Preparation method of piper nigrum root extract and application of piper nigrum root extract in pain-relieving cosmetics

Abstract

The invention belongs to the technical field of cosmetics, and provides a preparation method of a piper nigrum root extract and application of the piper nigrum root extract in pain relieving cosmetics. The kava pepper root extract is prepared by the following steps: crushing kava pepper roots, and performing alcohol extraction; carrying out secondary extraction by using a small molecular polyol solution containing isopropyl acetate; combining the extracting solutions, and adding compound enzymes (cellulase, pectinase and subtilisin) for enzymolysis to obtain a crude extract; and finally, carrying out subcritical extraction and purification on the crude extract to obtain the product. Through improvement of extraction and purification processes, the extraction rate of active ingredients is effectively increased, a certain amount of glutathione is obtained, and toxicity is reduced; the invention also discovers that the composition ratio of the active ingredients of the extract obtained by the optimized process is in a proper range, and the anti-inflammatory and analgesic effects of the Kava pepper root extract are further improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cosmetics, and provides a preparation method of Piper methysticum Forst root extract and application thereof in pain-relieving cosmetics. BACKGROUND

[0002] Piper methysticum Forst is a perennial erect shrub of Piperaceae, and the main pharmacodynamic components are Kavalactones, methysticin, Kawain and the like, which can bidirectionally regulate neurotransmitters and have multiple effects such as anti-anxiety and depression, sedation and hypnosis, local anesthesia, and anti-convulsion.

[0003] It has been found that the active component content and distribution of the root, stem and leaf of Piper methysticum Forst are obviously different, wherein the root and rhizome of Piper methysticum Forst mainly contain Kavain and Desmethoxyyangonin, which can be used as medicine, and the fresh leaves can also be used as medicine and have effects such as treating anxiety, depression, convulsion, asthma, and some skin diseases such as obesity, have significant curative effects on promoting sleep and improving sleep quality, have effects such as relieving mental stress, relaxing the body and local anesthesia, analgesia, and antifungal, and have small toxic and side effects.

[0004] At present, the extraction process of Piper methysticum Forst root extract mainly includes solvent such as alcohol solvent extraction and subsequent separation and purification treatment. For example, Chinese invention patent CN101239104B discloses a Piper methysticum Forst extract and a preparation method thereof, which comprises the following steps of sequentially collecting and sorting raw materials, washing, drying, crushing, first alcohol extraction, filtering, concentrating, drying and crushing, secondary alcohol extraction of residues, vacuum concentration of secondary extraction liquid, and mixing and crushing, to obtain a Piper methysticum Forst extract with a Kavalactones content of 20-50% and a glutathione content of 0.1-0.8%. Compared with the modern industrial standard extract of kava, the extract contains a certain amount of glutathione, and the toxicity is obviously reduced, but only alcohol extraction is used, without separation and purification treatment, so that the impurities are more and the extraction rate of effective components is low.

[0005] Chinese invention patent CN115006314B discloses a Piper methysticum Forst extract, a preparation method and application thereof. The method first crushes the Piper methysticum Forst raw material, then performs high-temperature and high-pressure treatment, and then performs alcohol extraction and decolorization, separates, and then performs secondary alcohol extraction, and the solvent used is butanediol. The method reduces impurities and achieves certain effects in skin anti-inflammatory, acne removal and analgesia, but needs to be combined with butanediol to form a composition to achieve the best effect.

[0006] In addition, Chinese invention patent application CN118217328A discloses a method for preparing and applying a high-content kava extract for soothing sensitive skin. The method involves extracting kava root powder using an organic solvent under reflux at 60-90°C, filtering, and concentrating the filtrate to obtain an extract; S2, the extract from step S1 is extracted using supercritical CO2 fluid extraction to remove impurities, the remaining extract after impurity removal is melted, centrifuged, the supernatant is collected, and cooled to room temperature to obtain the kava extract. The kava extract obtained using the method of this invention has a higher extraction yield and a higher content of kavalactone, with an extraction rate of over 99.7%.

[0007] Since kavalactones include core active ingredients such as capsaicin, dihydrocapsaicin, methoxycapsaicin, dihydrocapsaicin, and demethoxycapsaicin, the activity of kava pepper extract varies considerably due to their different compositions. Furthermore, there is currently no research on how to regulate the ratio of kavalactone-like active ingredients to glutathione in kava pepper extract to achieve further detoxification. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a method for preparing kava root extract. Using kava root as raw material, a higher extraction rate of kava root extract is obtained through improvements in the extraction and purification process. At the same time, the composition of the core active ingredient kava lactone and the relationship between glutathione in kava root extract are studied in depth, thereby amplifying the activity of kava root extract, which has potential applications in pain relief products such as cosmetics.

