A method for extracting active substances from biota based on enzymatic reaction and application thereof

Abstract

This invention discloses a method and application for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reactions, relating to the field of cosmetic technology. The method includes the following steps: S1. Providing Platycladus orientalis leaf extract; S2. Providing an enzyme activator, wherein the enzyme activator is selected from at least one of the following: β-glucosidase from Aspergillus niger, β-glucosidase from almonds, cellulase complex enzyme, malt extract, and citrus fruit fermentation broth; S3. Mixing the enzyme activator with the Platycladus orientalis leaf extract to carry out an enzymatic reaction, thereby obtaining Platycladus orientalis leaf active ingredients based on the enzymatic reaction. The method and application of this invention for extracting Platycladus orientalis leaf active ingredients based on enzymatic reactions utilize diversified enzyme activator sources, balancing efficient conversion and compliant application. By optimizing the enzyme powder reconstitution system, stable storage of enzyme activity is ensured, and targeted and efficient conversion is achieved through precise enzymatic hydrolysis processes.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a method for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reactions and its application. Background Technology

[0002] Hair loss has become a prevalent skin appendage problem worldwide and is increasingly affecting younger people, significantly impacting consumers' physical and mental health as well as their appearance. Its core pathogenesis revolves around three main pathways: activation of 5α-reductase leads to the production of dihydrotestosterone (DHT), inducing hair follicle miniaturization; decreased proliferation and differentiation capacity of hair follicle stem cells causes hair follicle cycle disorders; and increased oxidative stress on the scalp leads to reactive oxygen species (ROS) damaging hair follicle structure and accelerating hair loss.

[0003] Plant-derived ingredients, with their gentle and multi-functional advantages, have become the core of anti-hair loss product development. Platycladus orientalis leaf extract, due to its natural anti-hair loss potential, is widely used in various hair care products. However, current technology for its application remains at the rudimentary stage of "direct addition of raw materials." Problems such as misunderstandings of the mechanism, outdated technical solutions, and a disconnect between industry, academia, and research have prevented the full release of Platycladus orientalis leaf's anti-hair loss potential, resulting in weak and unstable product efficacy. The core technological gaps and the targeted solutions of this invention are as follows: First, the conversion efficiency of precursors is low, and the utilization rate of active ingredients is insufficient, failing to fully tap the value of raw materials. Existing technologies mistakenly add over 90% of the inactive glycoside precursors (quercetin, isoquercetin, etc.) from Platycladus orientalis leaf extract directly as active ingredients, ignoring the core logic that "precursors must be hydrolyzed into free flavonoids (quercetin, kaempferol, etc.) to possess targeted anti-hair loss activity." Furthermore, they rely solely on passive conversion by endogenous β-glucosidase in the human body, which has low activity and uneven distribution, resulting in an in vitro conversion efficiency of ≤25%, scalp penetration of ≤15%, and a core active ingredient utilization rate of ≤6.7%, wasting a large amount of potential anti-hair loss ingredients. Therefore, this invention aims to construct a targeted enzymatic hydrolysis system to achieve efficient conversion of precursors and maximize the release of active ingredients, significantly improving the utilization value of raw materials.

[0004] Secondly, free flavonoids are easily oxidized and deactivated after enzymatic hydrolysis, limiting product stability and shelf life. The phenolic hydroxyl groups of free flavonoids are easily oxidized by oxygen and metal ions, leading to decreased efficacy and yellowing of the product. Existing technologies only maintain the stability of small amounts of natural free active substances by adding a single antioxidant, which cannot cope with the oxidation requirements of high concentrations of active ingredients after enzymatic hydrolysis, making it difficult to guarantee the efficacy stability of the product after long-term storage. This invention aims to construct a complex stabilization system to specifically address the oxidation of enzymatic hydrolysis products and the yellowing problem, thereby extending the half-life of active ingredients and the product's shelf life.

[0005] Third, the enzyme and raw material premixing has poor compatibility, posing a dual risk of enzyme inactivation and sensitization. Existing technologies lack enzyme-raw material compatibility design, and the addition of exogenous enzymes can easily lead to premature premixing and enzyme degradation and inactivation. This invention will solve the problem of enzyme premixing and inactivation through process optimization, achieving a balance between efficacy and safety.

[0006] Fourth, the mismatch between product form and enzymatic hydrolysis requirements hinders the industrialization of the technology. Most existing anti-hair loss products are single-chamber pre-mixed forms, lacking an isolation structure between enzymes and raw materials, making them unsuitable for the "separate placement-once mixing" logic required for enzymatic hydrolysis. A few dual-chamber or cyclodextrin-encapsulated products are only used for ingredient preservation or simple separation, lacking precise ratios, efficient mixing, and triggering conversion designs for enzymes and precursors, thus failing to support the implementation of enzymatic hydrolysis technology. This invention aims to develop innovative product forms suitable for industrialization, enabling on-the-spot preparation and precise conversion of enzymes and raw materials, thus paving the way for the industrialization of the technology.

[0007] Fifth, the enzyme activation conditions and ratios are unclear, resulting in insufficient stability of the conversion effect. Existing technologies have not clearly defined the role of β-glucosidase as an anti-desorption activator, nor have they systematically explored the optimal parameters for its compatibility with Platycladus orientalis leaf precursors—there is a lack of optimized data on the pH range, temperature range, and ionic environment required for enzyme activation, and the precise ratios of enzyme, precursor, buffer system, and stabilizer are also unclear, leading to low enzyme activation efficiency and large fluctuations in conversion effects. This invention will clarify the core parameters of enzymatic hydrolysis, establish a scientific ratio system, and achieve targeted and efficient conversion of precursors.

[0008] In summary, existing technologies have not formed an integrated solution encompassing "mechanism understanding, component transformation, stable retention, and morphological adaptation," thus failing to address the core pain points in the application of Platycladus orientalis leaf extract. This invention addresses these gaps by constructing a targeted enzymatic hydrolysis system, a complex stabilization system, and innovative product forms, achieving a breakthrough from "passively using raw materials" to "actively creating active ingredients," filling the industry gap in the in-depth development and efficient utilization of Platycladus orientalis leaf extract. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a method and application for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reactions, aiming to solve the following technical issues: 1. Solving the problems of low conversion efficiency of glycoside precursors and insufficient utilization of active ingredients in Platycladus orientalis leaves, maximizing the release of potential anti-hair loss components, and improving the utilization value of raw materials; 2. Solving the problems of easy oxidation and inactivation of free flavonoid active ingredients and yellowing of products after enzymatic hydrolysis, extending the half-life of active ingredients and the shelf life of products; 3. Solving the problems of premature enzyme inactivation and degradation caused by pre-mixing enzymes with raw materials, and the risk of scalp sensitization caused by enzyme protein residues, balancing conversion efficiency and product safety; 4. Solving the problems of existing products... The present invention addresses the issue that the form cannot simultaneously achieve enzyme stability and efficient release of active substances, and aims to develop innovative product forms suitable for industrial applications to achieve on-the-spot preparation and precise conversion; 5. It solves the problem that existing technologies lack clear activation conditions and precise ratios for enzyme activators (β-glucosidase), breaking through the bottleneck of activation parameters and ratios—existing technologies have not explored the optimal pH range, temperature adaptation range, and ionic environment required for enzyme activation, and have not clearly defined the precise ratios of enzyme with precursors, buffer systems, and stabilizers, resulting in low enzyme activation efficiency and unstable conversion effects. This invention needs to clarify the enzyme activation conditions and scientific ratios suitable for Platycladus orientalis leaf precursors to achieve targeted and efficient conversion.

