Zanthoxylum bungeanum extract for anti-aging products and preparation method of zanthoxylum bungeanum extract

Abstract

The invention discloses a Chinese prickly ash extract for an anti-aging product and a preparation method of the Chinese prickly ash extract. The invention relates to application of a Chinese prickly ash extract in preparation of anti-aging cosmetics, and particularly provides a Chinese prickly ash extract which hardly contains Chinese prickly ash volatile oil and is rich in specific sanshool components. The extract is obtained through the processes of double-stage supercritical CO2 extraction, resin column purification and the like, the content of amide active substances is high, preferably, the extract contains hydroxyl-alpha-sanshool and hydroxyl-beta-sanshool, and the extract hardly contains hydroxyl-gamma-sanshool and hydroxyl-delta-sanshool. Cosmetics prepared from the extract have good skin compatibility, have no irritant reaction after being used for a long time, and are suitable for skin care purposes of removing wrinkles and increasing collagen content. The invention provides a natural, safe and efficient skin anti-aging active ingredient, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically relating to a Sichuan pepper extract for anti-aging products and its preparation method. Background Technology

[0002] Skin is the largest organ in the human body, and its aging not only affects appearance but also easily leads to various skin problems. As people pay increasing attention to health and beauty, safe and effective anti-aging methods are gaining widespread importance. In particular, products based on natural ingredients are more readily accepted by consumers due to their lower toxicity and side effects, making them an important development direction in the anti-aging field.

[0003] Studies have shown that skin aging is mainly influenced by factors such as oxidative stress, gene expression regulation, and collagen loss. Among these, inhibiting the degradation of matrix metalloproteinases (MMPs) and promoting collagen production are closely related to skin aging. Certain small molecules in natural plants can delay the skin aging process by regulating this pathway.

[0004] Sichuan pepper (Zanthoxylum bungeanum) is the pericarp of a plant belonging to the genus Zanthoxylum in the family Rutaceae. The *Chinese Materia Medica* records its effects as: warming the middle jiao (spleen and stomach), dispelling cold, eliminating dampness, relieving pain, killing parasites, treating indigestion, abdominal pain due to cold, toothache, vulvar itching, and sores and scabies. Amide compounds are the most characteristic class of alkaloids in Sichuan pepper, the source of its numbing sensation. More than 50 have been isolated and identified, with sanshool being a representative. Recent studies have found that sanshool has a wide range of biological effects, including promoting gastric motility, providing analgesia and anesthetic effects, expelling parasites, antitumor effects, and inhibiting platelet aggregation. However, which component of sanshool can combat aging while maintaining an acceptable level of stimulation remains unreported.

[0005] Given the growing prominence of balancing efficacy, safety, and cost in anti-aging products, exploring and developing natural anti-aging components derived from Sichuan pepper has significant research and application value. The realization of this technology will provide a more effective natural ingredient basis for the development of anti-aging products and may significantly improve the effectiveness of anti-aging treatments. This invention promises to provide a new and more effective option for those with anti-aging needs, and the Sichuan pepper extract is characterized by lower irritation and fewer side effects. Summary of the Invention

[0006] The purpose of this invention is to provide a new application for Sichuan pepper extract. Experimental screening has demonstrated that the Sichuan pepper extract of this invention achieves a better balance between anti-aging efficacy and safety.

[0007] This invention provides the application of Sichuan pepper extract in the preparation of anti-aging cosmetics, wherein the Sichuan pepper extract contains almost no Sichuan pepper volatile oil, thereby avoiding skin irritation reactions that may be caused by Sichuan pepper volatile oil components.

[0008] Furthermore, the Sichuan pepper extract contains sanshool.

[0009] Further, the sanshool is one or more of hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool, hydroxy-δ-sanshool, hydroxy-ε-sanshool, α-sanshool, β-sanshool, γ-sanshool, δ-sanshool, and ε-sanshool.

[0010] Furthermore, the sanshool is one or more of hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool and hydroxy-δ-sanshool.