[0009] Terminology Explanation: Unless otherwise specified, the term "multiple times" in this article refers to mass. For example, "adding 2-6 times the mass of kava root as the first solvent in a single extraction" means adding 2-6 times the mass of the raw material (kava root) as the first solvent.

[0010] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing a kava root extract, comprising the following steps: (1) Solvent extraction: After crushing the root of Pepper Kava, add the first solvent for a first extraction to separate the residue and extract 1; then add the residue to the second solvent for a second extraction to separate the filter residue 2 and extract 2. The first solvent is an alcohol solution; the second solvent is a C2-C6 small molecule polyol solution and isopropyl acetate; (2) Enzymatic hydrolysis with compound enzyme: Combine extract 1 and extract 2 to obtain a mixed extract, add compound enzyme to the mixed extract for enzymatic hydrolysis, and after inactivation, separation and concentration, obtain crude extract of pepper root; The complex enzyme is cellulase, pectinase, and subtilisin; (3) Subcritical extraction: The crude extract of kava root is added to an organic solvent for subcritical extraction. The extract is concentrated and dried to obtain the kava root extract.

[0011] In some specific embodiments of the present invention, during a single extraction, 2-6 times the mass of the kava pepper root is added as a first solvent, and the extraction is carried out at 55-65°C for 1-3 hours.

[0012] In some preferred embodiments of the present invention, the first extraction is performed by adding 5 times the amount of the first solvent and extracting at 55°C for 1.5 hours.

[0013] In some specific embodiments of the present invention, during the secondary extraction, a second solvent of 1-4 times the mass of the residue is added, and extraction is carried out at 60-80°C for 1-3 hours.

[0014] In some preferred embodiments of the present invention, the secondary extraction is performed by adding three times the amount of a second solvent and extracting at 80°C for 2 hours.

[0015] In some specific embodiments of the present invention, the alcohol solution includes one or more of methanol, ethanol, propanol, 1,3-butanediol, n-butanol, and 1,2-pentanediol solutions; Preferably, the concentration of the alcohol solution is 50-80 vol.%.

[0016] In some specific embodiments of the present invention, the C2-C6 small molecule polyol solution is one or more of ethylene glycol, 1,2-propanediol, glycerol, 1,3-butanediol, n-butanol, 1,2-pentanediol, and 1,6-hexanediol solutions; Preferably, the concentration of the C2-C6 small molecule polyol solution is 50-80 vol.%; and / or the concentration of isopropyl acetate in the 1,3-butanediol solution is 2-6 vol.%.

[0017] In some specific embodiments of the present invention, the mass ratio of cellulase, pectinase and subtilisin in the composite enzyme is 1:0.1-2:0.1-1.

[0018] In some specific embodiments of the present invention, the mass ratio of the compound enzyme to the mixed extract is 0.1-1:100.

[0019] In some specific embodiments of the present invention, the enzymatic hydrolysis of the compound enzyme is carried out at a temperature of 20-30°C and for a time of 1-2 hours.

[0020] In some specific embodiments of the present invention, before the compound enzyme hydrolyzes the solution, the pH of the mixed extract is adjusted to 4.0-8.0.

[0021] In some specific embodiments of the present invention, the organic solvent used in the subcritical extraction is one or more of methanol, butane, propane, isobutane, and ethyl acetate; preferably, the ratio of the crude extract of kava root to the organic solvent is 1:8-10.

[0022] In some specific embodiments of the present invention, the subcritical extraction temperature is 55-65°C and the pressure is 1-3 MPa.

[0023] Secondly, the present invention provides a kava root extract prepared by any of the preparation methods described above.

[0024] Furthermore, the active ingredient composition of the kava pepper root extract includes: capsaicin 32-35%, dihydrocapsaicin 18-20%, methoxycapsaicin 7.5-10%, dihydrocapsaicin 18.5-22%, demethoxycapsaicin 9-10%, and glutathione 0.05-0.2%.

[0025] Thirdly, the present invention provides a composition comprising any of the above-described kava pepper root extracts.

[0026] In some specific embodiments of the present invention, the composition further includes one or more of the following: cat's claw extract, calendula extract, purple daisy extract, lemon balm extract, rosemary extract, centella asiatica extract, licorice extract, scutellaria baicalensis extract, aloe vera extract, clove extract, peppermint extract, bisabolol, and dipotassium glycyrrhizate.

[0027] Fourthly, the present invention provides the use of the above-mentioned kava pepper root extract or the above-mentioned composition in pain-relieving cosmetics.

[0028] Furthermore, the dosage form of the cosmetic includes any one of the following: aqueous solution, emulsion, cream, gel, and spray.

