An enzyme-catalyzed reaction system and its application

Abstract

This invention discloses an enzymatic reaction system and its application, relating to the field of cosmetic technology. This enzymatic reaction system is used for the enzymatic hydrolysis of glycoside precursors in Platycladus orientalis leaf extract. It comprises Platycladus orientalis leaf extract and an enzyme activator 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. The enzymatic reaction system and its application of this invention, by introducing an enzyme activator, construct a highly efficient enzymatic reaction system for Platycladus orientalis leaf extract. Experiments show that this system can efficiently hydrolyze inactive glycoside precursors such as quercetin and isoquercetin in Platycladus orientalis leaf extract into free flavonoid active ingredients such as quercetin and kaempferol. The conversion rate is significantly better than the inefficient mode (≤25%) of existing technologies that rely on passive conversion by endogenous enzymes in the human body, fundamentally solving the core technical problem of low precursor utilization.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to an enzyme-catalyzed reaction system 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: Low precursor conversion efficiency and insufficient utilization of active ingredients result in underutilization of raw material value. 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, relying solely on passive conversion by endogenous β-glucosidase in the human body, which has low activity and uneven distribution, leads to 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 components. Therefore, this invention aims to construct a targeted enzymatic hydrolysis system to achieve efficient precursor conversion and maximize the release of active ingredients, significantly improving the utilization value of raw materials. Summary of the Invention

[0004] Existing technologies have not clearly defined the role of β-glucosidase as an anti-hair loss activator, nor have they systematically explored its optimal parameters for compatibility with Platycladus orientalis leaf precursors. Therefore, this invention aims to address the problems of low conversion efficiency and insufficient utilization of active ingredients in glycoside precursors from Platycladus orientalis leaves, maximizing the release of potential anti-hair loss components and enhancing the utilization value of raw materials. To solve the above technical problems, this invention provides an enzymatic reaction system and its application.

[0005] Specifically, the following technical solutions are included: In a first aspect, an enzymatic reaction system is provided for enzymatically hydrolyzing glycoside precursors in Platycladus orientalis leaf extract, comprising Platycladus orientalis leaf extract and 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.

[0006] Preferably, the enzyme activator is β-glucosidase derived from almonds, and the enzymatic reaction system further contains 0.01 mmol / L Mn. 2+ As an activator.

[0007] Preferably, the enzyme activator is selected from at least one of the following: β-glucosidase from Aspergillus niger, malt extract, and citrus fruit fermentation broth.

[0008] Furthermore, the enzyme activator is β-glucosidase derived from Aspergillus niger, and the β-glucosidase derived from Aspergillus niger accounts for 0.20-0.23% of the mass percentage of the enzyme-catalyzed reaction system.

[0009] Preferably, the β-glucosidase derived from Aspergillus niger accounts for 0.20% of the mass percentage of the enzymatic reaction system.

[0010] Furthermore, the mass ratio of the β-glucosidase derived from Aspergillus niger to the extract of Platycladus orientalis leaves is 1:(10-20).

[0011] Preferably, the mass ratio of the β-glucosidase derived from Aspergillus niger to the extract of Platycladus orientalis leaves is 1:15.

[0012] Furthermore, the enzyme activator is selected from at least one of malt extract and citrus fruit fermentation broth; when the enzyme activator contains malt extract, the malt extract accounts for 4%-8% of the mass percentage of the enzymatic reaction system; when the enzyme activator contains citrus fruit fermentation broth, the citrus fruit fermentation broth accounts for 6%-12% of the mass percentage of the enzymatic reaction system.

[0013] Furthermore, the enzyme activator is malt extract, and the malt extract accounts for 4%-8% of the mass percentage of the enzymatic reaction system.

[0014] Preferably, the enzyme activator is malt extract, and the malt extract accounts for 6% of the mass percentage of the enzymatic reaction system.

[0015] Furthermore, the enzyme activator is citrus fruit fermentation broth, and the citrus fruit fermentation broth accounts for 6%-12% of the mass percentage of the enzyme-catalyzed reaction system.

[0016] Preferably, the enzyme activator is citrus fruit fermentation broth, and the citrus fruit fermentation broth accounts for 9% of the mass percentage of the enzyme-catalyzed reaction system.

[0017] Furthermore, the enzyme activator is a combination of malt extract and citrus fruit fermentation broth, wherein the malt extract accounts for 4%-8% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 6%-12% of the mass percentage of the enzymatic reaction system.

[0018] Preferably, the enzyme activator is a combination of malt extract and citrus fruit fermentation broth, wherein the malt extract accounts for 6% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 9% of the mass percentage of the enzymatic reaction system.