[0010] Specifically, the following technical solutions are included: In a first aspect, a method for extracting active substances from Platycladus orientalis leaves based on enzymatic reactions is provided, comprising the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provide an enzyme activator, wherein the enzyme activator is selected from at least one of the following: β-glucosidase from Aspergillus niger, β-glucosidase from almond, cellulase complex enzyme, malt extract, and citrus fruit fermentation broth; S3. The enzyme activator is mixed with the Platycladus orientalis leaf extract to carry out an enzymatic reaction, thereby obtaining Platycladus orientalis leaf active product based on the enzymatic reaction.

[0011] The Platycladus orientalis leaf extract contains glycoside precursors, including at least one of quercetin, isoquercetin, afostigmoside, and myricetin. The enzymatically-catalyzed Platycladus orientalis leaf active ingredient contains free flavonoid active components, including at least one of quercetin, kaempferol, and myricetin.

[0012] Furthermore, the enzyme activator is β-glucosidase derived from Aspergillus niger, and its mass ratio with the extract of Platycladus orientalis leaves is 1:(10-20); the enzymatic reaction is carried out at a temperature of 25-40℃ for 4-4.5 hours.

[0013] Furthermore, the enzyme activator is malt extract and / or citrus fruit fermentation broth; the enzymatic reaction is carried out at a temperature of 25-40℃ for 5-7 hours.

[0014] Furthermore, the enzyme activator is composed of β-glucosidase derived from Aspergillus niger, malt extract, and citrus fruit fermentation broth; the enzymatic reaction is carried out at a temperature of 25-42℃ for 4-7 hours.

[0015] Furthermore, the enzyme activator is composed of a complex enzyme of almond-derived β-glucosidase and / or cellulase, malt extract, and citrus fruit fermentation broth; the enzymatic reaction is carried out at a temperature of 25-42°C for 4-7 hours.

[0016] Furthermore, the enzyme activator is an enzyme preparation; before participating in the enzymatic reaction, the enzyme preparation is first mixed with deionized water, a moisturizing stabilizer, and a chelating agent, and the pH value is adjusted with a buffer system to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract.

[0017] Furthermore, the enzyme preparation is selected from solid enzyme powder (i.e., enzyme powder) or liquid enzyme solution.

[0018] The enzyme activator is provided in the form of enzyme powder. Before participating in the enzymatic reaction, the enzyme powder is first mixed with deionized water, a moisturizing stabilizer, and a chelating agent, and the pH value is adjusted with a buffer system to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract. Furthermore, the moisturizing stabilizer is propylene glycol, the chelating agent is disodium EDTA, and the buffer system is a citrate-sodium citrate buffer solution.

[0019] Furthermore, the enzyme preparation accounts for 2.0%-17% of the mass percentage of the enzyme reconstitution system, the propylene glycol accounts for 3%-8% of the mass percentage of the enzyme reconstitution system, and the disodium EDTA accounts for 0.01%-0.1% of the mass percentage of the enzyme reconstitution system; the pH value of the citrate-sodium citrate buffer solution is adjusted to 5.0-5.2.

[0020] Furthermore, the mass ratio of the β-glucosidase derived from Aspergillus niger to the extract of Platycladus orientalis leaves is 1:15.

[0021] In a second aspect, an enzyme-catalyzed reaction-based active ingredient from Platycladus orientalis leaves is provided, which is prepared by the enzyme-catalyzed reaction-based extraction method for Platycladus orientalis leaves described in the first aspect.

[0022] Thirdly, the application of the enzymatically based active ingredient of Platycladus orientalis leaf as described in the second aspect in the preparation of anti-hair loss cosmetics is provided.

[0023] Fourthly, a hair loss prevention cosmetic is provided, comprising the enzymatically reacted Platycladus orientalis leaf active ingredient described in the second aspect; the dosage form of the cosmetic includes a dual-chamber shampoo, a single-use serum, or a cyclodextrin-encapsulated care product.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. Diversified sources of enzyme activators, balancing high efficiency and compliant application: This invention expands the enzyme activator into a diversified selection system containing at least one of the following: β-glucosidase from Aspergillus niger, β-glucosidase from almond, cellulase complex enzyme, malt extract, and citrus fruit fermentation broth, which has high conversion efficiency. 2. Optimized enzyme powder reconstitution system to ensure stable storage of enzyme activity: This invention has determined the optimal enzyme powder reconstitution system through systematic screening to ensure the enzyme activity retention rate after reconstitution; 3. Precise enzymatic hydrolysis process parameters for targeted and efficient conversion: This invention systematically optimizes the enzymatic hydrolysis process through single-factor and orthogonal experiments to improve conversion efficiency. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. 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.

[0026] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0027] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0028] It should also be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0029] To better understand the technical content of the present invention, the technical solution of the present invention will be further introduced and explained below with reference to specific embodiments.

[0030] The present invention aims to screen specific enzymes suitable for thuja leaf glycoside precursors and optimize enzymatic hydrolysis process parameters, and to determine the optimal enzymatic hydrolysis conditions through systematic experiments.

[0031] Unless otherwise stated, the concentration (%) referred to in this invention refers to mass concentration, content (%) refers to mass content, and amount added (%) refers to mass percentage.

[0032] I. The enzyme activator is β-glucosidase derived from Aspergillus niger. (1) Enzyme screening experiment Enzyme activity was determined using the DNS (3,5-dinitrosalicylic acid) method. Using glucose as a standard, one enzyme activity unit (U) was defined as the amount of enzyme required to catalyze the production of 1 μmol of glucose from the substrate (Thuja orientalis leaf extract) within 1 minute under conditions of pH 5.5 and a temperature of 25-40℃. The Thuja orientalis leaf extract contained 100% solids, more than 0.1% quercetin, and more than 1% total flavonoids.

[0033] Five potential glycoside hydrolases were initially screened: β-glucosidase from Aspergillus niger, β-glucosidase from almond, cellulase complex, pectinase, and xylanase. The core evaluation indicators were "precursor conversion efficiency, active ingredient yield, and compatibility with cosmetic systems." Each experiment had three replicates, and the experimental conditions were standardized: substrate concentration 45%, enzyme addition 0.2%, pH 5.5, temperature 25-40℃, and hydrolysis time 4 hours. The results of the enzyme screening experiments are shown in Table 1 below. Table 1 Results of enzyme screening experiments As shown in Table 1, pectinase and xylanase have no specific catalytic effect on the precursors of arborvitae leaf glycosides, and the total conversion efficiency is ≤10%, so they do not have the application value of enzymatic hydrolysis of arborvitae leaves and are directly excluded. Almond-derived β-glucosidase and cellulase complex enzymes can be used independently or alone as effective enzymes for the enzymatic hydrolysis of Platycladus orientalis leaf extract; when combined, they can form a synergistic combination and can also be used as an independent enzymatic hydrolysis system without relying on other enzyme components.

[0034] Among them, almond-derived β-glucosidase has high substrate specificity and can be efficiently hydrolyzed alone; cellulase complex enzyme has both cell wall disruption and glycoside hydrolysis capabilities and can complete enzymatic hydrolysis independently; when the two are combined, the total precursor conversion efficiency is ≥80%, achieving complementary advantages and the conversion efficiency is significantly higher than that of single cellulase complex enzyme, showing good application potential.

