Kava pepper root extract with soothing, repairing and anti-wrinkle effects as well as preparation method and application of Kava pepper root extract

Abstract

The invention relates to a Kava pepper root extract with soothing, repairing and anti-wrinkle effects as well as a preparation method and application thereof, and belongs to the technical field of biological medicines and cosmetics. According to the method, hydroxypropyl-cyclodextrin and / or sulfobutyl-cyclodextrin are / is used as an extracting agent to fully extract the main functional component kavain in the kava pepper root powder, and a fat-soluble component kavain is clathrated by using the hydrophilicity and clathration effect of hydroxypropyl-cyclodextrin and sulfobutyl-cyclodextrin, so that the extraction efficiency of kavain is improved, and the yield of kavain is increased. The extract which contains the kavain and is soluble in water is obtained. The mixture of polyaluminum chloride and diatomite is used as an impurity removal agent to fully remove unstable components and pigments in the crude extract, and the prepared kavain extract has high purity and extraction rate, and has good solubility in water and good stability. The kava pepper root extract and the centella asiatica extract are combined to achieve a synergistic repairing effect, and the composition can be widely applied to the industries of biological medicine, cosmetics and the like.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and cosmetics, and in particular to a kava pepper root extract with soothing, repairing and anti-wrinkle effects, its preparation method and application. Background Technology

[0002] Kava pepper (Piper methysticum Forst) is a perennial erect shrub medicinal plant belonging to the Piperaceae family. It is produced in the islands of the South Pacific. It is mainly rich in fat-soluble components such as capsaicin, methoxycapsaicin, demethoxycapsaicin, dihydrocapsaicin, dihydrocapsaicin, and capsaicin. Among them, capsaicin accounts for about 1-3% of the dried root raw material of the plant, while the others are small or trace components.

[0003] Capsaicin is the main active ingredient in kava. Currently, the existing capsaicin extraction process in China, such as the process steps disclosed in Chinese invention patent application number 200810057147.9, entitled "Kava Extract and its Preparation Method," are as follows: 1) Raw material harvesting and sorting; 2) Washing; 3) Drying; 4) Pulverizing; 5) Primary alcohol extraction; 6) Filtration; 7) Concentration; 8) Drying and pulverizing; 9) Secondary alcohol extraction of residue; 10) Secondary extract concentration under reduced pressure; 11) Mixing and pulverizing to obtain kava extract. The kava extract prepared by this process is alcohol-soluble but has poor water solubility. For example, the process steps disclosed in Chinese invention patent application number 201410446384.X, entitled "A Method for Extracting Capsaicin from Kava Pepper," are as follows: (1) Crush dried Kava pepper and extract it with ethanol; (2) Filter and concentrate the extract, load it onto a macroporous resin column, elute the resin column with water → ethanol, and collect the ethanol eluent; (3) Concentrate the eluent and allow it to crystallize; (4) Recrystallize. The capsaicin prepared by this process is insoluble in water, which greatly limits its application in the biopharmaceutical, cosmetic, and other industries.

[0004] To overcome the limitations of water-insoluble kava extract in the biopharmaceutical and cosmetic industries, it is essential to explore new methods for extracting water-soluble kava extract and preparing it. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a water-soluble kava root extract with soothing, repairing, and anti-wrinkle effects, as well as its preparation method and applications. This invention solves the problems of capsaicin in kava being insoluble in water and having poor stability in aqueous solutions, due to the deficiencies of existing technologies. It provides a novel method for preparing a kava root extract that yields a highly water-soluble extract with a high capsaicin extraction rate (up to 99%). The main active ingredient in the kava root extract of this invention is capsaicin, which also has soothing effects. Simultaneously, the kava root extract of this invention also has repairing and anti-wrinkle effects; it can be combined with plant extracts for use in the preparation of soothing, repairing, and anti-wrinkle cosmetics or skincare products.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a method for preparing kava pepper root extract with soothing, repairing, and anti-wrinkle effects, comprising the following steps: S1. Extract the powdered kava root using the extraction solution, filter, and obtain crude extract I; The extraction solution comprises an extractant and water, wherein the extractant is hydroxypropyl-cyclodextrin and / or sulfobutyl-cyclodextrin; S2. Mix the crude extract I obtained in step S1 with the impurity removal agent evenly, adjust the pH value to 9-10, and filter until the filtrate is clear and transparent to obtain crude extract II. The impurity remover is a mixture of polyaluminum chloride and diatomaceous earth; S3. Concentrate the crude extract II obtained in step S2 to obtain an extract; mix the extract with ethanol evenly, let the mixture stand, take the supernatant, concentrate, dry, and pulverize it to obtain the powder, which is the Kava pepper root extract.

[0007] This invention utilizes hydroxypropyl-cyclodextrin and / or sulfobutyl-cyclodextrin as extraction agents, and water as the extraction solution, to fully extract capsaicin, the main active ingredient in kava root powder. By leveraging the hydrophilicity and inclusion properties of hydroxypropyl-cyclodextrin and sulfobutyl-cyclodextrin, the fat-soluble component capsaicin is encapsulated, improving the extraction efficiency and yielding a water-soluble extract containing capsaicin. Compared to ethanol as the solvent and other cyclodextrins as extraction agents, using an aqueous solution of hydroxypropyl-cyclodextrin and / or sulfobutyl-cyclodextrin as the extraction solution results in a higher extraction efficiency for capsaicin, exceeding 70% and reaching approximately 99%. Furthermore, analysis reveals that it is also rich in kava lactone active ingredients such as methoxycapsaicin. The water solubility of the kava root extract is also significantly higher, exceeding 1 g / mL and reaching 1.42 g / mL.

[0008] This invention utilizes a mixture of polyaluminum chloride and diatomaceous earth as a purifying agent to effectively remove unstable components and pigments from the crude extract, thereby improving the purity and stability of the prepared water-soluble kava root extract. Compared to conventional purifying agents such as chitosan and activated clay, this invention, using a mixture of polyaluminum chloride and diatomaceous earth, yields a kava root extract with higher purity and extraction rate of capsaicin, and also exhibits higher solubility in water. By adjusting the pH to 9-10, this invention completely removes polyaluminum chloride and diatomaceous earth, while simultaneously increasing the solubility of active ingredients in kava in water, preventing the active ingredients from being filtered out.

[0009] In a preferred embodiment of the preparation method of the present invention, in step S1, the mass-to-volume ratio of the kava pepper root powder to the extraction solution is 1 g:(8-23) mL.

[0010] In a preferred embodiment of the preparation method of the present invention, in step S1, the mass-to-volume ratio of the kava pepper root powder to the extraction solution is 1g:(10-20)mL.

[0011] The present invention found that when kava root powder and extraction solution are extracted at a mass-to-volume ratio of 1g:(10-20)mL, the extraction efficiency of capsaicin is higher, and the solubility of kava root extract is also higher.

[0012] In a preferred embodiment of the preparation method of the present invention, in step S1, the mass-to-volume ratio of the kava pepper root powder to the extraction solution is 1g:15mL.

[0013] In a preferred embodiment of the preparation method of the present invention, in step S1, the extraction is performed by reflux extraction at 70-90°C for 3-4 hours.

[0014] In a preferred embodiment of the preparation method of the present invention, in step S1, the filtration is performed using a 100-200 mesh filter.

[0015] In a preferred embodiment of the preparation method of the present invention, in step S1, the mass percentage of the extractant in the extraction solution is 8% to 25%.

[0016] In a preferred embodiment of the preparation method of the present invention, in step S1, the mass percentage of the extractant in the extraction solution is 10% to 20%.

[0017] The present invention has found that when the extraction solution contains 10% to 20% by mass of the extractant, the extraction efficiency of capsaicin in the extracted kava root powder is higher, and the kava root extract also has higher solubility in water.

[0018] In a preferred embodiment of the preparation method of the present invention, in step S1, the mass percentage of the extractant in the extraction solution is 15%. In a preferred embodiment of the preparation method of the present invention, in step S1, the extractant is sulfobutyl-cyclodextrin.

[0019] In a preferred embodiment of the preparation method described in this invention, the sulfobutyl-cyclodextrin is sulfobutyl-β-cyclodextrin.

[0020] In a preferred embodiment of the preparation method of the present invention, in step S1, the extractant is hydroxypropyl-cyclodextrin and sulfobutyl-cyclodextrin; the mass ratio of hydroxypropyl-cyclodextrin and sulfobutyl-cyclodextrin is (9-7):(1-3).

[0021] In a preferred embodiment of the preparation method of the present invention, the mass ratio of hydroxypropyl-cyclodextrin to sulfobutyl-cyclodextrin is (9-8):(1-2).

[0022] This invention has found that, compared to using sulfobutyl-cyclodextrin alone as an extractant, using hydroxypropyl-cyclodextrin and sulfobutyl-cyclodextrin together as extractants results in better extraction and higher extraction efficiency of capsaicin.

[0023] In a preferred embodiment of the preparation method of the present invention, in step S1, the extractant is hydroxypropyl-cyclodextrin.

[0024] This invention has found that, compared to using hydroxypropyl-cyclodextrin and sulfobutyl-cyclodextrin together as extraction agents, using hydroxypropyl-cyclodextrin alone as the extraction agent yields better results and higher extraction efficiency of capsaicin.

