A composition for lightening and whitening spots and firming skin and a preparation method thereof

Abstract

This invention discloses a composition for fading dark spots, whitening, and firming the skin, and its preparation method, specifically belonging to the field of cosmetic technology. The composition contains extracts of *Bretschneidera sinensis* bark, hydroxypinazone 9-cis-retinate, sage lactone, tetrahydromagnoliol, and quercetin dimer. The composition provided by this invention can significantly reduce skin melanin content, improve skin brightness, and significantly improve skin elasticity and firmness. The synergistic effect of the components effectively achieves the effects of fading dark spots, whitening, and firming the skin. The preparation process provided by this invention is stable and controllable, suitable for large-scale production, and provides a novel and highly effective skincare composition and application solution for the cosmetic field.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a composition for fading spots, whitening, and firming the skin, and its preparation method. Background Technology

[0002] With rising living standards and evolving consumption concepts, consumers are demanding increasingly sophisticated skincare products, with whitening, brightening, firming, and anti-wrinkle effects becoming core requirements. However, existing skincare products still have many shortcomings in balancing efficacy and safety.

[0003] On the one hand, traditional whitening ingredients such as arbutin and vitamin C derivatives generally have the problem of a single mechanism of action. They mostly reduce melanin production by inhibiting tyrosinase activity, making it difficult to simultaneously address the metabolic excretion of existing pigmentation. In addition, some ingredients have poor stability and are prone to degradation and failure during storage or use, resulting in poor whitening effects and short duration.

[0004] On the other hand, existing firming and anti-wrinkle products often rely on the physical filling effect of ingredients such as hyaluronic acid and collagen, which can only temporarily improve fine lines on the skin surface and cannot fundamentally stimulate the synthesis of collagen and elastin fibers in the dermis, making it difficult to achieve long-lasting firming effects. While some anti-wrinkle ingredients containing retinol can promote cell renewal and collagen production, they are highly irritating and can easily lead to dry skin, peeling, and photosensitivity reactions, resulting in poor compatibility with sensitive skin and limiting their applicability.

[0005] In addition, most skin care products have the limitation of "single function". Whitening products often lack firming effects, and firming products cannot meet the needs of whitening. Consumers need to use multiple products at the same time, which not only increases the cost and steps of skin care, but may also cause skin discomfort due to the interaction between the ingredients of different products.

[0006] Therefore, developing a skincare composition that is safe in ingredients, has synergistic effects among its components, can simultaneously achieve highly effective whitening and brightening effects as well as long-lasting firming and anti-wrinkle effects, and is suitable for various skin types has become an urgent problem to be solved in the field of cosmetic technology. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the purpose of this invention is to provide a skin care composition that is safe in ingredients, has synergistic effects among its components, can simultaneously achieve highly effective spot-fading and whitening effects as well as long-lasting firming and anti-wrinkle effects, and is suitable for various skin types.

[0008] To achieve the above objectives, the present invention discloses the following technical solutions:

[0009] In a first aspect, the present invention provides a composition for fading dark spots, whitening the skin, and firming the skin, the composition comprising the following components in parts by weight:

[0010] 10-15 parts of bark extract from the variegated bellflower tree;

[0011] Hydroxypinazone 9-cis-retinate 2.5-2.7 parts;

[0012] Perilla lactone 1.0-2.0 parts;

[0013] Tetrahydromagnoliol 0.6-1.0 parts;

[0014] Quercetin dimer 2.0-2.4 parts.

[0015] Preferably, the method for preparing the bark extract of *Bretschneidera sinensis* in the composition includes the following steps:

[0016] 1-1. Take the bark of the variegated bellflower tree, dry it to constant weight, crush the dried bark, and sieve it to obtain bark powder;

[0017] 1-2. Mix the bark powder with anhydrous ethanol and soak it, then perform supercritical CO2 extraction for 2-4 hours to obtain the crude extract;

[0018] 1-3. The crude extract was concentrated under reduced pressure to remove ethanol, yielding the bark extract of the variegated bellflower tree.

[0019] More preferably, in steps 1-2, the bark powder is mixed with anhydrous ethanol at a mass-to-volume ratio of 1:(1-3) g / mL and soaked for 2-3 hours.

