Preparation method and application of soybean isoflavone extract and composition thereof

Abstract

The present invention relates to cosmetic application development field, there is provided a kind of soy isoflavones extract and the preparation method and application thereof of its composition. In the present invention, soy isoflavones raw material ethanol-water mixed solvent is diluted, macroporous resin is loaded and resin adsorption is carried out to saturation, obtains saturated resin;Then the saturated resin is carried out gradient elution, elution effluent is collected and concentrated, so as to obtain the soy isoflavones extract rich in genistein;And in the soy isoflavones extract rich in genistein (with dry weight basis), genistein content is up to more than 80%wt. In addition, in the present invention, with the genistein in soy isoflavones extract as main active ingredient, it is also possible to coordinate specific plant extract extract, obtain the compound composition with anti-wrinkle firming, nourishing effect, be highly suitable for preparing large health daily use or daily chemical products.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetic application development, and specifically relates to a method for preparing a genistein-rich soybean isoflavone extract and a composition thereof, and uses thereof. The present invention also provides a composite composition comprising the genistein-rich soybean isoflavone extract of the present invention and an extract of a plant extract other than soybeans, and uses thereof. Background Art

[0002] Isoflavones, with a 3-phenylchromone structural core, can bind to α- and β-estrogen receptors and mimic the effects of estrogen on target tissues, thereby exerting health benefits in certain diseases. Many natural plants and herbal isoflavones exhibit promising physiological activities, with isoflavones extracted from plants such as soybeans and kudzu root attracting extensive research attention.

[0003] Soybeans are rich in isoflavone compounds, such as soy isoflavones. These compounds have estrogen-like effects, which can regulate estrogen levels and promote the synthesis of collagen and elastin, thereby helping to improve skin firmness and elasticity.

[0004] The main components of soy isoflavones are daidzein, glycitin, and genistein. Genistein and its aglycones are the most abundant components of soy isoflavones. Furthermore, the aglycones of soy isoflavones are more active than the glycosides, with genistein being particularly active, exhibiting a bidirectional regulatory effect on phytoestrogens and holding promise for application in the development of daily chemical and consumer products for women's health.

[0005] Since soybeans are mostly isoflavones, which have high molecular similarity, similar polarity, and similar solubility, it is difficult to separate high-purity genistein. There are certain difficulties and challenges in the process of extracting and separating genistein from natural sources. The process steps are relatively numerous and the control conditions are strict, or difficult-to-remove irritants, strong acids and alkalis are introduced.

[0006] Currently, the preparation methods of natural genistein mostly adopt acid hydrolysis, alkali precipitation, recrystallization, or silica gel column chromatography. For example, CN101497594A adopts the method of acid hydrolysis, enzymatic hydrolysis, silica gel chromatography, and recrystallization; CN101709057A adopts the method of water precipitation, silica gel chromatography column, and recrystallization for purification. However, most of the currently disclosed purification methods require multiple steps to purify high-purity genistein, or use precision instruments for separation, which have complex processing processes and small yields. The processes involving acid-base precipitation and recrystallization have problems such as large processing materials, high equipment requirements, introduction of irritants, and difficult waste liquid treatment. Gel chromatography columns have problems such as low throughput, long processing time and low yield.

[0007] Therefore, there is an urgent need in this field to develop a preparation method for separating genistein from soybean isoflavones that is easy to scale, mature and stable, has efficient technical processes, is easy to implement, does not introduce irritating substances, has a high transfer rate, and can achieve rapid and continuous production, as well as the application of its composition. Summary of the Invention

[0008] The present invention aims to develop a method for preparing genistein from separated soybean isoflavones, a composition containing the same, and applications thereof, which are easy to scale, mature and stable, have efficient technical processes, are easy to implement, do not introduce irritating substances, have a high transfer rate, and can achieve rapid and continuous production. Specifically, it relates to a soybean isoflavone extract, a preparation method for the composition thereof, and applications thereof.

[0009] In a first aspect of the present invention, a method for preparing a soybean isoflavone extract is provided, the method comprising the following steps:

[0010] (1) diluting a soy isoflavone raw material with an ethanol-water mixed solution to obtain a sample solution; wherein the soy isoflavone raw material contains genistein and additional soy isoflavones selected from the group consisting of daidzein and glycitin; and the ethanol concentration in the sample solution is C1, and 40 v / v%≤C1≤70 v / v%;

[0011] (2) loading the sample solution onto a macroporous resin for resin adsorption, and performing gradient elution or elution with an ethanol / water mixed solvent, collecting the elution effluent containing genistein, thereby obtaining a soybean isoflavone extract rich in genistein; wherein the gradient elution is performed with water as phase A and ethanol as phase B, collecting the elution effluent with an ethanol concentration of C2, and 40v / v%≤C2≤70v / v%, wherein the ethanol concentration in the ethanol / water mixed solvent is C3, and 40v / v%≤C3≤70v / v%; and

[0012] (3) Optionally, the elution effluent is concentrated to obtain a soybean isoflavone extract rich in genistein.

[0013] In another preferred embodiment, the content of genistein in the soybean isoflavone raw material is 1-25wt%, preferably 2-20wt%, more preferably 5-15wt%, based on dry weight.

[0014] In another preferred embodiment, the total content of soy isoflavone compounds in the soy isoflavone raw material is ≥20%, preferably ≥30wt%; for example, 20-80wt%, 25-70wt%, 30-60wt%.

[0015] In another preferred embodiment, the soy isoflavone compounds include genistein, daidzein and glycitin.

[0016] In another preferred embodiment, the sum of the contents of genistein, daidzein and glycitin in the soy isoflavone raw material (based on dry weight) is ≥20%, preferably ≥30wt%; for example, 20-60wt%, 25-50wt%, 30-40wt%.

[0017] In another preferred embodiment, the sample solution is loaded onto a macroporous resin for resin adsorption, and then eluted with an eluent having an ethanol concentration of C3, and 40v / v%≤C3≤70v / v%.

[0018] In another preferred embodiment, the elution effluent corresponding to the ethanol concentration C2 is collected, and 40v / v%≤C2≤70v / v%.

[0019] In another preferred embodiment, the elution effluent corresponding to an elution volume of 3.0-6.0 BV is collected.

[0020] In another preferred embodiment, the content of genistein H0 in the soy isoflavone raw material is calculated according to the following formula Q0:

[0021] H0=W1 / W 原料 ×100% Q0

[0022] Where,

[0023] W1 is the mass of genistein in the soybean isoflavone raw material;

[0024] W 原料 The quality of soy isoflavone raw materials.

[0025] In another preferred embodiment, in the soy isoflavone raw material, the genistein content H0 is 8-12 wt % of the soy isoflavone raw material.

[0026] In another preferred embodiment, in the soybean isoflavone raw material, the relative abundance Y0 of genistein is 25-35 wt%, based on the total weight of soybean isoflavone compounds.

[0027] In another preferred embodiment, the relative abundance of genistein Y0 is calculated according to the following formula Q1:

[0028] Y0=Y1 / (Y1+Y2+Y3)×100% Q1

[0029] Where,

[0030] Y1 is the mass or concentration of genistein in the soybean isoflavone raw material;

[0031] Y2 is the mass or concentration of daidzein in the soy isoflavone raw material;

[0032] Y3 is the mass or concentration of glycitein in the soybean isoflavone raw material.

[0033] In another preferred embodiment, in the genistein-rich soybean isoflavone extract, the genistein content H1 is ≥80 wt%, based on the dry weight of the extract.

[0034] In another preferred embodiment, the genistein content H1 is calculated according to the following formula Q2:

[0035] H1=W1a / W 浸膏 ×100% Q2

[0036] Where,

[0037] W1a is the mass of genistein in the genistein-rich soybean isoflavone extract;

[0038] W 浸膏 The quality of soybean isoflavone extract rich in genistein.

[0039] In another preferred embodiment, the content of genistein in the dry matter of the genistein-rich soy isoflavone extract is ≥90wt%, more preferably ≥95wt%, most preferably ≥98wt%, such as 90-99wt%, 95-98wt%.

[0040] In another preferred embodiment, in the soybean isoflavone extract (or extract) rich in genistein, the relative abundance F1 of genistein is ≥80wt%.

[0041] In another preferred embodiment, the relative abundance F1 is calculated according to the following formula Q3:

[0042] F1=P1 / (P1+P2+P3)×100% Q3

[0043] Where,

[0044] P1 is the mass or concentration of genistein in the genistein-rich soybean isoflavone extract;

[0045] P2 is the mass or concentration of daidzein in the genistein-rich soybean isoflavone extract;

[0046] P3 is the mass or concentration of glycitein in the soybean isoflavone extract rich in genistein.

[0047] In another preferred embodiment, the enrichment ratio is the ratio of H1 to H0 (H1 / H0).

[0048] In another preferred embodiment, the enrichment ratio of genistein by the method is ≥4, preferably ≥8, and more preferably 5-15 or 8-12.

[0049] In another preferred embodiment, the relative abundance F1 of genistein in the soybean isoflavone extract (or extract) prepared by the method is greatly improved, that is, the ratio of F1 to Y0 (F1 / Y0) is ≥2, preferably ≥2.5, and more preferably ≥3.0.

[0050] In another preferred embodiment, the method comprises:

[0051] (S1) diluting a soybean isoflavone raw material with an ethanol-water mixed solvent, loading the material onto a macroporous resin for resin adsorption until saturation, to obtain a saturated resin; wherein the mass ratio of the soybean isoflavone raw material to the ethanol-water mixed solvent is ≤2.5%, the concentration of ethanol in the mixed solvent is C1, and 40 v / v% ≤ C1 ≤ 70 v / v%;

[0052] (S2) eluting the saturated resin in step (S1), collecting and concentrating the elution effluent to obtain a soybean isoflavone extract rich in genistein; wherein the gradient elution is performed with water as phase A and ethanol as phase B, wherein the elution is performed with an eluent having an ethanol concentration of C3, and 40v / v%≤C3≤70v / v%, and the elution volume is 3.0-6.0 BV.

[0053] In another preferred embodiment, the macroporous resin in step (S1) is selected from the following group: SP20SS macroporous resin, CHP20 macroporous resin.

[0054] In another preferred embodiment, the macroporous resin is SP20SS macroporous resin.

[0055] In another preferred embodiment, the mass volume ratio of the soy isoflavone raw material to the resin in step (S1) is 1g:20mL to 1g:100mL, preferably 1g:30mL to 1g:80mL, and more preferably 1g:40mL to 1g:60mL.

[0056] In another preferred embodiment, the mass volume ratio of the soy isoflavone raw material to the resin in step (S1) is 1 g:50 ml.

[0057] In the second aspect of the present invention, a soybean isoflavone extract enriched in genistein is provided. The soybean isoflavone extract is prepared by the method described in the first aspect of the present invention. After the soybean isoflavone extract is dried, the abundance or purity of genistein is ≥80wt%.

[0058] In another preferred embodiment, the genistein-enriched soybean isoflavone extract contains genistein and trace amounts of other substances.

[0059] In another preferred embodiment, the abundance of genistein in the genistein-enriched soybean isoflavone extract is ≥95wt%, preferably ≥98wt% (based on dry weight).

[0060] In a third aspect of the present invention, a composition is provided, comprising: (Z1) the soybean isoflavone extract enriched with genistein as described in the second aspect of the present invention; and

[0061] (Z2) Plant extracts other than soybean extracts;

[0062] The plant extract extract is composed of extracts selected from the following group: angelica extract, cimicifuga extract, kudzu root extract, kudzu root extract, clover extract, knotweed extract, and pomegranate fruit extract.

[0063] In another preferred embodiment, the mass percentage of the soybean isoflavone extract and the plant extract extract enriched with genistein in the composition is:

[0064] Soy isoflavone extract 0.5-5.0wt%

[0065] Angelica extract 0.2-2.0 wt%

[0066] Cimicifuga extract 0.5-4.0 wt%

[0067] Pueraria lobata extract 1-8 wt%

[0068] Pueraria lobata extract 1-5 wt%

[0069] Red Clover Extract 0.2-2.0 wt%

[0070] Polygonum cuspidatum extract 1-5 wt%

[0071] Pomegranate fruit extract 0.2-2.0 wt%.