[0011] Furthermore, the sanshool is hydroxy-α-sanshool or hydroxy-β-sanshool, and contains almost no hydroxy-γ-sanshool or hydroxy-δ-sanshool, which further reduces adverse reactions such as skin irritation while enhancing anti-aging effects.

[0012] In some specific embodiments, the hydroxy-α-sanshool in the Sichuan pepper extract has a mass percentage of not less than 70%, preferably not less than 75%, and more preferably not less than 85%.

[0013] In some specific embodiments, the hydroxy-β-sanshool in the Sichuan pepper extract is not less than 3% by mass, preferably not less than 8%, and more preferably not less than 10%.

[0014] In some specific embodiments, the hydroxy-γ-sanshool in the Sichuan pepper extract does not exceed 1.0% by mass.

[0015] In some specific embodiments, the hydroxy-δ-sanshool in the Sichuan pepper extract does not exceed 1.0% by mass.

[0016] In another aspect of the invention, the Sichuan pepper extract is used to prepare cosmetics for wrinkle reduction and / or collagen enhancement. Experimental results show that the Sichuan pepper extract can significantly reduce collagen loss and promote regeneration at the skin tissue level, while improving skin elasticity and fine lines. Furthermore, long-term use of the cosmetics does not cause common adverse skin reactions such as erythema, infiltration, edema, papules, vesicles, rashes, redness, burning, stinging, irritation, or dryness, demonstrating good biocompatibility and user experience.

[0017] The Sichuan pepper extract of the present invention can be obtained through the following process:

[0018] First, the dried Sichuan peppercorns are pulverized to 40 mesh and mixed thoroughly. 1000 grams of the Sichuan peppercorn powder is then transferred to the extraction vessel of a supercritical carbon dioxide extraction device, and extraction is performed in two steps as follows:

[0019] The first step, extraction, is used to remove volatile oils: the extraction pressure is 18 MPa, the temperature is 50°C, and the time is 2 hours. The resulting extract is processed in a separation tank (pressure 6 MPa, temperature 50°C) to separate and collect the volatile oils of Sichuan pepper, which is Sichuan pepper extract P1.

[0020] The second step is to extract and enrich the saponin-like components of Sichuan pepper: the extraction pressure is adjusted to 32 MPa and the temperature is 45℃. After 2 hours of extraction, the mixture is transferred to the second separation tank (pressure 7 MPa, temperature 45℃) to obtain 100 grams of mixed Sichuan pepper saponin.

[0021] The resulting mixture of Sichuan peppercorn extract and sanshool is further processed as follows:

[0022] Dissolve 100g of the mixed Sichuan peppercornin from the previous step in 800ml of 80% ethanol. Add 8g of activated carbon at 65℃, stir and decolorize for 1 hour, then filter. Concentrate the filtrate to dryness under vacuum at 60℃. Dissolve the residue in 200mL of 70% ethanol. Load the clear solution onto a 1000mL macroporous adsorption resin LK2MGL column for adsorption. First, wash with 3000mL of 45% ethanol to remove highly polar impurities, then elute with 3000mL of 70% ethanol. Collect only the 1000-2000ml eluent. Concentrate the eluent under vacuum at 60℃ to obtain 12g of purified Sichuan peppercornin, which is Sichuan peppercorn extract P2. It mainly contains hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool, and hydroxy-δ-sanshool.

[0023] Further, 12g of the first-refined xanthocyanin was dissolved in 50mL of 70% ethanol. The clear solution was then loaded onto a 250mL UniPS 40 polymer resin chromatography column for adsorption. First, 1000mL of 45% ethanol was used to remove small amounts of highly polar impurities, followed by 1500mL of 58% ethanol. The eluent rich in xanthocyanin amide was collected. The eluent was then concentrated under vacuum at 60℃ to finally obtain 8g of second-refined xanthocyanin, which is xanthocyanin extract P3. It mainly contains hydroxy-α-xanthocyanin and hydroxy-β-xanthocyanin.