[0020] More preferably, the parameters for supercritical CO2 extraction in steps 1-2 are: extraction temperature of 50-60℃, extraction pressure of 25-30MPa, separation vessel I temperature of 50-60℃, separation vessel I pressure of 6-8MPa, separation vessel II temperature of 30-40℃, separation vessel II pressure of 4-6MPa, CO2 flow rate of 25-35L / h, and extraction time of 2-4h.

[0021] More preferably, in steps 1-3, the crude extract is concentrated under reduced pressure at 40-50°C to remove ethanol.

[0022] Secondly, the present invention provides the application of the composition described in the first aspect in the preparation of skin care products with whitening, spot-fading, firming and anti-wrinkle effects.

[0023] Thirdly, the present invention provides a serum for fading spots, whitening, and firming the skin, wherein the serum contains the composition described in the first aspect;

[0024] The composition is added to the serum in an amount of 1-3 wt%.

[0025] Preferably, the essence further contains at least one of the following: a moisturizer, a skin emollient, a preservative, a pH adjuster, an emulsifier, a thickener, and a solvent.

[0026] More preferably, the moisturizer is at least one selected from glycerin, butylene glycol, and 1,2-pentanediol;

[0027] The emollient is at least one of polydimethylsiloxane and pentaerythritol tetra(ethylhexanoate);

[0028] The emulsifier is hydrogenated lecithin;

[0029] The preservative is p-hydroxyacetophenone;

[0030] The thickener is sodium polyacrylamide dimethyl taurate;

[0031] The solvent is deionized water.

[0032] Fourthly, the present invention provides a method for preparing the serum described in the third aspect, the method comprising the following steps:

[0033] 2-1. Heat deionized water to 70-80℃, add humectant, thickener and preservative, and stir at 200-300r / min until homogeneous to obtain mixture A;

[0034] 2-2. The emollient and emulsifier are stirred and mixed evenly at 80°C and 200 r / min. After cooling to 45°C, the composition is added and stirred and mixed evenly at 200-300 r / min to obtain mixture B.

[0035] 2-3. Add mixture B to mixture A, homogenize and disperse at 2000-5000 r / min for 10-20 min, cool to 35-40℃, add pH adjuster to adjust pH value to 6.0±0.2, sieve and bottle to obtain the essence.

[0036] In this invention:

[0037] The bark extract of the variegated bellflower tree, obtained using supercritical CO2 extraction technology, is rich in polyphenols, flavonoids, and terpenoids. On the one hand, it can inhibit the activity of tyrosinase and dopa tautomerase, reducing melanin synthesis at the source. On the other hand, it can promote the normal renewal rate of stratum corneum cells, accelerate the transfer of existing melanin to the stratum corneum and its shedding with the stratum corneum, thereby fading existing spots and dullness and improving the overall brightness of the skin.

[0038] Hydroxypinazone 9-cis-retinoic acid ester can penetrate the stratum corneum of the skin, bind to retinoic acid receptors in skin cells, activate cell proliferation and differentiation signaling pathways, promote epidermal cell renewal, and improve skin roughness and fine lines. At the same time, it can regulate the activity of matrix metalloproteinases (MMPs) in the dermis, reduce the degradation of collagen and elastin fibers, and stimulate fibroblasts to synthesize new collagen and elastin, increase the thickness of the dermis, and improve skin elasticity and firmness. Hydroxypinazone 9-cis-retinoic acid ester can indirectly promote the excretion of melanin granules by accelerating the renewal of the stratum corneum, forming a synergistic whitening effect with the bark extract of Campsis grandiflora.

[0039] Perilla lactone possesses anti-inflammatory activity, inhibiting the release of skin inflammatory factors (such as IL-1α and TNF-α), alleviating skin irritation caused by irritants, and protecting the integrity of the skin barrier function. Simultaneously, it promotes the synthesis of skin ceramides, enhancing the skin barrier's ability to retain moisture and resist external stimuli, thus improving the product's suitability for sensitive skin. Perilla lactone can also promote local blood circulation in the skin, delivering more oxygen and nutrients to skin cells, enhancing cellular metabolism, indirectly supporting fibroblast function, and helping to improve skin elasticity and firmness, thus alleviating dullness and sagging caused by poor blood circulation.