[0072] In another preferred embodiment, the mass percentage of the soybean isoflavone extract and the plant extract extract in the composition is:

[0073] Soy isoflavone extract 1-3 wt%

[0074] Angelica extract 0.3-1.8 wt%

[0075] Cimicifuga extract 0.8-3.2 wt%

[0076] Pueraria lobata extract 1.8-6.2 wt%

[0077] Pueraria lobata extract 1.3-4.7 wt%

[0078] Red Clover Extract 0.3-1.7 wt%

[0079] Polygonum cuspidatum extract 1.3-4.7 wt%

[0080] Pomegranate fruit extract 0.3-1.7 wt%.

[0081] In another preferred embodiment, the composition further comprises an auxiliary material.

[0082] In another preferred embodiment, the excipient is selected from the following group: butylene glycol, propylene glycol, glycerin, PEG40-hydrogenated castor oil, or a combination thereof.

[0083] In another preferred embodiment, the auxiliary material is butanediol.

[0084] In another preferred embodiment, the mass percentage of the auxiliary material in the composition is 60-95wt%, preferably 70-94wt%, and more preferably 75-92wt%.

[0085] In a fourth aspect of the present invention, there is provided a method for preparing the composition according to the third aspect of the present invention, comprising the steps of:

[0086] The composition as described in the third aspect of the present invention is prepared by mixing (Z1) the soybean isoflavone extract containing genistein as described in the second aspect of the present invention and (Z2) a plant extract extract other than soybeans, and optional auxiliary materials.

[0087] In another preferred embodiment, the method comprises:

[0088] (1) providing the soybean isoflavone extract with high genistein abundance as described above, and then dissolving it with an appropriate proportion of an organic or inorganic solvent to obtain a soybean isoflavone solution with high genistein abundance;

[0089] (2) providing the plant extract concentrate as described above, and then dissolving it with an appropriate proportion of an organic or inorganic solvent to obtain a plant extract solution;

[0090] (3) The soy isoflavone solution and the plant extract solution are mixed in an appropriate ratio to obtain the composition.

[0091] In another preferred embodiment, the method for preparing the plant extract comprises the steps of:

[0092] (1) Provide plant materials: angelica, cimicifuga, kudzu root, kudzu root, red clover, knotweed, pomegranate fruit;

[0093] (2) extracting each plant raw material in step (1) with an ethanol-water solution to obtain each plant extract;

[0094] (3) Concentrating the plant extracts obtained in step (2) to obtain the plant extract extracts: angelica extract, cimicifuga extract, kudzu root extract, thai kudzu root extract, clover extract, knotweed extract, and pomegranate fruit extract.

[0095] In a fifth aspect of the present invention, a composition is provided, comprising: (Z1) the soybean isoflavone extract enriched with genistein as described in the second aspect of the present invention; and

[0096] (Z2) Auxiliary materials.

[0097] In another preferred embodiment, in the composition, the content of genistein-enriched soybean isoflavone extract is 0.001-95 wt %, preferably 0.1-90 wt %, and more preferably 1-50 wt %.

[0098] In another preferred embodiment, the composition further comprises: one or more plant extracts other than soybeans. Preferably, the plant extract is selected from the group consisting of: angelica extract, cimicifuga extract, kudzu root extract, kudzu root extract, clover extract, knotweed extract, and pomegranate fruit extract.

[0099] In the sixth aspect of the present invention, there is provided a use of the composition as described in the third or fifth aspect for preparing a health daily product or a daily chemical product.

[0100] In another preferred embodiment, the use is for preparing anti-wrinkle, firming, anti-aging and nourishing daily use or daily chemical products.

[0101] In another preferred embodiment, the product has one or more characteristics selected from the following group:

[0102] (1) Increase the content of type I collagen (Collagen I);

[0103] (2) reduce the content of matrix metalloproteinase 1 (MMP-1);

[0104] (3) Improve skin moisture content;

[0105] (4) Increase ceramide / protein values;

[0106] (5) Increase the average carbon chain length of ceramide;

[0107] (6) Increase the value of fatty acid / protein;

[0108] (7) Increase the average carbon chain length of fatty acids;

[0109] (8) Increase collagen fiber content:

[0110] (9) Nourishing effect.

[0111] In another preferred embodiment, the product is a cosmetic.

[0112] In another preferred embodiment, the product comprises: (1) the composition described in the third aspect; and (2) pharmaceutically or cosmetically acceptable excipients.

[0113] In another preferred embodiment, the excipient is selected from the following group: one or more of butylene glycol, propylene glycol, glycerin, and PEG40-hydrogenated castor oil.

[0114] In another preferred embodiment, the cosmetic further contains additional ingredients that can be used in cosmetics.

[0115] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] Figure 1 The HPLC chromatogram of daidzein is shown.

[0117] Figure 2 The HPLC chromatogram of glycitein is shown.

[0118] Figure 3 The HPLC chromatogram of genistein is shown.

[0119] Figure 4 A mixed standard HPLC chromatogram is shown.

[0120] Figure 5 The HPLC chromatogram of soy isoflavone raw material is shown.

[0121] Figure 6 Shown is the chromatogram of genistein separation from a soybean isoflavone sample.

[0122] Figure 7 A chromatogram of the separation of standard genistein is shown.

[0123] Figure 8 The recovery rate of genistein in each section of the soy isoflavone sample is shown.

[0124] Figure 9 The relative peak area of ​​genistein under each section of the soy isoflavone sample is shown.

[0125] Figure 10 A cell viability curve graph of Sample Composition 1 based on fibroblasts is shown.

[0126] Figure 11 A cell morphology image of fibroblast-based sample composition 1 is shown.

[0127] Figure 12 A cell viability curve graph of fibroblast-based sample composition 2 is shown.

[0128] Figure 13Cell morphology images of fibroblast-based sample composition 2 are shown.

[0129] Figure 14 A cell viability curve graph of fibroblast-based sample composition 3 is shown.

[0130] Figure 15 Cell morphology images of fibroblast-based sample composition 3 are shown.

[0131] Figure 16 A comparison chart of Collagen I content is shown.

[0132] Figure 17 A comparison graph of MMP-1 content is shown.

[0133] Figure 18 A comparison chart of the anti-wrinkle and firming effects of composition 1, composition 2, and composition 3 is shown.

[0134] Figure 19 A cell viability curve graph of Sample Composition 1 based on keratinocytes is shown.

[0135] Figure 20 A cell morphology image of keratinocyte-based sample composition 1 is shown.

[0136] Figure 21 A cell viability curve graph of Sample Composition 2 based on keratinocytes is shown.

[0137] Figure 22 A cell morphology image of keratinocyte-based sample composition 2 is shown.

[0138] Figure 23 A cell viability curve graph of Sample Composition 3 based on keratinocytes is shown.

[0139] Figure 24 Cell morphology images of keratinocyte-based sample composition 3 are shown.

[0140] Figure 25 A bar graph showing the skin moisture test results for each group.

[0141] Figure 26 A bar graph showing the ceramide / protein test results for each group is shown.

[0142] Figure 27 A bar graph showing the average carbon chain length of ceramides in each group

[0143] Figure 28 A bar graph showing the fatty acid / protein test results for each group is shown.

[0144] Figure 29A bar graph showing the test results of the average carbon chain length of fatty acids in each group.

[0145] Figure 30 The test results of collagen fiber content in each group are shown.

[0146] Figure 31 A bar graph showing the relative area results of collagen fibers in each group. DETAILED DESCRIPTION

[0147] After extensive and in-depth research, through a large number of experimental screenings, a soybean isoflavone extract extraction process enriched in genistein was unexpectedly developed for the first time. In the present invention, the soybean isoflavone raw material is diluted with an ethanol-water mixed solvent, loaded on a macroporous resin for resin adsorption to saturation, and a saturated resin is obtained; wherein the mass ratio of the soybean isoflavone raw material to the ethanol-water mixed solvent is ≤2.5%, the concentration of ethanol in the mixed solvent is C1, and 40v / v%≤C1≤70v / v%; the saturated resin is then eluted with an ethanol concentration of 40v / v%-70v / v% (especially an ethanol concentration of 60v / v%), 3.0-6.0 BV outflow sections are collected, the eluted effluent is collected and concentrated, thereby obtaining a soybean isoflavone extract rich in genistein, and the soybean isoflavone extract rich in genistein is dried to an abundance of genistein ≥80wt%. The present invention also provides a composite composition comprising the genistein-rich soy isoflavone extract of the present invention and a plant extract other than soy, and its use. The composite composition is a plant-based composition with anti-wrinkle, firming, and nourishing properties, and is suitable for preparing daily health products or cosmetics, particularly skin care products, cosmetics, and body care products. This is based on the present invention.

[0148] Benefits of estrogen

[0149] Estrogen plays an important role in the female body, not only regulating the reproductive system but also having a significant impact on skin health.

[0150] Specifically, estrogen affects skin condition in the following ways: (1) Promote collagen synthesis: Estrogen can stimulate fibroblasts in the dermis of the skin, increasing collagen production. This helps maintain skin elasticity and firmness and reduces the formation of wrinkles. (2) Increase skin moisture: Estrogen regulates skin hydration and promotes the production of hyaluronic acid and other moisturizing factors, thereby maintaining the skin's moisture balance and making the skin look plumper and smoother. (3) Antioxidant effect: Estrogen has antioxidant properties that can reduce the damage of free radicals to skin cells, thereby slowing the aging process of the skin. (4) Promote skin healing: Estrogen helps accelerate the repair and regeneration of skin cells, promoting wound healing and skin renewal.

[0151] Application of soybean isoflavone extract with high genistein content

[0152] The high-genistein-rich soybean flavone extract of the present invention is rich in genistein, which has a similar structure to estrogen and is known as phytoestrogen. Therefore, the soybean flavone extract can be used alone to regulate and improve women's health.

[0153] In the theory of Traditional Chinese Medicine, many medicinal plants are not only traditionally believed to have the function of regulating hormones, but modern scientific research has also verified their potential in anti-aging and skin care.

[0154] In modern chemical research, some common medicinal plants such as ginseng, rhodiola rosea, raspberry, perilla, kudzu root, Thai wild kudzu root, pomegranate, red clover, angelica, cimicifuga, and knotweed have been found to contain flavonoids. These flavonoids have estrogen-like effects, that is, they can simulate the biological effects of estrogen, thereby regulating the hormone balance in the body. They can be considered as medicinal plants with hormone-regulating effects.

[0155] The soybean flavonoid extract with high genistein abundance of the present invention can be further compounded with plant extract extracts other than soybeans for use in conditioning and improving women's health.

[0156] In some preferred embodiments, the plant extract extract other than soybean is composed of medicinal plant extracts having hormone regulating effects.

[0157] In some preferred embodiments, the plant extract extract other than soybean is composed of kudzu root, kudzu root, pomegranate fruit, red clover, angelica root, cimicifuga, and knotweed.

[0158] Related medicinal plants with hormone regulating effects

[0159] Astragalus: The isoflavones in Astragalus have estrogen-like effects and can help maintain skin elasticity and moisture.

[0160] Ginseng: Ginsenosides not only have antioxidant and anti-aging effects, but can also improve skin condition through hormone regulation.

[0161] Rhodiola Rosea: Rhodiola Rosea contains a variety of bioactive ingredients, such as salidroside and phenolic compounds, which have estrogen-like effects and can help balance hormone levels in the body. Rhodiola Rosea has significant antioxidant and anti-inflammatory properties, which can reduce skin inflammation, protect skin cells from free radical damage, slow skin aging, and improve skin elasticity and firmness.

[0162] Raspberries: Raspberries are rich in phytoestrogens, such as isoflavones, which help regulate estrogen levels in the body and improve female hormone balance. The antioxidants in raspberries can protect skin cells, reduce free radical damage, promote skin repair and regeneration, and have moisturizing, anti-wrinkle and firming effects.

[0163] Perilla: Perilla is rich in flavonoids, which regulate hormone levels in the body and have estrogen-like effects, helping to improve hormone imbalances in women. Perilla also possesses powerful anti-inflammatory and antioxidant properties, alleviating skin inflammation, protecting skin cells from oxidative damage, promoting skin repair and regeneration, and improving skin's radiance and elasticity.

[0164] Pueraria root: Pueraria root is rich in phytoestrogens, such as puerarin and soy isoflavones, which can mimic the effects of estrogen in the body and help balance hormone levels. Pueraria root also contains a large number of flavonoids, such as isoflavones and flavonoid glycosides. These ingredients have antioxidant and anti-inflammatory properties, reducing free radical damage to the skin, promoting collagen production, helping the skin retain moisture, and helping to slow the aging process. Therefore, it is widely used in anti-wrinkle and firming cosmetics.