[0029] Fifthly, the present invention also provides an essence water containing the above-mentioned kava root extract.

[0030] Furthermore, the essence water comprises, by weight percentage: 0.1-0.5% kava root extract, 1-10% 1,2-propanediol, 0.01-5% sodium hyaluronate, 0.1-1% ceramide, 0.1-5% vitamin E, and the balance being deionized water.

[0031] Sixthly, the present invention also provides a method for preparing the above-mentioned essence water, comprising the following steps: (1) Preparation of aqueous phase: Sodium hyaluronate is added to part of the water to obtain an aqueous phase; (2) Preparation of oil phase: Mix kava root extract, ceramide and vitamin E evenly to obtain oil phase; (3) Preparation of Kava Pepper Root Extract Dispersion: Kava pepper root extract was added to 1,2-propanediol to obtain Kava pepper root extract dispersion; (4) Initial mixing: Slowly add the oil phase to the aqueous phase and stir to form a crude emulsion; (5) Homogenization: After homogenizing the crude emulsion, add the Kava pepper root extract dispersion and replenish the remaining deionized water, stir evenly, and the mixture is ready.

[0032] Furthermore, in step (2), the mixing temperature is 50-60℃.

[0033] Furthermore, in step (5), the homogenization is high-pressure homogenization, with a pressure of 10,000-30,000 psi and a number of homogenization cycles of 3-5.

[0034] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention uses pepper root as raw material. By adding an extraction aid (isopropyl acetate) during the solvent extraction process, especially during the secondary extraction process, the permeability of the solvent to plant cells is effectively improved, which promotes the dissolution of the active ingredient kavalactone and avoids the dissolution of other impurities. At the same time, in conjunction with the enzymatic hydrolysis process, under the action of the specific compound enzyme of this invention, the cellulose, gum and other substances that encapsulate the active ingredients of the Chinese herbal medicine are rapidly degraded, thereby destroying the dense structure of the cell wall, promoting the full exposure and release of the active ingredients, and through further subcritical extraction, impurities are removed, and the enrichment of kavalactone and glutathione is achieved. While obtaining a high extraction rate, the content of kavalactone is also significantly increased. (2) The present invention also found that the anti-inflammatory and analgesic effects of each kavalactone in the root extract of Pepper Kava at a specific content (or at a specific ratio of styraxol, dihydrostyraxol, methoxystyraxol, dihydrostyraxol and demethoxystyraxol) are further enhanced, and have potential applications in pain relief products such as cosmetics and pharmaceutical preparations. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1 This embodiment provides a kava pepper root extract, prepared by the following method: (1) After crushing the root of Pepper Kava, pass it through an 80-mesh sieve. Take 100g of Pepper Kava root powder and add 5 times the amount of 75 vol.% methanol solution. Heat to 55℃ and extract for 1.5h. After cooling to room temperature, perform solid-liquid separation. Add 3 times the amount of 70 vol.% 1,3-butanediol solution (containing isopropyl acetate, of which the concentration of isopropyl acetate in the 1,3-butanediol solution is 4 vol.%) to the filter residue. Heat to 80℃ and extract for 2h. After cooling to room temperature, perform solid-liquid separation. Combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.3% of a compound enzyme (the mass ratio of cellulase, pectinase and subtilisin Carslberg was 1:1:0.5) and the pH was adjusted to 6.0±0.5. After enzymatic hydrolysis at 25±2℃ for 2 hours, the enzyme was inactivated. The hydrolysate was centrifuged at 3000 rpm for 7 minutes to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of Peppercornia kava root. (3) Add the crude extract of kava root to the extraction tank, and add 10 times the amount of methanol / ethyl acetate (V / V=1:1). Set the temperature of the extraction tank to 60℃ and the pressure to 2MPa, and carry out subcritical extraction. After the solvent is recovered from the extract, it is dried to obtain 4.52g of the kava root extract.

[0037] Example 2 This embodiment provides a kava pepper root extract, prepared by the following method: (1) After crushing the kava root, pass it through an 80-mesh sieve. Take 100g of kava root powder and add 6 times the amount of 55 vol.% ethanol solution. Heat to 55℃ and extract for 3h. After cooling to room temperature, perform solid-liquid separation. Add 4 times the amount of 50 vol.% ethylene glycol solution (containing isopropyl acetate, of which the concentration of isopropyl acetate in 1,3-butanediol solution is 2 vol.%) to the filter residue. Heat to 75℃ and extract for 3h. After cooling to room temperature, perform solid-liquid separation. Combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.1% of a compound enzyme (the mass ratio of cellulase, pectinase and subtilisin Carslberg was 1:0.1:1) and the pH was adjusted to 6.0±0.5. After enzymatic hydrolysis at 25±2℃ for 1.5h, the enzyme was inactivated. The hydrolysate was centrifuged at 3000rpm for 8min to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of Peppercornia kava root. (3) Add the crude extract of kava root to the extraction tank, and add 8 times the amount of ethyl acetate. Set the temperature of the extraction tank to 60°C and the pressure to 2MPa for subcritical extraction. After the solvent is recovered from the extract, dry it to obtain 4.03g of the kava root extract.

