Preparation method of high-activity radix gentianae soothing anti-inflammatory active component

Abstract

The invention discloses a preparation method of a high-activity radix gentianae soothing anti-inflammatory active ingredient, and belongs to the technical field of natural plant extract preparation, and the preparation method comprises the steps of radix gentianae pretreatment, compound probiotic fermentation, compound enzymolysis, graded purification and low-temperature refining. The compound probiotics are formed by compounding lactobacillus plantarum and lactobacillus reuteri, the compound enzymes are formed by compounding cellulase, pectinase and papain, the molecular weight of the prepared active ingredients is below 3kDa, the retention rate of gentiopicroside is 95%, and the gentiopicroside-containing compound probiotic beverage also contains micromolecular peptides and organic acids fermented by the probiotics. A fermentation and enzymolysis combined process is adopted, deep degradation of radix gentianae is achieved, active ingredients are good in skin permeability, all substances synergistically improve the soothing and anti-inflammatory effects, and the whole preparation process is free of organic solvents, mild and free of irritation. The composition can be added into cosmetics such as facial masks and essence, the addition amount is 0.2%-5.0%, and the composition is suitable for various skin types and has a wide industrial prospect.

Description

Technical Field

[0001] This invention relates to the field of natural plant extract preparation technology, specifically to a method for preparing a highly active soothing and anti-inflammatory active ingredient from gentian root. Background Technology

[0002] Gentian root is the dried root and rhizome of a plant in the Gentianaceae family. Its main active ingredient is gentiopicrin, and it also contains swertiamarin, gentian polysaccharides, flavonoids, and other substances. It has significant anti-inflammatory, soothing, and antioxidant effects, can inhibit the release of inflammatory factors in the skin, reduce discomfort symptoms such as redness, swelling, itching, and stinging, and repair the skin barrier. It is a highly valuable natural plant raw material in the cosmetics industry.

[0003] Currently, the main methods for extracting active ingredients from gentian root are traditional water extraction, alcohol extraction, and ultrasound-assisted extraction. While these methods are simple to operate, they have several drawbacks: First, the extracted active ingredients have large molecular weights and are mostly crude extracts with poor skin permeability, making it difficult for the skin to absorb and utilize the effective ingredients, resulting in poor soothing and anti-inflammatory effects. Second, traditional extraction methods can only extract the inherent active ingredients in gentian root and cannot generate new active substances, resulting in limited functionality. Third, some extraction methods use organic solvents, posing a risk of solvent residue and easily damaging heat-sensitive active ingredients such as gentiopicrin, leading to low retention rates. Fourth, the extracted products have low purification levels and high impurity content, which can easily cause mild skin irritation when used in cosmetics, limiting their application in sensitive skin care products.

[0004] With consumers increasingly demanding natural, efficient, and gentle cosmetic raw materials, developing a preparation method that can improve the skin permeability and retention rate of gentian root active ingredients and generate new synergistic active substances has become an urgent need for the industry. The extraction method combining probiotic fermentation and enzymatic hydrolysis can degrade macromolecules in plant raw materials into smaller molecules. Simultaneously, probiotic fermentation produces small-molecule peptides, organic acids, vitamins, and other metabolites, which synergistically enhance the overall efficacy with the plant's inherent active ingredients. Furthermore, this method is environmentally friendly, leaving no organic solvent residue, and meets the requirements for cosmetic raw material preparation. However, there are currently no reports on applying the combination of compound probiotic fermentation and compound enzymatic hydrolysis to the preparation of highly active soothing and anti-inflammatory components from gentian root. Therefore, developing a method for preparing gentian root active ingredients based on compound probiotic fermentation and compound enzymatic hydrolysis has significant application value. Summary of the Invention

[0005] To address the problems of poor skin permeability, low retention rate of active ingredients, limited functionality, and easy solvent residue in existing methods for extracting active ingredients from gentian root, this invention provides a method for preparing highly active soothing and anti-inflammatory active ingredients from gentian root. This method combines gentian root pretreatment, compound probiotic fermentation, compound enzymatic hydrolysis, graded purification, and low-temperature refining to obtain highly active ingredients with a molecular weight below 3kDa. This process retains the inherent active ingredients such as gentiopicrin from gentian root while enriching small molecule peptides and organic acids from probiotic fermentation. The synergistic effect of these active substances significantly enhances the soothing and anti-inflammatory effects. Furthermore, it exhibits good skin permeability, leaves no solvent residue, and is gentle and non-irritating, making it suitable for various cosmetics and skincare products.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution: A method for preparing highly active soothing and anti-inflammatory active ingredients from gentian root includes gentian root pretreatment, compound probiotic fermentation, compound enzymatic hydrolysis, fractional purification, and low-temperature refining steps. The molecular weight of the obtained active ingredients is below 3 kDa. The compound probiotics are a compound bacterial agent of Lactobacillus plantarum and Lactobacillus reuteri, and the compound enzymes are a compound enzyme agent of cellulase, pectinase, and papain. The retention rate of gentiopicrin in the active ingredients is 95%, and it also contains small molecule peptides and organic acid active substances metabolized by probiotic fermentation.

[0007] The pretreatment steps for gentian root are as follows: Take fresh gentian root, remove impurities, wash, and cut into thin slices 2-4 mm thick. Vacuum dry at 45-55℃ until the moisture content is 8%, then pulverize through an 80-120 mesh sieve to obtain gentian root powder for later use. Vacuum drying can avoid high temperature damage to the heat-sensitive active ingredients in gentian root, and pulverizing to 80-120 mesh can increase the specific surface area of ​​the raw material and improve the efficiency of subsequent fermentation and enzymatic hydrolysis.

[0008] The ratio of *Lactobacillus plantarum* to *Lactobacillus reuteri* in the compound probiotics is (1.5~2.5):1, with both *Lactobacillus plantarum* and *Lactobacillus reuteri* having a bacterial count of 100~200 billion CFU / g. At this ratio, the two probiotics can grow synergistically, and the enzymes and active substances produced by their metabolism can effectively degrade the cell walls of gentian root, releasing internal active ingredients. Simultaneously, the small molecule peptides and organic acids produced can synergistically exert a soothing and anti-inflammatory effect with gentiopicrin.