[0165] Pueraria Mirifica: In traditional Thai herbal medicine, Pueraria Mirifica is used to treat various ailments and is also believed to nourish the skin, reduce wrinkles, firm the skin, enhance skin tenderness, and enhance breast size. Pueraria Mirifica contains a large amount of saponins, flavonoids, and polyphenols, which have strong antioxidant and anti-inflammatory properties, helping to protect the skin from environmental irritants and reduce the appearance of wrinkles.

[0166] Pomegranate: Rich in flavonoids, vitamins, and polyphenols, pomegranates have strong antioxidant and anti-inflammatory properties, helping to protect the skin from free radical damage and reduce the appearance of wrinkles. Traditionally used in Traditional Chinese Medicine, pomegranates are used to clear heat and detoxify, and to astringe the skin. They are also believed to help slow the aging process.

[0167] Red Clover: Red Clover is rich in genistein, flavonoids and polyphenols, which have strong antioxidant and anti-inflammatory effects, as well as female hormone regulating effects, helping to protect the skin from environmental irritants and reduce the formation of wrinkles.

[0168] Angelica: In traditional Chinese medicine, Angelica is known as the "holy medicine of gynecology" and is used to regulate female hormone levels. It is also believed to help promote blood circulation and nourish the skin. Angelica is rich in vitamins, flavonoids and polyphenols. These ingredients have strong nourishing and antioxidant effects, which help improve skin texture and reduce the formation of wrinkles.

[0169] Cimicifuga: Cimicifuga contains several active ingredients, such as cimicifuga glycosides and polyphenolic compounds, which have strong antioxidant and anti-inflammatory properties and may help slow the aging process of the skin. In traditional Chinese medicine, Cimicifuga is used to regulate female hormone levels and alleviate menopausal symptoms.

[0170] Japanese knotweed: Japanese knotweed contains several active ingredients, including resveratrol, which has estrogen-like properties that can help regulate hormone levels and improve female endocrine disorders. Japanese knotweed possesses powerful antioxidant and antibacterial properties, protecting skin cells from oxidative damage, reducing inflammation, and promoting skin repair and regeneration. Furthermore, resveratrol can improve skin elasticity and firmness, reducing wrinkles and dark spots.

[0171] Optimal application of soybean isoflavone extract with high genistein abundance

[0172] In the present invention, the soybean isoflavone extract with high abundance of genistein of the present invention, wherein genistein is the main active ingredient with abundance ≥80%, is combined with the plant extract extracts of Pueraria lobata, Pueraria lobata root, pomegranate fruit, red clover, angelica, Cimicifuga heracleifolia, and Polygonum cuspidatum, according to scientific cell platform and in vitro skin platform efficacy test data, thereby preparing a compound plant composition. The compound composition is used to prepare a kind of large health daily use or daily chemical product with anti-wrinkle firming and nourishing effects, preferably skin care products, cosmetics and body care products. The compound plant composition utilizes the rich antioxidant, anti-wrinkle nourishing and anti-inflammatory active ingredients in plant raw materials to protect the skin from external damage, improve skin texture, reduce the generation of wrinkles, and make the skin look younger and healthier.

[0173] Flavonoids and isoflavonoids

[0174] Flavonoids are a class of compounds composed of two benzene rings (A and B) with phenolic hydroxyl groups linked by a central three-carbon atom. Their basic nucleus is 2-phenylchromone. Flavonoids often contain functional groups such as phenolic hydroxyl, methoxy, methyl, and prenyl groups, and often combine with sugars to form glycosides. The main natural flavonoids can be classified into fifteen categories based on characteristics such as the degree of oxidation of the central three-carbon chain, the position of the B-ring attachment (2- or 3-position), and whether the three-carbon chain is cyclic: flavones, flavonols, flavonones, flavanonols, anthocyanidins, flavan-3,4-diols, xanthones, chalcones, and biflavonoids.

[0175] Isoflavones are a type of flavonoid compound, sharing a basic 3-phenylchromone skeleton. Unlike flavones, their B-ring connections differ. They are primarily found in leguminous plants, and soy isoflavones are secondary metabolites formed during soybean growth. Extracted from plants, they share a similar structure to estrogen, hence the name phytoestrogens. In the present invention, high-performance liquid chromatography (HPLC) is used for precise quantitative detection of the target compounds, thereby controlling product quality.

[0176] Because isoflavones are a type of flavonoid, and it is difficult to completely distinguish isoflavones from other flavonoids, mature methodologies, such as ultraviolet spectrophotometry, have been established for the quantitative determination of flavonoids. Therefore, ultraviolet spectrophotometry is used to quantitatively analyze and determine the flavonoid components in the plant composition extract, thereby providing a clue to the isoflavone content in the plant composition extract and providing chemical clues to the efficacy of the plant isoflavone composition of the present invention.

[0177] Active ingredients in soybeans

[0178] The active ingredients in soybeans are mostly isoflavones, which have high molecular similarity, similar polarity, and similar solubility. The main active ingredients in the soybean isoflavone raw materials described in the present invention are daidzein, glycitin, and genistein, and the structural formula is as follows:

[0179]

[0180] These three have similar structures and polarities and exist in two types: glycosides and aglycones, which makes it difficult to separate high-purity genistein.

[0181] Soy isoflavone extract containing high abundance of genistein of the present invention

[0182] In the present invention, the abundance of genistein in the soy isoflavone extract is ≥80wt%.

[0183] Preferably, the content of genistein in the dry matter of the genistein-rich soybean isoflavone extract is ≥90wt%, preferably ≥95wt%, and most preferably ≥98wt%, such as 90-99wt%, 95-98wt%. In the present invention, the soybean isoflavone extract is prepared by the following method:

[0184] (S1) diluting a soy isoflavone raw material with an ethanol-water mixed solvent, loading the material onto a macroporous resin for resin adsorption until saturation, to obtain a saturated resin; wherein the mass ratio of the soy isoflavone raw material to the ethanol-water mixed solvent is ≤2.5%, the concentration of ethanol in the mixed solvent is C1, and 40 v / v% ≤ C1 ≤ 70 v / v%;

[0185] (S2) eluting the saturated resin described in step (S1), collecting the elution effluent and concentrating it to obtain a soybean isoflavone extract rich in genistein; wherein the elution is performed with water as phase A and ethanol as phase B, wherein the elution is performed with an eluent having an ethanol concentration of C3, and 40v / v%≤C3≤70v / v%, and the elution volume is 3.0-6.0 BV.

[0186] Preferably, the soy isoflavone raw material contains daidzein at a content of 14-18 wt%, glycitein at a content of 5-9 wt%, and genistein at a content of 8-12 wt%.

[0187] Preferably, the soy isoflavone raw material contains daidzein at a content of 15-17 wt%, glycitein at a content of 6-8 wt%, and genistein at a content of 9-11 wt%.

[0188] Preferably, the volume of the sample loaded in step (S1) is 1-3 BV / h.

[0189] Preferably, the flow rate of the sample loading in step (S1) is 1-3 BV / h.

[0190] Preferably, the mass ratio of the soy isoflavone raw material to the ethanol-water mixed solvent in step (S1) is ≤2%, preferably ≤1.5%.

[0191] Preferably, the concentration of ethanol in step (S1) is C1, and 40v / v%≤C1≤60v / v%.

[0192] Preferably, the macroporous resin in step (S1) is selected from the group consisting of SP20SS macroporous resin, CHP20 macroporous resin, or a combination thereof.

[0193] Preferably, the macroporous resin is SP20SS macroporous resin.

[0194] Preferably, the mass volume ratio of the soy isoflavone raw material to the resin in step (S1) is 1:40-60 g / mL.

[0195] Preferably, the concentration of ethanol in step (S2) is C3, and 40v / v%≤C3≤60v / v%.

[0196] Preferably, after the soy isoflavone extract is dried, the genistein content is ≥80wt%, preferably ≥90wt%, more preferably ≥95wt%, and most preferably ≥98wt%, such as 90-99wt%, 95-98wt%.

[0197] Plant extract extract of the present invention

[0198] In existing research, genistein is mainly used in medicine, and there is no research on its use in anti-wrinkle, nourishing, and anti-aging cosmetics. The present invention is inspired by the effects of Chinese medicinal plants and genistein on human hormone regulation, as well as the effects of hormones on human skin. Seven Chinese medicinal plants with hormone-regulating effects (Pueraria lobata, Pueraria lobata in Thailand, Trifolium pratense, Pomegranate, Angelica sinensis, Cimicifuga heracleifolia, and Polygonum cuspidatum) are selected. By preparing a variety of medicinal plants in different proportions and conducting efficacy tests on each composition in different proportions, a plant extract extract with strong anti-wrinkle and nourishing effects is selected.

[0199] In the present invention, the plant extract extract consists of extracts selected from the group consisting of angelica extract, cimicifuga extract, kudzu root extract, kudzu root extract, clover extract, knotweed extract, and pomegranate fruit extract.

[0200] In the present invention, the method for preparing the plant extract comprises the steps of:

[0201] (1) Provide plant materials: angelica, cimicifuga, kudzu root, kudzu root, red clover, knotweed, pomegranate fruit;

[0202] (2) extracting each plant raw material in step (1) with an ethanol-water solution to obtain each plant extract;

[0203] (3) Concentrating the concentrated extracts of each plant obtained in step (2) to obtain the following plant extracts: angelica extract, cimicifuga extract, kudzu root extract, thai kudzu root extract, clover extract, knotweed extract, and pomegranate fruit extract.

[0204] Preferably, the flavonoid content in each of the plant extracts is as follows:

[0205] Angelica extract 60-80μg / mL

[0206] Cimicifuga extract 650-750 μg / mL

[0207] Pueraria lobata extract 50-150μg / mL

[0208] Pueraria lobata extract 280-380μg / mL

[0209] Trifolium pratense extract 80-180μg / mL

[0210] Polygonum cuspidatum extract 1900-1150 μg / mL

[0211] Pomegranate fruit extract 600-720μg / mL.

[0212] Preferably, the flavonoid content in each of the plant extracts is as follows:

[0213] Angelica extract 65-75μg / mL

[0214] Cimicifuga extract 670-730 μg / mL

[0215] Pueraria lobata extract 70-130 μg / mL

[0216] Pueraria lobata extract 300-360μg / mL

[0217] Trifolium erythrorhizome extract 100-160 μg / mL

[0218] Polygonum cuspidatum extract 1000-1100 μg / mL

[0219] Pomegranate fruit extract 650-700μg / mL.

[0220] Composition of the present invention and preparation method thereof

[0221] The present invention also provides a composition containing the genistein-rich extract of the present invention, which comprises: (Z1) the genistein-rich soybean isoflavone extract of the present invention; (Z2) optional plant extract extracts or other active ingredients other than soybeans, and (Z3) optional excipients.

[0222] Preferably, the plant extract extract other than soybeans consists of extracts selected from the group consisting of angelica extract, cimicifuga extract, kudzu root extract, kudzu root extract, clover extract, knotweed extract, and pomegranate fruit extract.

[0223] Preferably, the mass percentage of the soybean isoflavone extract and the plant extract extract in the composition is:

[0224] Soy isoflavone extract 0.5-5.0wt%

[0225] Angelica extract 0.2-2.0 wt%

[0226] Cimicifuga extract 0.5-4.0 wt%

[0227] Pueraria lobata extract 1-8 wt%

[0228] Pueraria lobata extract 1-5 wt%

[0229] Red Clover Extract 0.2-2.0 wt%

[0230] Polygonum cuspidatum extract 1-5 wt%

[0231] Pomegranate fruit extract 0.2-2.0 wt%.

[0232] Preferably, the mass percentage of the soy isoflavone extract and the plant extract extract in the composition is:

[0233] Soy isoflavone extract 1-3 wt%

[0234] Angelica extract 0.3-1.8 wt%

[0235] Cimicifuga extract 0.8-3.2 wt%

[0236] Pueraria lobata extract 1.8-6.2 wt%

[0237] Pueraria lobata extract 1.3-4.7 wt%

[0238] Red Clover Extract 0.3-1.7 wt%

[0239] Polygonum cuspidatum extract 1.3-4.7 wt%

[0240] Pomegranate fruit extract 0.3-1.7 wt%.