[0035] β-glucosidase derived from Aspergillus niger does not require metal ion activation, has the strongest substrate specificity, the highest conversion efficiency, the best cosmetic compatibility and safety, and the best overall performance, making it the preferred core enzyme.

[0036] In addition, malt extract and citrus fruit fermentation broth, two raw materials with enzymatic hydrolysis capabilities and eligible for registration, were selected to verify their release efficiency of active substances from Platycladus orientalis leaf extract. The malt extract was enzyme-active, containing ≥500 U / g β-glucosidase, ≥300 U / g amylase, ≥40% total solids, and pH 4.5-5.5. The citrus fruit fermentation broth was fermented, containing ≥400 U / g pectinase, ≥350 U / g β-glucosidase, ≤5% total acid, pH 3.5-4.5, and no alcohol residue.

[0037] (2) Enzymatic hydrolysis process Through single-factor experiments and orthogonal experiments (L9(3) 4 The systematic exploration of enzymatic hydrolysis methods was completed, clarifying the optimal schemes for enzyme types, reaction parameters, and enzyme powder reconstitution systems. This ensured that the hydrolysis efficiency, active ingredient yield, and enzyme activity stability all met the standards for industrial application. The specific exploration process is as follows: ① The process of exploring enzyme types Five potential glycoside hydrolases were initially screened: β-glucosidase from Aspergillus niger, β-glucosidase from almonds, cellulase complex, pectinase, and xylanase. The core evaluation indicators were "precursor conversion efficiency, active ingredient yield, and compatibility with the cosmetic system." Each experiment had three replicates, with standardized experimental conditions: substrate concentration 45%, enzyme addition 0.2%, pH 5.5, temperature 40℃, and hydrolysis time 4 hours. Results showed that pectinase and xylanase had no specific catalytic effect on thuja glycoside precursors, with a total conversion efficiency ≤10%, and were directly excluded. While the cellulase complex showed weak conversion ability, its low β-glucosidase activity (only 200 U / g) resulted in a total conversion efficiency ≤72%, and it was easily inactivated by binding with polyols in the formulation. Almond-derived β-glucosidase had a conversion efficiency ≥76%, but required Mn... 2+ Activation is required, but it is incompatible with cationic surfactants and has a low risk of allergenicity. β-glucosidase derived from Aspergillus niger does not require metal ion activation, has strong specificity for precursors such as quercetin and isoquercetin, has a total conversion efficiency of ≥88%, and has excellent compatibility with commonly used cosmetic ingredients and no allergenicity. It was ultimately identified as the core enzyme.

[0038] ② The process of exploring reaction parameters Using β-glucosidase derived from Aspergillus niger as the research subject, the parameter range was determined through single-factor experiments, and then the optimal value was optimized through orthogonal experiments (factors: temperature, time, enzyme dosage; levels: temperature 35 / 38 / 41℃, time 3 / 4 / 5h, enzyme dosage 0.15% / 0.2% / 0.25%), as detailed below: Temperature: At 35℃, enzyme activity is insufficient, with a total conversion efficiency of only 75%. Good enzymatic activity is maintained within the range of 25-40℃, with 38-40℃ being the peak in vitro activity range. At this temperature, the enzyme's active center structure is stable, and the conversion efficiency is ≥88%. This data serves as the benchmark parameter for optimizing enzymatic hydrolysis efficiency in the laboratory. Considering everyday room temperature use, this invention optimizes the enzyme stabilization system, ensuring the enzyme maintains high activity at scalp physiological temperatures (25-40℃, close to the actual effective temperature after contact with the scalp at room temperature), achieving a conversion efficiency ≥85%. No additional heating is required, perfectly suited for home use at room temperature. At enzymatic hydrolysis temperatures of 41℃ and above, the enzyme protein's spatial structure is destroyed, activity rapidly declines, and quercetin degradation rate is ≥10%. Therefore, the core effective temperature range for practical applications is clearly defined as the scalp physiological temperature range of 25-40℃, with a laboratory-optimized temperature of 38-40℃. The final product requires no deliberate temperature control, aligning with daily care habits.

[0039] Time: The reaction did not reach saturation within 3 hours of enzymatic hydrolysis, the precursor was not fully converted, and the conversion efficiency was ≤75%; the reaction reached saturation at 4 hours, the conversion efficiency was 88%, and the concentration of active ingredients reached its peak; extending to 5 hours, the conversion efficiency only increased by 1.5%, and the active ingredients were slightly reduced due to oxidation. Considering both efficiency and cost, the enzymatic hydrolysis time was determined to be 4 hours.

[0040] Enzyme addition amount: When the addition amount is 0.15%, the enzyme amount is insufficient, resulting in a conversion efficiency of 78%; when the enzyme and precursor concentration is matched at 0.2%, the conversion efficiency is 88% and the concentration of active ingredients meets the standard; when the addition amount is 0.25%, the conversion efficiency is only increased by 2%, but the residual amount of enzyme protein increases, making subsequent inactivation more difficult. The optimal enzyme addition amount is determined to be 0.2% (mass ratio of 1:15 with Platycladus orientalis leaf extract).

[0041] (3) The exploration process of enzyme powder reconstitution system formulation In this invention, the enzyme activator is stored only in the form of enzyme powder (lyophilized powder) or applied after being encapsulated with cyclodextrin, and is not used in the form of enzyme solution—enzyme solutions have extremely poor stability and are greatly affected by temperature and formulation components, which cannot meet the requirements of industrial storage and application. The core of this exploration is to optimize the enzyme powder reconstitution system to ensure that the enzyme powder is rapidly activated and stably effective after being added to the product base, and to clarify the enzyme powder storage conditions and specifications, as follows: ① Buffer system screening experiment Screening objective: To screen buffer systems that can maintain the optimal pH for enzymatic hydrolysis, promote enzyme powder dissolution, and ensure storage stability. All test concentrations were 0.05 mol / L. Experimental results are shown in Table 2 below. Table 2 Results of buffer system screening experiments ② Screening experiment for moisturizing stabilizers Screening objective: To screen for moisturizing stabilizers that can inhibit enzyme protein aggregation and improve enzyme powder dispersibility. The addition amount was 5% (based on the total mass of the enzyme reconstitution system). The experimental results are shown in Table 3 below. Table 3 Results of the screening experiment for moisturizing stabilizers ③ Chelating agent screening experiment Screening objective: To screen chelating agents that can chelate trace metal ions and protect enzyme structure. The addition amount was 0.05% (based on the total mass of the enzyme reconstitution system). The experimental results are shown in Table 4 below. Table 4 Results of chelating agent screening experiments (4) Determination of enzyme powder specifications and reconstitution formula Enzyme powder (lyophilized powder) specifications: β-glucosidase derived from Aspergillus niger, prepared using a modified freeze-drying process (freezing temperature -55℃, vacuum degree -0.12MPa, segmented drying time 30h, pre-freezing at low temperature for 8h followed by gradient temperature drying), enzyme activity 1200U / g, moisture content ≤2.5%, packaged in multi-layer composite vacuum aluminum foil (with built-in food-grade desiccant and oxygen absorbent, 0.1-1kg / bag, suitable for industrial feeding), packaging barrier properties: oxygen permeability ≤0.5cm. 3 / (m 2 • 24h • 0.1MPa), moisture permeability ≤ 0.3g / (m 2 •24h).