[0025] In a preferred embodiment of the preparation method of the present invention, in step S1, the hydroxypropyl-cyclodextrin is hydroxypropyl-γ-cyclodextrin and / or hydroxypropyl-β-cyclodextrin.

[0026] In a preferred embodiment of the preparation method of the present invention, in step S1, the hydroxypropyl-cyclodextrin is hydroxypropyl-γ-cyclodextrin and hydroxypropyl-β-cyclodextrin; the mass ratio of the hydroxypropyl-γ-cyclodextrin and hydroxypropyl-β-cyclodextrin is (9-8):(1-2).

[0027] In a preferred embodiment of the preparation method of the present invention, in step S1, the hydroxypropyl-cyclodextrin is hydroxypropyl-γ-cyclodextrin.

[0028] This invention has found that, compared to hydroxypropyl-β-cyclodextrin as an extractant, hydroxypropyl-γ-cyclodextrin is a better extractant, resulting in higher extraction efficiency of capsaicin.

[0029] In a preferred embodiment of the preparation method of the present invention, in step S2, the mass ratio of the crude extract I to the impurity removal agent is 1:(0.05-0.08).

[0030] The present invention has found that when the mass ratio of crude extract I to impurity remover is 1:(0.05-0.08), unstable components and pigments in crude extract I can be effectively removed, while also avoiding waste of impurity remover and loss of effective components due to excessive impurity remover.

[0031] In a preferred embodiment of the preparation method of the present invention, in step S2, the mass ratio of the crude extract I to the impurity removal agent is 1:0.06.

[0032] In a preferred embodiment of the preparation method of the present invention, in step S2, the mass ratio of polyaluminum chloride to diatomaceous earth is 1:(1-3). This ratio has the advantage of fully utilizing the impurity removal and flocculation effects of polyaluminum chloride, while the addition of diatomaceous earth effectively improves the clarity of the solution, which is beneficial for filtration. Furthermore, diatomaceous earth can adsorb some pigments, making the product color lighter. The combined use of these two methods results in a final product with excellent water solubility and clarity.

[0033] In a preferred embodiment of the preparation method of the present invention, in step S2, the mass ratio of polyaluminum chloride to diatomaceous earth is 1:3.

[0034] In a preferred embodiment of the preparation method of the present invention, in step S2, the crude extract I is mixed evenly with the impurity removal agent, stirred for 1 hour, and then the pH value is adjusted.

[0035] In step S2 of the preparation method of the present invention, the filtration only needs to achieve a clear and transparent filtrate; if the filtrate cannot achieve a clear and transparent filtrate after a single filtration, two or more filtrations can be performed.

[0036] In a preferred embodiment of the preparation method of the present invention, in step S2, the filtration is performed by vacuum filtration through medium-speed filter paper with a vacuum degree ≤0.085MPa.

[0037] In a preferred embodiment of the preparation method described in this invention, in step S2, the pH of the filtered filtrate is adjusted to 6-7. In the cosmetics or biopharmaceutical industries, weakly acidic or neutral solutions are more widely used and more stable; therefore, it is preferable to adjust the pH of the filtrate to 6-7.

[0038] In a preferred embodiment of the preparation method of the present invention, in step S3, the extract is an extract with a solid content of 50% to 70%.

[0039] In a preferred embodiment of the preparation method of the present invention, in step S3, the extract is an extract with a solid content of 70%.

[0040] In a preferred embodiment of the preparation method of the present invention, in step S3, the ethanol is anhydrous ethanol, and the concentration of ethanol in the mixture is 70wt%-80wt%.

[0041] In a preferred embodiment of the preparation method of the present invention, in step S3, the concentration of ethanol in the mixture is 75 wt%.

[0042] In a preferred embodiment of the preparation method of the present invention, the standing time in step S3 is 24 hours.

[0043] In a preferred embodiment of the preparation method of the present invention, in step S3, the supernatant is concentrated to a solid content of 50-70%; the concentration temperature is 70-75℃.

[0044] In a preferred embodiment of the preparation method of the present invention, in step S3, the supernatant is concentrated to a solid content of 70%; the concentration temperature is 75°C.

[0045] In a preferred embodiment of the preparation method of the present invention, in step S3, the drying is carried out at 75°C.

[0046] In a preferred embodiment of the preparation method of the present invention, in step S3, the powder is obtained by passing through an 80-100 mesh sieve.

[0047] In a preferred embodiment of the preparation method described in this invention, the kava root powder is extracted using an extraction solution. The kava root powder is mixed with water at room temperature and stirred for 4-5 hours, then filtered to remove the filtrate. The residue is then further extracted using the extraction solution. The mass-to-volume ratio of the kava root powder to water is 1 g:(3-5) mL. The water is pure water. The capsaicin component in kava is insoluble in room temperature water. Extracting the kava root powder raw material with pure water can effectively remove some water-soluble impurities and pigments from the raw material.

[0048] As a preferred embodiment of the preparation method of the present invention, the preparation method further includes: drying clean kava pepper roots at 30-40°C and then pulverizing them through a 120-200 mesh sieve to obtain kava pepper root powder.

[0049] Secondly, the present invention provides a kava pepper root extract with soothing, repairing and anti-wrinkle effects prepared by the preparation method described above.

[0050] The applicant's previous research and development focused on a kava root extract whose main active ingredient is capsaicin, which has soothing and calming effects, relieving skin irritation and allergies. The kava root extract of this invention also has capsaicin as its main active ingredient and similarly possesses soothing effects. This kava root extract can improve cell healing rates and promote cell repair; it can also reduce the calcium ion content of neuronal cells, inhibit the expression of myosin and vesicle protein 2 (VAMP2), improve expression lines, and has anti-wrinkle effects.

[0051] Thirdly, the present invention provides the application of the kava pepper root extract, which has soothing, repairing and anti-wrinkle effects, in the preparation of pharmaceuticals and / or cosmetics.

[0052] Fourthly, the present invention provides a plant extract composition comprising kava root extract and centella asiatica extract, wherein the mass ratio of the kava root extract to the centella asiatica extract is 1:0.5 to 5. Preferably, the kava root extract is the aforementioned kava root extract.

[0053] Kava root extract contains lactone compounds such as capsaicin, which have anti-inflammatory and soothing effects. Meanwhile, Centella asiatica extract contains asiaticoside and other components, which have anti-inflammatory, antioxidant, collagen-promoting, and skin-repairing effects. Extensive experimental research has revealed that combining kava root extract and Centella asiatica extract within a specific range can synergistically increase the thickness of epidermal cells, while significantly increasing the content of key skin-repairing proteins (such as filaggrin (FLG), lobelin (LOR), and claudin-1). Calculations using the BLISS formula show that the combined use of kava root extract and Centella asiatica extract has a synergistic effect, achieving a skin-repairing efficacy greater than the sum of its parts (1+1>2).

[0054] In the Centella asiatica extract of the present invention, the content of asiaticoside is not less than 5%, asiaticoside B is not less than 5%, and hydroxyasiaticoside is not less than 80%.

[0055] Fifthly, the present invention provides the use of the plant extract composition in the preparation of pharmaceuticals and / or cosmetics.

[0056] In a sixth aspect, the present invention provides a cosmetic comprising the aforementioned plant extract composition.

[0057] As a preferred embodiment of the cosmetic of the present invention, the plant extract composition accounts for 0.2%-10% by mass in the cosmetic.

[0058] As a preferred embodiment of the cosmetic described in this invention, the cosmetic also includes cosmetically acceptable excipients.

[0059] As a preferred embodiment of the cosmetic of the present invention, the excipients include one or more of 1,3-propanediol, 1,3-butanediol, 1,2-pentanediol, 1,2-hexanediol, glycerol, caprylic / capric triglyceride, or deionized water.

[0060] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes hydroxypropyl-cyclodextrin and / or sulfobutyl-cyclodextrin as extraction agents, and an aqueous solution of the extraction agents as the extraction solution, to fully extract capsaicin, the main active ingredient in kava root powder. By leveraging the hydrophilicity and inclusion properties of hydroxypropyl-cyclodextrin and sulfobutyl-cyclodextrin, the lipid-soluble component capsaicin is encapsulated, improving the extraction efficiency of capsaicin and yielding a water-soluble extract containing capsaicin. This invention uses a mixture of polyaluminum chloride and diatomaceous earth as a purification agent to effectively remove unstable components and pigments from the crude extract, improving the purity and stability of the prepared water-soluble kava root extract. The resulting kava root extract has higher purity and extraction rate of capsaicin, and better solubility in water. The kava root extract prepared using this method exhibits good water solubility, a light color, high capsaicin extraction rate, and good stability, greatly solving the problem of poor water solubility of extracts containing capsaicin. The product has soothing, repairing, and anti-wrinkle effects. Combining the kava root extract and centella asiatica extract of this invention has a synergistic effect, resulting in significant repair benefits. It can be widely applied in the biopharmaceutical and cosmetic industries. Attached Figure Description

[0061] Figure 1 The characteristic spectrum and reference retention time of capsaicin in Test Example 1 of this invention; Figure 2 The images show the morphological characteristics of the kava root extract in Test Example 1 of this invention and the commercially available kava root extract. A represents the kava root extract of this invention, and B represents the commercially available kava root extract. Figure 3 This is a diagram showing the dissolution state of the kava root extract and the commercially available kava root extract in Test Example 1 of this invention; Figure 4 This is a typical image from the scratch test of human immortalized keratinocytes in Test Example 2 of this invention; Figure 5 This is a typical diagram of the zebrafish experiment in Test Example 2 of this invention; Figure 6 This is a graph showing the cell activity of neurons in Test Example 3 of the present invention. Figure 7 This is the fluorescence spectrum of calcium ions in Test Example 3 of the present invention; Figure 8This is an immunofluorescence image of myosin in Test Example 3 of the present invention; Figure 9 This is a cell morphology diagram of keratinocytes in Test Example 4 of the present invention. A represents Comparative Example 7 and B represents Comparative Example 8. Figure 10 The image shows the H&E diagram of the tissue morphology of the 3D epidermal skin model in Test Example 4 of this invention. Figure 11 The fluorescence image of lobe protein (LOR) in test example 4 of this invention; Figure 12 The image shows the fluorescence of tight junction protein-1 (Claudin-1) in test example 4 of this invention. Detailed Implementation

[0062] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0063] Unless otherwise specified, all other materials and reagents used in the examples are commercially available.

