A skin external preparation extract, and a preparation method and application thereof

Abstract

This invention relates to a topical skin extract, its preparation method, and its application, belonging to the field of plant extraction and cosmetic application technology. The invention provides a method for preparing a topical skin extract derived from "Hibiscus Ointment," an optimized version of a traditional Chinese and Western medicine formula for treating acute abdominal pain. The formula includes Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron amurense, Coptis chinensis, Scutellaria baicalensis, and borneol. The resulting extract has a higher content of flavonoids, stronger anti-inflammatory and antioxidant capabilities, and is more stable. Furthermore, the combination of this extract and glycerol glucoside exhibits a synergistic effect in reducing skin inflammation and promoting the expression of barrier-related proteins and β-endorphins, thus simultaneously addressing the effects of endogenous and exogenous factors on the skin and improving skin condition.

Description

Technical Field

[0001] This invention relates to a topical skin extract, its preparation method, and its application, belonging to the field of plant extraction and cosmetic application technology. Background Technology

[0002] As the outermost barrier of the human body, the skin plays a vital role in protecting the body from external damage and maintaining homeostasis. External factors such as environmental pollution, ultraviolet radiation, and cooking, as well as internal factors like anxiety and work stress, can all harm the skin. Currently, most skincare products on the market focus on protecting or repairing skin damage caused by external factors; very few contain ingredients or products specifically targeting skin damage caused by internal factors, and even fewer address skin problems caused by both.

[0003] The skin is a reflection of emotions. Prolonged anxiety, stress, lack of sleep, and other negative emotions can all affect skin condition, manifesting as barrier dysfunction, reduced wound healing ability, and abnormal skin immune function. The main mechanism involves psychological stress stimulating the hypothalamus-pituitary-adrenal (HPA) axis to secrete various hormones. These hormones can reach skin tissues via blood circulation or other transport pathways, regulating skin condition by binding to corresponding receptors expressed by skin cells. β-Endorphin is one such hormone, processed from pro-opiomelanocortin (POMC), and is expressed in various tissues including the pituitary gland, brain, adrenal glands, and skin, participating in the repair process of various skin problems. For example, β-Endorphin can regulate Langerhans cell function by modulating the expression of interleukin 1β (IL-1β) and interleukin 10 (IL-10), participating in skin immune regulation; β-Endorphin can also promote the growth and migration of keratinocytes, promoting skin repair. When the body is in a state of prolonged negative emotions, it can affect the expression of β-endorphin, thereby impacting normal skin physiological function. Therefore, promoting β-endorphin expression is one of the important ways to address endogenous skin problems.

[0004] Patent application CN117357421A provides a composition containing ergothioneine and dipeptide-1 or its derivatives, which can synergistically promote the expression of pro-okratin or β-endorphin. However, ergothioneine has poor stability under light or high temperature conditions and is expensive, which is not conducive to its widespread application.

[0005] Patent application CN116785209A discloses an agarwood hydrosol extract that can repair skin problems caused by exogenous stimuli and endogenous stress. It can target and regulate the expression of TRPV1 receptors, improve skin sensory nerve dysfunction, and promote β-endorphin secretion to address stress-related skin problems. However, agarwood hydrosol itself contains aromatic components, and consumers have varying levels of acceptance of its scent, resulting in low market acceptance and hindering product promotion.

[0006] Traditional Chinese medicine (TCM) is a treasure of Chinese civilization and possesses high development value. The "Hibiscus Ointment" recorded in *Integrated Traditional Chinese and Western Medicine Treatment of Acute Abdominal Diseases* is composed of 8 liang of hibiscus leaves, 8 liang of rhubarb, 8 liang of *Eupatorium fortunei* leaves, 8 liang of phellodendron bark, 6 liang of scutellaria root, 6 liang of coptis root, and 2 qian of borneol. It has the effects of clearing heat, detoxifying, and reducing swelling, and can soothe skin redness and discomfort caused by mosquito bites, eczema, boils, etc. Existing technology, patent application CN106038780A, provides a TCM ointment for treating erysipelas and mumps, including hibiscus leaves, raw rhubarb, phellodendron bark, scutellaria root, *Eupatorium fortunei* leaves, coptis root, *Dapixiao* (a type of herb), and borneol. It has the effects of clearing heat, cooling blood, reducing swelling, and relieving pain, and can treat erysipelas, cellulitis, etc. However, this formula is for medicinal use and is difficult to apply in the cosmetic field. Patent application CN107007622A discloses a skin-regenerating composition that promotes the regeneration of soft tissue, comprising hibiscus leaves, rhubarb, eupatorium leaves, phellodendron bark, scutellaria root, coptis root, borneol, etc., which can improve the problem of redness and inflammation of wounds. However, it can only address skin problems caused by exogenous factors. Furthermore, the stability of traditional Chinese medicine extracts has always been a challenge hindering their application in cosmetics. How to improve the stability of raw materials through processing is also a problem that urgently needs to be solved in the industry. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a topical skin extract, its preparation method, and its applications. The topical skin extract prepared by this invention effectively combats free radical scavenging, reduces skin inflammation, and promotes the expression of barrier-related proteins. Through process optimization, the efficacy and stability of the topical skin extract are significantly improved, making it more suitable for application. Furthermore, this invention unexpectedly discovered that the composition of this topical skin extract and glycerol glucoside has a synergistic effect in reducing skin inflammation and promoting the expression of barrier-related proteins and β-endorphin, thereby simultaneously addressing the effects of endogenous and exogenous factors on the skin and improving skin condition.

[0008] The technical solution of the present invention is as follows:

[0009] In a first aspect, the present invention provides a method for preparing an extract of a topical skin preparation, comprising the following steps:

[0010] S1. Pulverize the medicinal materials of the topical skin formula separately, pass them through a 10-50 mesh sieve, take the sieve-less portion, mix the medicinal materials in a certain proportion and then mix them with the extraction solvent in a certain material-liquid ratio, and extract by gentle boiling and reflux to obtain crude extract;

[0011] S2. Filter the crude extract obtained in step S1 through a filter cloth to remove the residue, place it at room temperature, and then filter it through a filter plate. After sterilization, let the filtrate stand at room temperature or low temperature for 24 to 48 hours to obtain a secondary filtrate.