[0024] In summary, this invention provides a cosmetic active ingredient derived from natural plants, with controllable composition, high safety, and significant anti-aging effects, as well as its application. It fills the gaps in the safety and long-lasting effects of existing anti-aging products and has broad market application prospects and promotional value. Attached Figure Description

[0025] Figure 1This is a schematic diagram of the HPLC spectrum of Sichuan pepper extract P2. The elution times range from 15 min to 40 min. The four peaks from left to right are hydroxy-δ-salicornin, hydroxy-α-salicornin, hydroxy-β-salicornin, and hydroxy-γ-salicornin.

[0026] Figure 2 This is a schematic diagram of the HPLC spectrum of Sichuan pepper extract P3. Hydroxy-α-salicornin and hydroxy-β-salicornin have peak times ranging from 15 min to 40 min. The four peaks from left to right are hydroxy-δ-salicornin, hydroxy-α-salicornin, hydroxy-β-salicornin, and hydroxy-γ-salicornin.

[0027] Figure 3 The results show cell viability, indicating the effects of different concentrations of Sichuan pepper extracts P1, P2, and P3 on HSF cell activity.

[0028] Figure 4 This indicates the inhibition rate of the test substances (Sichuan pepper extracts P1, P2, P3) on MMP-1.

[0029] Figure 5 This shows a comparison of the IC50 values ​​of the inhibition rates of the test substances (Sichuan pepper extracts P1, P2, P3) against MMP-1.

[0030] Figure 6 This indicates the effect of the test substances (Sichuan pepper extracts P1, P2, and P3) on the total collagen content. Detailed Implementation

[0031] The present invention will be further described in conjunction with specific embodiments, but this does not limit the scope of the invention.

[0032] Unless otherwise specified, the methods used in the following examples are conventional methods. Unless otherwise specified, all materials and reagents required in the following examples are commercially available.

[0033] Example 1: Preparation of Sichuan pepper extract P1

[0034] The dried Sichuan peppercorns were pulverized to 40 mesh and mixed thoroughly. 1000 grams of the Sichuan peppercorn powder was then transferred to the extraction vessel of a supercritical carbon dioxide extraction device and extracted according to the following process:

[0035] Extraction was performed at a pressure of 18 MPa, a temperature of 50°C, and a time of 2 hours. The resulting extract was then processed in a separation tank (pressure 6 MPa, temperature 50°C) to separate and collect the volatile oil from the Sichuan pepper, which is Sichuan pepper extract P1.

[0036] Example 2: Preparation of Sichuan pepper extract P2

[0037] First, the dried Sichuan peppercorns are pulverized to 40 mesh and mixed thoroughly. 1000 grams of the Sichuan peppercorn powder is then transferred to the extraction vessel of a supercritical carbon dioxide extraction device, and extraction is performed in two steps as follows:

[0038] The first step is extraction to remove volatile oil: extraction pressure 18MPa, temperature 50℃, time 2 hours. The resulting extract is processed in a separation tank (pressure 6MPa, temperature 50℃) to separate and collect the volatile oil of Sichuan pepper.

[0039] The second step is to extract and enrich the saponin-like components of Sichuan pepper: the extraction pressure is adjusted to 32 MPa and the temperature is 45℃. After 2 hours of extraction, the mixture is transferred to the second separation tank (pressure 7 MPa, temperature 45℃) to obtain 100 grams of mixed Sichuan pepper saponin.