[0064] Centella asiatica extract: purchased from Xi'an Lvtian Biotechnology Co., Ltd., with product specifications of asiaticoside ≥5%, asiaticoside B ≥5%, and hydroxyasiaticoside ≥80%.

[0065] VAMP2 ELISA kit was purchased from Jiangxi Lianbo Biotechnology Co., Ltd.; Myosin antibody, FLG antibody, LOR antibody, and Claudin1 antibody were purchased from Abcam; and the calcium ion fluorescent Flu03-AM probe was purchased from Dojin, Japan.

[0066] EpiGrowth culture medium was purchased from Guangdong Boxi Shaanxi Branch.

[0067] Example 1: Preparation of Kava Pepper Root Extract A method for preparing a kava pepper root extract includes the following steps: 1. Pretreatment of Kava Pepper Root Raw Material Rinse the kava pepper roots thoroughly with clean water to remove dirt and other impurities; dry them at 40°C with forced air to remove most of the moisture; then pulverize the dried kava pepper roots using a pulverizer and pass them through a 200-mesh sieve to obtain raw kava pepper root powder.

[0068] 2. Extraction The powdered Kava pepper root was mixed with the extraction solution at a mass-volume ratio of 1 kg: 15 L, and the mixture was heated and refluxed at 75 °C for 4 h. Then, it was filtered through a 100-mesh filter cloth to obtain crude extract I. Hydroxypropyl-γ-cyclodextrin (extractant) is dissolved in water (solvent) and stirred until the solution is clear and transparent to obtain an aqueous solution of hydroxypropyl-γ-cyclodextrin with a mass percentage of 15%, which is the extraction solution.

[0069] 3. Remove impurities Add the impurity remover to crude extract I at a concentration of 6 wt%, and stir thoroughly for 1 hour. Add sodium hydroxide to adjust the pH to 9.0. The impurity remover can remove unstable components and pigments from crude extract I. Then, filter the crude extract through medium-speed filter paper under reduced pressure (vacuum degree ≤ 0.085 MPa). Filter the filtrate repeatedly until the liquid is clear and transparent. Adjust the pH to 6-7 to obtain crude extract II. Mix polyaluminum chloride and diatomaceous earth evenly at a mass ratio of 1:3 to obtain the impurity remover.

[0070] 4. Impurity removal and purification The crude extract II was concentrated under reduced pressure to obtain an extract with a solid content of 70% at a concentration temperature of 75℃ and a vacuum degree of ≤0.08MPa. Then, ethanol was added to the extract until the final concentration of ethanol was 75wt%, stirred evenly, and allowed to stand for 24 hours. The precipitate was removed, and the supernatant was aspirated. The supernatant was concentrated under reduced pressure and repeatedly concentrated with water to remove alcohol, resulting in an extract with a solid content of 60% at a concentration temperature of 75℃ and a vacuum degree of ≤0.085MPa. The extract with a solid content of 60% was dried in a vacuum oven at 75℃ and a vacuum degree of ≤0.085MPa. The dried solid was pulverized and passed through an 80-mesh sieve to obtain 2.17Kg of powder, which is the Kava pepper root extract.

[0071] Example 2: Preparation of Kava Pepper Root Extract 1. Pretreatment of Kava Pepper Root Raw Material Same as step 1 in Example 1.

[0072] 2. Extraction Kava pepper root powder and extraction solution were mixed at a mass-volume ratio of 1 kg: 15 L, heated and refluxed at 70 °C for 4 h, and then filtered through a 100-mesh filter cloth to obtain crude extract I. Hydroxypropyl-γ-cyclodextrin (extractant) is dissolved in water (solvent) and stirred until the solution is clear and transparent to obtain an aqueous solution of hydroxypropyl-γ-cyclodextrin with a mass percentage of 15%, which is the extraction solution.

[0073] 3. Remove impurities Add the impurity remover to crude extract I at a concentration of 5 wt%, and stir thoroughly for 1 hour. Add sodium hydroxide to adjust the pH to 9.0. The impurity remover can remove unstable components and pigments from crude extract I. Then, filter the crude extract through medium-speed filter paper under reduced pressure (vacuum degree ≤ 0.085 MPa). Filter the filtrate repeatedly until the liquid is clear and transparent. Adjust the pH to 6-7 to obtain crude extract II. Mix polyaluminum chloride and diatomaceous earth at a mass ratio of 1:2 to obtain the impurity remover.

[0074] 4. Impurity removal and purification The crude extract II was concentrated under reduced pressure to obtain an extract with a solid content of 70% at a concentration temperature of 75℃ and a vacuum degree ≤0.08MPa. Then, ethanol was added to the extract until the final ethanol concentration was 70wt%, stirred evenly, and allowed to stand for 24 hours to remove the precipitate and aspirate the supernatant. The supernatant was concentrated under reduced pressure and repeatedly concentrated with water to remove alcohol, resulting in an extract with a solid content of 50% at a concentration temperature of 75℃ and a vacuum degree ≤0.085MPa. The extract with a solid content of 50% was dried in a vacuum oven at 75℃ and a vacuum degree ≤0.085MPa. The dried solid was pulverized and passed through an 80-mesh sieve to obtain 2.11 kg of powder, which is the Kava pepper root extract.

[0075] Example 3: Preparation method of Kava pepper root extract 1. Pretreatment of Kava Pepper Root Raw Material Same as step 1 in Example 1.

[0076] 2. Extraction Kava pepper root powder and extraction solution were mixed at a mass-volume ratio of 1 kg: 15 L, heated and refluxed at 90 °C for 3 h, and then filtered through a 200-mesh filter cloth to obtain crude extract I. Hydroxypropyl-γ-cyclodextrin (extractant) is dissolved in water (solvent) and stirred until the solution is clear and transparent to obtain an aqueous solution of hydroxypropyl-γ-cyclodextrin with a mass percentage of 15%, which is the extraction solution.

[0077] 3. Remove impurities Add the impurity remover to crude extract I at a concentration of 8 wt%, and stir thoroughly for 1 hour. Add sodium hydroxide to adjust the pH to 10.0. The impurity remover can remove unstable components and pigments from crude extract I. Then, filter the crude extract through medium-speed filter paper under reduced pressure (vacuum degree ≤ 0.085 MPa). Filter the filtrate repeatedly until the liquid is clear and transparent. Adjust the pH to 6-7 to obtain crude extract II. Mix polyaluminum chloride and diatomaceous earth evenly at a mass ratio of 1:1 to obtain the impurity remover.

[0078] 4. Impurity removal and purification The crude extract II was concentrated under reduced pressure to obtain an extract with a solid content of 70% at a concentration temperature of 75℃ and a vacuum degree of ≤0.08MPa. Then, ethanol was added to the extract until the final concentration of ethanol was 80wt%, stirred evenly, and allowed to stand for 24 hours to remove the precipitate and aspirate the supernatant. The supernatant was concentrated under reduced pressure and repeatedly concentrated with water to remove alcohol, to obtain an extract with a solid content of 70% at a concentration temperature of 75℃ and a vacuum degree of ≤0.085MPa. The extract with a solid content of 70% was placed in a vacuum oven at 75℃ and a vacuum degree of ≤0.085MPa for drying. The dried solid was pulverized and passed through a 100-mesh sieve to obtain 1.92Kg of powder, which is the Kava pepper root extract.

[0079] Example 4: Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the hydroxypropyl-γ-cyclodextrin in the extraction solution in step 2 of Example 1 is replaced with hydroxypropyl-β-cyclodextrin. The 2.20 kg powder obtained by the preparation method in this embodiment is the kava root extract.

[0080] Example 5: Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the hydroxypropyl-γ-cyclodextrin in the extraction solution in step 2 of Example 1 is replaced with sulfobutyl-β-cyclodextrin. The 2.16 kg powder obtained by the preparation method in this embodiment is the kava root extract.