[0012] S3. Filter the secondary filtrate obtained in step S2 through a filter plate, add preservatives and stabilizers, and stir at 45℃~55℃ until completely dissolved to obtain the skin topical formula extract.

[0013] Preferably, the topical skin formula mentioned in step S1 is an optimized version of "Hibiscus Ointment" recorded in "Integrated Traditional Chinese and Western Medicine Treatment of Acute Abdominal Diseases". The medicinal materials include Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis, and borneol. The mass ratio of Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis, and borneol when mixed is 80:80:1:100:80:80:2.

[0014] Preferably, the material is passed through a 10-mesh sieve in step S1.

[0015] Preferably, the extraction solvent in step S1 is a 30% to 35% aqueous solution of 1,3-butanediol; more preferably, it is a 30% aqueous solution of 1,3-butanediol, all by mass percentage.

[0016] Preferably, the material-to-liquid ratio in step S1 is 1:(28-32); more preferably, it is 1:30.

[0017] Preferably, the temperature of the micro-boiling hot reflux extraction in step S1 is 90-100℃ and the time is 2-4h; more preferably, it is micro-boiling hot reflux extraction at 100℃ for 3.5h.

[0018] Preferably, the filter cloth in step S2 is a double-layered 200-400 mesh filter cloth; more preferably, it is a double-layered 300 mesh filter cloth.

[0019] Preferably, the filter plate in step S2 is a 0.22μm or 0.45μm filter plate.

[0020] Preferably, the sterilization in step S2 is sterilization at 121°C for 20 minutes.

[0021] Preferably, in step S2, the mixture is left to stand at room temperature for 36 hours.

[0022] Preferably, the low temperature in step S2 is 2 to 6°C.

[0023] Preferably, the filter plate in step S3 is a 0.22μm or 0.45μm filter plate.

[0024] Preferably, the preservative in step S3 is one or a combination of 1,2-hexanediol, p-hydroxyacetophenone, and 1,2-pentanediol; more preferably, the preservative is a combination of 1,2-hexanediol, p-hydroxyacetophenone, and 1,2-pentanediol.

[0025] More preferably, in the topical skin extract, the final concentration of p-hydroxyacetophenone is 0.3-0.6 wt%, the final concentration of 1,2-hexanediol is 0.5-2 wt%, and the final concentration of 1,2-pentanediol is 0.5-2 wt%.

[0026] Preferably, the stabilizer in step S3 is polysorbate-20.

[0027] More preferably, the final concentration of polysorbate-20 in the topical skin extract is 1-5 wt%.

[0028] Secondly, the present invention provides a topical skin extract prepared according to the above preparation method.

[0029] Thirdly, the present invention provides a composition for improving skin condition, comprising the above-mentioned topical skin extract and glyceryl glucoside.

[0030] In this invention, the glycerol glucoside is a commercially available product or prepared according to existing technology.

[0031] Preferably, in the system containing the skin topical formula extract and glyceryl glucoside, the final concentration of the skin topical formula extract is 0.5 to 6.0 wt%, and the final concentration of the glyceryl glucoside is 0.1 to 2.0 wt%.

[0032] More preferably, in the system containing the skin topical formula extract and glycerol glucoside, the final concentration of the skin topical formula extract is 1.0 to 4.0 wt%, and the final concentration of the glycerol glucoside is 1.0 to 2.0 wt%.

[0033] In this invention, the system containing the skin topical formula extract and glyceryl glucoside may also include purified water or other conventional cosmetic or skin care product bases, in addition to the skin topical formula extract and glyceryl glucoside.

[0034] In this invention, the combination of the above-mentioned topical skin extract and glyceryl glucoside can reduce skin inflammation and promote the expression of barrier-related proteins and β-endorphin through synergistic effects, thereby simultaneously addressing the effects of endogenous and exogenous factors on the skin and improving skin condition.

[0035] Fourthly, the application of the above-mentioned combination of topical skin extracts and glyceryl glucoside in the preparation of cosmetics or skin care products.

[0036] Preferably, the cosmetic or skin care product is in the form of liquid, gel, ointment, cream, lotion, mask or freeze-dried powder.

[0037] Fifthly, a cosmetic or skin care product comprising the above-mentioned topical skin extract and glyceryl glucoside.

[0038] In this invention, the cosmetics or skincare products can be applied to the skin surface to reduce skin inflammation and promote the expression of barrier-related proteins and β-endorphins through synergistic effects, thereby simultaneously addressing the effects of endogenous and exogenous factors on the skin and improving skin condition.

[0039] Beneficial effects:

[0040] 1. This invention provides a method for preparing a topical skin extract, which yields a topical skin extract with higher flavonoid content, stronger anti-inflammatory and antioxidant capabilities, and greater stability.

[0041] 2. This invention also provides a composition consisting of a topical skin extract and glyceryl glucoside. This composition: 1) can significantly reduce UVB-induced skin barrier damage by increasing the mRNA expression of filaggrin (FLG), thereby repairing the skin barrier and improving photodamage, with synergistic effects; 2) can significantly reduce UVB-induced skin inflammation by inhibiting the secretion of tumor necrosis factor α (TNF-α), thereby soothing skin inflammation and improving photodamage, with synergistic effects; 3) can address endogenous skin problems by increasing β-endorphin levels, thereby improving skin condition, with synergistic effects. Based on the above technical effects, it can simultaneously address the effects of endogenous and exogenous factors on the skin, thereby improving skin condition. Attached Figure Description

[0042] Figure 1 The standard curve for flavonoid content using rutin as the standard is shown.

[0043] Figure 2The bar chart shows the expression levels of FLG mRNA in HaCaT cells under the action of the compositions of Examples 4-8 and Comparative Examples 14-18.