[0038] Example 3 This embodiment provides a kava pepper root extract, prepared by the following method: (1) After crushing the kava root, pass it through an 80-mesh sieve. Take 100g of kava root powder and add it to 3 times the amount of 75 vol.% methanol solution. Heat the mixture to 65℃ and extract for 1h. After cooling to room temperature, perform solid-liquid separation. Add 2 times the amount of 70 vol.% 1,3-butanediol solution (containing isopropyl acetate, of which the concentration of isopropyl acetate in the 1,3-butanediol solution is 6 vol.%) to the filter residue. Heat the mixture to 80℃ and extract for 2h. After cooling to room temperature, perform solid-liquid separation. Combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.5% of a compound enzyme (the mass ratio of cellulase, pectinase and subtilisin Carslberg was 1:2:0.1) and the pH was adjusted to 6.0. After enzymatic hydrolysis at 25±2℃ for 1 h, the enzyme was inactivated. The hydrolysate was centrifuged at 3000 rpm for 8 min to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of Peppercornia kava root. (3) Add the crude extract of kava root to the extraction tank, and add 8 times the amount of methanol at the same time. Set the temperature of the extraction tank to 60°C and the pressure to 2MPa for subcritical extraction. After the solvent is recovered from the extract, it is dried to obtain 4.15g of the kava root extract.

[0039] Example 4 This embodiment provides a kava pepper root extract, prepared by the following method: (1) After crushing the root of Pepper Kava, pass it through an 80-mesh sieve. Take 100g of Pepper Kava root powder and add 5 times the amount of 75 vol.% methanol solution. Heat to 55℃ and extract for 1.5h. After cooling to room temperature, perform solid-liquid separation. Add 3 times the amount of 70 vol.% 1,3-butanediol solution (containing isopropyl acetate, of which the concentration of isopropyl acetate in the 1,3-butanediol solution is 4 vol.%) to the filter residue. Heat to 80℃ and extract for 2h. After cooling to room temperature, perform solid-liquid separation. Combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.3% of a compound enzyme (the mass ratio of cellulase, pectinase and subtilisin Carslberg in the compound enzyme was 1:1:2), the pH was adjusted to 6.0±0.5, and the enzyme was inactivated after 2h of enzymatic hydrolysis at 25±2℃. The hydrolysate was centrifuged at 3000rpm for 7min to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of Peppercornia kava root. (3) Add the crude extract of kava root to the extraction tank, and add 10 times the amount of methanol / ethyl acetate (V / V=1:1). Set the temperature of the extraction tank to 60℃ and the pressure to 2MPa, and carry out subcritical extraction. After the solvent is recovered from the extract, it is dried to obtain 4.52g of the kava root extract.

[0040] Comparative Example 1 This comparative example provides a kava root extract, differing from Example 1 only in that the first solvent contains 4 vol.% isopropyl acetate, while the second solvent does not contain isopropyl acetate; the specific preparation method is as follows: (1) After crushing the kava root, pass it through an 80-mesh sieve. Take 100g of kava root powder and add 5 times the amount of 75 vol.% methanol solution (containing isopropyl acetate, wherein the volume concentration of isopropyl acetate in the methanol solution is 4 vol.%). Heat to 55℃ and extract for 1.5h. After cooling to room temperature, perform solid-liquid separation. Add 3 times the amount of 70% 1,3-butanediol solution to the filter residue, heat to 80℃ and extract for 2h. After cooling to room temperature, perform solid-liquid separation and combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.3% of a compound enzyme (the mass ratio of cellulase, pectinase and subtilisin Carslberg was 1:1:0.5) and the pH was adjusted to 6.0±0.5. After enzymatic hydrolysis at 25±2℃ for 2 hours, the enzyme was inactivated. The hydrolysate was centrifuged at 3000 rpm for 7 minutes to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of Peppercornia kava root. (3) Add the crude extract of kava root to the extraction tank, and add 10 times the amount of methanol / ethyl acetate (V / V=1:1). Set the temperature of the extraction tank to 60℃ and the pressure to 2MPa, and carry out subcritical extraction. After the solvent is recovered from the extract, it is dried to obtain 3.98g of the kava root extract.

