Peony extract with scalp anti-aging and hair loss prevention and application

Abstract

The present application relates to the technical field of daily cosmetics, and in particular to a peony extract with scalp anti-aging and hair loss prevention and application. The peony extract disclosed by the present application is a peony flower extract, wherein the mass fraction of peony flower peptide in the peony flower extract is greater than or equal to 10%. The peony extract disclosed by the present application is found for the first time to have the functions of improving the superoxide dismutase (SOD) activity on human hair papilla cells, inhibiting the positive cell rate of human hair papilla cells of beta-galactosidase (SA-beta-Gal), and reducing the function of senescent cells. Meanwhile, the peony extract has the functions of promoting hair follicle expression of growth factors and promoting hair growth. The peony extract can be applied to the preparation of scalp care products to realize the functions of scalp anti-aging or hair loss prevention.

Description

Technical Field

[0001] This invention relates to the field of daily cosmetics technology, specifically to a peony extract with anti-aging and anti-hair loss properties for the scalp and its applications. Background Technology

[0002] Peony (Paeonia suffruticosa), a plant belonging to the genus Paeonia in the family Ranunculaceae, has a long history of being used for ornamental and medicinal purposes.

[0003] Among the ingredients listed in the cosmetics ingredient catalog, eight are related to peony: peony root bark extract, peony flower water, peony extract, peony seed oil, peony root extract, peony root water, peony flower / leaf / root extract, and peony branch / flower / leaf extract. The first three are more widely used in products. The application of peony-related products in cosmetics is not only diverse in form but also encompasses a wide range of effects, including skin whitening, anti-inflammation, and anti-oxidation, showing great promise. However, the efficacy and application of peony flower extract are limited.

[0004] With the fast pace of modern life, scalp aging and hair loss are becoming increasingly serious problems. Stress, environmental pollution, and unhealthy lifestyle habits accelerate scalp aging, cause hair follicle atrophy, and ultimately lead to hair loss. Statistics show that the number of people experiencing hair loss globally is increasing year by year, and scalp health issues have become a major problem troubling many people.

[0005] Against this backdrop, developing natural plant-based ingredients for scalp care is particularly necessary. Peony flowers are rich in flavonoids, phenols, and other active ingredients, which possess antioxidant and anti-inflammatory properties, laying a solid foundation for their application in the daily chemical industry. Currently, peony flowers have shown some application potential in anti-aging skincare products, perfumes, and other daily chemical products. For example, patent 202311551958.5 discloses "a short peptide protein from Danfeng peony petals and its application." This invention uses Danfeng peony petals as raw material, obtaining plant protein through an alkali-soluble acid precipitation method, followed by hydrolysis with dietary hydrolytic enzymes to obtain low-molecular-weight protein polypeptides. These polypeptides exhibit good α-glucosidase activity, can improve hyperglycemia symptoms in type 2 diabetic mice, and have certain antioxidant effects. However, the application of peony flower extracts in scalp care products is still in its early stages. Summary of the Invention

[0006] To address the gaps in existing technologies, this invention discloses a peony extract, specifically an extract of peony flowers. This peony extract is the first to be found to increase the activity of superoxide dismutase (SOD) on human dermal papilla cells, inhibit the rate of β-galactosidase (SA-β-Gal) positive cells in human dermal papilla cells, and reduce the function of senescent cells; simultaneously, it promotes the expression of growth factors in hair follicles, thereby promoting hair growth. It can be applied to the preparation of scalp care products to achieve anti-aging or hair loss prevention functions for the scalp. The specific details of this invention are as follows:

[0007] In a first aspect, the present invention provides a peony extract, wherein the peony extract is a peony flower extract, wherein the mass fraction of peony flower peptides in the peony flower extract is ≥10%, and optionally, the mass fraction of peony flower peptides is ≥15%.

[0008] Further, the peony flower peptide is a small molecule polypeptide. Optionally, the molecular weight of the peony flower peptide is ≤5000 Da, or ≤2500 Da, or ≤1000 Da.

[0009] In a second aspect, the present invention provides a composition comprising the peony extract and an additive acceptable in the cosmetics field, wherein the additive is optionally at least one of a surfactant, emulsifier, silicone oil, humectant, preservative, and fragrance.

[0010] Furthermore, the additive includes at least one of the following: polyacrylamide dimethyl taurate ammonium, dimethylsiloxane, PEG-40 hydrogenated castor oil, carbomer, cocamidopropyl betaine, and glucoside.

[0011] Further, the mass fraction of peony extract in the composition is 0.0001% to 20%, preferably 0.001% to 5%, and more preferably 0.001% to 2%.