[0044] Figure 3 The bar chart shows the amount of TNF-α secreted by HaCaT cells under the action of the compositions of Examples 4-8 and Comparative Examples 14-18;

[0045] Figure 4 Bar chart showing the β-endorphin content in HaCaT cells under the action of the compositions of Examples 4-8 and Comparative Examples 14-18;

[0046] In the figure, significance compared with the blank control group is indicated by #, 0.01 < P < 0.05 is indicated by #, 0.001 < P < 0.01 is indicated by ##, 0.0001 < P < 0.001 is indicated by ###, and P < 0.0001 is indicated by ####; significance compared with the negative control group is indicated by *, 0.01 < P < 0.05 is indicated by *, 0.001 < P < 0.01 is indicated by **, 0.0001 < P < 0.001 is indicated by ***, and P < 0.0001 is indicated by ****. Detailed Implementation

[0047] To better understand the present invention, further explanation is provided below with reference to embodiments. However, the scope of protection of the present invention is not limited to the following embodiments, and the embodiments should not be regarded as limiting the scope of protection of the present invention.

[0048] The topical skin formula described in the examples and comparative examples is an optimized version of "Hibiscus Ointment" recorded in "Integrated Traditional Chinese and Western Medicine Treatment of Acute Abdominal Diseases". The medicinal materials include: Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis, and borneol.

[0049] The rhubarb, hibiscus leaves, eupatorium leaves, phellodendron bark, coptis root, borneol, and scutellaria root used in the examples and comparative examples are all commercially available products. Other reagents and materials used in the examples and comparative examples are also commercially available products unless otherwise specified.

[0050] The glycerol glucosides described in this invention are commercially available products or prepared according to existing technologies. The glycerol glucosides used in the examples and comparative examples were purchased from Hubei Xinyuhong Biomedical Technology Co., Ltd.

[0051] Example 1

[0052] A method for preparing an extract from a topical skin treatment includes the following steps:

[0053] S1. Grind the medicinal materials of the topical skin formula into powder, pass them through a 10-mesh sieve, and take the sieve-less portion. Mix the leaves of Hibiscus mutabilis, rhubarb, Lycopus lucidus, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis and borneol in a mass ratio of 80:80:1:100:80:80:2. After mixing, add the mixture to an extraction tank with a 30% 1,3-butanediol aqueous solution at a material-to-liquid ratio of 1:30. Extract by gentle reflux at 100℃ for 3.5 hours to obtain the crude extract.

[0054] S2. The crude extract obtained in step S1 is filtered through a double-layer 300-mesh filter cloth to remove the residue, placed at room temperature, and then filtered through a 0.45μm filter plate. The filtrate is sterilized at 121℃ for 20 minutes and then allowed to stand at room temperature for 36 hours to obtain a secondary filtrate.

[0055] S3. After filtering the secondary filtrate obtained in step S2 through a 0.45μm filter plate, add 0.5wt% p-hydroxyacetophenone, 2wt% 1,2-hexanediol, and 1wt% 1,2-pentanediol as preservatives, and 1wt% polysorbate-20 as stabilizer. Stir at 45℃~55℃ until completely dissolved to obtain the topical skin extract.

[0056] Example 2

[0057] A method for preparing an extract from a topical skin treatment includes the following steps:

[0058] S1. Grind the medicinal materials of the external skin prescription into powder, pass them through a 30-mesh sieve, and take the sieve-below portion. Mix the leaves of Hibiscus mutabilis, rhubarb, Lycopus lucidus, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis and borneol in a mass ratio of 80:80:1:100:80:80:2. After mixing, add the mixture to an extraction tank with a 30% 1,3-butanediol aqueous solution at a material-liquid ratio of 1:30. Extract by gentle boiling and reflux at 100℃ for 2 hours to obtain the crude extract.

[0059] S2. The crude extract obtained in step S1 is filtered through a double-layer 200-mesh filter cloth to remove the residue, placed at room temperature, and then filtered through a 0.45μm filter plate. The filtrate is sterilized at 121℃ for 20 min and then allowed to stand at low temperature (4℃) for 24 h to obtain a secondary filtrate.

[0060] S3. After filtering the secondary filtrate obtained in step S2 through a 0.45μm filter plate, add 0.6wt% p-hydroxyacetophenone, 0.5wt% 1,2-hexanediol, and 2wt% 1,2-pentanediol as preservatives, and 3wt% polysorbate-20 as stabilizer. Stir at 45℃~55℃ until completely dissolved to obtain the topical skin extract.

[0061] Example 3

[0062] A method for preparing an extract from a topical skin treatment includes the following steps:

[0063] S1. Grind the medicinal materials of the topical skin formula into powder, pass them through a 50-mesh sieve, and take the sieve-underfill portion. Mix the leaves of Hibiscus mutabilis, rhubarb, Lycopus lucidus, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis and borneol in a mass ratio of 80:80:1:100:80:80:2. After mixing, add the mixture to an extraction tank with a 30% 1,3-butanediol aqueous solution at a material-to-liquid ratio of 1:30. Extract by gentle boiling and reflux at 100℃ for 4 hours to obtain the crude extract.

[0064] S2. The crude extract obtained in step S1 is filtered through a double layer of 400-mesh filter cloth to remove the residue, placed at room temperature, and then filtered through a 0.45μm filter plate. The filtrate is sterilized at 121℃ for 20 minutes and then allowed to stand at room temperature for 48 hours to obtain a secondary filtrate.

[0065] S3. After filtering the secondary filtrate obtained in step S2 through a 0.45μm filter plate, add 0.3wt% p-hydroxyacetophenone, 1wt% 1,2-hexanediol, and 0.5wt% 1,2-pentanediol as preservatives, and 5wt% polysorbate-20 as stabilizer. Stir at 45℃~55℃ until completely dissolved to obtain the topical skin extract.

[0066] Comparative Example 1

[0067] The method for preparing a topical skin extract differs from Example 1 in that the mass ratio of Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis, and borneol is 40:40:40:40:30:30:1.

[0068] Comparative Example 2

[0069] A method for preparing an extract of a topical skin formula differs from Example 1 in that the medicinal materials of the topical skin formula do not contain Hibiscus mutabilis leaves.