[0042] Storage conditions: Optimized for stable storage at room temperature. Unopened, store in a cool, dry environment below 25℃, protected from light, for a shelf life of 12 months (activity retention ≥75%). If stored in a cool place (15-20℃), the shelf life can be extended to 18 months (activity retention ≥80%). Once opened, use within 15 days at room temperature and in a dry environment. Refrigeration at 4℃ + drying can extend the shelf life to 1 month (expired enzyme activity ≤40%). This specification, through process optimization and packaging upgrades, breaks free from reliance on low-temperature storage, making it more suitable for industrial warehousing, transportation, and end-consumer usage scenarios in the cosmetics industry.

[0043] Example 1 An enzyme-catalyzed active ingredient from Platycladus orientalis leaves is prepared by the aforementioned enzyme-catalyzed extraction method for Platycladus orientalis leaves.

[0044] The method for extracting active substances from Platycladus orientalis leaves based on enzymatic reactions includes the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provide an enzyme activator, wherein the enzyme activator is a β-glucosidase derived from Aspergillus niger, and the mass ratio of the enzyme activator to the extract of Platycladus orientalis leaves is 1:15; S3. The enzyme activator is mixed with the Platycladus orientalis leaf extract to carry out an enzymatic reaction; the enzymatic reaction is carried out at a temperature of 38°C for 4 hours to obtain Platycladus orientalis leaf active product based on the enzymatic reaction.

[0045] The enzyme activator is provided in the form of enzyme powder. Before participating in the enzymatic reaction, the enzyme powder is mixed with deionized water, moisturizing stabilizer (propylene glycol), chelating agent (disodium EDTA), and the pH value is adjusted with a buffer system (citric acid-sodium citrate buffer) to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract. In the enzyme reconstitution system, the enzyme powder accounts for 2.0% of the total mass, the propylene glycol accounts for 5.0% of the total mass, and the disodium EDTA accounts for 0.05% of the total mass; the pH value of the citrate-sodium citrate buffer is adjusted to 5.2, and the remainder is deionized water.

[0046] Comparative Example 1 An extract of Platycladus orientalis leaves, without enzymatic hydrolysis.

[0047] Performance testing: Using Comparative Example 1 as the control group, the efficacy of Example 1 was verified by HPLC, human trials, and cost accounting. Specific data are shown in Tables 5-7 below: Table 5. Results of the active ingredient utilization rate test in Example 1 (with Comparative Example 1 as the control group). Table 6. Anti-hair loss effects in human trials of Example 1 and Comparative Example 1 Table 7. Raw material savings and industrialization benefits of Example 1 and Comparative Example 1 II. The enzyme activator is at least one of malt extract and citrus fruit fermentation broth. Two raw materials with enzymatic hydrolysis capabilities and registration requirements were selected: malt extract and citrus fruit fermentation liquid. Single-raw material enzymatic hydrolysis and compound raw material enzymatic hydrolysis experiments were designed to optimize the content of raw materials and verify their effects on the release efficiency of active substances in arborvitae leaf extract, anti-hair loss efficacy and scalp care effect.

[0048] (I) Specifications of core raw materials (% refers to mass content) 1. Arborvitae leaf extract: commercial powder, total content of quercetin + isoquercetin + afotiam + myricetin ≥15%, solids ≥30%, moisture ≤8%; 2. Malt extract: Enzymatic type, containing β-glucosidase ≥500U / g, amylase ≥300U / g, total solids ≥40%, pH 4.5-5.5; 3. Citrus fruit fermentation broth: fermented type, containing pectinase ≥400U / g, β-glucosidase ≥350U / g, total acid ≤5%, pH 3.5-4.5, and no alcohol residue; 4. Special excipients for compartmentalization: citrate-sodium citrate buffer, propylene glycol, disodium EDTA (all cosmetic grade, suitable for independent storage and compounding stability of enzyme hydrolysate); 5. Finished product formulation excipients: conventional cosmetic-grade surfactants, moisturizers, preservatives, etc. (consistent with the formulations of commercially available anti-hair loss products, and compatible with the compounded system).

[0049] (II) Core Evaluation Indicators of the Experiment 1. Specific indicators for enzymatic hydrolysate (chamber storage characteristics) • Enzyme activity retention rate (%, core enzyme activity retention of enzyme hydrolysate stored at 4℃ for 3 months); • pH fluctuation of the compounded system (pH difference before and after compounding, ≤0.5 is acceptable). • Separate compartment storage prevents layering / sedimentation / odor (visual and sensory evaluation).

[0050] 2. Core indicators of the finished product after compounding • Enzymatic hydrolysis efficiency: Total conversion efficiency of precursors (%), quercetin / kaempferol / myricetin content (mg / kg raw material), and utilization rate of active ingredients (%); • Anti-hair loss efficacy: Total inhibition rate of 5α-reductase (%), hair loss reduction rate in 28-day human trials (%), and hair density increase rate (%). • Scalp care benefits: TNF-α / IL-6 inhibition rate (%, anti-inflammatory), DPPH free radical scavenging rate (%, antioxidant), scalp transepidermal water loss (TEWL) improvement rate (%, barrier repair); • System compatibility: scalp irritation score (0-4 points, the lower the better), active ingredient retention rate of finished product after 6 months of room temperature storage (%), and no yellowing / stratification of the system (visual evaluation).

[0051] (III) Basic enzymatic hydrolysis process (adapted to compartment storage) The reaction conditions for malt extract / citrus fruit fermentation broth were adjusted to suit the specific requirements, while the storage process for the enzymatic hydrolysate was optimized to meet the needs for independent use in separate chambers. • Buffer system: Citric acid-sodium citrate buffer, pH 5.2 (fits the scalp microenvironment, adapts to the enzyme activity of the two raw materials, and facilitates stable storage in compartments). • Reaction temperature: 35-38℃; • Reaction time: 5 hours; • Add 2% propylene glycol and 0.05% disodium EDTA to stabilize the enzymatic hydrolysate system, then add citrate-sodium citrate buffer to adjust the pH to 5.2, dispense into the A chamber of the product (enzymatic hydrolysate chamber), seal and store in the dark; Compounding process: Before use, rapidly compound the enzymatic hydrolysate in cavity A (containing at least one of the following: arborvitae leaf extract, malt extract, and citrus fruit fermentation broth, propylene glycol, disodium EDTA, and citrate-sodium citrate buffer) with the product in cavity B (finished product formulation cavity) at a volume ratio of 1:4. Use immediately after compounding to extend the stability time of the active ingredients after compounding (addition / use should be completed within 2 hours).

[0052] (iv) Optimization of the content of Platycladus orientalis leaf extract by enzymatic hydrolysis of malt extract alone (with compartmentalized formulation) The content of Platycladus orientalis leaf extract was fixed at 45% (total mass of enzymatic hydrolysate). The addition of malt extract was set in gradients: 2%, 4%, 6%, 8%, 10%, and 12% (based on the total mass of enzymatic hydrolysate). The blank control group consisted of deionized water. Three replicates were set for each group. Separate enzymatic hydrolysates were prepared according to the basic enzymatic hydrolysis process (reaction temperature: 37℃; reaction time: 5h). After storage at 4℃ for 3 months, enzyme activity retention was measured. The core efficacy indicators were then measured after compounding. The results of the optimization experiment on the content of Platycladus orientalis leaf extract by enzymatic hydrolysis of malt extract alone are shown in Tables 8-9. Table 8. Results of the experiment on optimizing the content of Platycladus orientalis leaf extract by enzymatic hydrolysis of malt extract alone. Table 9. Results of the experiment on optimizing the content of Platycladus orientalis leaf extract by enzymatic hydrolysis of malt extract alone. As shown in Tables 8-9, the optimal addition amount of malt extract for enzymatic hydrolysis alone is 6%, with a content range of 4%-8%. Within this range, the enzyme activity retention rate of the hydrolysate after 3 months is ≥90.1%, there are no abnormalities in compartment storage, the pH fluctuation after compounding is ≤0.3, the precursor conversion efficiency is ≥58.3%, the quercetin content is ≥78.5mg / kg of raw material, the 5α-reductase inhibition rate is ≥59.6%, and the scalp irritation score is ≤0.3. This balances compartment storage stability, compound compatibility, and enzymatic hydrolysis efficacy. Adding an amount higher than 8% does not provide additional efficacy improvement and increases production costs and reduces compartment storage stability.