[0009] The fermentation process of the compound probiotics is as follows: 8-15 times the mass of purified water is added to the gentian root powder to adjust the pH to 6.0-6.8. Then, 0.8%-2.0% of the mass of the compound probiotics is added to the gentian root powder. Fermentation is carried out at 36-40℃ under anaerobic conditions for 24-30 hours, followed by sterilization at 85-95℃ for 15-25 minutes, and then cooled to room temperature to obtain the gentian root ferment. These fermentation conditions are the optimal growth conditions for the compound probiotics, ensuring sufficient fermentation. The sterilization step terminates the fermentation, preventing excessive growth of probiotics that could lead to the decomposition of active ingredients, while also killing other bacteria and ensuring product purity.

[0010] The mass ratio of cellulase, pectinase, and papain in the compound enzyme is (1~2):(1~2):1, with cellulase activity of 500,000 U / g, pectinase activity of 300,000 U / g, and papain activity of 200,000 U / g. Cellulase and pectinase can degrade cellulose, pectin, and other polysaccharides in the cell walls of gentian roots, breaking down the cell wall structure and releasing intracellular active ingredients. Papain can degrade proteins into small peptides, synergistically enhancing the soothing and anti-inflammatory effects with small peptides produced by probiotic fermentation. The combination of these three enzymes achieves comprehensive degradation of gentian root raw materials.

[0011] The steps of the compound enzymatic hydrolysis are as follows: 0.5% to 1.5% by mass of compound enzyme from gentian root powder is added to the gentian root fermentation product, the pH is adjusted to 5.0 to 6.0, and enzymatic hydrolysis is carried out at 45 to 55°C for 3 to 5 hours. Subsequently, the enzyme is inactivated at 85 to 95°C for 15 to 25 minutes, and then cooled to room temperature to obtain the gentian root enzymatic hydrolysate. These enzymatic hydrolysis conditions are the optimal operating conditions for the compound enzyme, ensuring enzymatic hydrolysis efficiency; the enzyme inactivation step terminates the enzymatic hydrolysis and prevents excessive enzyme action from destroying active components such as gentiopicrin.

[0012] The step of the graded purification is as follows: the gentian root enzymatic hydrolysate is first coarsely filtered through a 200-300 mesh filter cloth to remove the filter residue, obtaining a coarse filtrate; the coarse filtrate is then subjected to cross-flow filtration using a 3kDa ultrafiltration membrane at an operating pressure of 0.1-0.3 MPa and a temperature of 25-35℃, and the permeate is collected to obtain the purified gentian root solution. Coarse filtration can remove undegraded large molecular solid impurities, while cross-flow filtration using a 3kDa ultrafiltration membrane can retain large molecules with a molecular weight greater than 3kDa, ensuring that the molecular weight of the obtained active ingredients is below 3kDa, significantly improving skin permeability. At the same time, the cross-flow filtration method can prevent membrane clogging and ensure purification efficiency.

[0013] The low-temperature purification step is as follows: the purified gentian root extract is concentrated using a nanofiltration membrane to a solid content of 15%~25%, yielding a concentrated gentian root extract; the concentrated gentian root extract is then freeze-dried under vacuum at -40~-30℃, and after drying, it is pulverized through a 100~200 mesh sieve to obtain a powder containing highly active gentian root soothing and anti-inflammatory active ingredients. Nanofiltration membrane concentration can be carried out at room temperature, avoiding the destruction of active ingredients by high-temperature concentration, while removing some moisture and improving drying efficiency; vacuum freeze-drying can retain the structure and activity of active ingredients to the greatest extent, avoiding the decomposition of gentiopicrin and the inactivation of small molecule peptides caused by high-temperature drying; pulverization after drying yields a powder with uniform particle size, facilitating subsequent application in cosmetics.

[0014] Furthermore, the nanofiltration membrane has a molecular weight cutoff of 200-400 Da, a nanofiltration operating pressure of 0.5-1.0 MPa, and a temperature of 25-35°C. This nanofiltration membrane can retain small molecule active ingredients in gentian root, preventing them from being lost with water and ensuring the yield of active ingredients.

[0015] This invention also protects the application of the highly active gentian root soothing and anti-inflammatory active ingredient prepared by the above method in the preparation of cosmetics and skin care products. The cosmetics and skin care products are one or more of the following: face masks, serums, lotions, and creams. The mass fraction of the active ingredient added to the cosmetics and skin care products is 0.2% to 5.0%. This active ingredient has a small molecular weight and good skin permeability. The synergistic effect of gentianin with small molecule peptides and organic acids produced by probiotic fermentation results in a significant soothing and anti-inflammatory effect. Furthermore, it leaves no solvent residue, is gentle and non-irritating, and is suitable for the preparation of cosmetics for various skin types, including sensitive skin and acne-prone skin.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a process combining compound probiotic fermentation and compound enzymatic hydrolysis. Probiotic fermentation degrades the cell walls of gentian root, producing new active substances such as small molecule peptides and organic acids. Compound enzymatic hydrolysis further degrades macromolecules, achieving deep degradation of gentian root raw materials. The resulting active ingredients have molecular weights below 3kDa, significantly improving skin permeability and enabling rapid absorption and utilization of active ingredients by the skin. This solves the problem of poor absorption of active ingredients in traditional extraction methods.

[0017] The preparation process of this invention uses an aqueous system throughout, without the use of organic solvents, thus avoiding the risk of solvent residue. Furthermore, each step is controlled under mild temperature conditions. Vacuum drying, low-temperature freeze drying, and other processes can retain heat-sensitive active ingredients such as gentiopicrin to the greatest extent. The gentiopicrin retention rate is 95%, ensuring the effectiveness of the inherent active ingredients.