[0040] Tetrahydromagnoliol, as a natural whitening active ingredient, can effectively inhibit tyrosinase activity and reduce melanin synthesis. Compared with traditional whitening ingredients, it has a more precise target and is less irritating to the skin.

[0041] Quercetin dimer, as a flavonoid compound, has a strong ability to scavenge free radicals, reducing damage to skin cells from free radicals and delaying the skin aging process. It forms a synergistic effect of "multi-target inhibition of melanin synthesis" with the bark extract of *Bretschneidera sinensis* and tetrahydromagnoliol, further enhancing the whitening and spot-fading effect. Quercetin dimer reduces oxidative damage to collagen and elastin fibers by scavenging free radicals, protecting the dermal structure and helping to improve skin firmness.

[0042] The beneficial effects of this invention are:

[0043] 1. The composition of this invention achieves a synergistic dual effect of lightening and whitening, and firming and anti-wrinkle through the scientific ratio of five core ingredients, solving the problem of single efficacy in traditional skin care products. The bark extract of *Bretschneidera sinensis*, tetrahydromagnoliol, and quercetin dimer work synergistically to whiten the skin from three dimensions: inhibiting melanin synthesis, accelerating melanin excretion, and providing antioxidant protection. After 4 weeks of use, the skin's melanin content decreased by 12.17%, and brightness increased by 7.93%, demonstrating significant whitening and lightening effects. Hydroxypinazone 9-cis-retinate, *Bretschneidera sinensis* bark extract, quercetin dimer, and perilla lactone work synergistically to achieve firming and anti-wrinkle effects from four aspects: stimulating collagen synthesis, reducing collagen degradation, soothing and repairing, and promoting circulation. After 4 weeks, the skin's elasticity R2 value increased by 10.91%, and the firmness F4 value decreased by 25.05%, showing significant improvement in fine lines and sagging.

[0044] 2. This composition can not only be used to prepare serums, but also be adapted to various skin care formulations such as lotions, creams, and masks according to needs, making it flexible in application scenarios. At the same time, it has the characteristics of high efficacy and high safety, which meets the current consumer demand for natural, effective, and gentle skin care products, and has broad application and promotion prospects in the mid-to-high-end skin care market. Detailed Implementation

[0045] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0046] Unless otherwise specified, the test methods used in the examples and comparative examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the percentages mentioned in the examples and comparative examples are mass percentages unless otherwise specified.

[0047] In this invention

[0048] Bark of the variegated bellflower tree: purchased from Shanghai Qianfei Chemical Co., Ltd.

[0049] Perilla lactone: purchased from Shanghai Xinwo Biotechnology Co., Ltd.;

[0050] Tetrahydromagnoliol: Purchased from Shanghai Keqin Technology Co., Ltd.;

[0051] Quercetin dimer: purchased from Shanghai Qianfei Chemical Co., Ltd.;

[0052] Hydroxypinazone 9-cis-retinate: prepared according to Example 1 in patent CN116947723A.

[0053] Preparation of bark extract from *Bretschneidera sinensis*

[0054] Step 1. Place the bark of the variegated bellflower tree in an oven at 40-50℃ and dry it to constant weight. Use a pulverizer to pulverize the dried bark and then pass it through a 60-mesh sieve to obtain bark powder for later use.

[0055] Step 2. Mix the bark powder with anhydrous ethanol at a mass-to-volume ratio of 1:(1-3) g / mL and soak for 2-3 hours. Then transfer the mixture to a supercritical extraction vessel and set the extraction parameters as follows: extraction temperature 50-60℃, extraction pressure 25-30MPa, separation vessel I temperature 50-60℃, separation vessel I pressure 6-8MPa, separation vessel II temperature 30-40℃, separation vessel II pressure 4-6MPa, CO2 flow rate 25-35L / h, and extraction time 2-4 hours. Combine the extracts to obtain the crude extract.

[0056] Step 3. Place the crude extract in a rotary evaporator and remove ethanol under reduced pressure at 40-50℃ to obtain the bark extract of the variegated bellflower tree.