[0070] Comparative Example 3

[0071] A method for preparing an extract of a topical skin formula differs from Example 1 in that the topical skin formula does not contain rhubarb.

[0072] Comparative Example 4

[0073] A method for preparing an extract of a topical skin formula differs from Example 1 in that the medicinal materials in the topical skin formula do not contain Scutellaria baicalensis.

[0074] Comparative Example 5

[0075] The method for preparing a topical skin extract differs from Example 1 in that the mass ratio of Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis, and borneol is 40:40:10:50:40:40:1.

[0076] Comparative Example 6

[0077] The method for preparing a topical skin extract differs from Example 1 in that the mass ratio of Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis and borneol is 80:80:1:120:80:80:2.

[0078] Comparative Example 7

[0079] The method for preparing a topical skin extract differs from Example 1 in that the mass ratio of Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis and borneol is 80:80:1:100:100:80:2.

[0080] Comparative Example 8

[0081] A method for preparing a topical skin extract differs from Example 1 in that the extraction solvent in step S1 is 75% ethanol.

[0082] Comparative Example 9

[0083] A method for preparing a topical skin extract differs from Example 1 in that the material-to-liquid ratio in step S1 is 1:20.

[0084] Comparative Example 10

[0085] A method for preparing a topical skin extract differs from Example 1 in that the material-to-liquid ratio in step S1 is 1:40.

[0086] Comparative Example 11

[0087] A method for preparing a topical skin extract differs from Example 1 in that no stabilizer is added in step S3.

[0088] Comparative Example 12

[0089] A method for preparing a topical skin extract differs from Example 1 in that trehalose with a final concentration of 3% is added as a stabilizer in step S3.

[0090] Comparative Example 13

[0091] A method for preparing a topical skin extract differs from Example 1 in that xanthan gum with a final concentration of 0.1% is added as a stabilizer in step S3.

[0092] Examples 4-8 and Comparative Examples 14-18

[0093] A composition comprising the aforementioned topical skin extract and glyceryl glucoside. Table 1 shows the compositional composition of the skin-improving compositions of Examples 4-8. These compositions, through synergistic effects, jointly reduce skin inflammation, promote the expression of barrier-related proteins and β-endorphin, thereby simultaneously addressing the effects of endogenous and exogenous factors on the skin and improving skin condition. For comparison, Table 1 also provides Comparative Examples 14-18. Comparative Examples 14-15 differ from Examples 4-8 in that they contain only one substance from the composition; Comparative Examples 16-17 differ from Examples 4-8 in that one substance in the composition is outside the scope of protection; and Comparative Example 18 differs from Examples 4-8 in that the topical skin extract used is different.

[0094] Table 1. Raw material composition and proportions of the compositions of Examples 4-8 and Comparative Examples 14-18, unit: wt%.

[0095]

[0096]

[0097] Note: * indicates that the topical skin extract used in Comparative Example 18 was prepared according to the preparation method of Comparative Example 1. The topical skin extracts used in the other examples and comparative examples were all prepared according to the preparation method of Example 1.

[0098] Example 9 and Comparative Example 19

[0099] The combination of the topical skin extract and glyceryl glucoside prepared according to the preparation method of Example 1 was added to a skin cream to prepare a skin care product with the effect of improving skin condition, namely Example 9. At the same time, a placebo group was set up, namely Comparative Example 19. The difference between Comparative Example 19 and Example 9 is that Comparative Example 19 does not contain the combination of the topical skin extract and glyceryl glucoside. The specific raw material components and proportions are shown in Table 2. Human efficacy experiments were conducted to test its effect of improving skin condition.

[0100] Table 2. Ingredients and proportions of the skin creams in Example 9 and Comparative Example 19

[0101]

[0102]

[0103] The skin creams of Example 9 and Comparative Example 19 were both prepared using conventional methods in the art.

[0104] Experimental example:

[0105] The performance of the products in the above embodiments and comparative examples was tested, as follows:

[0106] Experimental materials:

[0107] The immortalized human keratinocytes (HaCaT cells) used in the following tests were available from the Shanghai Cell Bank of the Chinese Academy of Sciences; other reagents and materials, unless otherwise specified, are all commercially available products.

[0108] I. Antioxidant properties

[0109] The antioxidant properties of the topical skin extracts prepared in Examples 1-3 and Comparative Examples 1-10 were determined using a DPPH free radical scavenging assay.

[0110] Preparation of DPPH working solution (concentration 0.08 mg / mL): Accurately weigh 20 mg of DPPH powder, dissolve it in anhydrous ethanol, and dilute to 250 mL in a volumetric flask. Store at 0–4 °C protected from light. Prepare and use immediately.

[0111] Preparation of test solutions: The extract samples of topical skin formulas were diluted with purified water to obtain test solutions with sample concentrations of 0.5wt%, 0.8wt%, 1.0wt%, 1.5wt%, 2.5wt%, 3.0wt%, 5.0wt%, and 6.0wt%.

[0112] The detection method and steps are as follows:

[0113] S1. Take 1 mL of the test solution and mix it with 1 mL of 0.08 mg / mL DPPH solution (tube A);

[0114] S2. Mix 1 mL of anhydrous ethanol with 1 mL of 0.08 mg / mL DPPH solution (tube B);

[0115] S3. Take 1 mL of anhydrous ethanol and mix it with 1 mL of the test solution (tube C);

[0116] S4. After reacting in the dark for 30 min, take 200 μL of the reacted solution into a 96-well plate under dark conditions, and then measure the absorbance (OD) values ​​of tubes A, B, and C at 517 nm. Calculate the DPPH scavenging rate according to the following formula:

[0117] DPPH clearance rate (%) = (OD B +OD C -OD A ) / OD B ×100%

[0118] The DPPH free radical scavenging rate determination results of Examples 1-3 and Comparative Examples 1-7 are shown in Table 3; the DPPH free radical scavenging rate determination results of Examples 1-3 and Comparative Examples 8-10 are shown in Table 4.