[0040] The resulting mixture of Sichuan peppercorn extract and sanshool is further processed as follows:

[0041] Dissolve 100g of the mixed Sichuan peppercornin from the previous step in 800ml of 80% ethanol. Add 8g of activated carbon at 65℃, stir and decolorize for 1 hour, then filter. Concentrate the filtrate to dryness under vacuum at 60℃. Dissolve the residue in 200mL of 70% ethanol. Adsorb the clear solution onto a 1000mL macroporous adsorption resin LK2MGL column. First, wash with 3000mL of 45% ethanol to remove highly polar impurities, then elute with 3000mL of 70% ethanol. Collect only the 1000-2000ml eluent. Concentrate the eluent under vacuum at 60℃ to obtain 12g of purified Sichuan peppercornin, which is Sichuan peppercorn extract P2. It mainly contains hydroxy-α-sanshool, hydroxy-β-sanshool, hydroxy-γ-sanshool, and hydroxy-δ-sanshool. The HPLC spectrum is shown below. Figure 1 As shown. Wherein:

[0042] Hydroxy-α-piperidin content: 74.85%;

[0043] Hydroxy-β-sanshool: 6.28%;

[0044] Hydroxy-γ-sanshool: 16.79%;

[0045] Hydroxy-δ-sanshool: 1.48%.

[0046] Example 3: Preparation of Sichuan pepper extract P3

[0047] First, the dried Sichuan peppercorns are pulverized to 40 mesh and mixed thoroughly. 1000 grams of the Sichuan peppercorn powder is then transferred to the extraction vessel of a supercritical carbon dioxide extraction device, and extraction is performed in two steps as follows:

[0048] The first step is extraction to remove volatile oil: extraction pressure 18MPa, temperature 50℃, time 2 hours. The resulting extract is processed in a separation tank (pressure 6MPa, temperature 50℃) to separate and collect the volatile oil of Sichuan pepper.

[0049] The second step involves extraction and enrichment of the sanshool components from Zanthoxylum bungeanum: the extraction pressure was adjusted to 32 MPa, the temperature to 45°C, and after 2 hours of extraction, the mixture was transferred to a second separation tank (pressure 7 MPa, temperature 45°C), yielding 100 grams of mixed Zanthoxylum bungeanum components. The obtained mixed Zanthoxylum bungeanum components were then further processed as follows:

[0050] Dissolve 100g of the mixed Sichuan peppercornin from the previous step in 800ml of 80% ethanol. Add 8g of activated carbon at 65℃, stir and decolorize for 1 hour, then filter. Concentrate the filtrate to dryness under vacuum at 60℃. Dissolve the residue in 200mL of 70% ethanol. Load the clear solution onto a 1000mL macroporous adsorption resin LK2MGL column for adsorption. First, wash with 3000mL of 45% ethanol to remove highly polar impurities, then elute with 3000mL of 70% ethanol. Collect only the 1000-2000ml eluent. Concentrate the eluent under vacuum at 60℃ to obtain 12g of purified Sichuan peppercornin.

[0051] Further, 12g of the first-refined xanthocyanin was dissolved in 50mL of 70% ethanol. The clear solution was then loaded onto a 250mL UniPS 40 polymer resin column for adsorption. First, 1000mL of 45% ethanol was used to remove small amounts of highly polar impurities, followed by 1500mL of 58% ethanol. The eluent rich in xanthocyanin amide was collected. The eluent was concentrated under vacuum at 60℃ to finally obtain 8g of second-refined xanthocyanin, which is the xanthocyanin extract P3. The HPLC spectrum is shown below. Figure 2 As shown.

[0052] in:

[0053] Hydroxy-α-sanshool content: 87.48%;

[0054] Hydroxy-β-piperidin: 11.90%;

[0055] Hydroxy-γ-sanshool: 0.26%;

[0056] Hydroxy-δ-sanshool: 0.16%.

[0057] Example 4: MMP-1 activity inhibition assay

[0058] 1.1 Experimental Objective and Principle

[0059] MMP-1 is a member of the matrix metalloproteinase (MMP) family, a type of zinc-dependent proteolytic enzyme. MMPs can degrade almost all components of the extracellular matrix (ECM), including collagen and elastin. In the skin, MMP-1 primarily degrades type I and type III collagen secreted by dermal fibroblasts, which are the main structural support components of the skin. When MMP-1 is overexpressed, it specifically degrades extracellular matrix components, disrupting the normal structure of collagen and elastin fibers, leading to signs of aging such as wrinkles and decreased elasticity. This test aims to compare the anti-aging effects of Sichuan pepper extracts P1, P2, and P3. To comprehensively evaluate the anti-aging effects of Sichuan pepper extracts P1, P2, and P3, we will use the results of the matrix metalloproteinase-1 (MMP-1) activity inhibition assay as the primary evaluation criterion.