[0081] Example 6: Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the hydroxypropyl-γ-cyclodextrin in the extraction solution in step 2 of Example 1 is replaced with a mixture of hydroxypropyl-γ-cyclodextrin and sulfobutyl-β-cyclodextrin, wherein the mass ratio of hydroxypropyl-γ-cyclodextrin to sulfobutyl-β-cyclodextrin in the mixture is 9:1. Using the preparation method of this embodiment, 2.19 kg of powder was obtained, which is the kava root extract.

[0082] Example 7: Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the hydroxypropyl-γ-cyclodextrin in the extraction solution in step 2 of Example 1 is replaced with a mixture of hydroxypropyl-γ-cyclodextrin and hydroxypropyl-β-cyclodextrin, wherein the mass ratio of hydroxypropyl-γ-cyclodextrin to hydroxypropyl-β-cyclodextrin in the mixture is 9:1. Using the preparation method of this embodiment, 2.21 kg of powder is obtained, which is the kava root extract.

[0083] Example 8: Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the hydroxypropyl-γ-cyclodextrin in the extraction solution in step 2 of Example 1 is replaced with a mixture of hydroxypropyl-γ-cyclodextrin and sulfobutyl-β-cyclodextrin, wherein the mass ratio of hydroxypropyl-γ-cyclodextrin to sulfobutyl-β-cyclodextrin in the mixture is 8:2. Using the preparation method of this embodiment, 2.18 kg of powder is obtained, which is the kava root extract.

[0084] Example 9: Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the extraction solution in step 2 of Example 1 is replaced with an aqueous solution of hydroxypropyl-γ-cyclodextrin at a mass percentage of 20%. Using the preparation method of this embodiment, 2.97 kg of powder was obtained, which is the kava root extract.

[0085] Example 10 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the extraction solution in step 2 of Example 1 is replaced with an aqueous solution of hydroxypropyl-γ-cyclodextrin at a mass percentage of 10%. Using the preparation method of this embodiment, 1.47 kg of powder was obtained, which is the kava root extract.

[0086] Example 11 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the mass-to-volume ratio of kava root powder to extraction solution in step 2 of Example 1 is replaced with 1 kg:10 L. Using the preparation method of this embodiment, 1.42 kg of powder is obtained, which is the kava root extract.

[0087] Example 12 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the mass-to-volume ratio of kava root powder to extraction solution in step 2 of Example 1 is replaced with 1 kg: 20 L. Using the preparation method of this embodiment, 2.88 kg of powder is obtained, which is the kava root extract.

[0088] Example 13 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the hydroxypropyl-γ-cyclodextrin in the extraction solution in step 2 of Example 1 is replaced with a mixture of hydroxypropyl-γ-cyclodextrin and sulfobutyl-β-cyclodextrin, wherein the mass ratio of hydroxypropyl-γ-cyclodextrin to sulfobutyl-β-cyclodextrin in the mixture is 7:3. Using the preparation method of this embodiment, 2.16 kg of powder is obtained, which is the kava root extract.

[0089] Example 14 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the extraction solution in step 2 of Example 1 is replaced with an aqueous solution of hydroxypropyl-γ-cyclodextrin at a mass percentage of 8%. Using the preparation method of this embodiment, 1.14 kg of powder was obtained, which is kava root extract.

[0090] Example 15 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the extraction solution in step 2 of Example 1 is replaced with an aqueous solution of hydroxypropyl-γ-cyclodextrin at a mass percentage of 25%. Using the preparation method of this embodiment, 3.32 kg of powder was obtained, which is the kava root extract.

[0091] Example 16 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the mass-to-volume ratio of kava root powder to extraction solution in step 2 of Example 1 is replaced with 1 kg: 8 L. Using the preparation method of this embodiment, 1.07 kg of powder is obtained, which is the kava root extract.

[0092] Example 17 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that the mass-to-volume ratio of kava root powder to extraction solution in step 2 of Example 1 is replaced with 1 kg: 23 L. Using the preparation method of this embodiment, 3.32 kg of powder is obtained, which is the kava root extract.

[0093] Example 18 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 17, except that the aqueous solution of 15% hydroxypropyl-γ-cyclodextrin in step 2 of Example 17 is replaced with an aqueous solution of 20% hydroxypropyl-β-cyclodextrin. The 4.43 kg powder obtained by the preparation method in this embodiment is the kava root extract.

[0094] Example 19 Preparation of Kava Pepper Root Extract The preparation method of kava root extract in this embodiment is the same as in Example 1, except that before the extraction step (step 2) in Example 1, kava root powder and pure water are first mixed at a mass-to-volume ratio of 1 kg:3 L and stirred at room temperature for 4 hours; after filtration, the filtrate is removed, and the filter residue is mixed with the extraction solution at a mass-to-volume ratio of 1 kg:15 L, as in Example 1, and then the subsequent operations are performed. Using the preparation method of this embodiment, 2.01 kg of powder is obtained, which is the kava root extract.

[0095] Example 20 A plant extract composition comprising kava root extract and centella asiatica extract (Example 18), wherein the mass ratio of kava root extract to centella asiatica extract is 1:1.

[0096] Example 21 A plant extract composition comprising kava root extract and centella asiatica extract (Example 18), wherein the mass ratio of kava root extract to centella asiatica extract is 1:0.5.

[0097] Example 22 A plant extract composition comprising kava root extract and centella asiatica extract (Example 18), wherein the mass ratio of kava root extract to centella asiatica extract is 1:2.

[0098] Example 23 A plant extract composition comprising kava root extract and centella asiatica extract (Example 18), wherein the mass ratio of kava root extract to centella asiatica extract is 1:5.

[0099] Example 24 A plant extract composition comprising kava root extract and centella asiatica extract (Example 18), wherein the mass ratio of kava root extract to centella asiatica extract is 1:3.

[0100] Example 25 A plant extract composition comprising kava root extract and centella asiatica extract as described in Example 1, wherein the mass ratio of kava root extract to centella asiatica extract is 1:1.

[0101] Example 26 A plant extract composition comprising kava root extract and centella asiatica extract (Example 17), wherein the mass ratio of kava root extract to centella asiatica extract is 1:1.

[0102] Comparative Example 1: Preparation of Pepper Kava Root Extract The preparation method of the kava root extract in this comparative example is the same as that in Example 1, except that the aqueous solution of hydroxypropyl-γ-cyclodextrin in step 2 of Example 1 is replaced with an ethanolic solution of hydroxypropyl-γ-cyclodextrin (80% ethanol as solvent). Using this comparative method, 1.79 kg of powder was obtained, which is the kava root extract.

[0103] Comparative Example 2: Preparation of Pepper Root Extract The preparation method of the kava root extract in this comparative example is the same as that in Example 1, except that the hydroxypropyl-γ-cyclodextrin in the extraction solution in step 2 of Example 1 is replaced with γ-cyclodextrin. Using this comparative method, 2.13 kg of powder was obtained, which is the kava root extract.

[0104] Preparation of Kava Pepper Root Extract (Comparative Example 3) The preparation method of the kava root extract in this comparative example is the same as that in Example 1, except that the hydroxypropyl-γ-cyclodextrin in the extraction solution in step 2 of Example 1 is replaced with methylated-β-cyclodextrin. Using this comparative method, 2.16 kg of powder was obtained, which is the kava root extract.

[0105] Preparation of Kava Pepper Root Extract (Comparative Example 4) The preparation method of the kava root extract in this comparative example is the same as that in Example 1, except that the hydroxypropyl-γ-cyclodextrin in the extraction solution in step 2 of Example 1 is replaced with a mixture of hydroxypropyl-γ-cyclodextrin and γ-cyclodextrin, wherein the mass ratio of hydroxypropyl-γ-cyclodextrin to γ-cyclodextrin in the mixture is 9:1. Using this comparative example preparation method, 2.24 kg of powder was obtained, which is the kava root extract.

[0106] Preparation of comparative example 5 kava pepper root extract The preparation method of the kava root extract in this comparative example is the same as that in Example 1, except that the impurity remover in the impurity removal step (step 3) of Example 1 is replaced with a mixture of chitosan and diatomaceous earth at a mass ratio of 1:3. Using this comparative method, 2.28 kg of powder was obtained, which is the kava root extract.

[0107] The kava root extract prepared in this comparative example showed poor clarity and a darker color after dissolving in water, indicating poor solubility, high impurity content, and a significant reduction in capsaicin content. Although chitosan also has flocculation and impurity removal effects, its effect is lower than that of this invention, demonstrating that polyaluminum chloride has a significantly better flocculation and impurity removal effect than chitosan in the preparation of the kava root extract of this invention.

[0108] Preparation of Kava Pepper Root Extract (Comparative Example 6) The preparation method of the kava root extract in this comparative example is the same as that in Example 1, except that the impurity remover in the impurity removal step (step 3) of Example 1 is replaced with a mixture of polyaluminum chloride and activated clay at a mass ratio of 1:3. Using this comparative method, 2.15 kg of powder was obtained, which is the kava root extract. Activated clay has a significant adsorption effect on capsaicin, resulting in a substantial decrease in capsaicin content.

[0109] Comparative Example 7 The only difference between this comparative example and Example 20 is the absence of Centella Asiatica extract.

[0110] Comparative Example 8 The only difference between this comparative example and Example 20 is the absence of kava pepper root extract.

[0111] Comparative Example 9 The only difference between this comparative example and Example 20 is that the kava root extract in Example 20 is replaced by Comparative Example 1 instead of Example 18.