[0057] A composition for fading dark spots, whitening, and firming skin

[0058] The raw materials were accurately weighed according to the mass proportions in Table 1 to obtain the skin-lightening, whitening, and firming composition.

[0059] Table 1. Mass parts of raw materials for the composition

[0060]

[0061] Note: " / " in the table indicates no addition.

[0062] Preparation of a serum for fading dark spots, whitening, and firming the skin.

[0063] The composition of the serum is shown in Table 1, and the preparation method of the serum includes the following steps:

[0064] Step 1. Heat deionized water to 80°C, add glycerol, butanediol, 1,2-pentanediol, sodium polyacrylamide dimethyl taurate and p-hydroxyacetophenone, and stir and mix evenly at 200 r / min to obtain mixture A;

[0065] Step 2. Polydimethylsiloxane, pentaerythritol tetra(ethylhexanoate) ester and hydrogenated lecithin are stirred and mixed evenly at 80℃ and 200r / min. After cooling to 45℃, the raw materials in the composition, including the bark extract of *Cyclocarya paliurus*, hydroxypinazone 9-cis-retinate, succinate lactone, tetrahydromagnoliol and quercetin dimer, are added in sequence and stirred evenly at 200r / min to obtain mixture B.

[0066] Step 3. Add mixture B to mixture A, homogenize and disperse at 5000 r / min for 20 min, cool to 35℃, add pH adjuster to adjust pH value to 6.0±0.2, pass through a 200 mesh sieve and bottle to obtain the essence for lightening spots, whitening and firming skin.

[0067] Table 2. Percentage of each component in the serum by mass

[0068]

[0069] Note: " / " in the table indicates no addition.

[0070] Performance testing

[0071] Whitening, firming and anti-wrinkle test

[0072] Sixty participants aged 40-55 with dull facial skin, fine lines around the eyes, and lack of elasticity were randomly divided into five groups of ten each. They used the serum from Application Example 2 and the control Application Examples 1-4 for four weeks. The whitening, spot-fading, anti-wrinkle, and firming effects were objectively evaluated using a multifunctional skin tester, a skin redness and melanin tester, and a skin elasticity tester.

[0073] After cleansing their faces in the morning and evening, test subjects used the serums prepared in Application Example 2 and Comparative Application Examples 1-4 as described above, for 4 consecutive weeks. Using a skin color difference test probe and a multifunctional skin testing system (CL400 and MPA6, CK, Germany), a skin red and melanin tester and test probe (Mexameter MX18, CK, Germany), and a skin elasticity tester (Cutometer MPA580, CK, Germany), data were collected from the test subjects' faces before use (week 0), week 2, and week 4. Changes in skin whiteness (brightness) / L, skin melanin content / MI, skin elasticity R0 value, skin elasticity R2 value, and skin firmness F4 value before and after using the cosmetics were calculated.

[0074] in:

[0075] The L value represents the white balance of L*; the larger the value, the more the color leans towards white.

[0076] A higher MI measurement value indicates a higher melanin content in the skin;

[0077] The R0 value represents the firmness of the skin; the smaller the R0 value, the firmer the skin.

[0078] R2 value characterizes the viscoelasticity of the skin. The closer the R2 value is to 1 (100%), the greater the elasticity of the curve and the better the skin elasticity.

[0079] The F4 value is the area within the envelope curve formed during the application and removal of negative pressure; the smaller the value, the firmer the skin.

[0080] The average of the test results for each group was taken, and the specific results are shown in Table 3.