[0053] (v) Optimization of the content of Platycladus orientalis leaf extract by enzymatic hydrolysis of citrus fruit fermentation broth (with chamber adaptation) The content of Platycladus orientalis leaf extract was fixed at 45% (percentage of total mass of enzymatic hydrolysate). The addition of citrus fruit fermentation broth was set in gradients: 3%, 6%, 9%, 12%, 15%, and 18% (based on the total mass of the enzymatic hydrolysate, adapting the liquid fermentation broth to the characteristics of compartment filling). The control group consisted of deionized water. Three parallel samples were set for each group. Compartmentalized enzymatic hydrolysates were prepared according to the basic enzymatic hydrolysis process (reaction temperature: 36℃; reaction time: 5h). The compartment storage and compounding indicators were tested, with a focus on the effect of high fermentation broth addition on compartment pH and stability. The results of the optimization experiment on the content of Platycladus orientalis leaf extract in citrus fruit fermentation broth alone are shown in Tables 10-11. Table 10 Results of the experiment on optimization of the content of Platycladus orientalis leaf extract by enzymatic hydrolysis of citrus fruit fermentation broth alone Table 11 Results of the experiment on optimization of the content of Platycladus orientalis leaf extract by enzymatic hydrolysis of citrus fruit fermentation broth alone The experimental results in Tables 10 and 11 show that the optimal addition amount for the single enzymatic hydrolysis of citrus fruit fermentation broth is 9%, with a content range of 6%-12%. Within this range, the enzyme activity retention rate of the hydrolysate after 3 months is ≥91.0%, there are no abnormalities in compartment storage, the pH fluctuation after compounding is ≤0.3, the precursor conversion efficiency is ≥52.3%, the quercetin content is ≥68.5mg / kg of raw material, the 5α-reductase inhibition rate is ≥55.2%, and the scalp irritation score is ≤0.3. If the addition amount is less than 6%, the enzyme activity will be insufficient, and if it is more than 12%, it will easily lead to pH imbalance and yellowing / precipitation in compartment storage, affecting the user experience of compartment storage.

[0054] (vi) Optimization of enzymatic hydrolysis content in the compound of malt extract and citrus fruit fermentation broth (with compartmentalized formulation) Based on the optimal results for single raw materials, the arborvitae leaf extract was fixed at 45% (total mass percentage of the enzymatic hydrolysate). Using malt extract (A: 4%, 6%, 8%) and citrus fruit fermentation broth (B: 6%, 9%, 12%) as factors, an L9(3) model was designed. 2 Orthogonal experiment. The core evaluation index is the comprehensive score after compounding (weighted by precursor conversion efficiency, quercetin content, and 5α-reductase inhibition rate, with a maximum score of 100 points) + the cavity adaptation score (weighted by compounding pH fluctuation and system stability, with a maximum score of 50 points), for a total score of 150 points, to determine the optimal compounding ratio. The results of the optimization experiment of enzymatic hydrolysis content of malt extract + citrus fruit fermentation broth compound are shown in Table 12: Table 12 Results of the experiment on optimization of enzymatic hydrolysis content of malt extract + citrus fruit fermentation broth As shown in Table 12, the optimal ratio of malt extract to citrus fruit fermentation broth is 6% malt extract and 9% citrus fruit fermentation broth (45% Platycladus orientalis leaf extract, based on the total mass of the enzymatic hydrolysate). The ratio of malt extract to citrus fruit fermentation broth is 4%-8% to 6%-12%. Under this ratio, the enzyme activity retention rate of the enzymatic hydrolysate reaches 93.5% after 3 months, with no abnormalities during compartment storage. The pH fluctuation after compounding is only 0.2, and the compounding compatibility score is 49 out of 50. At the same time, a synergistic effect of enzyme activity is produced, with a precursor conversion efficiency of 82.6%, a quercetin content of 118.5 mg / kg raw material, and a 5α-reductase inhibition rate of 82.5%. The compounding efficacy score is 90.3, and the total score is 139.3, which is much higher than that of single-raw material enzymatic hydrolysis. The enzyme hydrolysis mechanisms are complementary (β-glucosidase from malt extract targets and hydrolyzes flavonoid glycosides, while pectinase from citrus fruit fermentation broth destroys the cell walls of Platycladus orientalis leaves), and the compartmentalized storage and compounding compatibility are excellent, with no risk of using unregistered enzyme preparations.

[0055] (vii) Comprehensive verification of the efficacy and performance of the optimal content compartmentalized enzymatic hydrolysate. Example 2 An enzyme-catalyzed active ingredient from Platycladus orientalis leaves is prepared by the aforementioned enzyme-catalyzed extraction method for Platycladus orientalis leaves.

[0056] The method for extracting active substances from Platycladus orientalis leaves based on enzymatic reactions includes the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provide an enzyme activator, wherein the enzyme activator is a malt extract; S3. The enzyme activator is mixed with the Platycladus orientalis leaf extract and an enzymatic reaction is carried out. The mixture is enzymatically hydrolyzed at 37°C for 5 hours to obtain Platycladus orientalis leaf active product based on the enzymatic reaction.

[0057] The enzyme activator is provided in the form of enzyme powder. Before participating in the enzymatic reaction, the enzyme powder is mixed with deionized water, moisturizing stabilizer (propylene glycol), chelating agent (disodium EDTA), and the pH value is adjusted with a buffer system (citric acid-sodium citrate buffer) to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract. In the enzyme reconstitution system, the enzyme powder accounts for 6.0% of the total mass, the propylene glycol accounts for 2.0%, and the disodium EDTA accounts for 0.05%. The pH of the citrate-sodium citrate buffer is adjusted to 5.2, and the remainder is deionized water. The mass of the Platycladus orientalis leaf extract is 45% of the total mass of the enzyme reconstitution system.

[0058] Example 3 An enzyme-catalyzed active ingredient from Platycladus orientalis leaves is prepared by the aforementioned enzyme-catalyzed extraction method for Platycladus orientalis leaves.

[0059] The method for extracting active substances from Platycladus orientalis leaves based on enzymatic reactions includes the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provide an enzyme activator, wherein the enzyme activator is citrus fruit fermentation broth; S3. The enzyme activator is mixed with the Platycladus orientalis leaf extract and subjected to an enzymatic reaction. The mixture is hydrolyzed at 36°C for 5 hours to obtain Platycladus orientalis leaf active ingredients based on the enzymatic reaction.