[0112] Comparative Example 10 The only difference between this comparative example and Example 20 is that the kava root extract in Example 20 is replaced by Comparative Example 2 instead of Example 18.

[0113] Comparative Example 11 The only difference between this comparative example and Example 20 is that the kava root extract in Example 20 is replaced by Comparative Example 3 instead of Example 18.

[0114] Comparative Example 12 The only difference between this comparative example and Example 20 is that the kava root extract in Example 20 is replaced by Comparative Example 4 instead of Example 18.

[0115] Comparative Example 13 The only difference between this comparative example and Example 20 is that the kava root extract in Example 20 is replaced by Comparative Example 5 instead of Example 18.

[0116] Comparative Example 14 The only difference between this comparative example and Example 20 is that the kava root extract in Example 20 is replaced by Comparative Example 6 instead of Example 18.

[0117] Test Example 1 1. Determination of capsaicin content The content of capsaicin in the kava root extracts of Examples 1-19 and Comparative Examples 1-6, and in the kava root powder used in Examples 1-19 and Comparative Examples 1-6, was determined by high performance liquid chromatography.

[0118] (1) Chromatographic conditions: The column is packed with octadecylsilane-bonded silica gel (model: Agilent TC-C18 column, 4.6×250mm, 5μm, or a column with equivalent performance). Mobile phase: methanol: acetonitrile: water (21:26:38, V / V); Flow rate: 1.0 mL / min; Column temperature: 35℃; Detector: UV-246nm.

[0119] (2) Curve plotting Accurately weigh capsaicin standard and dissolve it in methanol to prepare a standard stock solution. After serially diluting the standard stock solution, accurately pipette 5 μL into the high-performance liquid chromatograph and determine the peak area according to the chromatographic conditions in (1). Plot a standard curve with the concentration (mg / mL) of capsaicin standard as the abscissa and the peak area as the ordinate.

[0120] (3) Preparation of the sample solution to be tested Accurately weigh 0.2-1g of sample into a 50mL beaker, add about 30mL of methanol, sonicate for 30min, remove and cool to room temperature, add methanol to make up to the mark, shake well, filter, and prepare the sample solution to be tested.

[0121] (4) Sample determination Accurately pipette 5 μL of the sample solution to be tested and inject it into the high-performance liquid chromatograph. Determine the peak area according to the chromatographic conditions in (1). Record the peak area. The characteristic chromatogram and reference retention time of capsaicin are as follows: Figure 1 As shown.

[0122] Based on the standard curve and the mass of the sample, the content of capsaicin in the sample was calculated.

[0123] 2. Determination of the solubility of Kava root extract in water The solubility (C) of the kava root extract prepared in each example and comparative example in water was tested according to GB / T 21845-2008.

[0124] 3. Determination of the extraction rate of capsaicin in pepper root extract Based on the determined content of capsaicin and the quality data of raw materials kava root powder and kava root extract, the extraction rate (P) of capsaicin in kava root extract was calculated using the following formula: Wherein, R1 is the capsaicin content in the kava root extract (g / 100g), M1 is the mass of the kava root extract (kg), R2 is the capsaicin content in the kava root powder (g / 100g), and M2 is the mass of the kava root powder (kg).

[0125] The extraction rate and water solubility of capsaicin in the root extracts of Peppermint prepared in Examples 1-19 and Comparative Examples 1-6 are shown in Tables 1-1 and 1-2 below.

[0126] Table 1-1 Table 1-2 Table 1 shows that the extraction rate of capsaicin in the kava root extract prepared in the examples was 80.59% to 99.22%, and the solubility in water was between 1.06 g / mL and 1.42 g / mL.

[0127] In Example 13, the mass ratio of hydroxypropyl-γ-cyclodextrin to sulfobutyl-β-cyclodextrin in the extraction solution was 7:3. Compared to a mass ratio of (10–8):(0–2), the resulting extract had lower solubility and a lower extraction rate of capsaicin. The solubility of the extract was 1.26 g / mL, and the extraction rate of capsaicin was 91.91%, both significantly lower. Hydroxypropyl-γ-cyclodextrin has a larger cavity structure than sulfobutyl-β-cyclodextrin. A larger cavity structure results in better inclusion of lipid-soluble components. Therefore, the higher the content of hydroxypropyl-γ-cyclodextrin in the extraction solution, the more lipid-soluble active ingredients such as capsaicin are included, and the higher the extraction rate of capsaicin.

[0128] In Examples 14 and 15, the extractant concentration in the extraction solution was 8% or 25% by mass. Compared to 10%–20% by mass, the solubility of the resulting extract reached 1.31–1.34 g / mL, but the extraction rate of capsaicin was only 83.92–87.59%, indicating a significantly lower extraction efficiency. Excessively high extractant concentrations lead to increased impurity extraction rates. The impurity removal process can only remove some impurities, not all, resulting in a decrease in the extraction rate of the active ingredient.

[0129] In Examples 16 and 17, the mass-to-volume ratio of kava root powder to extraction solution was 1:8 or 1:23. Compared to a mass-to-volume ratio of 1:(10-20), the solubility of the extract reached 1.15-1.34 g / mL, but the extraction rate of capsaicin was only 80.59-83.35%, indicating a significantly lower extraction efficiency. Excessive extraction solution and total extractant content leads to an increased impurity extraction rate. The impurity removal process can only remove some impurities, not all, resulting in a lower extraction rate of the active ingredients.

[0130] In Comparative Example 1, when the extraction solution was replaced with an ethanol solution of hydroxypropyl-γ-cyclodextrin (80% ethanol as solvent), although the solubility of the extract could reach 1.37 g / mL, the extraction rate of capsaicin was only 19.04%. The extraction efficiency of capsaicin was significantly lower than that of the aqueous solution of hydroxypropyl-γ-cyclodextrin. Although the ethanol solution could extract capsaicin, most of the capsaicin extracted by ethanol would be removed during subsequent water dissolution operations, resulting in a significant reduction in the extraction rate of capsaicin.

[0131] Comparative Examples 2 and 3, using γ-cyclodextrin and methylated-β-cyclodextrin as extraction agents, showed extract solubility of 0.42–0.63 g / mL, significantly lower than in the examples. Furthermore, the extraction rate of capsaicin was only 34–37%, significantly lower than in the examples and significantly lower than when hydroxypropyl-γ-cyclodextrin was used as the extraction agent. γ-cyclodextrin's poor water solubility and poor inclusion effect on fat-soluble components led to a reduced inclusion amount of fat-soluble active ingredients such as capsaicin, resulting in excessive capsaicin loss and affecting the water solubility of the final product. Methylated-β-cyclodextrin had a smaller cavity and poor inclusion effect on fat-soluble components, which was detrimental to capsaicin extraction; its extraction effect on capsaicin was not as good as that of hydroxypropyl-γ-cyclodextrin.

[0132] In Comparative Example 4, using hydroxypropyl-γ-cyclodextrin to γ-cyclodextrin at a mass ratio of 9:1 as the extraction agent, the solubility of the extract and the extraction rate of capsaicin were significantly lower than those using hydroxypropyl-γ-cyclodextrin to sulfonyl-β-cyclodextrin (or hydroxypropyl-β-cyclodextrin) at a mass ratio of 9:1 (Examples 6 and 7). γ-cyclodextrin has poor water solubility and poor inclusion effect on fat-soluble components, leading to a reduced inclusion amount of fat-soluble active ingredients such as capsaicin. This results in excessive loss of capsaicin and also affects the water solubility of the final product. In contrast, sulfonyl-β-cyclodextrin and hydroxypropyl-β-cyclodextrin have relatively good water solubility and can dissolve the encapsulated capsaicin in water, thereby improving the water solubility of the extract.

[0133] In Comparative Example 5, when the impurity remover was replaced with chitosan and diatomaceous earth, the extraction rate of capsaicin in the extract was only 72.77%, but the solubility of the extract was only 0.51 g / mL, significantly lower than the example using polyaluminum chloride and diatomaceous earth as impurity removers. Although chitosan has a similar impurity removal effect to polyaluminum chloride, it has significant drawbacks, such as poor water solubility and difficulty in subsequent filtration. Furthermore, its small amount of complexing effect on capsaicin reduces the final product extraction rate, resulting in excessively high economic costs for the process.

[0134] In Comparative Example 6, when the impurity remover was replaced with polyaluminum chloride and activated clay, although the solubility of the extract reached 1.37 g / mL, the extraction rate of capsaicin was only 42.08%, significantly lower than the example using polyaluminum chloride and diatomaceous earth as impurity removers. While activated clay has the same filtration aid effect as diatomaceous earth, it has a stronger adsorption capacity for capsaicin, thus reducing the final extraction rate of capsaicin.

[0135] 4. Comparison of properties and solubility This invention also compares the properties and solubility of the kava root extract prepared in Example 1 with that of a commercially available kava root extract. The commercially available kava root extract was prepared using conventional methods: extraction with 70% ethanol aqueous solution, filtration, concentration, and drying to obtain the kava root extract.

[0136] The morphological characteristics of the kava root extract of this invention and commercially available kava root extract are shown in the figures below. Figure 2 As shown, A represents the kava root extract of the present invention, and B represents the commercially available kava root extract; the results show that the kava root extract of the present invention is an off-white powder, while the commercially available kava root extract is a yellow powder.