[0041] Comparative Example 2 This comparative example provides a kava root extract, differing from Example 1 only in that the second solvent is a 75 vol.% methanol solution; the specific preparation method is as follows: (1) After crushing the kava root, pass it through an 80-mesh sieve. Take 100g of kava root powder and add 5 times the amount of 75 vol.% methanol solution. Heat to 55℃ and extract for 1.5h. After cooling to room temperature, perform solid-liquid separation. Add 3 times the amount of 75 vol.% methanol solution (containing isopropyl acetate, of which the concentration of isopropyl acetate in the methanol solution is 4 vol.%) to the filter residue. Heat to 80℃ and extract for 2h. After cooling to room temperature, perform solid-liquid separation. Combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.3% of a compound enzyme (the mass ratio of cellulase, pectinase and subtilisin Carslberg was 1:1:0.5) and the pH was adjusted to 6.0±0.5. After enzymatic hydrolysis at 25±2℃ for 2 hours, the enzyme was inactivated. The hydrolysate was centrifuged at 3000 rpm for 7 minutes to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of Peppercornia kava root. (3) Add the crude extract of kava root to the extraction tank, and add 10 times the amount of methanol / ethyl acetate (V / V=1:1). Set the temperature of the extraction tank to 60℃ and the pressure to 2MPa, and carry out subcritical extraction. After the solvent is recovered from the extract, it is dried to obtain 3.61g of the kava root extract.

[0042] Comparative Example 3 This comparative example provides a kava root extract, differing from Example 1 only in that the enzymatic hydrolysis uses a composite enzyme consisting of cellulase and pectinase in a mass ratio of 1:1.5; the specific preparation method is as follows: (1) After crushing the root of Pepper Kava, pass it through an 80-mesh sieve. Take 100g of Pepper Kava root powder and add 5 times the amount of 75 vol.% methanol solution. Heat to 55℃ and extract for 1.5h. After cooling to room temperature, perform solid-liquid separation. Add 3 times the amount of 70 vol.% 1,3-butanediol solution (containing isopropyl acetate, of which the concentration of isopropyl acetate in the 1,3-butanediol solution is 4 vol.%) to the filter residue. Heat to 80℃ and extract for 2h. After cooling to room temperature, perform solid-liquid separation. Combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.3% of a compound enzyme (the mass ratio of cellulase and pectinase was 1:1.5) and the pH was adjusted to 6.0±0.5. After enzymatic hydrolysis at 25±2℃ for 2 hours, the enzyme was inactivated. The hydrolysate was centrifuged at 3000rpm for 7 minutes to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of Kava pepper root. (3) Add the crude extract of kava root to the extraction tank, and add 10 times the amount of methanol / ethyl acetate (V / V=1:1). Set the temperature of the extraction tank to 60℃ and the pressure to 2MPa, and carry out subcritical extraction. After the solvent is recovered from the extract, it is dried to obtain 3.72g of the kava root extract.

[0043] Comparative Example 4 This comparative example provides a Kava pepper root extract, differing from Example 1 only in that the enzymatic hydrolysis uses a composite enzyme with a mass ratio of cellulase and subtilisin Carslberg at 1:1.5; the specific preparation method is as follows: (1) After crushing the root of Pepper Kava, pass it through an 80-mesh sieve. Take 100g of Pepper Kava root powder and add 5 times the amount of 75 vol.% methanol solution. Heat to 55℃ and extract for 1.5h. After cooling to room temperature, perform solid-liquid separation. Add 3 times the amount of 70 vol.% 1,3-butanediol solution (containing isopropyl acetate, of which the concentration of isopropyl acetate in the 1,3-butanediol solution is 4 vol.%) to the filter residue. Heat to 80℃ and extract for 2h. After cooling to room temperature, perform solid-liquid separation. Combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.3% of a compound enzyme (the mass ratio of cellulase and Subtilisin Carslberg was 1:1.5) and the pH was adjusted to 6.0±0.5. After enzymatic hydrolysis at 25±2℃ for 2 hours, the enzyme was inactivated. The hydrolysate was centrifuged at 3000 rpm for 7 minutes to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of Peppercorns kava root. (3) Add the crude extract of kava root to the extraction tank, and add 10 times the amount of methanol / ethyl acetate (V / V=1:1). Set the temperature of the extraction tank to 60℃ and the pressure to 2MPa, and carry out subcritical extraction. After the solvent is recovered from the extract, it is dried to obtain 4.09g of the kava root extract.