[0241] In another preferred embodiment, the composition further comprises an auxiliary material.

[0242] In another preferred embodiment, the excipient is selected from the following group: butylene glycol, propylene glycol, glycerin, PEG40-hydrogenated castor oil, or a combination thereof.

[0243] In another preferred embodiment, the auxiliary material is butanediol.

[0244] In another preferred embodiment, in the composition of the present invention, the mass percentage of the auxiliary material in the composition is 60-95wt%, preferably 70-94wt%, and more preferably 75-92wt%.

[0245] In the present invention, the preparation method of the composition comprises the steps of:

[0246] (1) providing the soy isoflavone extract of the present invention and dissolving it in 1,3-butanediol, and fully mixing them to obtain a soy isoflavone solution, wherein the mass ratio of the soy isoflavone extract to 1,3-butanediol is 1-1.5:20;

[0247] (2) providing a plant extract extract dissolved in 1,3-butanediol, and fully mixing to obtain a plant extract solution, wherein the mass ratio of the plant extract extract to 1,3-butanediol is 7-25:20,

[0248] The plant extract extract is composed of extracts selected from the group consisting of angelica extract, cimicifuga extract, kudzu root extract, kudzu root extract, clover extract, knotweed extract, and pomegranate fruit extract.

[0249] (3) The soy isoflavone solution and the plant extract solution are mixed, 1,3-butanediol is added to the solution to make up the volume, and the mixture is stirred to obtain the composition.

[0250] Preferably, the mass percentages of soybean isoflavone extract, plant extract extract and 1,3-butanediol in the composition are:

[0251] Soy isoflavone extract 0.5-5.0wt%

[0252] Angelica extract 0.2-2.0 wt%

[0253] Cimicifuga extract 0.5-4.0 wt%

[0254] Pueraria lobata extract 1-8 wt%

[0255] Pueraria lobata extract 1-5 wt%

[0256] Red Clover Extract 0.2-2.0 wt%

[0257] Polygonum cuspidatum extract 1-5 wt%

[0258] Pomegranate fruit extract 0.2-2.0 wt%.

[0259] 1,3-Butanediol and the rest.

[0260] Preferably, the content of soybean isoflavone extract, plant extract extract and 1,3-butanediol in the composition is:

[0261] Soy isoflavone extract 1-3wt%

[0262] Angelica extract 0.3-1.8 wt%

[0263] Cimicifuga extract 0.8-3.2 wt%

[0264] Pueraria lobata extract 1.8-6.2 wt%

[0265] Pueraria lobata extract 1.3-4.7 wt%

[0266] Red Clover Extract 0.3-1.7 wt%

[0267] Polygonum cuspidatum extract 1.3-4.7 wt%

[0268] Pomegranate fruit extract 0.3-1.7 wt%

[0269] 1,3-Butanediol and the rest.

[0270] Compared with the prior art, the present invention has the following beneficial effects:

[0271] 1. In the present invention, an HPLC synchronous detection method for the main active ingredients in soy isoflavones, daidzein, glycitin, and genistein, was established, and the peak time of each compound was confirmed. The peak time of the mixed standard was consistent with that of the single standard, and the sample chromatographic peaks had good separation, providing a reliable method for the quantitative determination of the main active ingredients in soy isoflavones.

[0272] 2. In the present invention, soy isoflavones are used as raw materials, and a large number of experiments are conducted to provide a soy isoflavone extract extraction process enriched with genistein, wherein when the ethanol concentration in the macroporous resin loading solution is 40%-70% and the mass volume ratio of the soy isoflavones raw material to the resin is 1g:50mL, the adsorption rate of genistein is relatively high; in the elution procedure, elution with an ethanol concentration of 40v / v%-70v / v% (especially an ethanol concentration of 60v / v%) can obtain a better separation degree for genistein, and the chromatographic peak of genistein is completely separated from other compounds. The 3.0-6.0 BV effluent segment is collected, and the genistein content in the obtained soy isoflavone extract enriched with genistein is as high as more than 80% (by dry weight).

[0273] 3. According to the extraction process of the present invention, in the 3.0-6.0 BV range of 60% ethanol elution, all components of the soy isoflavones were well recovered at the end of the experiment, with a recovery rate greater than 90%. This method can be used as an industrial production means to achieve continuous production.

[0274] 4. In the present invention, genistein in soybean isoflavone extract is used as the main active ingredient, combined with a specific plant extract extract, and scientific cell platform and in vitro skin platform efficacy test data to prepare a compound plant composition. The composition is used to prepare a daily health or daily chemical product with anti-wrinkle, firming and nourishing effects, and is preferably suitable for skin care products, cosmetics and body care products.

[0275] 5. The method for separating genistein based on macroporous resin of the present invention has the following advantages over existing separation technologies:

[0276] (1) Efficient separation: Macroporous resins have larger pore sizes and surface areas, which can provide a larger contact area, facilitating the adsorption and separation between genistein and the resin. This allows for efficient separation and purification in a relatively short period of time.

[0277] (2) High selectivity: Macroporous resins can enhance their selective adsorption of target compounds by adjusting the chemical properties and functional groups of the resin, thereby achieving selective separation of genistein. This selectivity can improve the efficiency of separation and purification, reduce impurities while retaining the target product.

[0278] (3) Good separation ability and reduced operation steps: During the macroporous resin separation process, high-purity genistein can be obtained by appropriate elution conditions. Compared with other methods, macroporous resin separation can generally provide higher product purity and reduce subsequent processing steps.

[0279] (4) Reusability: Macroporous resins generally have good chemical resistance and mechanical stability and can be used multiple times. This makes macroporous resins highly economical and sustainable in laboratory and industrial production.

[0280] (5) Environmentally friendly and reduces the risk of use: The processing aids used in the present invention are only ethanol and water, and no acid, alkali, irritating solvents and other substances are introduced. Both the resin and the processing aid can be recycled, thereby reducing production costs, reducing waste emissions, and reducing the risk of product irritation.

[0281] In general, the macroporous resin adsorption method is used to directionally separate the main effective isoflavone substance, genistein, from soybean isoflavones. It has the characteristics of high transfer rate, continuous production, and reusable auxiliary materials. It effectively reduces production costs and environmental pollution during the production process, and solves the problem of low production efficiency.

[0282] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0283] Example 1 Separation and purification of genistein

[0284] 1. Determination of daidzein, glycitin, and genistein content

[0285] 1.1 Test Method

[0286] The test sample was soy isoflavone raw material purchased from Xi'an Haoxuan Biotechnology Co., Ltd. It was determined to contain about 35-40% soy isoflavones. The main soy isoflavone components are shown in Table 3.

[0287] Preparation of test solution: Accurately weigh about 10 mg of sample into a 10 mL volumetric flask, add 70% methanol solution to dissolve and dilute to 10 mL, shake well, filter through a 0.45 μm membrane, and take the filtrate.

[0288] Preparation of standard solution: Take appropriate amount of daidzein, glycitin, and genistein standard respectively, weigh accurately, add 70% ethanol to make a solution containing approximately 0.05 mg of daidzein, 0.05 mg of glycitin, and 0.05 mg of genistein per 1 mL, filter through a 0.45 μm filter membrane, and take the filtrate.

[0289] Chromatographic conditions:

[0290] Chromatographic column: octadecylsilane bonded silica gel as filler, size: 4.6 mm × 250 mm;

[0291] Mobile phase: 0.1% formic acid in water as phase A, methanol as phase B, gradient elution according to the requirements in Table 1; detection wavelength at 254 nm, column temperature at 30 °C.

[0292] Table 1 Mobile phase

[0293]

[0294] Determination method: Accurately aspirate 10 μL of standard solution and test solution respectively, inject into liquid chromatograph, and determine.

[0295] 1.2 Test Results

[0296] 1.2.1 Peak Time Positioning of Daidzein, Glycitein, and Genistein

[0297] HPLC chromatograms of daidzein, glycitin and genistein standards are shown in Figure 2. Figure 1 、 Figure 2 and Figure 3 The peak times of daidzein, glycitin and genistein are shown in Table 2. The HPLC results of soybean isoflavones raw material (test sample) are as follows: Figure 4 As shown, the HPLC of the mixed standard is as follows Figure 5 The contents of various substances in soybean isoflavones were calculated based on the peak areas as shown in Table 3.

[0298] Table 2 Peak time of each substance

[0299]

[0300] From the data in the table, it can be seen that the peak time of the mixed standard is consistent with that of the single standard.

[0301] Table 3 Content of various components in soybean isoflavones (by dry weight)

[0302]

[0303] Note: ① Concentrations of each substance in the mixed standard: daidzein 55.468 μg / mL; glycitin 58.016 μg / mL; genistein 50.568 μg / mL. ② Take 5.09 mg of soy isoflavones and dilute to 10 mL with 70% methanol.

[0304] In this experiment, an HPLC simultaneous detection method for the main active ingredients in soy isoflavone raw materials, namely daidzein, glycitein and genistein, was established. The peak time of each compound has been confirmed, and the peak time of the mixed standard is consistent with that of the single standard. The sample chromatographic peaks have good separation. The quantitative detection of the daidzein content in the soy isoflavone raw materials was 16.626%, the glycitein content was 7.541%, and the genistein content was 10.194%.

[0305] 2. Screening of Soy Isoflavone Concentration by Purification with Genistein Macroporous Resin

[0306] 2.1 Experimental Materials

[0307] 2.1.1 Macroporous resin: SP20SS (purchased from Mitsubishi Corporation)

[0308] 2.1.2 Prepare the sample solution and resin for testing according to Table 4 below

[0309] Table 4 Static adsorption of soybean isoflavone concentration and resin dosage

[0310]

[0311] Note: Use 40% ethanol to prepare the experimental samples

[0312] 2.1.3 Experimental methods

[0313] 1) Resin activation preparation: Take a certain amount of SP20SS macroporous adsorption resin and soak it in anhydrous ethanol overnight for activation treatment. After activation, elute with pure water until the eluate has no alcohol smell;

[0314] 2) Static adsorption test: Prepare 40% alcohol solution to dissolve soybean isoflavones according to the record in Table 4 of 2.1.2, and dilute to the recorded weight. Mix with macroporous resin, place on a shaker at 200 rpm for 2 hours, filter with a 300 mesh screen, and take the filtrate for testing.

[0315] 3) Test method:

[0316] Preparation of test solution: Take an appropriate amount of the filtrate prepared in step 2), dilute it with 70% methanol, and filter it through a 0.45 μm filter membrane;

[0317] The standard solution is the same as 1.1.

[0318] Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and perform the determination using the same chromatographic method as described in 1.1 of Example 1.

[0319] 2.1.4 Experimental Results

[0320] The total amount of each substance before and after adsorption was calculated by comparing the peak area with the standard. This was then divided by the amount of each substance in the supernatant after adsorption, and the adsorption rate was calculated as the ratio of the amount after adsorption to the amount before adsorption. The experimental results are shown in Table 5 below.

[0321] Table 5 Calculation results of adsorption rate of each part

[0322]

[0323] Note: The adsorption rate in the table indicates the ratio of each active ingredient transferred from the sample solution to the resin before loading. The test results of the content of each part in the sample solution before loading are shown in Table 3.

[0324] 2.1.5 Summary

[0325] The experimental results show that when the concentration of soy isoflavones increases, the adsorption rate of genistein shows a downward trend. When the soy isoflavones content is 1% and 2.50%, the adsorption rate of genistein can reach more than 85%. When the soy isoflavones content is 1%, the adsorption rate of genistein is as high as more than 97%. When the soy isoflavones content is greater than 5.00%, the adsorption rate of genistein drops rapidly. Therefore, the loading concentration of isoflavones is preferably 1% and 2.50%. The optimal 1% (W / W) isoflavones concentration was selected for subsequent experiments.

[0326] 3. Screening of Ethanol Concentration for Genistein Macroporous Resin Purification

[0327] 3.1.1 Resin model:

[0328] SP20SS (purchased from Mitsubishi Corporation)

[0329] 3.1.2 Prepare the sample solution and resin for testing according to Table 6 below

[0330] In this experiment, the loading concentration of soy isoflavones was 10%, so that the isoflavone concentration was high enough to achieve the purpose of saturated adsorption of the resin and eliminate the possibility of unsaturated adsorption, thereby ensuring the reliability of the experimental results.