[0012] A third aspect of the present invention provides the use of the peony extract or the composition in any of the following aspects:

[0013] A1. Application in the preparation of products that inhibit the rate of β-galactosidase (SA-β-Gal) positive cells in human dermal papilla cells;

[0014] A2. Application in the preparation of products that delay and / or inhibit the aging of human dermal papilla cells;

[0015] A3. Application in the preparation of products that enhance the activity of superoxide dismutase (SOD) in human dermal papilla cells;

[0016] A4. Application in the preparation of products that promote the expression of growth factors in hair follicles.

[0017] In a fourth aspect, the present invention provides the use of the peony extract or the composition in any of the following aspects:

[0018] B1. Applications in the preparation of anti-aging products;

[0019] B2. Applications in the preparation of products that inhibit and / or prevent hair loss;

[0020] B3. Applications in the preparation of products that promote hair growth;

[0021] B4. Applications in the preparation of antioxidant products.

[0022] Furthermore, the product is a cosmetic product. Optionally, the product includes at least one of scalp care products and skin care products. Optionally, the product includes at least one of shampoo, conditioner, hair mask, scalp pre-wash, or scalp serum.

[0023] In a fifth aspect, the present invention provides a method for preparing the peony extract, the method comprising at least the following steps:

[0024] S1: Crush the peony flowers and add them to deionized water, then steam at high temperature;

[0025] S2: Add protease to the peony flower aqueous solution obtained in step S1 for enzymatic hydrolysis and then filter to obtain a filtrate containing peony flower extract.

[0026] S3: Spray dry the filtrate obtained in step S2 to obtain the peony extract.

[0027] Furthermore, the specific steps in step S1 are as follows:

[0028] After pulverizing the peony flowers, add deionized water at a ratio of 1:10 to 1:100 and cook at high temperature for 20 to 180 minutes.

[0029] Furthermore, the specific steps in step S2 are as follows:

[0030] The added protease is one or more of alkaline protease, neutral protease, and acidic protease, and the filtration conditions are 200~5000 Da.

[0031] In a specific embodiment of the present invention, the specific steps of the method for preparing the peony extract are as follows: after pulverizing peony flowers, deionized water is added at a material-to-liquid ratio of 1:30, and the mixture is boiled at high temperature for 120 minutes. After adding neutral protease for a period of time, the mixture is filtered through a 1000 Da membrane and spray-dried to obtain the sample of the example.

[0032] The beneficial effects of the present invention include, but are not limited to:

[0033] This invention discloses a peony extract that, for the first time, has been found to increase the activity of superoxide dismutase (SOD) on human dermal papilla cells, inhibit the rate of β-galactosidase (SA-β-Gal) positive cells in human dermal papilla cells, and reduce the function of senescent cells; simultaneously, it promotes the expression of growth factors in hair follicles, thereby promoting hair growth. It can be applied to the preparation of scalp care products to achieve anti-aging or anti-hair loss functions for the scalp. Attached Figure Description

[0034] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0035] Figure 1 This is a schematic diagram illustrating the inhibitory effect of peony extract on superoxide dismutase (SOD) in human dermal papilla cells in an embodiment of the present invention.

[0036] Figure 2 This is a schematic diagram showing the results of human dermal papilla cells expressing β-galactosidase in an embodiment of the present invention.

[0037] Figure 3 This is a schematic diagram of the VEGF expression results in hair follicles in the embodiments and comparative examples of the present invention.

[0038] Figure 4 This is a schematic diagram showing the results of the whisker follicle length in the embodiments and comparative examples of the present invention. Detailed Implementation

[0039] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0040] Example 1: Preparation of Peony Extract

[0041] Example Sample: Peony flowers were pulverized and added to deionized water at a material-to-liquid ratio of 1:30. The mixture was then boiled at high temperature for 120 minutes. After adding neutral protease for a period of time, the mixture was centrifuged (4000-8000 rpm, 10-15 minutes), and the pH was adjusted to neutral. The mixture was then filtered through a 1000 Da membrane and spray-dried to obtain the example sample.

[0042] Comparative sample: Peony flower powder and purified water were mixed at a ratio of 1:20, heated in a water bath at 90°C for 20 min, centrifuged at 3600 rpm for 20 min, concentrated by vacuum rotary evaporation at 80°C, frozen at -18°C overnight, and vacuum-dried for 48 h.

[0043] Peptide content comparison:

[0044] Test method: Agilent 1260 Infinity II, mobile phase: 0.1M NaNO3, detector: RID, column: Waters Ultrahydrogel 1000 and Waters Ultrahydrogel 120 in series, temperature: 40℃, flow rate: 0.8 ml / min, standard: polyethylene oxide / polyethylene glycol (PEO / PEG), injection volume: 100 μl.