[0044] Comparative Example 5 This comparative example provides a kava root extract, differing from Example 1 only in that the enzymatic hydrolysis uses a complex enzyme composition of cellulase, pectinase, and papain in a mass ratio of 1:1:0.5; the specific preparation method is as follows: (1) After crushing the root of Pepper Kava, pass it through an 80-mesh sieve. Take 100g of Pepper Kava root powder and add 5 times the amount of 75 vol.% methanol solution. Heat to 55℃ and extract for 1.5h. After cooling to room temperature, perform solid-liquid separation. Add 3 times the amount of 70 vol.% 1,3-butanediol solution (containing isopropyl acetate, of which the concentration of isopropyl acetate in the 1,3-butanediol solution is 4 vol.%) to the filter residue. Heat to 80℃ and extract for 2h. After cooling to room temperature, perform solid-liquid separation. Combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.3% of a compound enzyme (the mass ratio of cellulase, pectinase and papain was 1:1:0.5) and the pH was adjusted to 6.0±0.5. After enzymatic hydrolysis at 25±2℃ for 2 hours, the enzyme was inactivated. The hydrolysate was centrifuged at 3000rpm for 7 minutes to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of pepper root. (3) Add the crude extract of kava root to the extraction tank, and add 10 times the amount of methanol / ethyl acetate (V / V=1:1). Set the temperature of the extraction tank to 60℃ and the pressure to 2MPa, and carry out subcritical extraction for 1h. After the solvent is recovered from the extract, it is dried to obtain 3.75g of the kava root extract.

[0045] Comparative Example 6 This comparative example provides a kava root extract, which differs from Example 1 only in that step (3) uses supercritical CO2 extraction; the specific preparation method is as follows: (1) After crushing the root of Pepper Kava, pass it through an 80-mesh sieve. Take 100g of Pepper Kava root powder and add 5 times the amount of 75 vol.% methanol solution. Heat to 55℃ and extract for 1.5h. After cooling to room temperature, perform solid-liquid separation. Add 3 times the amount of 70 vol.% 1,3-butanediol solution (containing isopropyl acetate, of which the concentration of isopropyl acetate in the 1,3-butanediol solution is 4 vol.%) to the filter residue. Heat to 80℃ and extract for 2h. After cooling to room temperature, perform solid-liquid separation. Combine the filtrates obtained from the two extractions. (2) After sterilization, the filtrate was inoculated with 0.3% of a compound enzyme (the mass ratio of cellulase, pectinase and Bacillus subtilis protease was 1:1:0.5) and the pH was adjusted to 6.0±0.5. After enzymatic hydrolysis at 25±2℃ for 2 hours, the enzyme was inactivated. The hydrolysate was centrifuged at 3000 rpm for 7 minutes to remove unreacted enzymes and substrates. The supernatant was concentrated to obtain crude extract of Peppermint root. (3) Add the crude extract of kava root to the extraction tank, extract for 1 hour under the conditions of extraction pressure of 6 MPa and extraction temperature of 30°C, recover the remaining raw material, heat and melt it at 50°C, and centrifuge at 3000 rpm for 5 minutes to obtain 4.30 g of kava root extract.

[0046] Test Example 1: Extraction Effect Study The extraction effect was evaluated by using the percentage of the final kava root extract mass to the kava root powder mass as the extraction rate. The results are shown in Table 1.

[0047] The content of calvalactone was detected by liquid chromatography under the following conditions: Chromatographic column: Waters XBridge C18 SUPERB (4.6 × 150 mm, 5 μm); Mobile phase: n-hexane-dioxane (82:18, V / V), isocratic elution; Flow rate: 1.0 mL / min; Detection wavelength: 350nm; Column temperature: 30 ℃.

[0048] Glutathione content was determined using the DTNB (5,5'-dithiobis-2-nitrobenzoic acid) colorimetric method, and the specific steps are as follows: (1) Preparation of DTNB stock solution: Dissolve 0.01 mol / L DTNB in ​​0.05 mol / L phosphate buffer (pH 7.0) to form DTNB stock solution; (2) Preparation of DTNB analysis solution: Dilute the DTNB stock solution 100 times with 0.5 mol / L Tris-HCl buffer solution at pH 8.0 to prepare the DTNB analysis solution. Store in the dark and use immediately after preparation. (3) Procedure: Take 0.5 mL of glutathione standard solution or the sample solution to be tested (0.1 ~ 3.0 mol / L) and add it to 1.5 mL of 0.15 mol / L NaOH solution. Then add 0.5 mL of 3% formaldehyde solution and react at pH 8.0 and 25℃ for 2 min. After the reaction is complete, take 0.5 mL of the reaction solution and add it to DTNB analytical solution. React at 25℃ for 5 min and measure the absorbance at a wavelength of 412 nm. Calculate the difference between the absorbances of the two solutions and substitute it into the standard curve to calculate the glutathione content.