[0331] Table 6 Sampling amount for each ethanol concentration test group

[0332]

[0333] Note: According to the experimental design concentration, 0%, 20%, 40%, and 60% ethanol solutions were prepared for experimental sample preparation.

[0334] 3.1.3 Experimental methods

[0335] The experimental method is the same as 2.1.3.

[0336] 3.1.4 Experimental Results

[0337] The contents of daidzein, glycitein, and genistein were calculated by comparing the peak areas of the chromatographic peaks in the HPLC graph with those of the standard product, and the adsorption rate (mg / g %) was obtained by dividing the contents by the amount of resin used.

[0338] The experimental results are shown in Table 7 below.

[0339] Table 7 Adsorption rate of each part

[0340]

[0341] Note: The results in the table indicate the amount of active ingredients that can be adsorbed per gram of resin.

[0342] 3.1.5 Summary

[0343] Based on the above experimental results, it can be concluded that at 40% and 60% ethanol concentrations, the adsorption of genistein by the resin is similar, with sample loadings of 10.031 mg / g and 9.910 mg / g, respectively. However, under 40% ethanol solution, the adsorption capacity of the macroporous resin for daidzein and glycitein is much greater than that under 60% ethanol conditions. In order to eliminate the interference of other substances on genistein, subsequent experiments were tested using 60% ethanol.

[0344] 4. Dynamic Testing of Genistein Macroporous Resin Purification

[0345] 4.1 Macroporous resin chromatography column: CHP20 macroporous resin chromatography column (purchased from Mitsubishi Corporation), size: 4.6 mm * 250 mm;

[0346] 4.2 Experimental methods:

[0347] 4.2.1 Preparation of test solution: Dissolve approximately 20 mg of soy isoflavones in 60% ethanol, filter through a 0.45 μm filter membrane, and obtain the filtrate.

[0348] 4.2.2 Mobile phase: Use water as phase A and ethanol as phase B, and perform gradient elution according to the requirements in Table 8 below; the detection wavelength is 254 nm, and the column temperature is 30°C.

[0349] Table 8 Mobile phase conditions

[0350]

[0351] Determination method: Accurately aspirate 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, and determine.

[0352] 4.4 Experimental Results

[0353] from Figure 6 、 Figure 7 It can be seen that by eluting with the mobile phase indicated in this experimental method, genistein can obtain a good separation degree, and the chromatographic peak of genistein is completely separated from other compounds. Subsequent experiments can refer to the above mobile phase ratio for scale-up testing.

[0354] 5. Separation and scale-up verification of genistein macroporous resin;

[0355] 5.1 Experimental Methods

[0356] 5.1.1 Macroporous resin Resin model: SP20SS

[0357] 5.1.2 Resin volume: 150 mL;

[0358] 5.1.3 Test Solution Preparation: Prepare the test solution by dissolving 3.15 g of soy isoflavones in 300 mL of 60% ethanol solution.

[0359] 5.1.4 Elution Conditions: Use water as phase A and ethanol as phase B, and perform gradient elution as specified in Table 9 below;

[0360] Table 9 Elution mobile phase conditions

[0361]

[0362] 5.1.5 Collect effluent every 0.5 BV for measurement

[0363] 5.1.6 Sample volume: The sample volume is 2 BV (300 mL), and the effluent (every 0.5 BV) is collected for testing.

[0364] 5.1.7 Flow rate: 2BV / h.

[0365] 5.1.8 Detection method:

[0366] 5.1.8.1 Preparation of Standards: Take appropriate amounts of daidzein, glycitein, and genistein standards and prepare a standard solution containing 50 μg of each of daidzein, glycitein, and genistein per mL using 70% methanol solution;

[0367] 5.1.8.2 Preparation of test solution: Take the test solution separately, dilute it with methanol according to the experimental requirements, and filter it through a 0.45 μm filter membrane;

[0368] 5.1.8.3 Chromatographic methods

[0369] Accurately pipette 10 μL of the reference solution and the test solution respectively, inject them into the liquid chromatograph, and perform the determination using the same chromatographic method as described in 1.1 of Example 1.

[0370] 5.2 Experimental Results

[0371] By comparing the peak areas of the chromatographic peaks in the HPLC graph with those of the standard, the content of daidzein in the raw material was calculated to be 1.681 (mg / mL), the content of glycitein was 0.847 (mg / mL), and the content of genistein was 0.920 (mg / mL).

[0372] 5.2.2 Effluent content and recovery rate of each section:

[0373] By comparing the peak area of ​​the chromatographic peak in the HPLC diagram with that of the standard, the content of each part of the effluent under each section was calculated as shown in Table 10, Table 11 and Table 12. The content of each component in the effluent was divided by the content of each component in the above sample solution to obtain the recovery rate of each component. The specific data are shown in Table 13. The recovery rate of genistein in the effluent under each section is shown in Figure 8 As shown, the relative peak area of ​​genistein in the effluent of each section is as follows Figure 9 shown.

[0374] Table 10 Calculation results of the content of each part of the effluent from each section (Test 1)

[0375]

[0376] Table 11 Results of content of each part of the effluent from each section (Test 2)

[0377]

[0378] Table 12 Results of content of each part of the effluent from each section (Test 3)

[0379]

[0380] Table 13 Recovery rate and relative peak area of ​​genistein in each section

[0381]

[0382] 5.3 Summary

[0383] 5.3.1 The experimental results indicate that the SP20SS has a good separation effect on genistein. The high-concentration section and the main outflow section are concentrated in the 60% ethanol elution range of 3.0-6.0 BV. The effluent from the 60% ethanol elution range of 3.0-6.0 BV can be collected for subsequent production.

[0384] 5.3.2 In the 3.0-6.0 BV range of the 60% ethanol elution, the relative peak area of ​​genistein was greater than 80%, and the effluent collected in this range had a high genistein content;

[0385] 5.3.3 Based on experimental results, the recovery rate of genistein in the 3.0-6.0 BV range of 60% ethanol elution was 99.44%, which is a high recovery rate and can be used as an industrial production method;

[0386] 5.3.4 The experimental results show that SP20SS resin has a good adsorption capacity for genistein when loaded with 60% ethanol, and no genistein leaks during the loading process.

[0387] 5.3.5 The experimental results show that all components of soy isoflavones were well recovered at the end of the experiment, with a recovery rate greater than 90%, indicating that the resin has good regeneration capacity and can achieve continuous production;

[0388] 5.3.6 Based on the above experimental results, it is not difficult to find that when the SP20SS resin is used for scaled-up testing, the experimental results are somewhat different from those of the CHP20 resin, but the separation effect is still good. This is because the SP20SS resin and the CHP20 resin have the same chemical composition, and only the difference in particle size causes the certain difference in their experimental results. In the subsequent preparation process, the mobile phase can be adjusted to simplify the process (see the preparation of the composition in Example 2).

[0389] 6. Separation of Genistein by SP20SS Column Chromatography - Determination of Genistein Content in Dry Matter

[0390] 6.1 Experimental Methods

[0391] 6.1.1 Test Solution Preparation: Based on the experimental data from 5, 60% ethanol elution in the 3.0-6.0 BV range shows a high concentration and recovery of genistein. Therefore, the lower effluent from this range was collected and rotary evaporated to dryness to obtain the purified genistein-rich soybean isoflavone extract. Prepare a solution containing approximately 50 μg of this dry matter per mL of genistein using 70% ethanol. Pass the solution through a 0.45 μm filter and dilute to 100 mL.

[0392] 6.1.2 Preparation of Standard Solution: Take an appropriate amount of genistein standard and prepare a solution containing approximately 50 μg of genistein per mL with 70% ethanol. Pass the solution through a 0.45 μm filter.

[0393] 6.1.3 Chromatographic conditions

[0394] Same as 1.1 in Example 1

[0395] Calculation: C0 = P0 ÷ P1 × C1 × V0 ÷ m × 100%

[0396] Where:

[0397] C0: Genistein content of original sample, unit (%)

[0398] P0: Sample genistein peak area detected by HPLC (mAu)

[0399] P1: Peak area of ​​standard genistein detected by HPLC (mAu)

[0400] C1: Standard genistein content (mg / mL)

[0401] V0: Volume of sample prepared and adjusted to (mL)

[0402] m: sampling mass of the sample (g)

[0403] 6.3 Experimental Results

[0404] In the HPLC spectrum, the peak area of ​​genistein detected by the standard was 1777.631 mAu, and the peak area of ​​genistein detected by the sample was 1687.054 mAu. The above formula showed that the content of genistein in the sample was 98.24%.

[0405] 6.4 Summary

[0406] From the above experimental results, it is not difficult to see that the dry matter prepared after collecting the 3.0-6.0BV effluent of 60% ethanol has a high genistein content and a genistein purity of 98.24%. This section of effluent can be collected in subsequent production as a raw material for the preparation of the composition. To improve production efficiency, after collecting this section of solution, a higher ethanol concentration can be used to rinse the resin to achieve the purpose of rapid regeneration of the resin and reduce the time required for a single production.

[0407] Example 2: Composition Preparation

[0408] 1.1 Plant materials:

[0409] Angelica, Cimicifuga, Pueraria lobata, Pueraria lobata, Trifolium pratense, Japanese knotweed, pomegranate fruit

[0410] 1.1.1 Extraction method

[0411] The following plants or herbs are provided: Angelica sinensis, Cimicifuga heracleifolia, Pueraria lobata, Pueraria lobata Thaia, Trifolium pratense, Japanese knotweed, and pomegranate fruit.

[0412] Different plants were taken respectively, and 10 to 80 times the mass of 40 to 60% ethanol-water solution was added for extraction based on dry weight. After the obtained extracts were concentrated (the solvent was removed), extracts from different plants (containing a variety of flavonoids) were obtained.

[0413] The content of flavonoids in the above plant extracts was determined by sodium nitrite-aluminum nitrate colorimetry using rutin as a reference substance. The calculation results are shown in the following table:

[0414] Table 14 Flavonoid content of extract

[0415]

[0416] As shown in Table 14 above, all plant extract ingredients in the composition contain plant flavonoid components, suggesting that isoflavone compounds are also highly distributed in the composition, which can effectively improve the efficacy of the composition in cosmetic applications and provide a certain chemical basis for the efficacy of the subsequent composition formed by compounding with genistein-rich rhubarb isoflavone extract.

[0417] 3. Preparation of Soy Isoflavone Extract

[0418] 3.1 Take a certain amount of soy isoflavones and dilute it with 60% ethanol solution to 100 times the sample volume, dissolve it and set aside;

[0419] 3.2 Take an appropriate amount of SP20SS resin that is 50 times (W / V) the amount of soy isoflavones fed, rinse with 60% ethanol until the ethanol concentration of the effluent reaches 60% and set aside;

[0420] 3.3 Adjust the sample flow rate to 2 BV / h and load the soy isoflavone solution prepared in 3.1 at a sample flow rate of 2 BV / h.

[0421] 3.4 Elution

[0422] The soy isoflavone solution was subjected to gradient elution according to the gradient elution conditions shown in Table 15.

[0423] Table 15 Elution conditions

[0424]

[0425] Note: It is not difficult to find from the technical solution of Example 1 that high-purity genistein flows out when eluted with 60% ethanol at 3-6 BV. Therefore, the mobile phase can be adjusted as shown in the table above to simplify the subsequent steps to quickly remove impurities in the macroporous resin and reduce the processing time.

[0426] 3.5 Effluent collection: discard the sample effluent and collect the eluate from 3BV to 6BV

[0427] 3.6 Preparation of soy isoflavone extract: Concentrate the eluate described in 3.5 to 0.2 times the amount of soy isoflavones fed to obtain genistein-rich soy isoflavone extract.

[0428] 3.7 Determination of genistein content and solid content in extracts

[0429] About 1g of sample was taken and measured by a rapid moisture meter at 135℃. The solid content in the extract was 51.07%.

[0430] The genistein content test method and calculation method are the same as those in 6.1 of Example 1.

[0431] The genistein content in the sample was 95.23% (based on the weight of solid matter in the extract).

[0432] 4. Composition Preparation

[0433] Composition 1:

[0434] Preparation steps: Take 20% (w / w) of 1,3-butanediol by weight of the finished product, heat it in a water bath to 80-83°C, add 1.25% (w / w) of soy isoflavone extract (the soy isoflavone extract prepared in 3.1-3.6 of Example 2) by weight of the finished product, mix and dissolve until clear and without turbidity, then take 20% of the 1,3-butanediol by weight of the finished product, weigh the various plant extract extracts prepared in 1.1.1-1.1.2 of Example 2 according to Table 16 below, and mix at room temperature until clear and without turbidity.