[0060] The enzyme activator is provided in the form of enzyme powder. Before participating in the enzymatic reaction, the enzyme powder is mixed with deionized water, moisturizing stabilizer (propylene glycol), chelating agent (disodium EDTA), and the pH value is adjusted with a buffer system (citric acid-sodium citrate buffer) to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract. In the enzyme reconstitution system, the enzyme powder accounts for 9.0% of the total mass, the propylene glycol accounts for 2.0%, and the disodium EDTA accounts for 0.05%. The pH of the citrate-sodium citrate buffer is adjusted to 5.2, and the remainder is deionized water. The mass of the Platycladus orientalis leaf extract is 45% of the total mass of the enzyme reconstitution system.

[0061] Example 4 An enzyme-catalyzed active ingredient from Platycladus orientalis leaves is prepared by the aforementioned enzyme-catalyzed extraction method for Platycladus orientalis leaves.

[0062] The method for extracting active substances from Platycladus orientalis leaves based on enzymatic reactions includes the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provide an enzyme activator, wherein the enzyme activator is malt extract and citrus fruit fermentation broth; S3. The enzyme activator is mixed with the Platycladus orientalis leaf extract and an enzymatic reaction is carried out. The mixture is enzymatically hydrolyzed at 38°C for 5 hours to obtain Platycladus orientalis leaf active product based on the enzymatic reaction.

[0063] The enzyme activator is provided in the form of enzyme powder. Before participating in the enzymatic reaction, the enzyme powder is mixed with deionized water, moisturizing stabilizer (propylene glycol), chelating agent (disodium EDTA), and the pH value is adjusted with a buffer system (citric acid-sodium citrate buffer) to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract. In the enzyme reconstitution system, the enzyme powder accounts for 15.0% of the total mass (6% malt extract and 9% citrus fruit fermentation broth), the propylene glycol accounts for 2.0%, and the disodium EDTA accounts for 0.05%. The pH of the citrate-sodium citrate buffer is adjusted to 5.2, and the remainder is deionized water. The arborvitae leaf extract accounts for 45% of the total mass of the enzyme reconstitution system.

[0064] Three optimal concentrations of enzymatic hydrolysates (i.e., the enzyme reconstitution systems of Examples 2, 3, and 4) were selected: 6% malt extract, 9% citrus fruit fermentation broth, and a 6%+9% compound solution. Deionized water was used as a blank control (blank group), and the enzyme reconstitution system of Example 1 was used as a positive control. The stability of the compartment storage, in vitro physicochemical experiments after compounding, and human clinical trials were carried out to comprehensively verify the compatibility of the compartments, the enzymatic hydrolysis effect, the anti-hair loss effect, and the scalp care effect.

[0065] Verification of the stability and compatibility of compartmentalized storage: Each group of enzyme hydrolysates (enzyme reconstitution systems of Examples 1-4) were dispensed into cavity A of the product and stored at 4℃, 25℃, and 45℃ for 3 months, respectively. The enzyme activity retention rate, system appearance, and pH value were tested. After reconstitution, the changes in pH and active ingredient content of the system were monitored within 2 hours to verify the compatibility of the reconstitution.

[0066] The experimental results of cavity storage stability and compatibility verification of Examples 1-4 and the blank group are shown in Table 13 below: Table 13 Results of experiments verifying the stability and compatibility of compartmentalized storage in Examples 1-4 and the blank group As shown in Table 13, the enzyme activity retention rate of all experimental compartment enzymatic hydrolysates was ≥88.6% after 3 months of storage at 4℃ / 25℃ and ≥75.3% after storage at 45℃. There was no stratification / precipitation / odor after compartment storage, which meets the storage requirements for compartmentalized cosmetic products. The 6%+9% compounding group showed the best compatibility, with a pH difference of only 0.2 before and after compounding. The active ingredient retention rate reached 96.5% after 2 hours of compounding, which is comparable to the compatibility of the pure enzyme preparation in Example 1 with compartments. Moreover, it does not have the risk of unregistered enzyme preparations and is fully compatible with compartment-type product forms.

[0067] Validation of active ingredient release efficiency and raw material utilization (after compounding): The samples from Examples 2-4 were tested within 2 hours after compounding. The contents of quercetin, kaempferol, and myricetin in the extracts of Platycladus orientalis leaves after enzymatic hydrolysis were detected by HPLC. The raw material utilization rate (content of active ingredient / total content of precursor) was calculated and compared with the blank group and the positive control group (Example 1).

[0068] The results of the verification of active ingredient release efficiency and raw material utilization rate in Examples 1-4 and the blank group are shown in Table 14: Table 14. Verification results of active ingredient release efficiency and raw material utilization rate in Examples 1-4 and the blank group. As shown in Table 14, the test results indicate that the enzymatic hydrolysis of malt extract and citrus fruit fermentation broth alone can significantly improve the release efficiency of active substances from Platycladus orientalis leaf extract. The raw material utilization rate is more than 10 times higher than that of the blank group, approaching 80%-90% of that of the pure enzyme preparation in Example 1. The compound group showed the best enzymatic hydrolysis effect, with a precursor conversion efficiency of 82.6% and a raw material utilization rate of 78.5%, which was only slightly lower than the positive control group. Moreover, the compartment storage and compounding compatibility were excellent, meeting the needs of compartment product development in the cosmetic industry.

[0069] Core efficacy verification for hair loss prevention (after compounding) – In vitro experiment: 5α-reductase inhibition rate The inhibition rates of type I and II 5α-reductase in each group of samples (Examples 1-4) after compounding were detected by in vitro enzyme activity assay. The results are shown in Table 15 below: Table 15 In vitro experimental results of Examples 1-4 and the blank group As shown in Table 15, the three types of enzymatically digested samples can significantly inhibit 5α-reductase activity after being combined. The total inhibition rate of the combined group reached 82.5%, which is 2.9 times that of the blank group. It can effectively reduce the production of dihydrotestosterone (DHT) and inhibit hair follicle miniaturization.

[0070] Core efficacy verification for anti-hair loss (after compounding) – Human trial: Anti-hair loss effect in 28 days Ninety participants aged 25-45 with androgenetic alopecia (45 males and 45 females) were randomly divided into five groups (malt group, citrus group, compound group, blank group, and positive control group), with 18 participants in each group. The product used a combination of "Cavity A enzymatic hydrolysate (the active ingredients of Platycladus orientalis leaf based on the enzymatic reaction of Examples 1-4) + Cavity B finished product formula," which was added to the base shampoo (6% by mass) once daily for 28 consecutive days. The rate of hair loss reduction and the rate of increase in hair density were measured, and the results are shown in Table 16 below. Table 16 Results of human trials in Examples 1-4 and the control group As shown in Table 16, the hair loss reduction rate of the compound group reached 45.2% and the hair density increase rate reached 16.8% after 28 days, which is close to the anti-hair loss effect of the pure enzyme preparation in Example 1 (above 48%), and far superior to the blank group. Moreover, the anti-hair loss effect of male subjects was slightly higher than that of female subjects, which is consistent with the pathogenesis of androgenetic alopecia.

[0071] In addition, the compartmentalized form avoids long-term contact between the enzymatically hydrolyzed active ingredients and preservatives, surfactants and other ingredients in the finished product formula, further ensuring the stability of the active ingredients and that the efficacy does not decrease after compounding.