[0049] Table 1

[0050] As can be seen from the table above, the extraction rates of the processes in Examples 1-4 of this invention are relatively high, and the total content of active ingredients all reach more than 90%, resulting in the best overall extraction effect.

[0051] Comparative Example 1 added isopropyl acetate as an extraction aid during the first extraction, resulting in a higher extraction rate. However, the total content of kavalactone decreased significantly, indicating that isopropyl acetate did not play a significant role in the extraction of kavalactone during the first extraction (when the kavalactone content was high), and instead promoted the extraction of other impurities. Only during the second extraction (or when the kavalactone content in the raw material was low) did it play a major role in the extraction of kavalactone and glutathione. Comparative Example 2 used methanol instead of C2-C6 small molecule polyols during the second extraction, and both the extraction rate and the kavalactone content of the final extract were unsatisfactory. This shows that C2-C6 small molecule polyols play an important role in the second extraction.

[0052] After adjusting the complex enzymes used in Comparative Examples 3-5, the extraction rate and total active ingredient content both decreased. Comparative Examples 3 and 5 showed the largest decrease, indicating that Bacillus subtilis protease is indispensable in the complex enzyme; otherwise, the extraction rate and total active ingredient content would be significantly reduced. Although Comparative Example 4 contained Bacillus subtilis protease, it did not contain pectinase, which was an improvement compared to Comparative Examples 3 and 5, but still far lower than Example 1.

[0053] Comparative Example 6 used supercritical CO2 extraction, which further improved the extraction rate. However, similar to Comparative Example 1, the total content of kavalactone in the extracted material decreased significantly. This also indicates that while using supercritical CO2 extraction to extract kavalactone, more other components were also extracted.

[0054] Test Example 2: Study on in vitro analgesic effect One hundred and twenty KM mice, weighing 20±2g, were randomly divided into 12 groups of 10 mice each, with half males and half females. These groups were: model group, positive group, and sample groups 1-10. The positive group was administered 1% naproxen injection, while sample groups 1-10 were administered the root extracts of *Peperomia kava* from Examples 1-4 and Comparative Examples 1-6, respectively. Hair was removed from the backs of the mice. The mice were administered 22mg / kg bw daily for 7 consecutive days. One hour after the last administration, 160ppm capsaicin was injected into the right paw of all mice in the model group, positive group, and sample groups 1-10 to induce pain. The duration of pain responses such as paw lifting, licking, and shaking was observed and recorded for 5 minutes. The results are shown in Table 2.

[0055] Table 2

[0056] Note: Compared to the model group, ## p < 0.01; compared with sample group 1, && p < 0.01.

[0057] Application Example 1 Essence Water A comprises: 0.3 wt% of Kava pepper root extract prepared in Example 1, 5.5 wt% of 1,2-propanediol, 2.8 wt% of sodium hyaluronate, 0.8 wt% of ceramide, 3.5 wt% of vitamin E, and the balance being deionized water. The preparation method is as follows: (1) Preparation of aqueous phase: Sodium hyaluronate is added to water at approximately 80% of the formulation amount to obtain an aqueous phase; (2) Preparation of oil phase: Kava pepper root extract, ceramide and vitamin E were mixed evenly at 60°C to obtain a premixed oil phase; (3) Preparation of Kava Pepper Root Extract Dispersion: Kava pepper root extract was added to 1,2-propanediol to obtain Kava pepper root extract dispersion; (4) Initial mixing: Slowly add the oil phase to the aqueous phase and stir to form a crude emulsion; (5) Homogenization: After homogenizing the crude emulsion three times under a pressure of 15000psi, add the Kava pepper root extract dispersion and replenish the remaining deionized water, stir evenly, and the product is obtained.

[0058] Essence Water B: Compared with Essence Water A, only the kava root extract prepared in Example 4 is used instead of the kava root extract in Example 1. The dosage and preparation method of other components are the same as those of Essence Water A.

[0059] Test Example 3: Human Efficacy Trial Twenty volunteers aged 18-60 years who reported symptoms of impaired skin barrier function, such as dryness, flaking, or redness, and who tested positive for lactic acid stinging, were recruited and randomly divided into two groups of 10 each. After cleansing their faces daily, each group applied approximately 2g of the corresponding essence water to their face and gently patted it until fully absorbed. During this period, no cosmetics or medications with similar effects were used. After 7 consecutive days of use, the anti-inflammatory and analgesic effects of the essence water were evaluated. The evaluation indicators and standards are as follows: Stinging / burning sensation: 0 indicates no stinging or burning sensation, 1 indicates mild stinging or burning sensation, 2 indicates moderate stinging or burning sensation, and 3 indicates severe stinging or burning sensation. Transepidermal water loss, stratum corneum moisture content, and skin redness were tested using a skin tester (model: Courage+Khazaka electronic GmbH, manufacturer: CK, Germany). The results are expressed as mean ± standard deviation, as shown in Table 3.