[0119] Table 3. Results of DPPH free radical scavenging rate determination in Examples 1-3 and Comparative Examples 1-7

[0120]

[0121] Table 3 shows that, under the same process, the DPPH free radical scavenging effect was best when the mass ratio of Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis, and borneol in Examples 1-3 was 80:80:1:100:80:80:2. Lacking any one of these substances or changing their proportions resulted in lower DPPH free radical scavenging effects in Comparative Examples 1-4 compared to Examples 1-3, under the same detection conditions. Increasing the proportion of a particular substance, under the same detection conditions, did not significantly improve the DPPH free radical scavenging effect of Comparative Examples 5-7 compared to Examples 1-3; in fact, it decreased it.

[0122] Table 4. Results of DPPH free radical scavenging rate determination in Examples 1-3 and Comparative Examples 8-10

[0123]

[0124]

[0125] Table 4 shows that, under the same ratio of medicinal materials, the DPPH free radical scavenging effect of the skin topical formula extracts prepared by the methods of Examples 1-3 is better. When the extraction solvent is changed or the material-liquid ratio is altered, the DPPH free radical scavenging effect of the skin topical formula extracts of Comparative Examples 8-10 is lower than that of Examples 1-3 under the same detection conditions.

[0126] II. Anti-inflammatory performance testing

[0127] COX-2 (cyclooxygenase-2) is an inflammatory factor. The topical skin extracts obtained in Examples 1-3 and Comparative Examples 1-10 were diluted with DMSO to 0.5 wt%, 0.8 wt%, 1.0 wt%, 1.5 wt%, 2.5 wt%, 3.0 wt%, 5.0 wt%, and 6.0 wt%, respectively. The COX-2 inhibition rate was determined according to the kit instructions to reflect its anti-inflammatory activity. The COX-2 inhibitor screening kit used was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0128] The COX-2 inhibition rate measurement results of Examples 1-3 and Comparative Examples 1-7 are shown in Table 5; the COX-2 inhibition rate measurement results of Examples 1-3 and Comparative Examples 8-10 are shown in Table 6.

[0129] Table 5. Results of COX-2 inhibition rate determination in Examples 1-3 and Comparative Examples 1-7

[0130]

[0131] Table 5 shows that, under the same process, the best COX-2 inhibition effect was observed in Examples 1-3 when the mass ratio of Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis, and borneol was 80:80:1:100:80:80:2. The absence of any one of these substances or changes in their proportions resulted in lower COX-2 inhibition rates in Examples 1-4 compared to Examples 1-3, under the same detection conditions. Increasing the proportion of any one substance, under the same detection conditions, did not significantly improve the COX-2 inhibition rate of Examples 5-7 compared to Examples 1-3; in fact, it decreased it.

[0132] Table 6. Results of COX-2 inhibition rate determination in Examples 1-3 and Comparative Examples 8-10

[0133]

[0134] The results in Table 6 show that, under the same ratio of medicinal materials, the extracts of the topical skin formula prepared by the methods in Examples 1-3 have a better inhibitory effect on COX-2. When the extraction solvent is changed or the material-liquid ratio is altered, the inhibitory effect of Comparative Examples 8-10 on COX-2 is lower than that of Examples 1-3 under the same detection conditions.

[0135] III. Flavonoid Content Detection

[0136] Take 1 mL of the topical skin extract samples from Examples 1-3 and Comparative Examples 8-10 into test tubes, and add 4 mL of purified water and 0.3 mL of 5% NaNO2 aqueous solution sequentially. After mixing for 5 min, add 0.3 mL of 10% Al(NO3)3 aqueous solution and react for 6 min. Then add 2 mL of 1 mol / L NaOH aqueous solution and 2.4 mL of purified water sequentially. After 10 min, measure the absorbance at 510 nm. Plot a standard curve with absorbance value A as the ordinate and rutin standard concentration as the abscissa. Calculate the total flavonoid content of the sample based on the standard curve.

[0137] Figure 1 The standard curve for the flavonoid content of rutin standard is given by equation y = 0.1636x + 0.1624, R0. 2 =0.9962. The total flavonoid content of the topical skin extracts of Examples 1-3 and Comparative Examples 8-10 was calculated based on the absorbance value, and the results are shown in Table 7.

[0138] Table 7. Results of total flavonoid content determination in Examples 1-3 and Comparative Examples 8-10

[0139] Group Example 1 Example 2 Example 3 Comparative Example 8 Comparative Example 9 Comparative Example 10 Total flavonoid content 2.95±0.01 2.43±0.01 2.64±0.01 0.38±0.04 0.65±0.04 0.42±0.01

[0140] The results in Table 7 show that the total flavonoid content was higher under the preparation methods of Examples 1-3. In Comparative Examples 8-10, the total flavonoid content was lower than that of Examples 1-3 when the extraction solvent was changed or the material-liquid ratio was altered.

[0141] IV. Stability Comparison

[0142] The skin extracts prepared in Examples 1-3 and Comparative Examples 8-13 were placed under low temperature (-20℃), refrigeration (4℃), high temperature (50℃), alternating hot and cold conditions (55℃ / -20℃, every other day), and light exposure (4500±500LX) for 90 days to observe their stability under different storage conditions. The results are shown in Table 8. Within 90 days, the skin extracts of Examples 1-3 and Comparative Examples 8-10 remained stable under different storage conditions. Comparative Examples 11-13 showed precipitation at different time points, and the precipitation gradually increased with time, affecting their application.