[0060] 1.2 Instruments and Reagents

[0061] 1.2.1 Instruments

[0062] ELISA reader

[0063] centrifuge

[0064] 1.2.2 Reagents and Consumables

[0065] MMP-1 enzyme

[0066] Human skin fibroblasts (HSF)

[0067] High-glucose DMEM medium

[0068] Fetal bovine serum (FBS)

[0069] Penicillin and streptomycin (0.99 U / mL)

[0070] 25cm 2 Culture flasks

[0071] 96-well plates, 6-well plates, 100mm cell culture dishes

[0072] PBS (phosphate buffered saline)

[0073] Human MMP-1 ELISA Kit

[0074] TMB colorimetric solution

[0075] DMSO

[0076] 1.3 Cytotoxicity test

[0077] The safe dosage concentrations (P1, P2, and P3) of Sichuan pepper extract for human skin fibroblasts (HSF) were evaluated using the CCK-8 assay. The specific steps were as follows: Cells uniformly suspended in the culture medium were incubated at 5.0 × 10⁻⁶... 3 Cells were seeded at a density of 1 / well in 96-well plates and incubated for 24 hours. Then, the cells were treated with different concentrations (0, 10, 20, 40, 80, 100, and 160 μM) of Sichuan pepper extract P1, P2, and P3 for 24 hours. Subsequently, CCK-8 reagent was added, and the optical density (OD) value was measured at a wavelength of 450 nm.

[0078] 1.4 MMP-1 inhibition rate test

[0079] (I) Cell resuscitation and culture:

[0080] Human skin fibroblasts (HSF) were seeded in a 25cm² incubator. 2 Culture flasks were prepared using high-glucose DMEM medium (containing 10% FBS and 1% penicillin / streptomycin) and cultured in a 37°C, 5% CO2 incubator. The medium was changed every other day, and cells were passaged when they reached 80% confluence. Only cells in the logarithmic growth phase (generations 4-10) were used to ensure experimental consistency.

[0081] (II) Cell Seeding and Grouping Treatment:

[0082] HSF at 2×10 5 Inoculate the cells at a density of 1 / 2 well in 96-well plates and incubate for 24 hours until adherence. Discard the culture medium and wash once with PBS. According to the experimental groups (Table 1), add 200 μL of H2O2 (200 μmol / L, prepared with serum-free DMEM) to the model group and the Sichuan pepper extract treatment group, and add an equal volume of serum-free DMEM to the blank group. Incubate at 37°C for 1 hour. Discard the H2O2 and wash once with PBS. Add 10% FBSDMEM containing the corresponding concentration of Sichuan pepper extract to each Sichuan pepper extract treatment group, and add normal culture medium to the model group and the blank group. Continue incubation for 24 hours.

[0083] Table 1 Experimental Groups

[0084]

[0085] (III) ELISA detection of MMP-1:

[0086] Collect the supernatant and centrifuge at 2000–3000 rpm for 20 minutes. Aliquot the supernatant. Serially dilute the MMP-1 standard (0–1000 pg / mL), adding 100 μL of each concentration to a row of 7 wells in an ELISA plate. Include a zero well (containing only 100 μL of sample dilution) and a TMB blank well. Label each sample group and add 100 μL to the corresponding well. Strictly follow the ELISA kit instructions. After the reaction, measure the absorbance at 450 nm using a microplate reader. Plot a standard curve and calculate the MMP-1 concentration by substituting the sample absorbance values ​​into the standard curve equation.