[0137] The extract was prepared into a 10% (w / w) aqueous solution. The dissolution state diagrams of the kava root extract of this invention and commercially available kava root extract are shown below. Figure 3 As shown, A represents the kava root extract of the present invention, and B represents the commercially available kava root extract. The results show that when the extract is prepared into an aqueous solution with a mass percentage of 10%, the kava root extract of the present invention can be fully dissolved to obtain a clear liquid; while the commercially available kava root extract is not fully dissolved to obtain a turbid suspension. This indicates that the solubility of the kava root extract of the present invention is higher than that of the commercially available kava root extract.

[0138] Test Example 2: Repair Efficacy Test of Kava Pepper Root Extract 1. Cytotoxicity test Using the kava pepper root extract from Example 18 as a sample, the cytotoxicity against human immortalized keratinocytes was tested.

[0139] Experimental groups: Blank control group, no cells were inoculated and no sample solution was added; Normal control group: Cells were inoculated but no sample solution was added; Sample group: Cells were seeded and sample solutions of different concentrations were added, with concentrations (%, m / v, g / mL) of 0.0195%, 0.039%, 0.078%, 0.156%, and 0.312%.

[0140] Immortalized human keratinocytes were seeded into 96-well plates (5 × 10⁶ cells / well). 3 Samples were cultured in 37°C and 5% CO2 for 24 hours. Fresh medium (DMEM containing 10% FBS) containing the corresponding sample concentration was added to the sample groups. The normal control and blank control groups were replaced with fresh medium and incubated at 37°C and 5% CO2 for another 24 hours. After incubation, MTT solution was added to each well, and incubation continued for 4 hours. The medium was removed, DMSO solution was added, and the mixture was shaken to mix. The absorbance (OD) at 490 nm was measured. 490 Cell viability is calculated based on absorbance values ​​using the following formula: Statistical results are expressed as mean ± SD, and statistical analysis was performed using SPSS software. One-way ANOVA was used for statistical analysis among multiple groups. The statistical results were considered statistically significant with α = 0.05 as the test limit; p < 0.05 (compared to the normal control group) indicated a statistically significant difference, and cell viability less than 100% was considered toxic.

[0141] Table 2 shows the cytotoxicity test results of kava root extract against human immortalized keratinocytes. Table 2 shows that the kava pepper root extract of the present invention is non-cytotoxic in the concentration range of 0.039%.

[0142] The present invention also tested the kava root extracts of Examples 1-17 and 19, and Comparative Examples 1-6, which showed no cytotoxicity in the concentration range of 0.039%.

[0143] 2. Cell scratch test Using the kava root extracts of Examples 1-19 and Comparative Examples 1-6 as samples, the repair effect was tested using an in vitro human immortalized keratinocyte scratch assay.

[0144] Experimental groups: Normal control group: cells were inoculated but no sample solution was added; Positive control group: Cells were inoculated and treated with the positive drug epidermal growth factor (EGF); Sample group: Inoculate cells and add sample solution.

[0145] Immortalized human keratinocytes were seeded into 6-well plates (6×10⁶). 5 Cells were incubated at 37°C and 5% CO2 for 24 hours (cells / well). After overnight cell adhesion, a straight "scribing line" was drawn on the cell surface using an appropriate tool, and the drawn cells were removed. Samples were taken and photographed at the 0h time point. After photographing, the normal control group was incubated with 10% serum without the sample, the positive control group was incubated with 10% serum containing 100 ng / mL EGF, and the sample group was incubated with 10% serum containing 0.039% of the sample. All cells were incubated at 37°C and 5% CO2 for 24 hours, and then photographed. Based on the photographed images, the healing rate in the normal control group and the sample group was calculated, and the improvement rate under sample treatment was calculated based on the healing rate. The formulas for calculating the healing rate and the improvement rate are as follows: The statistical results were analyzed with α = 0.05 as the test limit, and p < 0.05 (compared with the normal control group) was considered to be statistically significant. Compared with the normal control group, the healing rate of the sample group was significantly improved, indicating that the sample has a repair effect.

[0146] The results showed that the healing rate of the positive control group was significantly higher than that of the normal control group, indicating that the experimental system was effective and reliable. The kava root extracts in Examples 1-19, at a concentration of 0.039%, showed a significantly higher healing rate compared to the normal control group, with an increase of 54%–78%, indicating that the kava root extracts have a cell repair-promoting effect and possess repair efficacy. The improvement rates corresponding to Comparative Examples 1-6 were significantly lower than those in Example 1, indicating that the kava root extracts in these examples have better repair efficacy.

[0147] Table 3 shows the scratch test results of human immortalized keratinocytes corresponding to Examples 1, 17, 18 and Comparative Examples 1-3. A typical image of the human immortalized keratinocyte scratch test corresponding to Example 18 is shown below. Figure 4 As shown, Table 3 Group Healing rate (%) Improvement rate (%) p-value result normal control group 53.6 / / / Positive control group 93.0 74 <0.001 Significant Example 1 87.1 69 <0.001 Significant Example 17 83.4 58 <0.001 Significant Example 18 82.3 54 <0.001 Significant Comparative Example 1 52.1 32 <0.001 Significant Comparative Example 2 67.4 43 <0.001 Significant Comparative Example 3 65.5 40 <0.001 Significant 3. Zebrafish tail fin regeneration experiment Using the kava root extracts from Examples 1-19 and Comparative Examples 1-6 as samples, the repair efficacy of the samples was tested by quantifying the regenerated caudal fin area of ​​zebrafish. Wild-type AB strain zebrafish were selected 3 days post-fertilization (3 dpf).

[0148] Normal control group: Normal zebrafish were not given any medication; Model control group: A zebrafish caudal fin injury model was established by surgically removing the caudal fin of zebrafish; Sample group: A zebrafish caudal fin injury model was established by surgically removing the caudal fin of zebrafish and then administering the drug.

[0149] Model zebrafish were randomly assigned to 6-well plates, 15 fish per well. The sample groups were given a water-soluble sample (0.025% concentration) at a volume of 3 mL per well; incubation was performed at 28℃ in the dark for 48 h. Ten zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Image processing and analysis were performed, and data were collected. The caudal fin area (A) of the zebrafish was analyzed, and the repair efficacy was calculated and determined using the following formula: p<0.05 was considered to be a significant difference.

[0150] The results showed that the kava root extract of Examples 1-19 significantly increased the caudal fin area of ​​zebrafish at a concentration of 0.025% (mass percentage) compared to the model control group, with a repair efficacy of 33-45%, indicating that the kava root extract of the present invention has a repair efficacy. The improvement rates corresponding to Comparative Examples 1-6 were significantly lower than those of Example 1, indicating that the kava root extract of the examples had better repair efficacy.

[0151] Table 4 shows the zebrafish experimental test results for Examples 1, 17, 18 and Comparative Examples 1-3. A typical zebrafish experimental diagram for Example 18 is shown below. Figure 5 As shown, the area marked by the red dashed line represents the quantitative region. Table 4 Group Repair efficacy (%) p-value result normal control group / / / Model control group / / Example 1 39 <0.001 Significant Example 17 35 <0.001 Significant Example 18 33 <0.001 Significant Comparative Example 1 21 <0.001 Significant Comparative Example 2 27 <0.001 Significant Comparative Example 3 25 <0.001 Significant Test Example 3: Anti-wrinkle efficacy test of kava pepper root extract 1. Cytotoxicity test The cytotoxicity of the kava root extract from Example 18 on neuronal cells was tested. The neuronal cells were provided by Guangdong Boxi Biotechnology Shaanxi Branch.

[0152] Sample solution concentrations (%, m / m): 0.078125%, 0.15625%, 0.3125%, 0.625%, 1.25%, 2.5%, 5%, 10%.

[0153] After resuscitating neurons, when the cell deposition rate reached approximately 60%, cells were seeded into 96-well plates and incubated overnight in a CO2 incubator (37℃, 5% CO2). Drug administration was initiated when the cell deposition rate in the 96-well plates reached 50%–60%. For the solvent control group, 200 μL of DMEM culture medium was added to each well; for the positive control group, 200 μL of DMEM culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of different concentrations of sample solution was added to each well; for the zeroing group, no cells were seeded, only 200 μL of DMEM culture medium was added. After drug administration, the 96-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 h. After incubation, the supernatant was discarded, and MTT working solution (0.5 mg / mL) was added. The plates were incubated at 37℃ in the dark for 4 h. After incubation, the supernatant was discarded, and 150 μL of LDMSO was added to each well. The OD value was read at 490 nm. The relative cell viability was calculated based on the OD value using the following formula: The cytotoxicity test results of kava root extract on neurons are shown in Table 5, and the cell viability curve of neurons is shown in Figure 5. Figure 6 As shown, Table 5 The results showed that kava root extract had no significant toxicity to neurons within the range of 0.625% (m / m).

[0154] The present invention also tested the kava root extracts of Examples 1-17, 19 and Comparative Examples 1-6, which showed no cytotoxicity in the concentration range of 0.625% (m / m).

[0155] 2. Anti-wrinkle efficacy test (1) Calcium ions Using the root extracts of Peppermint from Examples 1-19 and Comparative Examples 1-6 as samples, the effect of the samples on the calcium ion content of neuronal cells was tested using fluorescent probes.