[0435] Table 16 Amount of plant extract added in composition 1

[0436]

[0437] Mix all the materials, dilute to 100% (based on the total amount of finished product) with 1,3-butanediol, and stir at room temperature until the mixture is clear and free of turbidity.

[0438] Composition 2:

[0439] Preparation steps: Take 20% (w / w) of the finished product's mass of 1,3-butanediol, heat it in a water bath to 80-83°C, add 1.75% (w / w) of the finished product's mass of soy isoflavone extract (the soy isoflavone extract prepared in 3.1-3.6 in Example 2), mix and dissolve until clear and without turbidity, then take 20% of the mass of the finished product's mass of 1,3-butanediol, weigh the plant extract extracts prepared in 1.1.1-1.1.2 in Example 2 according to Table 17 below, and mix at room temperature until clear and without turbidity.

[0440] Table 17 Amount of plant extract added in composition 2

[0441]

[0442] Mix all the materials, dilute to 100% (based on the total amount of finished product) with 1,3-butanediol, and stir at room temperature until the mixture is clear and free of turbidity.

[0443] Composition 3:

[0444] Preparation steps: Take 20% (w / w) of the finished product's weight of 1,3-butanediol, heat it in a water bath to 80-83°C, add 2.5% (w / w) of the finished product's weight of soy isoflavone extract (the soy isoflavone extract prepared in 3.1-3.6 of Example 2), mix and dissolve until clear and free of turbidity, then take 20% of the weight of the finished product's weight of 1,3-butanediol, weigh each plant extract extract prepared in 1.1.1-1.1.2 of Example 2 according to Table 18, and mix at room temperature until clear and free of turbidity.

[0445] Table 18 Amount of plant extract added in composition 3

[0446]

[0447] Mix all the materials, dilute to 100% (based on the total amount of finished product) with 1,3-butanediol, and stir at room temperature until the mixture is clear and free of turbidity.

[0448] Example 3: Comparative test of composition efficacy

[0449] 1.1 Test Materials

[0450] The cells used in this test were fibroblasts, batch number: Fb19052002, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0451] 1.2 Sample Information

[0452] Composition 1, composition 2 and composition 3 prepared in Example 2 were taken as samples for subsequent experiments.

[0453] 1.3 Main Reagents

[0454] DMEM culture medium (Gibco), PBS (Solyb), MTT (Sigma), DMSO (Sigma), TGF-β1 (Peprotech), CollagenI ELISA kit (CUSABIO), MMP-1 ELISA kit (Abcam).

[0455] 1.4 Main Equipment

[0456] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), microplate reader (BioTek, Epoch), UVA irradiator (Philips), inverted microscope (Olympus, CKX53)

[0457] 1.5 Fibroblast-based cytotoxicity assay

[0458] 1.5.1 Test Method

[0459] 1.5.1.1 Cell viability test method

[0460] 1) Cell seeding: After thawing cells, when the plating rate reaches approximately 60%, the cells are seeded into 96-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0461] 2) Experimental Grouping: The experiment was divided into a zero adjustment group, a solvent control group, a positive control group, and a sample group. Within the sample group, each sample was set at 8 concentrations, with 3 replicate wells at each concentration.

[0462] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration gradient table (Table 19).

[0463] Table 19 Test concentration setting table

[0464]

[0465] 4) Dosing: Dosing was performed when the cell plating rate in the 96-well plate reached 50%-60%. For the solvent control group, 200 μL of culture medium was added to each well; for the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of culture medium containing the corresponding sample concentration was added to each well; for the zero adjustment group, no cells were seeded, and only 200 μL of cell culture medium was added. After dosing, the 96-well plate was placed in a CO2 incubator (37°C, 5% CO2) for 24 hours.

[0466] 5) Detection: After incubating the cells for 24 hours, discard the supernatant and add MTT working solution (0.5 mg / mL). Incubate at 37°C in the dark for 4 hours. After incubation, discard the supernatant and add 150 μL of DMSO to each well. Read the OD value at 490 nm.

[0467] 6) Calculation of relative cell viability: Calculate relative cell viability according to the formula:

[0468] .

[0469] 1.5.1.2 Cell morphology test method

[0470] 1) Cell seeding: After thawing cells, when the plating rate reaches approximately 60%, seed the cells into a 24-well plate and incubate overnight in a CO2 incubator (37°C, 5% CO2).

[0471] 2) Experimental Grouping: The experiment set up a solvent control group and a sample group. In the sample group, each sample was set up with 5 concentration gradients.

[0472] 3) Dosing: Dosing was performed when the cell plating rate in the 24-well plate reached 50% to 60%. For the solvent control group, 1 mL of culture medium was added to each well; for the sample group, 1 mL of culture medium containing the corresponding concentration of the test sample was added to each well. After dosing, the 24-well plate was placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours.

[0473] 4) Photography: After incubation, discard the supernatant and take photos under an inverted microscope.

[0474] 1.6 Test Results

[0475] The cell viability curve of composition 1 is shown in FIG. Figure 10 As shown, the cell morphology of sample composition 1 is as follows Figure 11 According to the cell viability curve and morphological results, it is believed that composition 1 does not show obvious cytotoxicity in the concentration range of 0.125% based on fibroblasts.

[0476] The cell viability curve of composition 2 is shown in FIG. Figure 12 As shown, the cell morphology of sample composition 2 is as follows Figure 13 According to the cell viability curve and morphological results, it is believed that composition 2 does not show obvious cytotoxicity in the concentration range of 0.125% based on fibroblasts.

[0477] The cell viability curve of composition 3 is shown in FIG. Figure 14 As shown, the cell morphology of sample composition 3 is shown in FIG. Figure 15 According to the cell viability curve and morphological results, it is believed that composition 3 does not show obvious cytotoxicity in the concentration range of 0.0625% based on fibroblasts.

[0478] 1.7 Anti-wrinkle and firming efficacy test

[0479] 1.7.1 Test Method

[0480] Cell seeding: After thawing cells, when the plating rate reaches about 60%, seed the cells into 6-well plates and incubate in a CO2 incubator (37°C, 5% CO2) overnight.

[0481] Solution preparation: Prepare the working solution of the test substance according to the test group (Table 20).

[0482] Table 20 Test Grouping

[0483]

[0484] Dosing: Assemble the test groups according to Table 20. Dosing was performed when the cell plating rate in the 6-well plate reached 30% to 50%. Each group had three replicate wells. For the blank and negative control groups, add 2 mL of culture medium to each well; for the positive control group, add 2 mL of culture medium containing TGF-β1 to each well; for the sample groups, add 2 mL of culture medium containing the corresponding sample concentration to each well. After dosing, incubate the 6-well plates in a CO2 incubator (37°C, 5% CO2) for 24 hours.

[0485] UVA irradiation: According to the test groups in Table 20, except for the blank control group, the other groups were irradiated with UVA at a dose of 30 J / cm². After irradiation, the cells were placed in a CO2 incubator (37°C, 5% CO2) and cultured for 24 hours.

[0486] ELISA test: After the incubation, the cell culture supernatant was collected and ELISA test was performed according to the ELISA kit instructions.

[0487] Calculation of promotion rate and inhibition rate:

[0488]

[0489] Statistical analysis of results: GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. The summary table of COL I content detection results is shown in Table 21, the summary table of MMP-1 content detection results is shown in Table 22, and the comparison chart of COL I (Collagen I) content is shown in Table 23. Figure 16 As shown in the figure, the comparison of MMP-1 content is shown in Figure 17 As shown, the anti-wrinkle and firming effect comparison of composition 1, composition 2 and composition 3 is shown in Figure 18 shown.

[0490] Table 21 Test results (summary of COL I content test results)

[0491]

[0492] Note: When using the t-test method for statistical analysis, compared with the BC group, the significance is indicated by #, P value <0.05 is indicated by #, and P value <0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P value <0.05 is indicated by *, and P value <0.01 is indicated by **;

[0493] Compared with the BC group, the content of Collagen I in the NC group was significantly decreased, indicating that the stimulation conditions of this test were effective.

[0494] Compared with the NC group, the Collagen 1 content in the PC group increased significantly, indicating that the positive control in this test was effective.

[0495] Compared with the NC group, the Collagen I content of composition 1-0.0625%, composition 2-0.0625%, and composition 3-0.0625% were significantly increased, with increase rates of 15.10%, 11.53%, and 20.33%, respectively.

[0496] Table 22 Summary of MMP-1 content detection results

[0497]

[0498] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0499] Compared with the BC group, the MMP-1 content in the NC group increased significantly, indicating that the stimulation conditions of this test were effective.

[0500] Compared with the NC group, the MMP-1 content in the PC group was significantly decreased, indicating that the positive control in this test was effective.

[0501] Compared with the NC group, the MMP-1 contents of composition 1-0.0625%, composition 2-0.0625%, and composition 3-0.0625% were significantly decreased, with inhibition rates of 12.23%, 11.94%, and 16.47%, respectively.

[0502] 1.7.2 Conclusion

[0503] Based on fibroblasts, compared with the control group, the content of type I collagen (Collagen I) in composition 1 at a concentration of 0.0625% (v / v) was significantly increased by 15.10%, and the content of matrix metalloproteinase 1 (MMP-1) was significantly decreased, with an inhibition rate of 12.23%. This shows that the sample can increase the content of type I collagen (Collagen I) and reduce the content of matrix metalloproteinase 1 (MMP-1) at this concentration, and has anti-wrinkle and firming effects.

[0504] Composition 2, at a concentration of 0.0625% (v / v), significantly increased type I collagen (Collagen I) content by 11.53%, and significantly decreased matrix metalloproteinase-1 (MMP-1) content by 11.94%. This indicates that at this concentration, the sample can increase type I collagen (Collagen I) content and reduce matrix metalloproteinase-1 (MMP-1) content, demonstrating anti-wrinkle and firming effects.

[0505] Composition 3 at a concentration of 0.0625% (v / v) significantly increased the type I collagen (Collagen I) content by 20.33%, and significantly decreased the matrix metalloproteinase 1 (MMP-1) content by 16.47%. This indicates that the sample can increase the type I collagen (Collagen I) content and reduce the matrix metalloproteinase 1 (MMP-1) content at this concentration, demonstrating anti-wrinkle and firming effects.

[0506] 1.8 Summary

[0507] Based on the above results, it can be seen that Composition 1, Composition 2, and Composition 3 all have certain anti-wrinkle and firming effects, and Composition 3 is the best in terms of both the Collagen I enhancement rate and the MMP-1 inhibition rate. Therefore, Composition 3 was selected for the anti-wrinkle and firming efficacy test on ex vivo skin tissue to verify its anti-wrinkle and firming efficacy.

[0508] 2 Nourishing effect comparison test

[0509] 2.1 Test Basis

[0510] Testing was conducted according to the "Method for Determining Skin Water, Fatty Acid, and Ceramide Content Based on a 3D Epidermal Skin Model (EpiKutis*)."

[0511] 2.2 Test Purpose

[0512] This test is divided into two parts: the first part is based on keratinocytes, conducting cytotoxicity testing to determine the sample's dosage concentration on keratinocytes; the second part is based on a 3D epidermal skin model (EpiKutis), by detecting changes in skin water content, fatty acids, and ceramide content to evaluate the nourishing effect of the test sample.

[0513] 2.3 Test materials

[0514] 2.3.1 Test System

[0515] The 3D epidermal skin model (EpiKutis%) used in this test, batch number ES231203, was provided by Guangdong Boxi Biotechnology Co., Ltd.

[0516] 2.3.2 Sample Information

[0517] Composition 1, composition 2 and composition 3 prepared in Example 2 were taken as samples for subsequent experiments.

[0518] 2.3.3 Main reagents

[0519] KeGrowth culture medium (Guangdong Boxi Biotechnology), PBS (Solaibao), MTT (Sigma), DMSO (Sigma), EpiGrowth culture medium (Guangdong Boxi Biotechnology), glycerol (Sinopharm), WY14643 (Sigma), isopropanol (Sinopharm), nonadecanoic acid (CH 19 H 38 02,CAS74713-60-3,Sigma),C 12 Cermide(C 30 H 59 NO3, CAS74713-60-3, Avanti Polar Lipids, INC.), protein detection kit (Biovision, K814-2500), proteinase K (Thermo, 17916).