[0087] 1.5 Result Calculation

[0088] The 2-ΔΔCt method was used to process the data. The analysis was repeated three times (n=3), and the results are expressed as mean. One-way ANOVA was used for comparisons among multiple groups, and Tukey's test was used for pairwise comparisons between groups. Compared with the model group, #P<0.05, ##P<0.01, ###P<0.001; compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

[0089] 1.6 Data Analysis

[0090] 2.6.1 Cytotoxicity test

[0091] The effects of different concentrations of Sichuan pepper extracts (P1, P2, and P3) on HSF cell viability were evaluated using the CCK-8 assay. Experimental data are shown below. Figure 3 As shown, when the concentrations of Sichuan pepper extract P1 ≥ 40 μM, Sichuan pepper extract P2 ≥ 80 μM, and Sichuan pepper extract P3 ≥ 160 μM, cell viability was significantly lower than that of the blank control group (P < 0.001). Based on the balance between cell activity and safety, a non-toxic dosage gradient of 0-40 μM was ultimately determined for subsequent studies. Among them, Sichuan pepper extract P3 showed relatively low cytotoxicity among the three test substances.

[0092] 1.6.2 MMP-1 Inhibition Rate Test

[0093] like Figures 4-5The results of this test showed that in cells under oxidative stress stimulated by H2O2, compared with the model group, all the Sichuan pepper extract treatment groups significantly inhibited the expression of MMP-1 (P < 0.001). Comparing the same test concentration, the inhibition rate of Sichuan pepper extract P3 on MMP-1 was much higher than other treatment groups: when the concentration of Sichuan pepper extract P1 was 40 μM, its inhibition rate of MMP-1 was 62.7%; when the concentration of Sichuan pepper extract P2 was 40 μM, its inhibition rate of MMP-1 was 68.4%; and when the concentration of Sichuan pepper extract P3 was 40 μM, its inhibition rate of MMP-1 was as high as 88.3%. The IC50 value of the MMP-1 inhibition rate curve of Sichuan pepper extract P3 was calculated to be 6.727 μM, which is 2.8 times different from that of Sichuan pepper extract P1 and 2.1 times different from that of Sichuan pepper extract P2. Within the tested concentration range, Sichuan pepper extract P3 has better efficacy and advantages. All the Sichuan pepper extract treatment groups were able to effectively inhibit the high expression of MMP-1, a photoaging-related factor in HSF under oxidative stress, and had excellent anti-aging effects. Among them, the anti-aging effect of Sichuan pepper extract P3 was more prominent.

[0094] Example 5: Effect on total collagen content

[0095] 1.1 Experimental Objective and Principle Human fibroblasts (HSF) are the main effector cells of the dermis, producing various extracellular matrix (ECM) components such as collagen, laminin, and elastin. The moisture retained by fibroblasts and their ECM is the material basis for maintaining skin elasticity. Total collagen accounts for 70%–80% of the total protein in the dermis, making it the most important protein component in the dermal layer and a key component in maintaining skin structure and strength. This study used ELISA to detect the effects of Sichuan pepper extracts P1, P2, and P3 on total collagen synthesis in cultured human skin fibroblasts (HSF), comparing the anti-aging effects of Sichuan pepper extracts P1, P2, and P3.

[0096] 1.2 Main Instruments, Reagents and Consumables

[0097] 1.2.1 Instruments

[0098] CO2 incubator

[0099] ELISA reader

[0100] centrifuge

[0101] UVB generator

[0102] 1.2.2 Reagents and Consumables: Human skin fibroblasts (HSF)

[0103] Fetal bovine serum (FBS) in high-glucose DMEM medium

[0104] Penicillin-streptomycin dual antibody (1%)

[0105] Total Collagen ELISA Kit

[0106] PBS buffer

[0107] Cell lysis buffer (RIPA buffer)

[0108] Dimethyl sulfoxide (DMSO)

[0109] 1.3 Test Procedure

[0110] HSF cell resuscitation: Cultured in high-glucose DMEM medium (containing 10% FBS and 1% penicillin antibiotics) at 37°C and 5% CO2. Passaged when cells reached 80%–90% confluence. HSF cells in logarithmic growth phase were cultured at 2 × 10⁶ cells / well. 5 The samples were seeded at a density of 10 μM in 6-well plates. Experimental groups: the control group contained no test substance or irritant; the model control group was treated with only the irritant (UVB irradiation); and the P1, P2, and P3 treatment groups of Sichuan pepper extract were selected with test concentrations of 10, 20, and 40 μM, respectively.