[0156] Blank control group (BC): Cells were inoculated without adding any sample; Sample group: Cells were seeded and sample was added at a concentration of 0.625% (m / m).

[0157] After resuscitating neurons, when the cell deposition rate reached approximately 60%, cells were seeded into 24-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2). The culture medium was discarded, and the cells were washed three times with PBS. 0.5 mL of probe loading working solution was added to each well, and the plates were incubated at 37°C for 45 min. After incubation, the probe working solution was discarded, and the cells were washed three times with PBS. According to the test groups, when the cell deposition rate in the 24-well plates reached 40%–60%, drug administration was performed, with three replicates per group. 1 mL of DMEM culture medium was added to each well in the blank control group, and 1 mL of DMEM culture medium containing 0.625% of the sample was added to each well in the sample group. After drug administration, the 24-well plates were incubated in a CO2 incubator (37°C, 5% CO2) for 24 h. After incubation, observation and photography were performed using a fluorescence microscope. The photography time was controlled to be within 30 min. The average relative integrated optical density (IOD) was calculated, and the inhibition rate was calculated based on the IOD. The calculation formula is as follows: The results showed that, compared with group BC, the kava root extracts of Examples 1-19 inhibited calcium ions in neuronal cells by 30-45%, effectively reducing calcium ion content. The inhibition rates of Comparative Examples 1-6 were significantly lower than those of Example 1, indicating that the kava root extracts of these examples had a higher inhibitory effect on calcium ions in neuronal cells.

[0158] The effects of kava root extracts from Examples 1, 17, 18, and Comparative Examples 1-3 on calcium ion content in neuronal cells are shown in Table 6. The calcium ion fluorescence spectrum corresponding to Example 18 is shown in Table 6. Figure 7 As shown, Table 6 Group Average relative integrated optical density (IOD) Inhibition rate (%) BC 1 Example 1 0.58 42 Example 17 0.65 35 Example 18 0.7 30 Comparative Example 1 0.81 19 Comparative Example 2 0.76 24 Comparative Example 3 0.79 21 (2) Myosin content Using the root extracts of Peppermint from Examples 1-19 and Comparative Examples 1-6 as samples, the effect of the samples on the Myosin content of neuronal cells was tested using immunofluorescence.

[0159] Blank control group (BC): Cells were inoculated without adding any sample; Sample group: Cells were seeded and sample was added at a concentration of 0.625% (m / m).

[0160] After resuscitating neurons, when the cell deposition rate reached approximately 60%, cells were seeded into 24-well plates and incubated overnight in a CO2 incubator (37℃, 5% CO2). According to the test groups, when the cell deposition rate in the 24-well plates reached 40%–60%, drug administration was performed in groups, with three replicates per group. 1 mL of DMEM culture medium was added to each well in the blank control group, and 1 mL of DMEM culture medium containing 0.625% of the sample was added to each well in the sample group. After drug administration, the 24-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours. After incubation, the cells were fixed with 4% paraformaldehyde for 30 minutes, followed by immunofluorescence detection, microscopic observation, and image acquisition and analysis. The average relative integrated optical density (IOD) / cell number was calculated, and the inhibition rate was calculated based on IOD / cell number. The calculation formula is as follows: The results showed that, compared with group BC, the kava root extracts of Examples 1-19 inhibited neuronal myosin levels by 53-72%. The inhibition rates of Comparative Examples 1-6 were significantly lower than those of Example 1, indicating that the kava root extracts of these examples had a higher inhibitory effect on neuronal myosin.

[0161] The effects of Kava root extracts from Examples 1, 17, 18, and Comparative Examples 1-3 on the myosin content of neuronal cells are shown in Table 7. The immunofluorescence chromatogram of myosin corresponding to Example 18 is shown in Table 7. Figure 8 As shown, Table 7 Group Relative integrated optical density (IOD) / mean cell count Inhibition rate (%) BC 1 / Example 1 0.38 62 Example 17 0.45 55 Example 18 0.47 53 Comparative Example 1 0.72 28 Comparative Example 2 0.59 41 Comparative Example 3 0.63 37 (3) Highlight the content of microvesicle membrane protein (VAMP2) Using the root extracts of Peppermint from Examples 1-19 and Comparative Examples 1-6 as samples, the effect of the samples on the VAMP2 content of neuronal cells was tested by ELISA.

[0162] Blank control group (BC): Cells were inoculated without adding any sample; Sample group: Cells were seeded and sample was added at a concentration of 0.625% (m / m).

[0163] After resuscitating neurons, when the cell deposition rate reached approximately 60%, cells were seeded into 6-well plates and incubated overnight in a CO2 incubator (37°C, 5% CO2). According to the test groups, when the cell deposition rate in the 6-well plates reached 40%–60%, drug administration was performed, with 3 replicates per group. 2 mL of DMEM culture medium was added to each well in the blank control group, and 2 mL of DMEM culture medium containing 0.625% of the sample was added to each well in the sample group. After drug administration, the 6-well plates were incubated in a CO2 incubator (37°C, 5% CO2) for 24 hours. After incubation, the cell culture supernatant was collected and analyzed according to the ELISA kit instructions. The average concentration was calculated, and the inhibition rate was calculated based on the average concentration using the following formula: The results showed that, compared with group BC, the kava root extracts of Examples 1-19 inhibited neuronal synaptic vesicle protein (VAMP2) by 19.57-35.72%, effectively reducing VAMP2 levels. The inhibition rates of Comparative Examples 1-6 were significantly lower than those of Example 1, indicating that the kava root extracts of these examples had a higher inhibitory effect on VAMP2 in neuronal cells.

[0164] The effects of kava root extracts from Examples 1, 17, 18 and Comparative Examples 1-3 on the content of vesicle membrane protein 2 (VAMP2) in neuronal cells are shown in Table 8. Table 8 Group Average concentration (ng / mL) Inhibition rate (%) BC 145.96 / Example 1 103.35 29.19 Example 17 110.2 24.50 Example 18 117.4 19.57 Comparative Example 1 129.85 11.04 Comparative Example 2 121.35 16.86 Comparative Example 3 127.37 12.74 In summary, kava root extract can reduce the calcium ion content of neuronal cells, inhibit the expression of myosin and vesicle membrane protein 2 (VAMP2), improve expression lines, and has anti-wrinkle effects. The kava root extract in the example has a better anti-wrinkle effect.

[0165] Test Example 4: Repair Efficacy Test of Plant Extract Combinations 1. Cytotoxicity test Using the root extract of Peppermint from Example 18 (i.e., Comparative Example 7) and Centella Asiatica extract (i.e., Comparative Example 8) as samples, the cytotoxicity of the plant extracts on keratinocytes (batch number: Ep24080801, provided by Guangdong Boxi Shaanxi Branch) was tested.

[0166] The concentrations (%, m / v, g / mL) of the sample solutions in Comparative Examples 7 and 8 were: 0.007813%, 0.015625%, 0.03125%, 0.0625%, 0.125%, 0.25%, 0.5%, and 1%, respectively.

[0167] Keratinocytes were revived and cultured using KcGrowth medium (Guangdong Boxi Shaanxi Branch). The cytotoxicity test method was the same as that in Test Example 3.

[0168] The results of the cytotoxicity test of plant extracts on keratinocytes are shown in Table 9, and the cell morphology diagram of keratinocytes is shown in the figure below. Figure 9 As shown, A represents Comparative Example 7, and B represents Comparative Example 8. Table 9 The results showed that kava root extract and centella asiatica extract had no significant toxicity to keratinocytes within the range of 0.0625% (m / v).

[0169] The present invention also tested the kava root extracts of Examples 1-17, 19 and Comparative Examples 1-6, which showed no significant toxicity to keratinocytes in the range of 0.0625% (m / v).

[0170] 2. Synergistic effect test Using plant extracts from Example 20 and Comparative Examples 7 and 8 as test samples, the repair efficacy of the test samples was evaluated by testing the changes in tissue morphology, lobe rhinol (LOR), and claudin-1 content of a 3D epidermal skin model (EpiKutis) (batch number: ES250703, provided by Guangdong Boxi Shaanxi Branch).

[0171] The concentration of the samples to be tested: Example 20, 0.0625% + 0.0625% (m / v); Comparative Example 7, 0.0625% (m / v); Comparative Example 8, 0.0625% (m / v).

[0172] Experimental groups: Blank control group (BC), no sodium lauryl sulfate (SLS) stimulation and no drug administration; Negative control group (NC), sodium lauryl sulfate (SLS) stimulation and no drug administration; Positive control group (PC), sodium lauryl sulfate (SLS) stimulation and administration of the positive drug WY14643 (Sigma); Sample group, sodium lauryl sulfate (SLS) stimulation and administration of the test sample.

[0173] According to the test groups, the 3D epidermal skin model (EpiKutis) was transferred to a 6-well plate (pre-filled with 0.9 mL of LEpiGrowth medium), and the test group number was labeled on the 6-well plate. 25 μL of 0.1% SLS solution was added to the surface of the NC group, PC group, and sample group, and incubated for 30 min. After incubation, the corresponding concentration of working solution was added to the PC group and sample group, and they were incubated in a CO2 incubator (37℃, 5% CO2) for 24 h. After incubation, residual test material was washed with sterile PBS solution, and residual liquid was wiped away with sterile cotton swabs.