[0018] In the active ingredients obtained by this invention, gentiopicroside works synergistically with small molecule peptides and organic acid active substances produced by probiotic fermentation. Compared with gentiopicroside alone, its inhibition rate of skin inflammatory factors is increased by more than 30%, and its soothing and anti-inflammatory effects are significantly enhanced. At the same time, it also has certain antioxidant and skin barrier repair effects, making its functions more comprehensive.

[0019] The preparation process of this invention is clear and easy to operate. The graded purification and low-temperature refining process significantly reduces the impurity content in the product. The resulting active ingredient has high purity and is mild and non-irritating. Patch tests have verified that there are no adverse reactions such as skin redness, swelling, or itching. It is suitable for the preparation of various cosmetics and skin care products, especially for sensitive skin skin care products, and has broad application prospects.

[0020] The compound probiotics and compound enzymes of this invention are compounded in a specific ratio and optimized through multiple experiments to maximize the efficiency of fermentation and enzymatic hydrolysis. Compared with single probiotic fermentation or single enzymatic hydrolysis, the yield of active ingredients is increased by more than 25%, the production cost is lower, and it is suitable for large-scale industrial production. Detailed Implementation

[0021] The present invention will be described in detail below with reference to specific embodiments. The illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0022] In a first aspect, the present invention provides a method for preparing highly active soothing and anti-inflammatory active ingredients from gentian root, comprising the steps of gentian root pretreatment, compound probiotic fermentation, compound enzymatic hydrolysis, fractional purification, and low-temperature refining. The molecular weight of the obtained active ingredients is all below 3kDa. The compound probiotics are a compound bacterial agent of Lactobacillus plantarum and Lactobacillus reuteri, and the compound enzymes are a compound enzyme agent of cellulase, pectinase, and papain. The retention rate of gentiopicrin in the active ingredients is 95%, and it contains small molecule peptides and organic acid active substances metabolized by probiotic fermentation.

[0023] This invention utilizes a continuous process of pretreatment, fermentation with compound probiotics, enzymatic hydrolysis, fractional purification, and low-temperature refining to deeply process gentian root. The fermentation process employs a compound probiotic strain of *Lactobacillus plantarum* and *Lactobacillus reuteri* to degrade macromolecules and produce small peptides, organic acids, and other metabolically active substances. Further enzymatic hydrolysis using a combination of cellulase, pectinase, and papain further degrades cell walls and macromolecules. Fractional purification via a 3kDa ultrafiltration membrane enriches the small-molecule active ingredients. Finally, low-temperature refining preserves the structure and activity of the active ingredients, yielding active ingredients with molecular weights all below 3kDa. This process achieves a high retention rate of gentiopicrin and the synergistic presence of multiple active substances.

[0024] This invention achieves deep resource utilization of gentian root raw materials, retaining the inherent highly active ingredient gentiopicrin with a retention rate of no less than 95%, while also enriching novel active substances such as small molecule peptides and organic acids produced by probiotic fermentation. The synergistic effect of various active ingredients significantly enhances the soothing and anti-inflammatory effects. The small molecular structure below 3kDa gives the active ingredients excellent skin permeability, effectively solving the problem of poor absorption of traditionally extracted ingredients. The entire process is processed using an aqueous phase system without the use of organic solvents, avoiding the risk of solvent residue from the source of the process. The resulting active ingredients are highly safe and suitable for the preparation of various cosmetic raw materials.

[0025] In some embodiments, the pretreatment steps of the gentian root are as follows: take fresh gentian root, remove impurities, wash and cut into thin slices of 2-4 mm thickness, vacuum dry at 45-55℃ to a moisture content of 8%, pulverize and pass through an 80-120 mesh sieve to obtain gentian root powder for later use.

[0026] It should be noted that after the basic treatment of cleaning and removing impurities from fresh gentian roots, they are cut into 2-4mm thin slices to increase the drying contact area. Under vacuum drying conditions at 45-55℃, the moisture content of the gentian roots is reduced to below 8%, avoiding damage to heat-sensitive active ingredients such as gentiopicrin by high temperatures. The roots are then pulverized through an 80-120 mesh sieve to obtain gentian root powder with uniform particle size, further increasing the contact area between the raw materials and the raw materials in subsequent fermentation and enzymatic hydrolysis processes, ensuring the efficiency of these reactions. The vacuum drying process maximizes the retention of heat-sensitive active ingredients in the gentian roots, avoiding component decomposition caused by traditional high-temperature drying. The specific mesh size pulverization process allows for more thorough contact between the raw materials and probiotics and enzymes, effectively improving the reaction rate and extent of subsequent fermentation and enzymatic hydrolysis, and reducing raw material waste. Standardized pretreatment steps make subsequent process parameters easier to control, improving batch-to-batch stability of the product.

[0027] In some embodiments, the ratio of Lactobacillus plantarum to Lactobacillus reuteri in the compound probiotics is (1.5~2.5):1, and the bacterial counts of both Lactobacillus plantarum and Lactobacillus reuteri are 100~200 billion CFU / g.

[0028] It should be noted that when *Lactobacillus plantarum* with a viable count of 100-200 billion CFU / g and *Lactobacillus reuteri* at a viable count ratio of 1.5-2.5:1, the two probiotics form a synergistic microecological system. The metabolites of *Lactobacillus plantarum* provide the nutrients needed for the growth of *Lactobacillus reuteri*, while *Lactobacillus reuteri* regulates the pH of the system to suit the growth of *Lactobacillus plantarum*. After compounding, the enzyme secretion capacity and metabolic activity of the probiotics are significantly enhanced, making it easier to degrade the cell wall structure of *Gentiana scabra* root. This specific viable count ratio allows the two probiotics to achieve synergistic effects in growth and metabolism. Compared to single probiotics, the compound probiotic agent improves the degradation efficiency of *Gentiana scabra* root cell walls, allowing for a more complete release of intracellular active ingredients. The high viable count of the compound probiotic agent ensures the reaction rate of the fermentation process, shortens the fermentation cycle, and reduces the time cost of industrial production. The significantly increased variety and content of active substances produced by synergistic metabolism provide a stronger material basis for the soothing and anti-inflammatory effects of the active ingredients.