[0111] Except for the blank control group, the other groups were pretreated with the corresponding concentrations of Sichuan pepper extract P1, P2, P3 or positive control drugs for 4 hours. The model control group and the Sichuan pepper extract P1, P2, P3 treatment groups were given a stimulating factor (UVB irradiation) and cultured for another 24 hours.

[0112] After the experiment, discard the culture medium and wash the cells twice with PBS. Add 200 μL of cell lysis buffer (RIPA buffer) to each well and lyse on ice for 30 minutes. Collect the lysis buffer, centrifuge at 12000 rpm for 10 minutes, aliquot the supernatant, and store at -80℃ for later use. Strictly follow the ELISA kit instructions. After the reaction, measure the absorbance (OD value) at 450 nm using a microplate reader, and plot a standard curve based on the OD values ​​of the standards.

[0113] Substitute the OD values ​​of the samples into the standard curve equation to calculate the total collagen content. Compare the total collagen content of each group to evaluate the effects of Sichuan pepper extracts P1, P2, and P3 on collagen synthesis.

[0114] 1.4 Repeatability

[0115] The absolute difference between at least three independent measurements obtained under repeatability conditions shall not exceed 20% of the arithmetic mean.

[0116] 1.5 Data Processing

[0117] All experimental data were analyzed using Student's t-tests, using the mean and standard deviation of three measurements. A p-value less than 0.05 was considered statistically significant. Compared to the control group, #P < 0.05, ##P < 0.01, ###P < 0.001; compared to the model group, *P < 0.05, **P < 0.01, ***P < 0.001.

[0118] 1.6 Results Analysis

[0119] Depend on Figure 6 It was found that each Sichuan pepper extract treatment group significantly promoted the synthesis of total collagen in fibroblasts in a dose-dependent manner. At the same concentration, Sichuan pepper extract P3 was more effective than Sichuan pepper extracts P1 and P2 in promoting the synthesis of total collagen in cells. Combined with the MMP-1 inhibition rate test results, Sichuan pepper extract P3 has outstanding anti-aging effects.

[0120] Example 6: Irritation Test - Human Skin Patch Test

[0121] 1.1 Precautions for the test

[0122] 1.1.1 Ethical compliance requires approval from the institution's ethics committee (approval number must be noted) and signing of an informed consent form.

[0123] 1.1.2 Quality control: Environmental temperature and humidity control (22±2℃, 50±10% RH);

[0124] All scoring is completed by the same operator to reduce bias.

[0125] 1.2 Basic Principles of the Experiment

[0126] 1.2.1 Select qualified volunteers as experimental subjects

[0127] 1.2.2 Subject selection: Volunteers aged 18-60 years who meet the trial requirements should be selected as subjects, with a number of 80.

[0128] (20 cases in each group)

[0129] 1.2.3 Individuals with the following conditions should not be selected as subjects:

[0130] a) Those who have used antihistamines in the past week or immunosuppressants in the past month;

[0131] b) Those who have used any anti-inflammatory drugs on the test site within the past two months;

[0132] c) Subjects with clinically unhealed inflammatory skin diseases;

[0133] d) Patients with insulin-dependent diabetes mellitus;

[0134] e) Patients with asthma or other chronic respiratory diseases who are currently receiving treatment;

[0135] f) Individuals who have received anti-cancer chemotherapy within the past 6 months;

[0136] g) Patients with immunodeficiency or autoimmune diseases;

[0137] h) Women who are breastfeeding or pregnant;

[0138] i) Patients who have undergone bilateral mastectomy or bilateral axillary lymph node dissection;

[0139] j) Those whose judgment of test results is affected by scars, pigmentation, atrophy, port-wine stains or other blemishes at the skin test site; k) Those participating in other clinical studies;

[0140] l) Individuals with highly sensitive constitutions;

[0141] m) Non-volunteer participants or those who cannot complete the prescribed tasks as required by the experiment.