[0060] Table 3

[0061] Note: Compared to Essence Water A, ## p < 0.01.

[0062] As can be seen from the table above, the kava root extract prepared in Example 1 of this invention has better anti-inflammatory and analgesic effects, indicating that the specific content of kava lactone (or capsaicin, dihydrocapsaicin, methoxycapsaicin, dihydrocapsaicin, and demethoxycapsaicin in a specific ratio) can further enhance the anti-inflammatory and analgesic effects of the extract.

[0063] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing a Kava pepper root extract, characterized in that, Includes the following steps: (1) Solvent extraction: After crushing the root of Pepper Kava, add the first solvent for a first extraction to separate the residue and extract 1; then add the residue to the second solvent for a second extraction to separate the extract 2. The first solvent is an alcohol solution; the second solvent is a C2-C6 small molecule polyol solution and isopropyl acetate; (2) Enzymatic hydrolysis with compound enzyme: Combine extract 1 and extract 2 to obtain a mixed extract, add compound enzyme to the mixed extract for enzymatic hydrolysis, and after inactivation, separation and concentration, obtain crude extract of pepper root; The complex enzyme is cellulase, pectinase, and subtilisin; (3) Subcritical extraction: The crude extract of kava root is added to an organic solvent for subcritical extraction. The extract is concentrated and dried to obtain the kava root extract.

2. The preparation method according to claim 1, characterized in that, In one extraction, add 2-6 times the weight of the first solvent (based on the mass of the Kava pepper root) and extract at 55-65℃ for 1-3 hours; preferably, in one extraction, add 5 times the weight of the first solvent (based on the mass of the Kava pepper root) and extract at 55℃ for 1.5 hours. In the secondary extraction and / or the second extraction, a second solvent of 1-4 times the mass of the residue is added, and the extraction is carried out at 60-80℃ for 1-3 hours; preferably, in the secondary extraction, a second solvent of 3 times the mass of the residue is added, and the extraction is carried out at 80℃ for 2 hours.

3. The preparation method according to claim 1, characterized in that, The alcohol solution comprises one or more of methanol, ethanol, propanol, 1,3-butanediol, n-butanol, and 1,2-pentanediol; preferably, the concentration of the alcohol solution is 50-80 vol.%. And / or the C2-C6 small molecule polyol solution is one or more of ethylene glycol, 1,2-propanediol, glycerol, 1,3-butanediol, n-butanol, 1,2-pentanediol, and 1,6-hexanediol; preferably, the concentration of the C2-C6 small molecule polyol solution is 50-80 vol.%; and / or the concentration of isopropyl acetate in the 1,3-butanediol solution is 2-6 vol.%.

4. The preparation method according to claim 1, characterized in that, The mass ratio of cellulase, pectinase and subtilisin in the compound enzyme is 1:0.1-2:0.1-1; and / or the mass ratio of the compound enzyme to the mixed extract is 0.1-1:

100.

5. The preparation method according to claim 1, characterized in that, In the enzymatic hydrolysis of the compound enzyme, the hydrolysis temperature is 20-30℃ and the hydrolysis time is 1-2h; Preferably, before enzymatic hydrolysis by the compound enzyme, the pH of the mixed extract is adjusted to 4.0-8.

0.

6. The preparation method according to claim 1, characterized in that, The organic solvent used in the subcritical extraction is one or more of methanol, butane, propane, isobutane, and ethyl acetate; preferably, the ratio of the crude extract of kava root to the organic solvent is 1:8-10. And / or the subcritical extraction temperature is 55-65℃ and the pressure is 1-3MPa.

7. Kava pepper root extract prepared by any one of the preparation methods described in claims 1-6.

8. The kava root extract according to claim 7, characterized in that, The active ingredient composition of the kava pepper root extract is as follows: The active ingredients of the kava pepper root extract include: capsaicin 32-35%, dihydrocapsaicin 18-20%, methoxycapsaicin 7.5-10%, dihydrocapsaicin 18.5-22%, demethoxycapsaicin 9-10%, and glutathione 0.05-0.2%.

9. A composition comprising the kava root extract according to claim 7 or 8, characterized in that, The composition further includes one or more of the following: cat's claw extract, calendula extract, purple daisy extract, lemon balm extract, rosemary extract, centella asiatica extract, licorice extract, scutellaria baicalensis extract, aloe vera extract, clove extract, peppermint extract, bisabolol, and dipotassium glycyrrhizate.

10. The use of the kava pepper root extract as described in claim 7 or 8, or the composition as described in claim 9, in pain-relieving cosmetics.

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