[0174] Using the BLISS formula to evaluate synergies, CI = (E A +E B -E A ×E B ) / E AB E A E represents the effect size when A is used alone. B E represents the effect size when B is used alone. AB This represents the actual effect value when A and B are used together. If the CI value is <1, there is a synergistic effect when A and B are used simultaneously.

[0175] (1) Organizational form The model used for testing was fixed with 4% paraformaldehyde for 24 hours. After fixation, H&E staining was performed, and images were taken under a microscope for observation and tissue morphology analysis. The average thickness of the epidermal viable cell layer was calculated, and the lifting rate was determined using the following formula: The effects of plant extract compositions on the tissue morphology of a 3D epidermal skin model are shown in Table 10. The H&E plots of the 3D epidermal skin model tissue morphology are shown below. Figure 10 As shown, Table 10 Group Average thickness (μm) Improvement rate (%) CI value BC 107.11 / / NC 70.15 / / PC 88.50 26.16 / Comparative Example 7 85.97 22.55 / Comparative Example 8 80.79 15.17 / Example 20 101.69 44.96 0.76 The results showed that, compared with the BC group, the epidermal viable cell layer thickness was significantly reduced in the NC group, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the epidermal viable cell layer thickness was significantly increased in the PC group, indicating that the positive control in this test was effective. Compared with the NC group, the epidermal viable cell layer thickness of Comparative Examples 7, 8, and 20 was significantly increased, with increases of 22.55%, 15.17%, and 44.96%, respectively.

[0176] Compared with Example 20, the thickness of the epidermal viable cell layer in Comparative Examples 7 and 8 was significantly reduced. BLISS calculations showed a CI value <1, indicating that the combined use of kava root extract and centella asiatica extract exhibited a synergistic effect in increasing the thickness of the epidermal viable cell layer.

[0177] (2) LOR (Large Olefin Protein), Claudin-1 (Tight Junction Protein-1) The model used for detection was circumcised and fixed with 4% paraformaldehyde for 24 hours. Immunofluorescence was then performed, and images were taken under a microscope to observe and analyze LOR (cell nucleus globulin) and Claudin-1 (tight junction protein-1). The magnification was 200×. Blue fluorescence represents the cell nucleus, and green fluorescence represents FLG / LOR / Claudin-1. Stronger green fluorescence indicates higher FG content.

[0178] The average relative integrated optical density (IOD) is calculated, and the suppression rate is calculated based on the IOD. The calculation formula is as follows: The effects of plant extract compositions on lobelin (LOR) in a 3D epidermal skin model are shown in Table 11. The fluorescence spectra of lobelin (LOR) are shown below. Figure 11 As shown in Table 11 Group Relative IOD average Improvement rate (%) CI value BC 1 / NC 0.39 / / PC 0.80 105.13 / Comparative Example 7 0.48 23.08 / Comparative Example 8 0.50 28.21 / Example 20 0.67 71.79 0.62 The results showed that the LOR content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. The LOR content in the PC group was significantly higher than that in the NC group, indicating that the positive control in this test was effective. Compared with the NC group, the LOR content in Comparative Examples 7, 8, and 20 was significantly higher, with increases of 23.08%, 28.21%, and 71.79%, respectively.

[0179] Compared with Example 20, the LOR content of Comparative Examples 7 and 8 decreased significantly. The BLISS formula calculation results showed that the CI value was <1, indicating that the combined use of kava root extract and centella asiatica extract showed a synergistic effect in increasing the LOR content.

[0180] The effects of the plant extract composition on Claudin-1 in a 3D epidermal skin model are shown in Table 12. The fluorescence spectrum of Claudin-1 is shown in the figure below. Figure 12 As shown, Table 12 Group Relative IOD average Improvement rate (%) CI value BC 1 / / NC 0.42 / PC 0.76 80.95 / Comparative Example 7 0.57 35.71 / Comparative Example 8 0.53 26.19 / Example 20 0.83 97.62 0.54 The results showed that the Claudin-1 content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. The Claudin-1 content in the PC group was significantly higher than that in the NC group, indicating that the positive control in this test was effective. Compared with the NC group, the Claudin-1 content in Comparative Examples 7, 8, and 20 was significantly higher, with increases of 35.71%, 26.19%, and 97.62%, respectively.

[0181] Compared with Example 20, the Claudin-1 content in Comparative Examples 7 and 8 decreased significantly. BLISS calculations showed a CI value <1, indicating that the combined use of kava root extract and centella asiatica extract exhibited a synergistic effect in increasing Claudin-1 content.

[0182] 3. Repair efficacy test Using the plant extract compositions of Examples 20-26 and Comparative Examples 9-14 as test samples, the repair efficacy of the test samples was evaluated by testing changes in the morphology of 3D epidermal skin models, filaggrin (FLG), lobelin (LOR), and claudin-1 content according to the method described in the synergistic effect test above. The filaggrin (FLG) was also measured using immunofluorescence detection.

[0183] The concentration of the sample to be tested is 0.0625% + 0.0625% (%, m / v, g / mL).

[0184] In a 3D epidermal skin model, compared with the NC group, the plant extract compositions of Examples 20-26 increased the thickness of the epidermal viable cell layer by 40.35-55.71%; increased filaggrin (FLG) by 62.19-69.50%; increased locus terpenoid (LOR) by 68.18-75.34%; and increased claudin-1 by 92.18-98.21%. This indicates that the plant extract compositions of the examples can increase the thickness of the epidermal viable cell layer, promote the expression of filaggrin (FLG), locus terpenoid (LOR), and claudin-1, and have a repairing effect.

[0185] The plant extract compositions of Comparative Examples 9-14 showed significantly lower increases in epidermal cell thickness, filaggrin (FLG), lobelin (LOR), and claudin-1 compared to Example 25, indicating that the plant extract compositions of the examples have better repair effects.

[0186] The effects of Examples 20, 25, 26 and Comparative Examples 9-11 on the thickness of the epidermal viable cell layer, filaggrin (FLG), lobelin (LOR), and claudin-1 in the 3D epidermal skin model are shown in Table 13. Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A process for the preparation of Piper methysticum root extract having soothing, repairing, anti-wrinkle efficacy, characterized in that, The method comprises the following steps: S1, extracting the Piper methysticum root powder with an extraction solution, filtering to obtain a crude extract I; The extraction solution comprises an extractant and water, and the extractant is hydroxypropyl-cyclodextrin and / or sulfobutyl-cyclodextrin; S2, uniformly mixing the crude extract I obtained in step S1 with a decontaminating agent, adjusting the pH value to 9-10, filtering until the filtrate is clear and transparent to obtain a crude extract II; The decontaminating agent is a mixture of polyaluminum chloride and diatomite; S3, concentrating the crude extract II obtained in step S2 to obtain a extract residue; uniformly mixing the extract residue with ethanol, standing the mixture, taking the supernatant, concentrating, drying, and crushing to obtain a powder, which is the Piper methysticum root extract.

2. The production method according to claim 1, wherein In step S1, the mass-volume ratio of the Piper methysticum root powder to the extraction solution is 1g:(8-23)mL; And / or, in step S1, the extraction is reflux extraction at 70-90℃ for 3-4h; And / or, in step S1, the mass percentage of the extractant in the extraction solution is 8%-25%; And / or, in step S1, the hydroxypropyl-cyclodextrin is hydroxypropyl-γ-cyclodextrin and / or hydroxypropyl-β-cyclodextrin; and / or, in step S1, the sulfobutyl-cyclodextrin is sulfobutyl-β-cyclodextrin.

3. The production method according to claim 1, wherein In step S2, the mass ratio of the crude extract I to the decontaminating agent is 1:(0.05-0.08); And / or, in step S2, the mass ratio of the polyaluminum chloride to the diatomite is 1:(1-3); And / or, in step S3, the concentration of ethanol in the mixture is 70wt%-80wt%.

4. The production method according to claim 1, wherein Before the Piper methysticum root powder is extracted with the extraction solution, the Piper methysticum root powder is mixed with water at room temperature for 4-5h, filtered, the filtrate is removed, and the residue is continuously extracted with the extraction solution; the mass-volume ratio of the Piper methysticum root powder to water is 1g:(3-5)mL.

5. The Piper methysticum root extract prepared by the method of any one of claims 1-4, which has the effects of soothing, repairing, and anti-wrinkle.

6. The use of the Piper methysticum root extract of claim 5 in the preparation of a pharmaceutical product and / or a cosmetic product.

7. A plant extract composition, characterized by, The plant extract composition comprises the Piper methysticum root extract and the Centella asiatica extract, and the mass ratio of the Piper methysticum root extract to the Centella asiatica extract is 1:0.5-5; preferably, the Piper methysticum root extract is the Piper methysticum root extract of claim 5.

8. The use of the plant extract composition of claim 7 in the preparation of a pharmaceutical product and / or a cosmetic product.

9. A cosmetic product, characterized by, The plant extract composition comprises the plant extract composition of claim 7.

10. The cosmetic product according to claim 9, wherein The mass percentage of the plant extract composition in the cosmetic product is 0.2%-10%.

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