[0142] 1.3 Test Methods and Standards The Sichuan pepper extracts P1, P2, and P3 were diluted to a 1% solution with physiological saline, using physiological saline as a negative control. The skin on both sides of the spine on the upper back (avoiding the scapular region) was disinfected with alcohol swabs, and four 2cm × 2cm test areas were marked. Samples were loaded using a Finn Chamber, with approximately 15 μL of the test substance applied in a random order. The patch containing the test substance was secured to the subject's back with breathable adhesive tape, and gently pressed with the palm of the hand to ensure even application to the skin, ensuring no air bubbles remained, for 24 hours. After 24 hours, the patch was removed, and the skin reaction was observed after a 30-minute interval until the pressure marks disappeared. If the result was negative, observations were repeated at 24 hours and 48 hours after the patch test, and the reaction results were recorded according to the skin reaction grading criteria in Table 2.

[0143] Table 2. Skin Reaction Grading Standards

[0144]

[0145] 1.4 Results Evaluation: Record adverse reactions and assess acceptability. The positive reaction rate is the number of people experiencing adverse reactions in each test group and negative control group divided by 20.

[0146] 1.5 Results Analysis

[0147] Table 3 shows that the positive reaction rate of Sichuan pepper extract P1 was 40%, indicating high irritation; the positive reaction rate of Sichuan pepper extract P2 was 15%, also indicating a risk of adverse reactions; Sichuan pepper extract P3 did not cause any adverse reactions in the subjects. At the same test concentration, Sichuan pepper extract P3 showed higher safety, with almost no irritation to human skin, making it a more reliable and safer product.

[0148] Table 3 Summary of adverse reaction data for each test substance treatment group

[0149]

[0150]

[0151] The above embodiments are merely illustrative of the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments, or substitutions made by those skilled in the art based on the content of this specification are all within the scope of protection claimed by the present invention.

Claims

1. The application of Sichuan pepper extract in the preparation of anti-aging cosmetics, characterized in that, The Sichuan pepper extract contains almost no Sichuan pepper volatile oil.

2. The application according to claim 1, characterized in that, The Sichuan pepper extract contains sanshool.

3. The application according to claim 1 or 2, characterized in that, The Sichuan pepper extract contains hydroxy-α-salicornin, hydroxy-β-salicornin, hydroxy-γ-salicornin and hydroxy-δ-salicornin.

4. The application according to claim 3, characterized in that, The Sichuan pepper extract contains hydroxy-α-salicornin and hydroxy-β-salicornin, but contains almost no hydroxy-γ-salicornin and hydroxy-δ-salicornin.

5. The application according to claim 4, characterized in that, The hydroxy-α-piperidine content in the Sichuan pepper extract is 70-85% by mass.

6. The application according to claim 5, characterized in that, The hydroxy-β-piperidine content in the Sichuan pepper extract is 3-10% by mass.

7. The application according to claim 5 or 6, characterized in that, The hydroxy-γ-piperidine content in the Sichuan pepper extract does not exceed 1% by mass.

8. The application according to claim 5 or 6, characterized in that, The hydroxy-δ-piperidine content in the Sichuan pepper extract does not exceed 1% by mass.

9. The application according to any one of claims 1-8, characterized in that, The cosmetic product is used for anti-aging and can inhibit MMP-1 expression and promote collagen production.

10. According to claim 9, long-term use of the cosmetic will not cause erythema, infiltration, edema, papules, or vesicles.

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