[0029] In some embodiments, the fermentation step of the compound probiotic is as follows: add 8 to 15 times the mass of purified water to gentian root powder, adjust the pH to 6.0 to 6.8, add 0.8% to 2.0% of the mass of compound probiotics to gentian root powder, ferment at 36 to 40°C under anaerobic conditions for 24 to 30 hours, then sterilize at 85 to 95°C for 15 to 25 minutes, and cool to room temperature to obtain gentian root fermentation product.

[0030] It should be noted that 8-15 times the mass of purified water is added to the gentian root powder to form a homogeneous suspension system. The pH is adjusted to 6.0-6.8 to suit the growth and metabolic environment of the compound probiotics. After adding 0.8%-2.0% of the compound probiotics, fermentation is carried out under anaerobic conditions at 36-40℃ for 24-30 hours, allowing the probiotics to fully grow, metabolize, and degrade the gentian root raw material. After fermentation, sterilization is carried out at 85-95℃ for 15-25 minutes to terminate the fermentation reaction and kill miscellaneous bacteria and probiotics, avoiding over-fermentation that could lead to the decomposition of active ingredients. After cooling, a stable gentian root fermentation product is obtained. The specific material-to-liquid ratio and pH value provide the optimal growth environment for the compound probiotics, ensuring fermentation efficiency and metabolic activity. The anaerobic fermentation conditions are adapted to the growth characteristics of *Lactobacillus plantarum* and *Lactobacillus reuteri*, increasing the number of viable probiotics and their metabolic capacity. The mild sterilization conditions effectively terminate fermentation and kill miscellaneous bacteria while avoiding high temperatures that could damage the already generated active ingredients, ensuring the content and stability of active ingredients in the fermentation product.

[0031] In some embodiments, the mass ratio of cellulase, pectinase and papain in the complex enzyme is (1~2):(1~2):1, with cellulase activity of 500,000 U / g, pectinase activity of 300,000 U / g, and papain activity of 200,000 U / g.

[0032] It should be noted that by combining cellulase, pectinase, and papain in a mass ratio of 1-2:1-2:1, cellulase and pectinase work synergistically to degrade polysaccharides such as cellulose and pectin in the cell walls of gentian roots, breaking down the dense structure of the cell walls. This allows papain to penetrate deep into the raw material and degrade proteins into small peptides. The complementary enzymatic sites of the three enzymes achieve comprehensive degradation of macromolecules in the gentian root raw material, while controlling the lower limit of enzyme activity to ensure the efficiency of the enzymatic hydrolysis reaction. The complementary enzymatic sites of the compound enzymes achieve deep degradation of macromolecules in the cell walls and interior of gentian roots. Compared with single enzymatic hydrolysis, the proportion of small molecule components in the hydrolysis products is significantly increased, further optimizing the skin permeability of active ingredients. The specific mass ratio of the compound enzymes optimizes the hydrolysis efficiency of each enzyme, avoiding the increased hydrolysis cost and subsequent enzyme inactivation pressure caused by excessive use of a single enzyme. The high enzyme activity ensures complete enzymatic hydrolysis in a short time, improving the efficiency of industrial production.

[0033] In some embodiments, the compound enzymatic hydrolysis step is as follows: add 0.5% to 1.5% by mass of compound enzyme from gentian root powder to gentian root fermentation product, adjust the pH to 5.0 to 6.0, enzymatically hydrolyze at 45 to 55°C for 3 to 5 hours, then inactivate the enzyme at 85 to 95°C for 15 to 25 minutes, and cool to room temperature to obtain gentian root enzymatic hydrolysate.

[0034] It should be noted that 0.5%–1.5% by mass of a compound enzyme is added to the gentian root ferment, and the pH is adjusted to 5.0–6.0 to suit the optimal enzymatic hydrolysis environment for the compound enzyme. Enzymatic hydrolysis is carried out at 45–55℃ for 3–5 hours to allow the compound enzyme to fully degrade macromolecules in the ferment. After enzymatic hydrolysis, the enzyme is inactivated at 85–95℃ for 15–25 minutes to denature and inactivate the enzyme protein, thus terminating the enzymatic hydrolysis reaction and preventing excessive enzyme action that could damage active ingredients such as gentiopicrin. After cooling, the gentian root enzymatic hydrolysate is obtained. The optimal pH and temperature conditions maximize the enzymatic hydrolysis activity of the compound enzyme, ensuring the efficiency and extent of the enzymatic hydrolysis reaction. The specific amount of enzyme added and the enzymatic hydrolysis time achieve full enzymatic hydrolysis while avoiding enzyme waste and over-enzymatic hydrolysis. The mild enzyme inactivation conditions effectively inactivate the enzyme while protecting the structure and activity of active ingredients such as gentiopicrin and small molecule peptides, ensuring the efficacy and quality of the enzymatic hydrolysate.

[0035] In some embodiments, the step of graded purification is as follows: the gentian root enzymatic hydrolysate is first coarsely filtered through a 200-300 mesh filter cloth to remove the filter residue and obtain a coarse filtrate; the coarse filtrate is then subjected to cross-flow filtration using a 3kDa ultrafiltration membrane at an operating pressure of 0.1-0.3MPa and a temperature of 25-35℃, and the permeate is collected to obtain a purified gentian root solution.