[0081] Table 3

[0082] Group Testing items 0 weeks 2 weeks 4 weeks Week 4 Change Rate Application Example 2 Skin melanin MI 186.44 171.20 163.75 -12.17% Application Example 2 Skin brightness L* value 62.16 65.42 67.09 7.93% Application Example 2 Skin elasticity R0 value 0.251 0.222 0.208 -17.13% Application Example 2 Skin elasticity R2 value 0.614 0.661 0.681 10.91% Application Example 2 Skin firmness F4 value 5.310 4.260 3.980 -25.05% Comparative Application Example 1 Skin melanin MI 187.23 180.71 177.41 -5.24% Comparative Application Example 1 Skin brightness L* value 62.84 64.39 65.53 4.28% Comparative Application Example 1 Skin elasticity R0 value 0.262 0.246 0.243 -7.25% Comparative Application Example 1 Skin elasticity R2 value 0.609 0.628 0.637 4.60% Comparative Application Example 1 Skin firmness F4 value 5.186 4.709 4.554 -12.19% Comparative Application Example 2 Skin melanin MI 185.27 178.15 174.64 -5.74% Comparative Application Example 2 Skin brightness L* value 63.39 64.96 66.08 4.24% Comparative Application Example 2 Skin elasticity R0 value 0.258 0.245 0.241 -6.59% Comparative Application Example 2 Skin elasticity R2 value 0.620 0.636 0.648 4.52% Comparative Application Example 2 Skin firmness F4 value 5.261 4.763 4.667 -11.29% Comparative Application Example 3 Skin melanin MI 190.12 187.24 186.16 -2.08% Comparative Application Example 3 Skin brightness L* value 59.25 60.53 61.21 3.31% Comparative Application Example 3 Skin elasticity R0 value 0.270 0.247 0.238 -11.85% Comparative Application Example 3 Skin elasticity R2 value 0.598 0.631 0.645 7.86% Comparative Application Example 3 Skin firmness F4 value 5.305 4.644 4.451 -16.10% Comparative Application Example 4 Skin melanin MI 186.19 182.08 179.72 -3.47% Comparative Application Example 4 Skin brightness L* value 61.94 63.71 64.37 3.92% Comparative Application Example 4 Skin elasticity R0 value 0.273 0.251 0.241 -11.72% Comparative Application Example 4 Skin elasticity R2 value 0.608 0.642 0.653 7.40% Comparative Application Example 4 Skin firmness F4 value 5.307 4.661 4.508 -15.06%

[0083] Results Analysis

[0084] As shown in Table 3, there are significant differences between Application Example 2 and Comparative Application Examples 1-4 in terms of whitening indicators (skin melanin MI, skin brightness L* value):

[0085] After 4 weeks of use, the melanin MI value in Application Example 2 decreased from 186.44 to 163.75, a change rate of -12.17%, which is significantly higher than that in Comparative Application Examples 1, 2, 3, and 4. This indicates that when any one of the core ingredients is missing from the composition, the synergistic effect of inhibiting melanin synthesis and accelerating melanin excretion is broken, and the melanin reduction effect is greatly weakened. In particular, the lack of tetrahydromagnoliol in Comparative Application Example 3 resulted in its targeted inhibition of tyrosinase, with a melanin reduction rate of only 2.08%, further demonstrating the synergistic necessity of the five ingredients in the whitening process.

[0086] In Application Example 2, the L* value increased from 62.16 to 67.09 after 4 weeks of use, with a change rate of 7.93%, which was significantly higher than that of the comparative application examples. This proves that the absence of any ingredient will lead to a decrease in the effect of improving brightness, and verifies the importance of ingredient synergy in improving skin radiance.

[0087] Based on skin elasticity (R0 value, R2 value) and firmness (F4 value) indicators, the firming and anti-wrinkle effect of application example 2 is significantly better than that of the comparison application example:

[0088] In Application Example 2, after 4 weeks of use, the R0 value decreased from 0.251 to 0.208, a change rate of -17.13%, significantly lower than the control application example. The R2 value increased from 0.614 to 0.681, a change rate of 10.91%, higher than the control application example. In Control Application Example 1, due to the lack of hydroxypinazone 9-cis-retinate, the improvement in both R0 and R2 values ​​was significantly reduced, indicating that hydroxypinazone 9-cis-retinate is crucial for stimulating collagen synthesis and improving skin elasticity. In Control Application Example 2, the lack of perillaldehyde also resulted in a significant decrease in the firming effect, verifying the role of perillaldehyde in maintaining healthy skin and ensuring anti-wrinkle efficacy.

[0089] In Application Example 2, the F4 value decreased from 5.310 to 3.980 after 4 weeks of use, a change rate of -25.05%, significantly higher than that of the control application example. In Control Application Example 4, the lack of quercetin dimer resulted in the loss of the protective effect of free radical scavenging on collagen, leading to accelerated collagen degradation and a weakened firmness-enhancing effect. This further demonstrates the synergistic effect of the five ingredients in maintaining the structure of the dermal layer and improving firmness.