[0072] Anti-inflammatory and antioxidant (in vitro) efficacy verification: The inhibition rate of anti-inflammatory factors was detected using an LPS-induced RAW264.7 macrophage model, and antioxidant activity was detected using the DPPH / ABTS method. The results are shown in Table 17 below: Table 17. Results of anti-inflammatory and antioxidant efficacy verification in Examples 1-4 and the blank group. As shown in Table 17, the combined enzymatic hydrolysate has significant anti-inflammatory and antioxidant effects. The combined group showed a TNF-α inhibition rate of 78.5% and a DPPH scavenging rate of 85.3%, which are close to the anti-inflammatory and antioxidant effects of the pure enzyme preparation in Example 1.

[0073] Verification of scalp barrier repair efficacy (after compound formulation): Thirty participants with sensitive scalps and redness were randomly divided into three groups (malt group, citrus group, and compound group), with 10 participants in each group. The compounded products were formulated into a 1.0% (w / w) serum solution and used continuously for 14 days. Scalp TEWL values ​​and redness scores were measured, and the results are shown in Table 18 below. Table 18. Verification results of scalp barrier repair efficacy in Examples 2-4 As shown in Table 18, the enzyme-based active ingredients of Platycladus orientalis leaves in Examples 2-4 can effectively repair the scalp barrier. The TEWL improvement rate of the compound group reached 44.2%, and the scalp redness score was significantly reduced. The compartmentalized form did not affect the scalp care efficacy. Moreover, both raw materials are natural plant / fermentation-derived, mild and non-irritating, and suitable for sensitive scalps.

[0074] Stability test: The finished products from Examples 1-4 were subjected to accelerated stability tests at 4°C, 25°C, and 45°C for 6 months. The retention rate of active ingredients, appearance, pH, and viscosity were measured, and the results are shown in Table 19 below. Table 19 Stability test results of Examples 1-4 As shown in Table 19, the enzyme-catalyzed reaction-based active ingredients of Platycladus orientalis leaf in Examples 1-4 have excellent compatibility with conventional cosmetic formulations. After 6 months of accelerated storage, the retention rate of active ingredients is ≥76.8%, and the retention rate of active ingredients in the compound group is 80.5%.

[0075] Safety experiment: A skin patch experiment was conducted on 20 healthy subjects. The finished product (10% mass concentration) of the enzymatic reaction-based active ingredients of Platycladus orientalis leaf from Examples 1-3 was applied to the back of the subjects. Skin reactions were observed after 48 hours. The results showed that no sensitization reactions such as redness, swelling, stinging, or itching occurred in any of the experimental groups. The scalp irritation score was ≤0.3 points, which met the requirements of the "Cosmetic Safety Technical Specifications (2022 Edition)". There was no risk of enzyme protein residue (natural raw materials do not have the protein sensitization problem of pure enzyme preparations).

[0076] Therefore, the compartmentalized form effectively avoids the long-term interaction between enzymatic active ingredients and the finished product formula, further improving the stability of the finished product. Moreover, the raw materials are natural, mild and non-irritating, with no risk of allergies or residues, meeting the safety and industrial production requirements of compartmentalized cosmetic products.

[0077] III. The enzyme activator consists of β-glucosidase derived from Aspergillus niger, malt extract, and citrus fruit fermentation broth. Example 5 An enzyme-catalyzed active ingredient from Platycladus orientalis leaves is prepared by the aforementioned enzyme-catalyzed extraction method for Platycladus orientalis leaves.

[0078] The method for extracting active substances from Platycladus orientalis leaves based on enzymatic reactions includes the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provide an enzyme activator, said enzyme activator being composed of β-glucosidase derived from Aspergillus niger, malt extract and citrus fruit fermentation broth; The enzyme activator is provided in the form of enzyme powder. Before participating in the enzymatic reaction, the enzyme powder is mixed with deionized water, moisturizing stabilizer (propylene glycol), chelating agent (disodium EDTA), and the pH value is adjusted with a buffer system (citric acid-sodium citrate buffer) to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract. In the enzyme reconstitution system, the enzyme powder accounts for 17.0% of the total mass (2% β-glucosidase from Aspergillus niger, 6% malt extract, and 9% citrus fruit fermentation broth), the propylene glycol accounts for 2.0% of the total mass, and the disodium EDTA accounts for 0.05% of the total mass. The pH of the citrate-sodium citrate buffer is adjusted to 5.2, and the remainder is deionized water. The mass of the Platycladus orientalis leaf extract is 45% of the total mass of the enzyme reconstitution system.

[0079] S3. The enzyme activator is mixed with the Platycladus orientalis leaf extract, and the mixture is subjected to enzymatic hydrolysis at 38°C for 5 hours to obtain Platycladus orientalis leaf active product based on enzymatic reaction.

[0080] Performance tests were conducted on Example 5, and the results are shown in Table 20 below: Table 20 Performance test results of Example 5 IV. Using almond-derived β-glucosidase, cellulase complex, or their compound as enzymes to replace β-glucosidase from Aspergillus niger, and combining it with malt extract and citrus fruit fermentation broth as enzyme activators. Based on the identification of β-glucosidase from Aspergillus niger as the core enzyme, in order to broaden the range of raw material selection and improve process adaptability, we further verified that three forms of β-glucosidase from almonds, cellulase complex enzyme, and β-glucosidase + cellulase complex enzyme from almonds can all replace β-glucosidase from Aspergillus niger and can be combined with malt extract and citrus fruit fermentation broth to construct a highly efficient enzymatic reaction system.

[0081] In this system, the amount of Platycladus orientalis leaf extract added was 45% (mass percentage), the amount of malt extract added was 6%, the amount of citrus fruit fermentation broth added was 9%, the enzymatic hydrolysis temperature was 38~40℃, and the enzymatic hydrolysis time was 5h.

[0082] Example 6 An enzyme-catalyzed active ingredient from Platycladus orientalis leaves is prepared by the aforementioned enzyme-catalyzed extraction method for Platycladus orientalis leaves.

[0083] The method for extracting active substances from Platycladus orientalis leaves based on enzymatic reactions includes the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provides 0.01 mmol / L of Mn 2+ (Activator) and enzyme activator, wherein the enzyme activator is composed of almond-derived β-glucosidase, malt extract and citrus fruit fermentation broth; The enzyme activator is provided in the form of enzyme powder. Before participating in the enzymatic reaction, the enzyme powder is mixed with deionized water, moisturizing stabilizer (propylene glycol), chelating agent (disodium EDTA), and the pH value is adjusted with a buffer system (citric acid-sodium citrate buffer) to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract. In the enzyme reconstitution system, the enzyme powder accounts for 15.2% of the total mass (0.2% almond-derived β-glucosidase, 6% malt extract, and 9% citrus fruit fermentation broth), the propylene glycol accounts for 2.0% of the total mass, and the disodium EDTA accounts for 0.05% of the total mass. The pH of the citrate-sodium citrate buffer is adjusted to 5.2, and the remainder is deionized water. The arborvitae leaf extract accounts for 45% of the total mass of the enzyme reconstitution system.

[0084] S3. Add 0.01 mmol / L Mn 2+ The enzyme activator is mixed with the Platycladus orientalis leaf extract and enzymatically hydrolyzed for 5 hours at pH 5.2-5.6 and temperature 40°C to obtain Platycladus orientalis leaf active product based on enzymatic reaction.

[0085] Example 7 An enzyme-catalyzed active ingredient from Platycladus orientalis leaves is prepared by the aforementioned enzyme-catalyzed extraction method for Platycladus orientalis leaves.