[0520] 2.3.4 Main equipment

[0521] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), microplate reader (BioTek, Epoch), inverted microscope (Olympus, CKX53), Corncometer°CM 825 skin moisture content tester (Courage+Khazakaelectronic), LC-MS (Thermo Fisher Scientific, Q Exactive), KQ3200E ultrasonic cleaner (Kunshan Shumei), low-temperature high-speed centrifuge (Hunan Xiangyi).

[0522] 2.4 Keratinocyte-based cytotoxicity test

[0523] 2.4.1 Test Method

[0524] 2.4.1.1 Cell viability test method

[0525] 1) Cell seeding: After thawing, when the plating rate reaches approximately 60%, the cells are seeded into 96-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2).

[0526] 2) Experimental Grouping: The experiment was divided into a zero adjustment group, a solvent control group, a positive control group, and a sample group. In the sample group, each sample was set at 8 concentrations, and 3 replicate wells were set at each concentration.

[0527] 3) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration gradient table (Table 23).

[0528] Table 23 Test concentration setting table

[0529]

[0530] 4) Dosing: Dosing was performed when the cell plating rate in the 96-well plate reached 50%-60%. For the solvent control group, 200 μL of culture medium was added to each well; for the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of culture medium containing the corresponding sample concentration was added to each well; for the zero-adjustment group, no cells were seeded, and only 200 μL of cell culture medium was added. After dosing, the 96-well plate was placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours.

[0531] 5) Detection: After incubating the cells for 24 h, discard the supernatant and add MTT working solution (0.5 mg / mL). Incubate at 37°C in the dark for 4 h. After incubation, discard the supernatant and add 150 μL of DMSO to each well. Read the OD value at 490 nm.

[0532] 6) Calculation of relative cell viability: According to the formula, the relative cell viability

[0533]

[0534] 2.4.1.2 Cell morphology test method

[0535] 1) Cell seeding: After thawing cells, when the plating rate reaches approximately 60%, seed the cells into a 24-well plate and incubate overnight in a CO2 incubator (37°C, 5% CO2).

[0536] 2) Experimental Grouping: The experiment set up a solvent control group and a sample group. In the sample group, each sample was set up with 5 concentration gradients.

[0537] 3) Dosing: Dosing was performed when the cell plating rate in the 24-well plate reached 50%-60%. For the solvent control group, 1 mL of culture medium was added to each well; for the sample group, 1 mL of culture medium containing the test sample at the corresponding concentration was added to each well. After dosing, the 24-well plate was placed in a CO2 incubator (37°C, 5% CO2) for 24 hours.

[0538] 4) Photography: After incubation, discard the supernatant and take photos under an inverted microscope.

[0539] 2.5 Test Results

[0540] The cell viability curve of composition 1 is shown in FIG. Figure 19 As shown, the cell morphology of sample composition 1 is as follows Figure 20 According to the cell viability curve and morphological results, it is believed that composition 1 does not show obvious cytotoxicity based on keratinocytes within the concentration range of 0.0078%.

[0541] The cell viability curve of composition 2 is shown in FIG. Figure 21 As shown, the cell morphology of sample composition 2 is as follows Figure 22 According to the cell viability curve and morphological results, it is believed that composition 2 does not show obvious cytotoxicity based on keratinocytes within the concentration range of 0.0078%.

[0542] The cell viability curve of composition 3 is shown in FIG. Figure 23 As shown, the cell morphology of sample composition 3 is shown in FIG. Figure 24 According to the cell viability curve and morphological results, it is believed that composition 3 does not show obvious cytotoxicity in the concentration range of 0.0078% based on keratinocytes.

[0543] 2.6 Nourishing Efficacy Test

[0544] 2.6.1 Skin Moisture Content Test Method

[0545] 2.6.1 Test Grouping

[0546] Table 24 Test Grouping

[0547]

[0548] 2.6.2 Preparation of working fluid

[0549] Preparation of positive control group (glycerol) working solution: Add 400 μL of glycerol stock solution to 1.6 mL of PBS to prepare a 20% working solution.

[0550] 2.6.3 Medication

[0551] 1) Divide the test groups according to Table 24, transfer the models into a 6-well plate (add 0.9 mL EpiGrowth culture medium in advance), and mark the test group number on the 6-well plate.

[0552] 2) Add 20% glycerol working solution to the PC group, apply the sample to be tested on the surface of the sample group, evenly distribute the sample on the model surface, and incubate in a CO2 incubator (37°C, 5% CO2) for 24 hours.

[0553] 3) After incubation, clean the test substance remaining on the surface of the model with sterile PBS solution, and wipe away the residual liquid inside and outside the model with a sterile cotton swab.

[0554] 2.6.4 Skin Moisture Content Test

[0555] 1) Measurement Preparation: After cleaning, prepare a 24-well plate according to the number of models and label them accordingly. Add 0.3 L of EpiGrowth culture medium to each well. Place the 24-well plate containing the models in a clean bench, open the lid, and let it sit for 30 minutes before measuring.

[0556] 2) Measurement: Wipe the bottom of the model dry, remove the model ring, place it on the probe position of the tester, press the probe to measure, measure each model three times, and take the average value.

[0557] 2.6.5 Calculation of promotion rate

[0558]

[0559] 2.6.6 Statistical analysis of results

[0560] GraphPad Prism was used for graphing, and the results were expressed as Mean ± SD.

[0561] 2.7 Fatty Acid and Ceramide Test Methods

[0562] 2.7.1 Test Grouping

[0563] Table 25 Test Grouping

[0564]

[0565] 2.7.2 Preparation of working fluid

[0566] Preparation of the positive control group (WY14643) working solution: Pipette 10 μL of 30 mM WY14643 stock solution and dissolve it in 6 mL of culture medium to prepare 50 μM WY14643.

[0567] 2.7.3 Model drug administration

[0568] 1) Assign the test groups according to Table 25, transfer the model to a 6-well plate (add 0.9 mL EpiGrowth culture medium in advance), and mark the test group number on the 6-well plate.

[0569] 2) The BC group received no treatment. The PC group received 0.9 mL of 50 μM WY14643 working solution. The surface of the sample group was smeared with the sample to be tested, and the sample was evenly distributed on the model surface. The model was placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 h.

[0570] 3) After the incubation, clean the test substance remaining on the surface of the model with sterile PBS solution, and wipe away the residual liquid inside and outside the model with a sterile cotton swab.

[0571] 2.7.4 Extraction and detection of ceramide and fatty acids

[0572] 1) Lipid Sample Extraction: Each model was cut in half, with one half used for protein determination and the other half for lipid extraction. Each half was placed in a centrifuge tube, and proteinase K solution was added. The tubes were incubated at 50°C for 1 hour. The sticky material on the stratum corneum was removed with tweezers, and the water was blotted dry. The stratum corneum was then placed in a glass tube. 1 mL of a chloroform-methanol mixture was added to each tube. The tubes were sonicated on ice, and the supernatant was transferred to a sample vial and air-dried.

[0573] 2) Pretreatment of lipid samples: A mixture of acetonitrile and isopropanol (1:1) was added to a nitrogen-dried sample bottle, followed by 20 μL of ceramide C12 internal standard solution. Dissolved by ultrasonication, the sample was transferred to a sample centrifuge tube and centrifuged at 12,000 rpm for 10 min. 100 μL of the upper layer was collected and placed in an inner liner tube for analysis.

[0574] 2.7.5 Calculation of promotion rate

[0575]

[0576] 2.7.6 Statistical analysis of results

[0577] GraphPad Prism was used for graphing, and the results were expressed as Mean ± SD.

[0578] 2.8 Test Results

[0579] 2.8.1 Skin Moisture Content Test Results

[0580] Table 26 Summary of skin moisture test results

[0581]

[0582] Note: When the t-test method was used for statistical analysis, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by ** compared with the BC group.

[0583] The summary table of skin moisture content test results is shown in Table 26, and the bar graph of skin moisture content test results of each group is shown in Figure 25 shown.

[0584] Compared to the BC group, the PC group showed a significant increase in skin hydration, demonstrating the effectiveness of the positive control in this test. Compared to the BC group, the skin hydration levels of the 1-2.0%, 2-2.0%, and 3-2.0% compositions all increased significantly, with increases of 143.87%, 91.76%, and 187.89%, respectively.

[0585] 2.8.2 Fatty acid and ceramide test results

[0586] Table 27 Summary of Ceramide / Protein Test Results

[0587]

[0588] Note: When the t-test method was used for statistical analysis, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by ** compared with the BC group.

[0589] The summary of ceramide / protein test results is shown in Table 27, and the bar graph of ceramide / protein test results of each group is shown in Figure 26 shown.

[0590] Compared with the BC group, the ceramide / protein values ​​in the stratum corneum of the PC group skin model were significantly increased.

[0591] Compared with the BC group, the ceramide / protein values ​​in the stratum corneum of the skin models of composition 1-2.0%, composition 2-2.0%, and composition 3-2.0% were significantly increased, with the increase rates being 9.40%, 9.54%, and 14.09%, respectively.

[0592] Table 28 Summary of the test results of average carbon chain length of ceramide

[0593]

[0594] Note: When the t-test method was used for statistical analysis, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by ** compared with the BC group.

[0595] The summary of the test results of the average carbon chain length of ceramide is shown in Table 28, and the bar graph of the test results of the average carbon chain length of ceramide in each group is shown in Figure 27 shown.

[0596] Compared with the BC group, the average carbon chain length of ceramide in the stratum corneum of the PC group skin model did not change significantly.

[0597] Compared with the BC group, the average carbon chain length of ceramide in the stratum corneum of the skin model with composition 3-2.0% was significantly increased, with an increase rate of 0.83%; the average carbon chain length of ceramide in the stratum corneum of the skin model with composition 1-2.0% and composition 2-2.0% did not change significantly.

[0598] Table 29 Summary of fatty acid / protein test results

[0599]

[0600] Note: When using the t-test method for statistical analysis, compared with the BC group, significance is indicated by *, P value <0.05 is indicated by *, and P value <0.01 is indicated by **.

[0601] The summary of fatty acid / protein test results is shown in Table 29, and the bar graph of fatty acid / protein test results of each group is shown in Figure 28 shown.

[0602] Compared with the BC group, the values ​​of fatty acids / proteins in the stratum corneum of the skin model in the PC group were significantly increased.

[0603] Compared with the BC group, the values ​​of fatty acids / proteins in the stratum corneum of the skin models with composition 2-2.0% and composition 3-2.0% were significantly increased, with increase rates of 12.34% and 8.75%, respectively; the values ​​of fatty acids / proteins in the stratum corneum of the skin model with sample composition 1-2.0% did not change significantly.

[0604] Table 30 Summary of the test results of average carbon chain length of fatty acids

[0605]

[0606] Note: When using the t-test method for statistical analysis, compared with the BC group, significance is indicated by *, P value <0.05 is indicated by *, and P value <0.01 is indicated by **.

[0607] The summary table of the average carbon chain length test results of fatty acids is shown in Table 30, and the bar graph of the average carbon chain length test results of fatty acids in each group is shown in Figure 29 shown.

[0608] Compared with the BC group, the average carbon chain length of fatty acids in the stratum corneum of the PC group skin model did not change significantly.

[0609] Compared with the BC group, the average carbon chain length of fatty acids in the stratum corneum of the skin models with composition 2-2.0% and composition 3-2.0% increased significantly, with increase rates of 0.60% and 0.87%, respectively; the average carbon chain length of fatty acids in the stratum corneum of the skin model with sample composition 1-2.0% did not change significantly.

[0610] 2.8 Summary

[0611] Based on a 3D epidermal skin model (EpiKutis), compared to the control group, Composition 1 at a concentration of 2.0% (v / v) significantly increased skin moisture content and ceramide / protein values ​​by 143.87% and 9.40%, respectively. This indicates that the sample can increase skin moisture content and ceramide / protein values ​​at this concentration, demonstrating a nourishing effect.