[0036] In practice, the gentian root enzymatic hydrolysate is first coarsely filtered using a 200-300 mesh filter cloth to remove undegraded solid residue and large molecular impurities, resulting in a clear coarse filtrate. This coarse filtrate is then subjected to cross-flow filtration through a 3kDa ultrafiltration membrane. The operating pressure is controlled at 0.1-0.3 MPa and the temperature at 25-35°C. Utilizing the molecular retention characteristics of the ultrafiltration membrane, large molecules with a molecular weight greater than 3kDa are retained, allowing smaller active ingredients to permeate through the membrane. The permeate is collected to obtain the purified gentian root solution. The coarse filtration step rapidly removes solid impurities, reducing the risk of subsequent ultrafiltration membrane clogging and improving purification efficiency. Cross-flow filtration with the 3kDa ultrafiltration membrane enables precise enrichment of small-molecule active ingredients, ensuring that the molecular weight of active ingredients in the purified solution is below 3kDa, significantly improving the skin permeability of the active ingredients. Specific operating pressure and temperature prevent membrane damage and ensure that the active ingredients do not undergo structural changes during purification, thus improving the activity and quality of the purified solution.

[0037] In some embodiments, the low-temperature purification step is as follows: the purified gentian root solution is concentrated through a nanofiltration membrane to a solid content of 15% to 25% to obtain a gentian root concentrate; the gentian root concentrate is freeze-dried under vacuum at -40 to -30°C, and after drying, it is pulverized and passed through a 100 to 200 mesh sieve to obtain a powder of highly active gentian root soothing and anti-inflammatory active ingredients.

[0038] In practice, the purified gentian root extract is first concentrated using a nanofiltration membrane to remove some water, increasing the solid content of the purified extract to 15%–25%, resulting in a concentrated gentian root extract. This extract is then subjected to vacuum freeze-drying at -40 to -30°C. Utilizing the low-temperature dehydration properties of freeze-drying, water is removed without damaging the structure of the active ingredients. After drying, the extract is pulverized through a 100–200 mesh sieve to obtain a highly active powder of gentian root soothing and anti-inflammatory active ingredients with uniform particle size. Nanofiltration concentration is performed at room temperature, avoiding the decomposition of active ingredients caused by traditional heating concentration, while also improving the efficiency of the subsequent drying process and reducing energy consumption. The vacuum freeze-drying process preserves the structure and bioactivity of the active ingredients to the greatest extent, avoiding problems such as the decomposition of gentiopicrin and the inactivation of small molecule peptides caused by high-temperature drying. The pulverization process after drying forms a powder with uniform particle size, facilitating subsequent dispersion and addition in cosmetics and improving application convenience.

[0039] In some embodiments, the nanofiltration membrane has a molecular weight cutoff of 200-400 Da, a nanofiltration operating pressure of 0.5-1.0 MPa, and a temperature of 25-35°C.

[0040] It should be noted that a nanofiltration membrane with a molecular weight cutoff of 200-400 Da was used to concentrate the purified gentian root solution. The operating pressure was controlled at 0.5-1.0 MPa and the temperature at 25-35℃. While removing water, the nanofiltration membrane retained small-molecule active ingredients such as gentiopicrin, small peptides, and organic acids from the gentian root, preventing the loss of active ingredients with the water and achieving both enrichment of active ingredients and efficient water removal. The specific molecular weight cutoff of the nanofiltration membrane enables precise separation of water and active ingredients, effectively retaining active ingredients such as gentiopicrin and small peptides during concentration, thus improving the yield of active ingredients. The specific operating pressure and temperature are adapted to the separation characteristics of the nanofiltration membrane, improving concentration efficiency while avoiding membrane damage and extending the service life of the membrane module. The room-temperature concentration conditions ensure that the active ingredients do not undergo structural changes during concentration, maintaining their high biological activity.

[0041] Secondly, the present invention provides a method for preparing the above-mentioned highly active gentian root soothing and anti-inflammatory active ingredient, and its application in the preparation of cosmetics and skin care products. The cosmetics and skin care products are one or more of masks, serums, lotions, and creams, and the mass fraction of the active ingredient added to the cosmetics and skin care products is 0.2% to 5.0%.

[0042] It should be noted that the highly active gentian root soothing and anti-inflammatory active ingredients prepared by the above-mentioned method can be added to the formulation system of cosmetics and skin care products such as masks, serums, lotions, and creams at a mass fraction of 0.2% to 5.0%. Utilizing the excellent skin permeability of its small molecular structure, the active ingredients such as gentianin, small molecule peptides, and organic acids can quickly penetrate into the deep layers of the skin to exert a synergistic effect of soothing and anti-inflammatory properties and repairing the skin barrier. An addition level of 0.2% to 5.0% allows the active ingredient to fully exert its efficacy in the cosmetic system, while avoiding increased costs and skin burden caused by excessive addition. The active ingredient is compatible with various cosmetic formulations such as masks and serums, with a wide range of applications to meet different cosmetic production needs. The mild and non-irritating nature of the active ingredient, along with its excellent soothing and anti-inflammatory effects, makes it suitable for various skin types, including sensitive and acne-prone skin, solving the problem of limited application of traditional gentian root extract in sensitive skin skincare products. Cosmetics containing this active ingredient can effectively relieve skin inflammation such as redness, itching, and stinging, while repairing the skin barrier and enhancing the skincare efficacy and market competitiveness of cosmetics.

[0043] The present invention will be further described below with reference to several embodiments, but the present invention is not limited to these embodiments.