[0143] Table 8. Stability test results of Examples 1-3 and Comparative Examples 8-13

[0144]

[0145] V. Composition Efficacy Testing

[0146] (1) Effects of the compositions of Examples 4-8 and Comparative Examples 14-18 on FLG mRNA expression levels in HaCaT cells

[0147] Human immortalized keratinocytes (HaCaT cells) in good growth condition were collected and a cell suspension was prepared using DMEM complete culture medium. The cell concentration of the suspension was adjusted to 1.3 × 10⁻⁶ cells / mL. 5 Cells were cultured at a concentration of 10 cells / mL, with 2 mL of cell suspension added to each well of a 6-well cell culture plate. The experiment included a blank control group (DMEM high-glucose medium), a negative control group (UVB + DMEM high-glucose medium), and a sample group (UVB + DMEM high-glucose medium containing the compositions of Examples 4-8 and Comparative Examples 14-18), with three replicates for each group. The seeded 6-well cell culture plates were placed in a cell culture incubator (5% CO2, 37℃) and cultured for 24 h. When cell confluence reached 40-60%, the culture medium was discarded, and PBS buffer was added. Except for the blank control group, each group received 43 mJ / cm² of PBS buffer. 2UVB irradiation was performed at specific doses. After irradiation, PBS buffer was discarded, and DMEM high-glucose culture medium was added to the blank control group and negative control group. The remaining groups were added to DMEM high-glucose culture medium containing the compositions of Examples 4-8 and Comparative Examples 14-18, respectively. The cells were cultured in a cell incubator for 24 h, the culture medium was discarded, and total RNA was extracted from each group. The mRNA expression level of FLG was detected by RT-qPCR.

[0148] The test concentrations of the compositions in Examples 4-8 and Comparative Examples 14-18 are shown in Table 1, and the solvent is DMEM high-glucose culture medium.

[0149] FLG is an important molecule in the stratum corneum of human skin that connects keratin fibers. With the assistance of FLG monomers, keratin fibers aggregate regularly, forming a solid physical barrier on the outermost layer of the epidermis, which can prevent the loss of epidermal moisture and the invasion of harmful external substances. The test results are shown in Table 9 and... Figure 2 As shown, after UVB stimulation, the FLG mRNA expression level in the negative control group was significantly lower than that in the blank control group (P<0.0001), proving the successful modeling of photodamage. After UVB stimulation, compared to the negative control group, the expression level of FLG mRNA was significantly increased after adding the compositions of Examples 4-8 and Comparative Examples 14-18 to the damaged cells (P<0.001). Specifically, the improvement rates of FLG mRNA expression levels by the compositions of Examples 4-8 were 473.90%, 400.97%, 372.91%, 352.05%, and 362.75%, respectively, while the improvement rates of FLG mRNA expression levels by the compositions of Comparative Examples 14-18 were 197.71%, 101.93%, 314.37%, 317.07%, and 259.75%, respectively. The results of Comparative Examples 14 and 15 show that both the topical skin extract and glycerol glucoside alone have a certain effect on increasing FLG mRNA expression. However, compared with the results of Examples 4-8, it can be seen that the combined use of the two has a better effect, indicating that the combination of the topical skin extract and glycerol glucoside can significantly alleviate UVB-induced skin barrier damage by increasing FLG mRNA expression, thereby repairing the skin barrier and improving photodamage, and can have a synergistic effect. Comparative Examples 16 and 17, compared with the results of Examples 4-8, show that when either the topical skin extract or glycerol glucoside exceeds a certain concentration range, increasing the concentration does not increase the effect of the combination on increasing FLG mRNA expression. Meanwhile, compared with the results of Example 4, Comparative Example 18 shows that the combined use of the topical skin extract and glycerol glucoside in the embodiments of the present invention has a better effect.

[0150] Table 9. Effects of the compositions of Examples 4-8 and Comparative Examples 14-18 on FLG mRNA expression levels in HaCaT cells.

[0151]

[0152]

[0153] (2) Effects of the compositions of Examples 4-8 and Comparative Examples 14-18 on TNF-α secretion in HaCaT cells

[0154] Human immortalized keratinocytes (HaCaT cells) in good growth condition were collected, and a cell suspension was prepared using DMEM complete culture medium. The cell density of the suspension was adjusted to 1.3 × 10⁻⁶ cells / mL. 5 Cells were cultured at a concentration of 1 / mL, with 2 mL of cell suspension added to each well of a 6-well cell culture plate. The experiment included a blank control group (DMEM high-glucose medium), a negative control group (UVB + DMEM high-glucose medium), and a sample group (UVB + DMEM high-glucose medium containing the compositions of Examples 4-8 and Comparative Examples 14-18), with three replicates per group. The seeded 6-well cell culture plates were placed in a cell culture incubator (5% CO2, 37℃) for 24 h. When the cell confluence reached 40-60%, the culture medium was discarded, and PBS buffer was added. Except for the blank control group, each group received 43 mJ / cm² of PBS buffer. 2 After UVB irradiation, the PBS buffer was discarded. Fresh DMEM high-glucose culture medium was added to the blank control group and the negative control group. The remaining groups were added with DMEM high-glucose culture medium containing the compositions of Examples 4-8 and Comparative Examples 14-18, respectively. After culturing in a cell incubator for 24 h, the cell culture medium was aspirated into 1.5 mL EP tubes, centrifuged at 12000 r / min for 10 min, and the supernatant was collected. The secretion of TNF-α was measured according to the instructions of the ELISA kit.

[0155] The test concentrations of the compositions in Examples 4-8 and Comparative Examples 14-18 are shown in Table 1, and the solvent is DMEM high-glucose culture medium.

[0156] TNF-α, acting as an inflammatory sentinel in the upstream initiation stage of the inflammatory cascade, is an endogenous pyrogen that can cause skin redness and heat. Test results are shown in Table 10. Figure 3As shown, after UVB stimulation, the secretion of TNF-α in the negative control group was significantly increased compared with the blank control group (P<0.0001), proving the successful modeling of photodamage. After UVB stimulation, compared with the negative control group, the secretion of TNF-α was significantly reduced after adding the compositions of Examples 4-8 and Comparative Examples 14-18 to the damaged cells (P<0.01). Specifically, the inhibition rates of TNF-α secretion by the compositions of Examples 4-8 were 84.36%, 88.75%, 72.68%, 76.60%, and 83.84%, respectively, while the inhibition rates of TNF-α secretion by the compositions of Comparative Examples 14-18 were 53.27%, 10.99%, 70.96%, 70.89%, and 60.44%, respectively. The results of Comparative Examples 14 and 15 show that both the topical skin extract and glycerol glucoside have certain anti-inflammatory effects when used alone. However, compared with the results of Examples 4-8, the anti-inflammatory effect is significantly enhanced when the two are used in combination. This indicates that the combination of the topical skin extract and glycerol glucoside can significantly reduce UVB-induced skin inflammation by inhibiting TNF-α secretion, thereby soothing skin inflammation, improving photodamage, and exhibiting synergistic effects. Comparative Examples 16 and 17, compared with the results of Examples 4-8, show that when either the topical skin extract or glycerol glucoside exceeds a certain concentration range, increasing the concentration does not improve the inhibitory effect of the combination on TNF-α. Furthermore, compared with the results of Example 4, Comparative Example 18 shows that the combined use of the topical skin extract and glycerol glucoside in the embodiments of this invention is more effective.