[0612] Composition 2 at a concentration of 2.0% (v / v) significantly increased skin moisture content, ceramide / protein ratio, fatty acid / protein ratio, and average carbon chain length of fatty acids, with increases of 91.76%, 9.54%, 12.34%, and 0.60%, respectively. This indicates that the sample can increase skin moisture content, ceramide / protein ratio, fatty acid / protein ratio, and average carbon chain length of fatty acids at this concentration, demonstrating a nourishing effect.

[0613] The skin moisture content, ceramide / protein value, average ceramide carbon chain length, fatty acid / protein value, and average fatty acid carbon chain length of composition 3 at a concentration of 2.0% (vv) were significantly increased, with increase rates of 187.89%, 14.09%, 0.83%, 8.75%, and 0.87%, respectively. This indicates that the sample can increase the skin moisture content, ceramide / protein value, average ceramide carbon chain length, fatty acid / protein value, and average fatty acid carbon chain length at this concentration, and has a nourishing effect.

[0614] From the above test results, it can be seen that all the results except the ceramide flat carbon chain length test result show that composition 3 has the strongest nourishing effect, so it can be further determined that composition 3 is the optimal composition.

[0615] 3 Composition 3 In vitro skin tissue anti-wrinkle and firming test

[0616] 3.1 Test System

[0617] The ex vivo skin tissue used in this test was produced and provided by Guangdong Boxi Biotechnology Co., Ltd.

[0618] 3.2 Sample Information

[0619] The composition 3 prepared in Example 2 was used as the experimental sample.

[0620] 3.3 Main reagents

[0621] In vitro skin tissue culture medium (Guangdong Boxi Biological), vitamin C (VC, Sigma), vitamin E (VE, Sigma), PBS (Solaibao), and paraformaldehyde (Biosharp).

[0622] 3.4 Main equipment

[0623] CO2 incubator (Thermo, 150I), clean bench (Suzhou Antai, SW-CJ-1F), UVA irradiator (Philips), UVB irradiator (Philips), upright microscope (Olympus, BX53).

[0624] 3.5 Test Method

[0625] 3.5.1 Test Grouping

[0626] Table 31 Test Grouping

[0627]

[0628] 3.5.2 Model drug administration

[0629] 1) Tissue Processing: Freshly obtained skin tissue was immersed in 75% alcohol for 30 seconds and then washed three times with sterile PBS buffer. The skin was then cut into 24 ± 2 mm² blocks with the epidermis facing up and the dermis facing down. The blocks were then placed in a culture mold. The mold was then transferred to a 6-well plate. 3.7 mL of culture medium was added to each well. The plates were incubated at 37°C in a 5% CO2 incubator, with the medium changed daily.

[0630] 2) Irradiation and Dosing: After two days of in vitro skin tissue culture, irradiation and dosing were initiated according to the test groups and treatment conditions listed in Table 31. Irradiation doses were UVA (30 J / cm²) and UVB (50 mJ / cm²) for four consecutive days. Fresh culture medium was replaced after each irradiation session, and dosing was administered. The positive control (VC+VE) group received submerged dosing, while the sample group received topical dosing. Following four days of continuous irradiation, the ex vivo skin tissue was cultured for an additional three days, during which no irradiation was performed, with only sample dosing administered.

[0631] 3) Cleaning: After the incubation is completed, clean the model with a sterile PBS solution in a washing bottle and gently wipe away any residual liquid inside and outside the model with a sterile cotton swab.

[0632] 4) Collagen fiber detection: After washing, the ex vivo skin tissue was fixed with 4% paraformaldehyde, embedded, and sliced ​​for Masson staining. The slices were collected, photographed using a microscope, and analyzed using Imagc-Pro&Plus image processing software.

[0633] 5) Calculation of promotion rate:

[0634] 3.6 Statistical analysis of results

[0635] GraphPad Prism was used for graphing, and the results were expressed as Mean ± SD. 3.7 Test results

[0636] The summary table of collagen fiber relative area is shown in Table 32, and the test results of collagen fiber content in each group are shown in Figure 30 As shown in the bar graph, the relative area of ​​collagen fibers in each group is shown in Figure 31 shown.

[0637] Table 32 Summary of relative areas of collagen fibers

[0638]

[0639] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0640] Compared with the BC group, the collagen fiber content in the NC group was significantly decreased, indicating that the stimulation conditions of this test were effective.

[0641] Compared with the NC group, the collagen fiber content in the PC group increased significantly, indicating that the positive control in this test was effective.

[0642] Compared with the NC group, the collagen fiber content of sample composition 3 at a concentration of 2% was significantly increased, with an increase rate of 166.67%.

[0643] The above test results show that the composition 3 of the present invention has significant anti-wrinkle, firming, anti-aging and nourishing effects.

[0644] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for preparing a soybean isoflavone extract rich in genistein, characterized in that: The method comprises the following steps: (1) diluting a soy isoflavone raw material with an ethanol-water mixed solution to obtain a sample solution; wherein the soy isoflavone raw material contains genistein and additional soy isoflavones selected from the group consisting of daidzein and glycitin; and the ethanol concentration in the sample solution is C1, and 40 v / v%≤C1≤70 v / v%; (2) loading the sample solution onto a macroporous resin for resin adsorption, and performing gradient elution or elution with an ethanol / water mixed solvent, collecting the elution effluent containing genistein, thereby obtaining a soybean isoflavone extract rich in genistein; wherein the gradient elution is performed with water as phase A and ethanol as phase B, collecting the elution effluent with an ethanol concentration of C2, and 40v / v%≤C2≤70v / v%, wherein the ethanol concentration in the ethanol / water mixed solvent is C3, and 40v / v%≤C3≤70v / v%; The mass ratio of the soy isoflavone raw material to the resin is 1 g: 20 mL to 1 g: 100 mL, and the elution effluent corresponding to the elution volume of 3.0-6.0 BV is collected; (3) concentrating the eluted effluent to obtain an extract of soybean isoflavones rich in genistein; The macroporous resin in step (2) is selected from the following group: SP20SS macroporous resin; wherein the genistein-rich soybean isoflavone extract has a genistein content of ≥95 wt%, based on the dry weight of the extract; The content of genistein in the soy isoflavone raw material is H0, which is calculated according to the following formula Q0: H0=W1 / W raw material × 100% Q0 Wherein, W1 is the mass of genistein in the soybean isoflavone raw material; W raw material is the quality of soybean isoflavone raw material; In the extract of soybean isoflavones rich in genistein, the content of genistein is H1, and H1 is calculated according to the following formula Q2: H1=W1a / W extract×100% Q2 Wherein, W1a is the mass of genistein in the genistein-rich soybean isoflavone extract; W extract is the quality of genistein-rich soy isoflavone extract; Moreover, the enrichment ratio of genistein by the method is 5-12, and the enrichment ratio is the ratio of H1 to H0, H1 / H0.

2. The method according to claim 1, wherein The mass ratio of the soy isoflavone raw material to the ethanol-water mixed solvent is ≤2.5%.

3. The method according to claim 1, wherein The mass ratio of the soybean isoflavone raw material to the volume ratio of the resin is 1g:30mL to 1g:80mL.

4. A method for preparing a composition, characterized in that The method comprises: (1) diluting a soy isoflavone raw material with an ethanol-water mixed solution to obtain a sample solution; wherein the soy isoflavone raw material contains genistein and additional soy isoflavones selected from the group consisting of daidzein and glycitin; and the ethanol concentration in the sample solution is C1, and 40 v / v%≤C1≤70 v / v%; (2) loading the sample solution onto a macroporous resin for resin adsorption, and performing gradient elution or elution with an ethanol / water mixed solvent, collecting the elution effluent containing genistein, thereby obtaining a soybean isoflavone extract rich in genistein; wherein the gradient elution is performed with water as phase A and ethanol as phase B, collecting the elution effluent with an ethanol concentration of C2, and 40v / v%≤C2≤70v / v%, wherein the ethanol concentration in the ethanol / water mixed solvent is C3, and 40v / v%≤C3≤70v / v%; The mass ratio of the soy isoflavone raw material to the resin is 1 g: 20 mL to 1 g: 100 mL, and the elution effluent corresponding to the elution volume of 3.0-6.0 BV is collected; (3) concentrating the eluted effluent to obtain an extract of soybean isoflavones rich in genistein; The macroporous resin in step (2) is selected from the following group: SP20SS macroporous resin; wherein the genistein-rich soybean isoflavone extract has a genistein content of ≥95 wt%, based on the dry weight of the extract; The content of genistein in the soy isoflavone raw material is H0, which is calculated according to the following formula Q0: H0=W1 / W raw material × 100% Q0 Wherein, W1 is the mass of genistein in the soybean isoflavone raw material; W raw material is the quality of soybean isoflavone raw material; In the extract of soybean isoflavones rich in genistein, the content of genistein is H1, and H1 is calculated according to the following formula Q2: H1=W1a / W extract×100% Q2 Wherein, W1a is the mass of genistein in the genistein-rich soybean isoflavone extract; W extract is the quality of genistein-rich soy isoflavone extract; Furthermore, the enrichment ratio of genistein by the method is 5-12, and the enrichment ratio is the ratio of H1 to H0, H1 / H0; (4) dissolving the soybean isoflavone extract as described above in 1,3-butanediol, and mixing to obtain a soybean isoflavone solution, wherein the mass ratio of the soybean isoflavone extract to 1,3-butanediol is 1-1.5:20; (5) providing a plant extract extract dissolved in 1,3-butanediol, and mixing to obtain a plant extract solution, wherein the mass ratio of the plant extract extract to 1,3-butanediol is 7-25:20; The plant extract extract is composed of extracts selected from the group consisting of angelica extract, cimicifuga extract, kudzu root extract, kudzu root extract, clover extract, knotweed extract, and pomegranate fruit extract; (6) The soy isoflavone solution and the plant extract solution are mixed, 1,3-butanediol is added to make up the volume, and the mixture is stirred to obtain the composition.

5. The method according to claim 4, wherein The mass percentages of soybean isoflavone extract and plant extract extract in the composition are: Soy isoflavone extract 0.5-5.0wt% Angelica extract 0.2-2.0 wt% Cimicifuga extract 0.5-4.0 wt% Pueraria lobata extract 1-8 wt% Pueraria lobata extract 1-5 wt% Red Clover Extract 0.2-2.0 wt% Polygonum cuspidatum extract 1-5 wt% Pomegranate fruit extract 0.2-2.0 wt%.

6. The method according to claim 4, wherein The mass percentages of soybean isoflavone extract and plant extract extract in the composition are: Soy isoflavone extract 1-3wt% Angelica extract 0.3-1.8 wt% Cimicifuga extract 0.8-3.2 wt% Pueraria lobata extract 1.8-6.2 wt% Pueraria lobata extract 1.3-4.7 wt% Red Clover Extract 0.3-1.7 wt% Polygonum cuspidatum extract 1.3-4.7 wt% Pomegranate fruit extract 0.3-1.7 wt%.

7. The method according to claim 4, wherein The preparation method of the plant extract comprises the steps of: (1) Provide plant materials: angelica, cimicifuga, kudzu root, kudzu root, red clover, knotweed, pomegranate fruit; (2) extracting each plant raw material in step (1) with an ethanol-water solution to obtain each plant extract; (3) Concentrating the concentrated extracts of each plant obtained in step (2) to obtain the following plant extracts: angelica extract, cimicifuga extract, kudzu root extract, thai kudzu root extract, clover extract, knotweed extract, and pomegranate fruit extract.

8. The method according to claim 4, wherein The flavonoid content in the plant extract is as follows: Angelica extract 60-80μg / mL Cimicifuga extract 650-750 μg / mL Pueraria lobata extract 50-150μg / mL Pueraria lobata extract 280-380μg / mL Trifolium pratense extract 80-180μg / mL Polygonum cuspidatum extract 1900-1150 μg / mL Pomegranate fruit extract 600-720μg / mL.

9. A method for preparing a health-related daily chemical product, characterized in that: The method comprises: A method for preparing a composition as described in any one of claims 4 to 8; and preparing the composition into a health and daily chemical product.

10. The method according to claim 9, wherein The product has nourishing properties.

11. The method according to claim 9, wherein The product has one or more characteristics selected from the following group: (1) Increase the content of type I collagen; (2) reduce the content of matrix metalloproteinase 1; (3) Improve skin moisture content; (4) Increase ceramide / protein values; (5) Increase the average carbon chain length of ceramide; (6) Increase the value of fatty acid / protein; (7) Increase the average carbon chain length of fatty acids; (8) Increase collagen fiber content.

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