[0044] Example 1 A method for preparing a highly active soothing and anti-inflammatory active ingredient from gentian root includes the following steps: Gentian root pretreatment: Take fresh gentian root, remove impurities, wash and cut into 3mm thick slices, vacuum dry at 50℃ until the moisture content is 6%, pulverize and pass through a 100-mesh sieve to obtain gentian root powder for later use. Fermentation with compound probiotics: The compound probiotics are a compound bacterial agent of Lactobacillus plantarum and Lactobacillus reuteri, with a bacterial activity ratio of 2:1 and a bacterial activity of 15 billion CFU / g for each. 12 times the mass of purified water was added to gentian root powder to adjust the pH to 6.5. 1.5% of the mass of compound probiotics was added to the gentian root powder. Fermentation was carried out at 38℃ under anaerobic conditions for 27 hours, followed by sterilization at 90℃ for 20 minutes and cooling to room temperature to obtain gentian root fermentation product. Compound enzymatic hydrolysis: The compound enzyme is a mixture of cellulase, pectinase and papain in a mass ratio of 1.5:1.5:1. The cellulase activity is 600,000 U / g, the pectinase activity is 400,000 U / g, and the papain activity is 250,000 U / g. 1.0% by mass of the compound enzyme was added to the gentian root fermentation product, the pH was adjusted to 5.5, and enzymatic hydrolysis was carried out at 50℃ for 4 hours. Then, the enzyme was inactivated at 90℃ for 20 minutes and cooled to room temperature to obtain the gentian root enzymatic hydrolysate. Fractional purification: The gentian root enzymatic hydrolysate was coarsely filtered through a 250-mesh filter cloth to remove the filter residue and obtain a coarse filtrate; the coarse filtrate was then subjected to cross-flow filtration using a 3kDa ultrafiltration membrane at an operating pressure of 0.2MPa and a temperature of 30℃, and the permeate was collected to obtain the purified gentian root solution. Low-temperature purification: The purified gentian root extract was concentrated to a solid content of 20% using a nanofiltration membrane with a molecular weight cutoff of 300 Da. The nanofiltration operation pressure was 0.8 MPa and the temperature was 30°C to obtain a concentrated gentian root extract. The concentrated gentian root extract was then freeze-dried under vacuum at -35°C. After drying, it was pulverized and passed through a 150-mesh sieve to obtain a powder containing highly active gentian root soothing and anti-inflammatory active ingredients.

[0045] Tests showed that the molecular weight of the active ingredients was all below 3kDa, the retention rate of gentiopicrin was 98.2%, and it contained small molecule peptides (molecular weight 200~1000Da), citric acid, lactic acid and other organic acid active substances.

[0046] Example 2 A method for preparing a highly active soothing and anti-inflammatory active ingredient from gentian root includes the following steps: Gentian root pretreatment: Take fresh gentian root, remove impurities, wash and cut into 2mm thick slices, vacuum dry at 45℃ until the moisture content is 7%, pulverize and pass through an 80-mesh sieve to obtain gentian root powder for later use; Fermentation with compound probiotics: The compound probiotics are a compound bacterial agent of Lactobacillus plantarum and Lactobacillus reuteri, with a bacterial activity ratio of 1.5:1 and a bacterial activity of 10 billion CFU / g. Eight times the mass of purified water was added to gentian root powder to adjust the pH to 6.0. 0.8% of the mass of compound probiotics was added to gentian root powder and fermented at 36℃ under anaerobic conditions for 24 hours. Then, it was sterilized at 85℃ for 25 minutes and cooled to room temperature to obtain gentian root fermentation product. Compound enzymatic hydrolysis: The compound enzyme is a mixture of cellulase, pectinase and papain in a mass ratio of 1:1:1. The cellulase activity is 500,000 U / g, the pectinase activity is 300,000 U / g, and the papain activity is 200,000 U / g. 0.5% by mass of the compound enzyme was added to the gentian root fermentation product, the pH was adjusted to 5.0, and enzymatic hydrolysis was carried out at 45℃ for 3 hours. Then, the enzyme was inactivated at 85℃ for 25 minutes and cooled to room temperature to obtain the gentian root enzymatic hydrolysate. Fractional purification: The gentian root enzymatic hydrolysate was coarsely filtered through a 200-mesh filter cloth to remove the filter residue and obtain a coarse filtrate; the coarse filtrate was then subjected to cross-flow filtration using a 3kDa ultrafiltration membrane at an operating pressure of 0.1MPa and a temperature of 25℃, and the permeate was collected to obtain a purified gentian root solution. Low-temperature purification: The purified gentian root extract was concentrated to a solid content of 15% by passing it through a nanofiltration membrane with a molecular weight cutoff of 200 Da. The nanofiltration operation pressure was 0.5 MPa and the temperature was 25°C to obtain a concentrated gentian root extract. The concentrated gentian root extract was then freeze-dried under vacuum at -40°C. After drying, it was pulverized and passed through a 100-mesh sieve to obtain a powder of highly active gentian root soothing and anti-inflammatory active ingredients.

[0047] Tests showed that the molecular weight of the active ingredients was all below 3kDa, the retention rate of gentiopicrin was 95.6%, and it contained small molecule peptides (molecular weight 200~1000Da), citric acid, lactic acid and other organic acid active substances.

[0048] Example 3 A method for preparing a highly active soothing and anti-inflammatory active ingredient from gentian root includes the following steps: Gentian root pretreatment: Take fresh gentian root, remove impurities, wash and cut into 4mm thick slices, vacuum dry at 55℃ until the moisture content is 8%, pulverize and pass through a 120-mesh sieve to obtain gentian root powder for later use. Fermentation with compound probiotics: The compound probiotics are a compound bacterial agent of Lactobacillus plantarum and Lactobacillus reuteri, with a bacterial activity ratio of 2.5:1 and a bacterial activity of 20 billion CFU / g. Add 15 times the mass of purified water to gentian root powder, adjust the pH to 6.8, add 2.0% by mass of compound probiotics to gentian root powder, ferment at 40℃ under anaerobic conditions for 30h, then sterilize at 95℃ for 15min, cool to room temperature, and obtain gentian root fermentation product; Compound enzymatic hydrolysis: The compound enzyme is a mixture of cellulase, pectinase and papain in a mass ratio of 2:2:1. The cellulase activity is 700,000 U / g, the pectinase activity is 500,000 U / g, and the papain activity is 300,000 U / g. 1.5% by mass of the compound enzyme was added to the gentian root fermentation product, the pH was adjusted to 6.0, and enzymatic hydrolysis was carried out at 55℃ for 5 hours. Then, the enzyme was inactivated at 95℃ for 15 minutes and cooled to room temperature to obtain the gentian root enzymatic hydrolysate. Fractional purification: The enzymatic hydrolysate of gentian root was coarsely filtered through a 300-mesh filter cloth to remove the filter residue and obtain a coarse filtrate; the coarse filtrate was then subjected to cross-flow filtration using a 3kDa ultrafiltration membrane at an operating pressure of 0.3MPa and a temperature of 35℃, and the permeate was collected to obtain a purified gentian root solution. Low-temperature purification: The purified gentian root extract was concentrated to a solid content of 25% by passing it through a nanofiltration membrane with a molecular weight cutoff of 400 Da. The nanofiltration operation pressure was 1.0 MPa and the temperature was 35°C to obtain a concentrated gentian root extract. The concentrated gentian root extract was then freeze-dried under vacuum at -30°C. After drying, it was pulverized and passed through a 200-mesh sieve to obtain a powder of highly active gentian root soothing and anti-inflammatory active ingredients.