[0045] Example sample peptide content: 23.6%.

[0046] Peptide content of the comparative sample: 6.2%.

[0047] Example 2: DPPH Antioxidant Test

[0048] 1. Experimental Methods

[0049] Add 100 μL of the test substance and 100 μL of DPPH sequentially to a 96-well plate. Set up 3 replicates for each reaction and shake at room temperature for 5 min.

[0050] Meanwhile, a blank control and a self-control group were set up; 100 μL LPPH working solution + 100 μL pure water solution served as the blank control group; 100 μL sample solution + 100 μL pure water solution served as the self-control group; the absorbance value was measured at a wavelength of 517 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0051] Calculate the clearance rate using the following formula:

[0052]

[0053] In the formula:

[0054] A t - Sample tube absorbance, i.e., the absorbance of the solution after the sample reacts with DPPH.

[0055] A r -Absorbance of self-control group

[0056] A0 - Average absorbance value of the blank control group

[0057] The experimental results are shown in Table 1. As can be seen from Table 1, the antioxidant effect of the peony extract of the present invention is significantly better than that of the conventional water-extracted peony extract.

[0058] Table 1. DPPH removal rate results

[0059]

[0060] Example 3: Inhibitory effect of peony extract on superoxide dismutase (SOD) in human dermal papilla cells

[0061] 1. Experimental Methods

[0062] This test is based on a DHT-stimulated human dermal papilla cell model. After drug administration, the antioxidant efficacy of the test samples was evaluated by detecting the superoxide dismutase (SOD) content. The specific experimental procedure is as follows:

[0063] 1) Cell seeding: at 1×10 6 Cells were seeded at a density of cells / well into 6-well plates and cultured overnight in DMEM / F12 medium + 10% fetal bovine serum (FBS) in an incubator (37°C, 5% CO2).

[0064] 2) Solution preparation: Prepare sample working solutions of different concentrations according to the test concentration setting table (Table 2).

[0065] 3) Drug administration: Add the drug according to Table 2 and continue incubation at 37℃ and 5% CO2 for 24 h.

[0066] 4) Sample Collection: Add 0.1 mL of RIPA lysis buffer (containing protease / phosphatase inhibitor) to each well and lyse by pipetting. Collect the cell lysate in an EP tube. After collection, transfer the sample to the appropriate detection tube.

[0067] Store in a freezer at -80°C.

[0068] 5) Detection: Superoxide dismutase activity was detected and analyzed according to the instructions of the superoxide dismutase detection kit (purchased from Shanghai Jinpan Biotechnology Co., Ltd.).

[0069] Table 2 Concentration Setting Table

[0070]

[0071] 2. Experimental Results

[0072] The results are as follows Figure 1 As shown, the significance of the NC group compared with the BC group is indicated by #, p-value < 0.05 is indicated by #, and p-value < 0.01 is indicated by ##; the significance of the sample group and PC group compared with the NC group is indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **. Compared with the control group, the SOD activity in human dermal papilla cells was significantly reduced after DHT stimulation, but the SOD activity was significantly increased after treatment with peony extract, indicating that the peony extract in Example 1 has antioxidant capacity, reduces oxidative stress, and delays scalp cell aging.

[0073] Example 4: Peony extract anti-hair loss test

[0074] 1. Experimental Methods

[0075] Previous studies have shown that the aging of hair follicle stem cells leads to the gradual atrophy of the hair follicles themselves, resulting in thinning and hair loss. Therefore, inhibiting the aging of hair follicle stem cells can delay hair loss. β-galactosidase (SA-β-Gal) is a commonly used marker of cellular aging, exhibiting increased activity during cellular aging. By detecting the activity level of SA-β-Gal in cells, the degree of cellular aging can be assessed.

[0076] This test is based on a DHT-stimulated human dermal papilla cell model. The changes in SA-β-Gal expression levels in human dermal papilla cells were detected using SA-β-Gal staining to evaluate whether the test sample had an anti-hair loss effect. The specific experimental procedure is as follows:

[0077] 1) Cell seeding: Seed at an appropriate seeding density (8 × 10⁻⁶). 4 Human dermal papilla cells were seeded into 24-well plates and incubated overnight in DMEM / F12 medium + 10% fetal bovine serum (FBS) in an incubator (37°C, 5% CO2).

[0078] 2) Administration: Add 1 mL of culture medium containing different concentrations of samples according to Table 2.

[0079] 3) Staining: Staining was performed according to the instructions of the β-galactosidase staining kit (Beyotime SA-β-Gal staining kit (C0602)).