[0090] In summary, the five core components of the composition provided by this invention—Cephalotaxus fortunei bark extract, hydroxypinazone 9-cis-retinate, sage lactone, tetrahydromagnoliol, and quercetin dimer—exhibit irreplaceable synergistic effects. The absence of any one of these components will significantly reduce the whitening, spot-fading, or firming and anti-wrinkle effects. Only when these five components are combined in specific mass proportions can they simultaneously achieve multi-target inhibition of melanin and multi-pathway repair of skin structure, ultimately achieving highly effective spot-fading and whitening, and long-lasting firming and anti-wrinkle effects.

[0091] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for lightening and firming skin, characterized by comprising, The composition is composed of the following components in mass fraction: 10-15 parts of the extract of the bark of the tree Planchonella obovata; 2.5-2.7 parts of hydroxyl pinacolone 9-cis retinoic acid; 1.0-2.0 parts of sclareolide; 0.6-1.0 parts of tetrahydro magnolia alcohol; 2.0-2.4 parts of quercetin dimer; The preparation method of the extract of the bark of the tree Planchonella obovata in the composition comprises the following steps: 1-1. Dry the bark of the tree Planchonella obovata to constant weight, crush the dried bark, sieve, and obtain bark powder; 1-2. Mix and soak the bark powder with anhydrous ethanol, then perform supercritical CO2 extraction for 2-4 hours to obtain a crude extract; 1-3. Concentrate and remove ethanol from the crude extract under reduced pressure to obtain the extract of the bark of the tree Planchonella obovata; The parameters of the supercritical CO2 extraction in step 1-2 are as follows: the extraction temperature is 50-60℃, the extraction pressure is 25-30 MPa, the temperature of separation kettle I is 50-60℃, the pressure of separation kettle I is 6-8 MPa, the temperature of separation kettle II is 30-40℃, the pressure of separation kettle II is 4-6 MPa, the CO2 flow rate is 25-35 L / h, and the extraction time is 2-4 h.

2. The composition of claim 1, wherein, In step 1-2, the bark powder is mixed and soaked with anhydrous ethanol at a mass-to-volume ratio of 1: (1-3) g / mL for 2-3 h.

3. The composition of claim 1, wherein, In step 1-3, the crude extract is concentrated and ethanol is removed under reduced pressure at 40-50℃.

4. Use of the composition of any one of claims 1-3 in the preparation of a skin care product having the effects of whitening, lightening spots, and tightening and anti-wrinkling.

5. A skin whitening and firming essence, characterized in that, The serum contains the composition of any one of claims 1-3; The addition amount of the composition in the serum is 1-3 wt%.

6. The serum of claim 5, wherein, The serum further contains at least one of a humectant, an emollient, a preservative, a pH regulator, an emulsifier, a thickening agent, and a solvent.

7. The serum of claim 6, wherein the serum is characterized by, The serum contains a humectant, an emollient, a preservative, a pH regulator, an emulsifier, a thickening agent, and a solvent; The humectant is at least one of glycerol, butylene glycol, and 1,2-pentanediol; The emollient is at least one of dimethicone and pentaerythrityl tetraethylhexanoate; The emulsifier is hydrogenated lecithin; The preservative is p-hydroxyacetophenone; The thickening agent is sodium polyacryloyldimethyl taurate; The solvent is deionized water.

8. The method of claim 7, wherein the serum is prepared by the steps of: The preparation method comprises the following steps: 2-1. Heat deionized water to 70-80℃, add a humectant, a thickening agent, and a preservative, and mix uniformly under stirring at 200-300 r / min to obtain a mixed solution A; 2-2. Mix and uniformly stir an emollient and an emulsifier at 80℃ and 200 r / min, cool to 45℃, then add the composition, and mix uniformly under stirring at 200-300 r / min to obtain a mixed solution B; 2-3. Add the mixed solution B to the mixed solution A, homogenously disperse at 2000-5000 r / min for 10-20 min, cool to 35-40℃, add a pH regulator to adjust the pH value to 6.0±0.2, sieve, and pack to obtain the serum.

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