[0049] Tests showed that the molecular weight of the active ingredients was all below 3kDa, the retention rate of gentiopicrin was 97.8%, and it contained small molecule peptides (molecular weight 200~1000Da), citric acid, lactic acid and other organic acid active substances.

[0050] Comparative Example 1 Gentian root extract was prepared using a traditional water extraction method. The steps were as follows: Gentian root powder (same as in Example 1) was taken, and 12 times its weight of purified water was added. The mixture was boiled and extracted for 2 hours, filtered, and the filtrate was concentrated under reduced pressure at 60°C and freeze-dried to obtain the gentian root water extract. Analysis showed that the extract retained 82.5% of gentiopicrosides, and most of the molecules were above 10 kDa.

[0051] Comparative Example 2 Gentian root extract was prepared using only a single Lactobacillus plantarum fermentation method. The steps were identical to those in Example 1, except that the compound probiotics were replaced with a single Lactobacillus plantarum. Testing revealed that the extract retained 92.3% of gentiopicroside, and the content of small molecule peptides and organic acids was 65% of that in Example 1.

[0052] Comparative Example 3 Gentian root extract was prepared using only a single cellulase enzymatic hydrolysis. The steps were identical to those in Example 1, except that the complex enzyme was replaced with a single cellulase. The extract showed a gentiopicroside retention rate of 93.1%, with a molecular weight fraction between 3 and 5 kDa.

[0053] Performance testing Patch test Thirty healthy subjects (15 men and 15 women) were selected. The active ingredient powder prepared in Examples 1-3 was formulated into a 2% aqueous solution, placed in a spot treatment device chamber, and applied to the inner arm of the subjects. The spot treatment device was removed after 24 hours, and the skin condition was observed within 48 hours. The results showed that none of the subjects experienced adverse reactions such as itching, redness, swelling, rash, or stinging, indicating that the active ingredient prepared in this invention is mild, non-irritating, and highly safe.

[0054] Anti-inflammatory effect test The inhibition rates of the samples from Examples 1-3 and Comparative Examples 1-3 on the inflammatory factors TNF-α and IL-6 released by macrophages were detected using cell culture methods. The samples were prepared into solutions with a mass concentration of 0.5 mg / mL and applied to lipopolysaccharide-induced macrophages. After 24 h of culture, the content of inflammatory factors was measured using enzyme-linked immunosorbent assay (ELISA), and the inhibition rate was calculated. The results are shown in Table 1.

[0055] Table 1. Inhibition rate of inflammatory factors (%) Sample TNF inhibition rate IL-6 inhibition rate Example 1 78.5 76.2 Example 2 75.3 73.1 Example 3 77.8 75.6 Comparative Example 1 45.2 42.8 Comparative Example 2 62.5 60.3 Comparative Example 3 60.8 58.5 As shown in Table 1, the active ingredients prepared in Examples 1-3 of this invention showed significantly higher inhibition rates against inflammatory factors TNF-α and IL-6 than those in the comparative example. This indicates that the process combining compound probiotic fermentation and compound enzymatic hydrolysis can significantly enhance the anti-inflammatory effect of the active ingredients in gentian root, and the synergistic effect of each active substance is significant.

[0056] Skin permeability test In vitro transdermal diffusion assays were performed. Samples from Example 1 and Comparative Example 1 were prepared into 2% (w / w) aqueous solutions and placed in the supply chamber of a transdermal diffusion cell. The receiving chamber was a phosphate buffer solution, and porcine skin was used as the transdermal barrier. After 8 hours of incubation, the content of gentiopicroside in the receiving chamber was measured, and the transdermal permeability was calculated. The results showed that the transdermal permeability of gentiopicroside from Example 1 was 68.3%, while that from Comparative Example 1 was 15.6%, indicating that the active ingredient prepared by this invention has a small molecular weight and significantly improved skin permeability.

[0057] Skincare product application testing The active ingredient prepared in Example 1 was added to the mask essence at a mass fraction of 1.0% to prepare a soothing and anti-inflammatory mask. Sixty subjects with sensitive skin experiencing redness and mild itching were randomly divided into two groups. The experimental group used the mask, while the control group used a blank mask without the added gentian root active ingredient. The masks were used every two days for 20 minutes each time, for one month. The results showed that 85% of the subjects in the experimental group experienced significant improvement in skin redness and itching, demonstrating a significant skin barrier repair effect. Only 10% of the subjects in the control group experienced slight symptom relief, indicating that the active ingredient prepared in this invention has excellent soothing and anti-inflammatory effects in skincare products.

[0058] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention employs a process combining compound probiotic fermentation and compound enzymatic hydrolysis. Probiotic fermentation degrades the cell walls of gentian root, producing new active substances such as small molecule peptides and organic acids. Compound enzymatic hydrolysis further degrades macromolecules, achieving deep degradation of gentian root raw materials. The resulting active ingredients have molecular weights below 3kDa, significantly improving skin permeability and enabling rapid absorption and utilization of active ingredients by the skin. This solves the problem of poor absorption of active ingredients in traditional extraction methods.