[0157] Table 10. Effects of the compositions of Examples 4-8 and Comparative Examples 14-18 on TNF-α secretion in HaCaT cells

[0158] Group Secretion volume (pg / mL) SD value Inhibition rate / % P-value Blank control 424.38 14.07 / / negative control 903.97 18.22 / <0.0001 Example 4 141.38 12.24 84.36 <0.0001 Example 5 101.68 3.64 88.75 <0.0001 Example 6 246.93 16.43 72.68 <0.0001 Example 7 211.50 16.57 76.60 <0.0001 Example 8 146.07 12.46 83.84 <0.0001 Comparative Example 14 422.43 13.59 53.27 <0.0001 Comparative Example 15 804.59 11.27 10.99 0.0013 Comparative Example 16 262.53 15.45 70.96 <0.0001 Comparative Example 17 263.12 10.15 70.89 <0.0001 Comparative Example 18 357.65 11.23 60.44 <0.0001

[0159] (3) Effects of the compositions of Examples 4-8 and Comparative Examples 14-18 on β-endorphin content in HaCaT cells

[0160] Human immortalized keratinocytes (HaCaT cells) in good growth condition were collected, and a cell suspension was prepared using DMEM complete culture medium. The cell density of the suspension was adjusted to 3 × 10⁻⁶ cells / mL. 5Cells were cultured at a density of 1 mL / mL in 12-well cell culture plates and incubated for 24 h in a cell culture incubator (5% CO2, 37℃). The experiment included a blank control group (DMEM high-glucose culture medium) and a sample group (DMEM high-glucose culture medium containing the compositions of Examples 4-8 and Comparative Examples 14-18), with three replicates in each group. After inoculation, the 12-well cell culture plates were placed in a cell culture incubator (5% CO2, 37℃) and cultured for 24 h. When the cell confluence rate reached 40-60%, the culture medium was discarded. Fresh DMEM high-glucose culture medium was added to the blank control group, and DMEM high-glucose culture medium containing the compositions of Examples 4-8 and Comparative Examples 14-18 were added to the sample groups. After culturing for 24 h, cells were collected, and cells were lysed by repeated freeze-thaw cycles. The cells were centrifuged at 1500g for 10 min, and the supernatant was used to detect β-endorphin content.

[0161] The test concentrations of the compositions in Examples 4-8 and Comparative Examples 14-18 are shown in Table 1, and the solvent is DMEM high-glucose culture medium.

[0162] β-Endorphins are one of the important "happy molecules" in the skin, possessing local analgesic, immunomodulatory, repairing, anti-inflammatory, and soothing effects. They are an important target for addressing endogenous skin problems. Test results are shown in Table 11. Figure 4 As shown, compared with the blank control group, after the addition of the compositions of Examples 4-8 and Comparative Examples 14-18 to the cells, the β-endorphin content of all groups except Comparative Example 14 was significantly increased (P<0.01). Specifically, the increase rates of β-endorphin content by the compositions of Examples 4-8 were 66.51%, 54.58%, 46.43%, 58.98%, and 64.99%, respectively, while the increase rates of β-endorphin content by the compositions of Comparative Examples 14-18 were 0.3%, 18.87%, 45.76%, 44.36%, and 24.10%, respectively. Comparative Example 14 showed that using the topical skin extract alone had no significant effect on increasing β-endorphin levels. Comparative Example 15 showed that using glyceryl glucoside alone had a certain effect on increasing β-endorphin levels, but compared with the results of Examples 4-8, the combined use of the two showed a better effect in increasing β-endorphin levels. This indicates that the combination of the topical skin extract and glyceryl glucoside can address endogenous skin problems by increasing β-endorphin levels, thus improving the skin and exhibiting synergistic effects. Comparative Examples 16 and 17, compared with the results of Examples 4-8, showed that exceeding a certain concentration range for either the topical skin extract or glyceryl glucoside did not increase the effect of the combination on increasing β-endorphin levels. Furthermore, Comparative Example 18, compared with the results of Example 4, showed that the combined use of the topical skin extract and glyceryl glucoside in the embodiments of this invention was more effective.

[0163] Table 11. Effects of the compositions of Examples 4-8 and Comparative Examples 14-18 on β-endorphin content in HaCaT cells

[0164]

[0165]

[0166] (4) Human efficacy testing

[0167] ① Selection and requirements of volunteers: Thirty outdoor workers (18 women and 12 men) were selected, exhibiting facial redness and impaired skin barrier. They were required to sleep after 3 a.m. for three days prior to the test. All participants had no history of skin or systemic diseases, no abnormalities at the test sites, and did not apply any medications or cosmetics unrelated to the experiment during the test period.

[0168] ②Testing environment: The testing site is kept at a constant temperature and humidity, with an ambient temperature of 20℃~22℃ and a relative humidity of 40%~60%. Before the test, the subject should keep their body in a stable state. After washing the subject's face with clean water at around 35℃, the subject should sit quietly in the testing environment for 30 minutes before the test begins.

[0169] ③ Sample Usage Method: The test samples were the product of Example 9 and the product of Comparative Example 19 (placebo). The matrix of Comparative Example 19 was the same as that of Example 9, except that Comparative Example 19 did not contain the composition of extracts from topical skin preparations and glyceryl glucoside. Self-administered half-face control: Approximately 1g of sample was used twice daily, once in the morning and once in the evening after cleansing, for 28 consecutive days.