[0086] The method for extracting active substances from Platycladus orientalis leaves based on enzymatic reactions includes the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provide an enzyme activator, said enzyme activator being composed of a cellulase complex enzyme, malt extract and citrus fruit fermentation broth; The enzyme activator is provided in the form of enzyme powder. Before participating in the enzymatic reaction, the enzyme powder is mixed with deionized water, moisturizing stabilizer (propylene glycol), chelating agent (disodium EDTA), and the pH value is adjusted with a buffer system (citric acid-sodium citrate buffer) to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract. In the enzyme reconstitution system, the enzyme powder accounts for 15.4% of the total mass (0.4% cellulase complex, 6% malt extract, and 9% citrus fruit fermentation broth), the propylene glycol accounts for 2.0%, and the disodium EDTA accounts for 0.05%. The pH of the citrate-sodium citrate buffer is adjusted to 5.2, and the remainder is deionized water. The arborvitae leaf extract accounts for 45% of the total mass of the enzyme reconstitution system.

[0087] S3. The enzyme activator is mixed with the Platycladus orientalis leaf extract, and the mixture is enzymatically hydrolyzed for 5 hours at pH 4.8-5.3 and temperature 42°C to obtain Platycladus orientalis leaf active product based on enzymatic reaction.

[0088] Example 8 An enzyme-catalyzed active ingredient from Platycladus orientalis leaves is prepared by the aforementioned enzyme-catalyzed extraction method for Platycladus orientalis leaves.

[0089] The method for extracting active substances from Platycladus orientalis leaves based on enzymatic reactions includes the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provides 0.01 mmol / L of Mn 2+ (Activator) and enzyme activator, wherein the enzyme activator is composed of almond-derived β-glucosidase, cellulase complex enzyme, malt extract and citrus fruit fermentation broth; The enzyme activator is provided in the form of enzyme powder. Before participating in the enzymatic reaction, the enzyme powder is mixed with deionized water, moisturizing stabilizer (propylene glycol), chelating agent (disodium EDTA), and the pH value is adjusted with a buffer system (citric acid-sodium citrate buffer) to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract. In the enzyme reconstitution system, the enzyme powder accounts for 15.3% of the total mass (0.10% almond-derived β-glucosidase, 0.20% cellulase complex, 6% malt extract, and 9% citrus fruit fermentation broth), the propylene glycol accounts for 2.0% of the total mass, and the disodium EDTA accounts for 0.05% of the total mass. The pH of the citrate-sodium citrate buffer is adjusted to 5.2, and the remainder is deionized water. The arborvitae leaf extract accounts for 45% of the total mass of the enzyme reconstitution system.

[0090] S3. The enzyme activator is reacted with the 0.01 mmol / L Mn 2+ The active ingredient of Platycladus orientalis leaf was obtained by enzymatic hydrolysis of the activator and Platycladus orientalis leaf extract for 5 hours under the conditions of pH 5.0~5.4 and temperature 40℃.

[0091] Performance tests were conducted on Examples 6-8, and the test results are shown in Table 21 below: Table 21 Performance test results of Examples 6-8 The test results in Table 21 show that: 1. Almond-derived β-glucosidase, cellulase complex enzyme, and a combination of both can effectively replace β-glucosidase from Aspergillus niger and form a stable enzymatic reaction system when combined with malt extract and citrus fruit fermentation broth. 2. The total precursor conversion efficiency of the three compound systems is ≥82%, which is much higher than the conversion efficiency of human endogenous enzymes (≤25%), and can efficiently convert inactive glycoside precursors in Platycladus orientalis leaves into free flavonoid active ingredients; 3. The β-glucosidase + cellulase complex enzyme group derived from almonds showed synergistic effects, with a higher conversion efficiency than the single cellulase complex enzyme group, but slightly lower than the single almond enzyme group, and the process was highly flexible. 4. All three alternative systems can be used to prepare anti-hair loss cosmetics, with controllable safety and compatibility that meets the requirements of cosmetic formulations.

[0092] The above description is merely a specific embodiment of the present invention, but... The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reactions, characterized in that, Includes the following steps: S1. Provides Platycladus orientalis leaf extract; S2. Provide an enzyme activator, wherein the enzyme activator is selected from at least one of the following: β-glucosidase from Aspergillus niger, β-glucosidase from almond, cellulase complex enzyme, malt extract, and citrus fruit fermentation broth; S3. The enzyme activator is mixed with the Platycladus orientalis leaf extract to carry out an enzymatic reaction, thereby obtaining Platycladus orientalis leaf active product based on the enzymatic reaction.

2. The method for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reaction according to claim 1, characterized in that, The enzyme activator is β-glucosidase derived from Aspergillus niger, and its mass ratio with the extract of Platycladus orientalis leaves is 1:(10-20); the enzymatic reaction is carried out at a temperature of 25-40℃ for 4-4.5 hours.

3. The method for extracting active substances from Platycladus orientalis leaves based on enzymatic reaction according to claim 1, characterized in that, The enzyme activator is malt extract and / or citrus fruit fermentation broth; the enzymatic reaction is carried out at a temperature of 25-40℃ for 5-7 hours.

4. The method for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reaction according to claim 1, characterized in that, The enzyme activator consists of β-glucosidase derived from Aspergillus niger, malt extract, and citrus fruit fermentation broth; the enzymatic reaction is carried out at a temperature of 25-42℃ for 4-7 hours.

5. The method for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reaction according to claim 1, characterized in that, The enzyme activator is composed of a complex enzyme of almond-derived β-glucosidase and / or cellulase, malt extract, and citrus fruit fermentation broth; the enzymatic reaction is carried out at a temperature of 25-42℃ for 4-7 hours.

6. The method for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reaction according to claim 1, characterized in that, The enzyme activator is an enzyme preparation; before participating in the enzymatic reaction, the enzyme preparation is first mixed with deionized water, a moisturizing stabilizer, and a chelating agent, and the pH value is adjusted with a buffer system to prepare an enzyme reconstitution system before being mixed with the Platycladus orientalis leaf extract.

7. The method for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reaction according to claim 6, characterized in that, The enzyme preparation is selected from solid enzyme powder or liquid enzyme solution.

8. The method for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reaction according to claim 6, characterized in that, The moisturizing stabilizer is propylene glycol, the chelating agent is disodium EDTA, and the buffer system is citrate-sodium citrate buffer.

9. The method for extracting active ingredients from Platycladus orientalis leaves based on enzymatic reaction according to claim 8, characterized in that, The enzyme preparation accounts for 2.0%-17% of the mass percentage of the enzyme reconstitution system, the propylene glycol accounts for 3%-8% of the mass percentage of the enzyme reconstitution system, and the disodium EDTA accounts for 0.01%-0.1% of the mass percentage of the enzyme reconstitution system; the pH value of the citrate-sodium citrate buffer solution is adjusted to 5.0-5.

2.

10. An active ingredient from Platycladus orientalis leaves based on an enzymatic reaction, characterized in that, It is prepared by the enzymatic reaction-based extraction method for active substances from Platycladus orientalis leaves as described in any one of claims 1-9.

11. The application of the enzymatically reacted Platycladus orientalis leaf active ingredient according to claim 10 in the preparation of anti-hair loss cosmetics.

12. A hair loss prevention cosmetic, characterized in that, The product contains the enzyme-catalyzed arborvitae leaf active ingredient as described in claim 10; the dosage form of the cosmetic includes a dual-chamber shampoo, a single-use serum, or a cyclodextrin-encapsulated care product.

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