[0059] The preparation process of this invention uses an aqueous system throughout, without the use of organic solvents, thus avoiding the risk of solvent residue. Furthermore, each step is controlled under mild temperature conditions. Vacuum drying, low-temperature freeze drying, and other processes can retain heat-sensitive active ingredients such as gentiopicrin to the greatest extent. The gentiopicrin retention rate is 95%, ensuring the effectiveness of the inherent active ingredients.

[0060] In the active ingredients obtained by this invention, gentiopicroside works synergistically with small molecule peptides and organic acid active substances produced by probiotic fermentation. Compared with gentiopicroside alone, its inhibition rate of skin inflammatory factors is increased by more than 30%, and its soothing and anti-inflammatory effects are significantly enhanced. At the same time, it also has certain antioxidant and skin barrier repair effects, making its functions more comprehensive.

[0061] The preparation process of this invention is clear and easy to operate. The graded purification and low-temperature refining process significantly reduces the impurity content in the product. The resulting active ingredient has high purity and is mild and non-irritating. Patch tests have verified that there are no adverse reactions such as skin redness, swelling, or itching. It is suitable for the preparation of various cosmetics and skin care products, especially for sensitive skin skin care products, and has broad application prospects.

[0062] The compound probiotics and compound enzymes of this invention are compounded in a specific ratio and optimized through multiple experiments to maximize the efficiency of fermentation and enzymatic hydrolysis. Compared with single probiotic fermentation or single enzymatic hydrolysis, the yield of active ingredients is increased by more than 25%, the production cost is lower, and it is suitable for large-scale industrial production.

[0063] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for preparing a highly active soothing and anti-inflammatory active ingredient from gentian root, characterized in that, The process includes steps such as gentian root pretreatment, compound probiotic fermentation, compound enzymatic hydrolysis, fractionation purification, and low-temperature refining. The molecular weight of the obtained active ingredients is below 3kDa. The compound probiotics are a combination of Lactobacillus plantarum and Lactobacillus reuteri, and the compound enzymes are a combination of cellulase, pectinase, and papain. The retention rate of gentiopicroside in the active ingredients is 95%, and it also contains small molecule peptides and organic acid active substances metabolized by probiotic fermentation.

2. The method for preparing the highly active anti-inflammatory and soothing active ingredient from gentian root according to claim 1, characterized in that, The steps for pre-treatment of gentian root are as follows: take fresh gentian root, remove impurities, wash and cut into thin slices of 2-4 mm thickness, vacuum dry at 45-55℃ to a moisture content of 8%, pulverize and pass through an 80-120 mesh sieve to obtain gentian root powder for later use.

3. The method for preparing the highly active soothing and anti-inflammatory active ingredient from gentian root according to claim 1, characterized in that, The ratio of *Lactobacillus plantarum* to *Lactobacillus reuteri* in the compound probiotics is (1.5~2.5):1, and the bacterial counts of both *Lactobacillus plantarum* and *Lactobacillus reuteri* are 100~200 billion CFU / g.

4. The method for preparing the highly active soothing and anti-inflammatory active ingredient from gentian root according to claim 1, characterized in that, The steps of the compound probiotic fermentation are as follows: add 8 to 15 times the mass of purified water to gentian root powder, adjust the pH to 6.0 to 6.8, add 0.8% to 2.0% of the mass of compound probiotics to gentian root powder, ferment at 36 to 40°C under anaerobic conditions for 24 to 30 hours, then sterilize at 85 to 95°C for 15 to 25 minutes, and cool to room temperature to obtain gentian root fermentation product.

5. The method for preparing the highly active soothing and anti-inflammatory active ingredient from gentian root according to claim 1, characterized in that, The mass ratio of cellulase, pectinase and papain in the compound enzyme is (1~2):(1~2):1, with cellulase activity of 500,000 U / g, pectinase activity of 300,000 U / g, and papain activity of 200,000 U / g.

6. The method for preparing the highly active soothing and anti-inflammatory active ingredient from gentian root according to claim 1, characterized in that, The steps of the compound enzymatic hydrolysis are as follows: add 0.5% to 1.5% by weight of compound enzyme from gentian root powder to the gentian root fermentation product, adjust the pH to 5.0 to 6.0, enzymatically hydrolyze at 45 to 55°C for 3 to 5 hours, then inactivate the enzyme at 85 to 95°C for 15 to 25 minutes, and cool to room temperature to obtain gentian root enzymatic hydrolysate.

7. The method for preparing the highly active soothing and anti-inflammatory active ingredient from gentian root according to claim 1, characterized in that, The step of the graded purification is as follows: the gentian root enzymatic hydrolysate is first coarsely filtered through a 200-300 mesh filter cloth to remove the filter residue and obtain a coarse filtrate; the coarse filtrate is then subjected to cross-flow filtration using a 3kDa ultrafiltration membrane at an operating pressure of 0.1-0.3MPa and a temperature of 25-35℃, and the permeate is collected to obtain the purified gentian root solution.

8. The method for preparing the highly active soothing and anti-inflammatory active ingredient from gentian root according to claim 1, characterized in that, The low-temperature purification step is as follows: the purified gentian root solution is concentrated through a nanofiltration membrane to a solid content of 15%~25% to obtain a gentian root concentrate; the gentian root concentrate is freeze-dried under vacuum at -40~-30℃, and after drying, it is pulverized and passed through a 100~200 mesh sieve to obtain a powder of highly active gentian root soothing and anti-inflammatory active ingredients.

9. The method for preparing the highly active anti-inflammatory and soothing active ingredient from gentian root according to claim 8, characterized in that, The nanofiltration membrane has a molecular weight cutoff of 200~400 Da, a nanofiltration operating pressure of 0.5~1.0 MPa, and a temperature of 25~35℃.

10. The application of the highly active gentian root soothing and anti-inflammatory active ingredient prepared by any one of claims 1 to 9 in the preparation of cosmetics and skin care products, characterized in that, The cosmetics and skincare products mentioned are one or more of the following: facial masks, serums, lotions, and creams. The mass fraction of active ingredients added to the cosmetics and skincare products is 0.2% to 5.0%.