[0170] ④ Test items:

[0171] a. Experimental Methods: Self-controlled half-face observation; products from Example 9 and Comparative Example 19 were randomly distributed. Transdermal water loss (TEWL), epidermal moisture content, and facial redness were measured on days 0 and 28 after continuous use of products from Example 9 or Comparative Example 19 using a skin moisture loss meter (Tewameter, TM300, Courage and Khazaka, Germany), a corneometer (Corneometer, CM825, Courage and Khazaka, Germany), and a facial imaging system (VISIA-CR, Canfield Imaging Systems, Fairfield, NY, USA). The facial redness was analyzed using Image Pro Plus image analysis software.

[0172] b. Experimental Results: The test results are shown in Table 12. After 28 days of use of the test samples, the transdermal water loss (TEWL) in the Example 9 product group significantly decreased by 14.40%, the epidermal moisture content significantly increased by 23.80%, and the skin redness a* value significantly decreased by 12.22%, while the improvement in the Comparative Example 19 product group was not significant. This indicates that the product containing a composition of topical skin extracts and glyceryl glucoside can improve skin redness and dryness caused by endogenous and exogenous factors, enhance skin barrier function, and thus improve skin condition.

[0173] Table 12. Results of skin physiological parameters before and after sample use

[0174]

[0175]

[0176] Note: Compared with Comparative Example 19, P < 0.05 is considered to be statistically significant.

[0177] The above results demonstrate that the topical skin extract prepared in this invention can effectively resist oxidative free radicals, reduce skin inflammation, and promote the expression of barrier-related proteins. The efficacy and stability of the topical skin extract obtained through process optimization have been significantly improved, making it more suitable for application. Furthermore, the combination of this topical skin extract and glycerol glucoside exhibits a synergistic effect in reducing skin inflammation and promoting the expression of barrier-related proteins and β-endorphin, thereby simultaneously addressing the effects of endogenous and exogenous factors on the skin and improving skin condition.

Claims

1. A method for preparing an extract from a topical skin treatment, characterized in that, Includes the following steps: S1. Pulverize the medicinal materials of the topical skin formula separately, pass them through a 10-50 mesh sieve, take the sieve-undersized portion, mix the medicinal materials in a certain proportion and mix them with the extraction solvent in a certain material-liquid ratio, and extract by gentle boiling and hot reflux to obtain crude extract; The medicinal materials in the topical skin formula include Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis, and borneol. The mass ratio of Hibiscus mutabilis leaves, rhubarb, Lycopus lucidus leaves, Phellodendron chinense, Coptis chinensis, Scutellaria baicalensis, and borneol when mixed is 80:80:1:100:80:80:

2. The extraction solvent is a 30%~35% aqueous solution of 1,3-butanediol, which is a mass percentage; The material-to-liquid ratio is 1:(28~32); The temperature for the micro-boiling hot reflux extraction is 90~100℃, and the time is 2~4h; S2. Filter the crude extract obtained in step S1 through a filter cloth to remove the residue. The filter cloth is a double-layered 200-400 mesh filter cloth. Place it at room temperature and filter it through a filter plate. After sterilization, let the filtrate stand at room temperature or low temperature for 24-48 hours to obtain a secondary filtrate. S3. Filter the secondary filtrate obtained in step S2 through a filter plate, add preservatives and stabilizers, wherein the stabilizer is polysorbate-20, and stir at 45℃~55℃ until completely dissolved to obtain the skin topical formula extract.

2. The preparation method according to claim 1, characterized in that, One or more of the following conditions must be met: i. Pass through a 10-mesh sieve in step S1; ii. The extraction solvent in step S1 is a 30% aqueous solution of 1,3-butanediol, by mass percentage; iii. The material-to-liquid ratio in step S1 is 1:30; iv. The temperature for the micro-boiling reflux extraction in step S1 is 100℃ for 3.5 h. v. The filter cloth mentioned in step S2 is a double-layer 300-mesh filter cloth; vi. The filter plate mentioned in steps S2 and S3 is a 0.22μm or 0.45μm filter plate; vii. The sterilization described in step S2 is sterilization at 121°C for 20 minutes; viii. In step S2, let it stand at room temperature for 36 hours; ix. The low temperature mentioned in step S2 is 2~6℃; x. The preservative mentioned in step S3 is one or a combination of 1,2-hexanediol, p-hydroxyacetophenone, and 1,2-pentanediol; xi. In step S3, the final concentration of polysorbate-20 in the topical skin extract is 1~5 wt%.

3. The preparation method according to claim 2, characterized in that, The preservative mentioned in step S3 is a combination of 1,2-hexanediol, p-hydroxyacetophenone and 1,2-pentanediol.

4. The preparation method according to claim 2, characterized in that, In step S3, the final concentration of p-hydroxyacetophenone in the topical skin extract is 0.3~0.6wt%, the final concentration of 1,2-hexanediol is 0.5~2wt%, and the final concentration of 1,2-pentanediol is 0.5~2wt%.

5. A topical skin extract prepared according to the method of claim 1.

6. A composition for improving skin condition, characterized in that, It contains the topical skin extract as described in claim 5 and glyceryl glucoside.

7. The composition according to claim 6, characterized in that, In the system containing the composition, the final concentration of the topical skin extract is 0.5-6.0 wt%, and the final concentration of the glycerol glucoside is 0.1-2.0 wt%.

8. The composition according to claim 6, characterized in that, In the system containing the composition, the final concentration of the topical skin extract is 1.0 to 4.0 wt%, and the final concentration of the glycerol glucoside is 1.0 to 2.0 wt%.

9. The use of the composition according to claim 6 in the preparation of cosmetics or skin care products.

10. The application as described in claim 9, characterized in that, The cosmetics or skincare products are in the form of liquids, gels, ointments, creams, lotions, masks, or freeze-dried powders.

11. A cosmetic or skin care product comprising the composition of claim 6.

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