese, voluntarily sign the informed consent form for the test study, and voluntarily participate; be able to cooperate in participating in the test and abide by the research requirements and experimental measurement time arrangements of this research institute;

[0125] (5) Those who have not used hormonal drugs or immunosuppressants within the past month;

[0126] (6) The test site for the current or recent three months has not participated in other clinical test experiments.

[0127] 4. Test steps:

[0128] (1) Preparation before the test: The subjects clean the inner side of both forearms and wipe them clean with a dry paper towel. Sit quietly in the clinical room for 30 minutes, do not drink water during this period, expose the forearms, present the test state, and keep a relaxed state;

[0129] (2) Mark a 3×3 cm area on the inner side of the subject's arm 2Test area, with an interval of 1 cm between areas, a total of 4 test areas, including 1 blank area and 3 test areas. The water content of the stratum corneum of the skin in each area was measured by the capacitance method, and the results of 5 measurements in each area were averaged.

[0130] (3) The test sample was applied at a rate of (2 ± 0.1) mg / cm 2 Once, and evenly applied to the test area of the latex glove.

[0131] 5. Test period:

[0132] Short-term test: The test sample was applied at a rate of (2 ± 0.1) mg / cm 2 Once, and evenly applied to the test area of the latex glove. The water content of the stratum corneum of the skin in each area before application was recorded as the initial value; the values were measured again 4 h and 8 h after application.

[0133] Long-term test: The test samples were distributed to the subjects, and a sample the size of a rice grain was required to be applied to the test area once in the morning and once in the evening. The water content of the stratum corneum of the skin in each area before application was recorded as the initial value; the values were measured once after 7 d, 14 d, and 28 d respectively.

[0134] 6. Test samples:

[0135] Blank group: Butylene glycol + water;

[0136] Sample group: A1: 1% dilution of the rose extracellular matrix extract provided in Example 1 (diluted with butylene glycol + water), A2: 3% dilution of the rose extracellular matrix extract provided in Example 1, A3: 5% dilution of the rose extracellular matrix extract provided in Example 1.

[0137] The results are shown in Table 6.

[0138] Table 6 Water content of the stratum corneum of the skin

[0139]

[0140]

[0141] Note: In Table 6 * represents vs 0 h, 0.01 < p < 0.05; ** represents vs 0 h, 0.001 < p < 0.01, *** represents vs 0 h, p < 0.001.

[0142] As can be seen from the results in Table 6, compared with the initial value, after 28 days of using the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1, the moisture content of the stratum corneum of the subjects increased significantly, and the short-term moisturizing effect was not significant. This shows that the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 with a concentration above 1% has a long-term moisturizing effect.

[0143] Test Example 6

[0144] In this test example, the anti-aging effect of the rose extracellular matrix extract provided in Example 1 was detected, and the specific steps are as follows:

[0145] The Soft Plus instrument was used to detect the depth of the eye wrinkles of the subjects who used sample A2 in Test Example 5, and the depth of the wrinkles before and 28 days after one cycle of use by the subjects was measured. The results are shown in Table 7 and Figure 1-2 as follows.

[0146] Table 7 Wrinkle Depth

[0147]

[0148] As can be seen from Table 7 and Figure 1-2 the results, after 28 days of using the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 with a concentration of 3%, the depth of the wrinkles under the eyes of the subjects became smaller and the skin became smoother. This shows that the rose extracellular matrix extract prepared by the preparation method of the rose extracellular matrix extract provided in Example 1 with a concentration above 1% has an anti-aging effect.

[0149] The embodiments described above are some, but not all, of the embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the present application claimed, but merely represents selected embodiments of the present application. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present application without creative efforts fall within the scope of protection of the present application.

Claims

1. A method for preparing a rose extracellular matrix extract, characterized in that: include: (1) taking withered rose petals, mixing them with water and extracting them to obtain rose extract; (2) freeze-drying and crushing fresh rose petals to obtain rose petal crushed material, mixing the rose petal crushed material with water and performing ultrasonic extraction to obtain a rose extract; (3) The rose extract and the rose extract solution are mixed for reaction, and the rose extracellular matrix extract is obtained through separation, impurity removal and sterilization.

2. The preparation method according to claim 1, characterized in that: The withered rose petals are rose petals that have withered for 1 to 10 days.

3. The preparation method according to claim 1, characterized in that: The withered rose petals are mixed with water in a weight ratio of 1:5-15, and extracted with stirring at room temperature for 2-4 hours.

4. The preparation method according to claim 1, characterized in that: The fresh rose petals are selected from rose petals that are about to bloom.

5. The preparation method according to claim 1, characterized in that: In the step (2), the freeze-dried fresh rose petals are crushed at 4500-5000 rpm, and the crushed rose petals are screened through 50-80 meshes to obtain rose petal crushed material.

6. The preparation method according to claim 1, characterized in that: The crushed rose petals are mixed with water at a weight ratio of 1:10-50, and ultrasonic extraction is performed at a power of 200-300 W and 30-50° C. for 20-50 minutes.

7. The preparation method according to claim 1, characterized in that: The rose extract and the rose extract solution are mixed at a weight ratio of 1:5-10 and reacted at 35-37° C. for 40-55 minutes.

8. The preparation method according to claim 1, characterized in that: In the step (3), separation and impurity removal are performed sequentially through 0.45-0.5 μm microfiltration, 0.22-0.3 μm microfiltration, 100-300 kDa ultrafiltration, and 10-20 kDa membrane filtration.

9. A rose extracellular matrix extract prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the rose extracellular matrix extract according to claim 9 in preparing skin care products.

Images