[0080] 4) Results analysis: Image Pro Plus software was used to perform quantitative analysis on SA-β-Gal positive cells.

[0081] 2. Experimental Results

[0082] The results are as follows Figure 2 As shown, significant differences between the sample group and the BC group are indicated by *, p-value < 0.05 is indicated by *, and p-value < 0.01 is indicated by **. Based on the DHT-stimulated human dermal papilla cell model, the sample significantly inhibited the SA-β-Gal positive cell rate of human dermal papilla cells at all three concentrations, and the difference was statistically significant compared with the NC group (p < 0.05), indicating that the sample has an anti-hair loss effect.

[0083] Example 4: Peony extract promotes the expression of growth factors in hair follicles, thereby promoting hair growth.

[0084] 1. Laboratory animals and environment

[0085] 1) In vitro hair follicle experiment: Animal species and strain: SPF-grade C57BL / 6J mice (age: 5 weeks old, sex: male); Number of animals: 10; Quality certificate number: 440072000142022.

[0086] 2) Animal source: Guangdong Provincial Center for Medical Laboratory Animals: Production license number: SCXK (Guangdong) 2022-0002.

[0087] 3) Feeding environment: Temperature: 20-26°C, Relative humidity: 30-70%.

[0088] 2. Experimental methods

[0089] In vitro hair follicle culture and hair growth experiment: The hair growth cycle of mouse vibrissae is synchronized with their age, and the isolated vibrissa hair follicles can be maintained in vitro for up to 23 days. Using the organ culture method to evaluate hair follicle growth is considered to have a high correlation with the in vivo system because the degree of hair growth can be observed as the sum of each cell function. An in vitro model was used to study the promoting effect of the formulation on hair growth. The specific experimental process is as follows:

[0090] 1) Isolation of hair follicles

[0091] Pretreatment of culture plate: The 24-well plate was pre-coated with 1% agarose (300 μL / well) to prevent hair follicles from adhering to the wall, and the hair follicles were placed in the center (hair bulb facing down).

[0092] Hair follicle culture: Incubate for 14 days at 37°C and 5% CO2, each well containing 1 mL of culture medium (Williams E medium + 2 mM L-glutamine + 10 μg / mL insulin + 10% FBS + 1% antibiotic), placed in a 24-well plate.

[0093] Sample addition: Sample addition was carried out on the next day of culture. The culture media containing the samples of different concentrations (mass fraction 0.001%, 0.05%) of the examples and the comparative example were added to the corresponding wells. Each concentration was set with 3 parallel replicates, and the medium was changed every 3 days. The blank control group (BC) only added the culture medium, and the positive control group (PC) added the culture medium containing 0.2% minoxidil.

[0094] 2) Determination of VEGF content: After collecting the mouse vibrissa hair follicles on the 15th day of culture, the hair follicles of each group were transferred to cryopreservation tubes, PBS was added, and they were kept in the refrigerator at 80°C for standby. After the specimens were thawed, they were kept at 2-8°C, PBS was added, the hair follicles were ground by hand, and the cells were broken by repeated freezing and thawing, centrifuged (12000 rpm, 4°C, 15 min), and the supernatant was carefully collected. The ELISA method was used to detect the VEGF content in the specimens.

[0095] 3) Image analysis: The length of the whisker hair follicles was photographed every 7 days, and the morphological observation and measurement of the whisker hair follicle length were performed.

[0096] 2. Experimental Results

[0097] The results are as follows Figure 3 , Figure 4 As shown, the peony extract in the examples, at concentrations of 0.001% and 0.005%, significantly increased the VEGF content in hair follicles, and the relative hair growth rate was significantly better than that of the control group, indicating that the samples have a good anti-hair loss effect. Compared with the comparative example, the examples showed significant advantages in promoting VEGF expression and promoting hair length growth.

[0098] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of peony extract in the preparation of products for preventing hair loss and / or promoting hair growth, characterized in that, The peony extract is a peony flower extract, wherein the peony flower extract contains ≥10% peony flower peptides by mass, the peony flower peptides are small molecule peptides, and the molecular weight of the peony flower peptides is ≤5000 Da. The peony flower extract was prepared by the following method: S1: After crushing the peony flowers, add deionized water at a ratio of 1:10 to 1:100 and cook at high temperature for 20 to 180 minutes. S2: Add neutral protease to the peony flower aqueous solution obtained in step S1 for enzymatic hydrolysis and then filter. The filtration conditions are 200~5000 Da to obtain a filtrate containing peony flower extract. S3: Spray dry the filtrate obtained in step S2 to obtain the peony extract.

2. The application according to claim 1, characterized in that, The product in question is a cosmetic product.

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