[0019] Furthermore, the enzyme activator is composed of β-glucosidase derived from Aspergillus niger, malt extract, and citrus fruit fermentation broth. The β-glucosidase derived from Aspergillus niger accounts for 0.20-0.23% of the mass of the enzymatic reaction system, the malt extract accounts for 4%-8% of the mass of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 6%-12% of the mass of the enzymatic reaction system.

[0020] Preferably, the enzyme activator is composed of β-glucosidase from Aspergillus niger, malt extract, and citrus fruit fermentation broth. The β-glucosidase from Aspergillus niger accounts for 0.20% of the mass percentage of the enzyme-catalyzed reaction system, the malt extract accounts for 6% of the mass percentage of the enzyme-catalyzed reaction system, and the citrus fruit fermentation broth accounts for 9% of the mass percentage of the enzyme-catalyzed reaction system.

[0021] Furthermore, the enzyme activator is composed of almond-derived β-glucosidase, malt extract, and citrus fruit fermentation broth. The almond-derived β-glucosidase accounts for 0.10-0.20% of the mass of the enzyme-catalyzed reaction system, the malt extract accounts for 4%-8% of the mass of the enzyme-catalyzed reaction system, and the citrus fruit fermentation broth accounts for 6%-12% of the mass of the enzyme-catalyzed reaction system.

[0022] Furthermore, the enzyme activator is composed of a cellulase complex, malt extract, and citrus fruit fermentation broth. The cellulase complex accounts for 0.20-0.40% of the enzymatic reaction system by mass, the malt extract accounts for 4%-8% of the enzymatic reaction system by mass, and the citrus fruit fermentation broth accounts for 6%-12% of the enzymatic reaction system by mass.

[0023] Furthermore, the enzyme activator is composed of almond-derived β-glucosidase, cellulase complex enzyme, malt extract, and citrus fruit fermentation broth. The almond-derived β-glucosidase accounts for 0.10-0.20% of the mass of the enzymatic reaction system, the cellulase complex enzyme accounts for 0.20-0.40% of the mass of the enzymatic reaction system, the malt extract accounts for 4%-8% of the mass of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 6%-12% of the mass of the enzymatic reaction system.

[0024] Preferably, the arborvitae leaf extract accounts for 3-45% of the mass percentage of the enzymatic reaction system.

[0025] In a second aspect, a method for enzymatically hydrolyzing arborvitae leaf extract is provided, comprising the following steps: mixing arborvitae leaf extract with the enzymatic reaction system described in the first aspect, reacting under enzymatic hydrolysis conditions, and obtaining the enzymatic hydrolysis product.

[0026] Furthermore, the enzymatic hydrolysis conditions include: an enzymatic hydrolysis temperature of 25-42℃ and an enzymatic hydrolysis time of 4-7 hours.

[0027] Furthermore, when the enzyme activator is β-glucosidase derived from Aspergillus niger, the enzymatic hydrolysis temperature is 28-38°C, and the enzymatic hydrolysis time is 4 hours.

[0028] Furthermore, when the enzyme activator is selected from at least one of malt extract and citrus fruit fermentation broth, the enzymatic hydrolysis temperature is 36-38°C, and the enzymatic hydrolysis time is 5 hours.

[0029] The preferred enzymatic hydrolysis temperature is 25-40℃. This temperature can prevent the activity of fermentation broth raw materials from decaying, and the hydrolysis time can compensate for the problem that the enzyme activity of natural enzymatic hydrolysate raw materials is lower than that of pure enzyme preparations.

[0030] Thirdly, an enzymatic hydrolysis product of Platycladus orientalis leaves is provided, which is prepared by the enzymatic hydrolysis method of Platycladus orientalis leaf extract described in the second aspect.

[0031] Fourthly, the application of the enzymatic hydrolysate of Platycladus orientalis leaves as described in the third aspect in the preparation of cosmetics with anti-hair loss function is provided.

[0032] The beneficial effects of this invention are as follows: By introducing an enzyme activator, this invention constructs a highly efficient enzymatic reaction system for Platycladus orientalis leaf extract. Experiments show that this system can efficiently hydrolyze inactive glycoside precursors such as quercetin and isoquercetin in Platycladus orientalis leaf extract into free flavonoid active components such as quercetin and kaempferol. The conversion rate is significantly better than the inefficient mode (≤25%) of existing technologies that rely on passive conversion by endogenous enzymes in the human body, fundamentally solving the core technical problem of low precursor utilization. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] Unless otherwise stated, the concentration (%) referred to in this invention refers to mass concentration, the content (%) refers to mass content, and the amount added (%) refers to mass percentage.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 in 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.

[0045] I. Enzymatic reaction system using β-glucosidase from Aspergillus niger as enzyme activator 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 25-40℃, time 3 / 4 / 5h, enzyme dosage 0.15% / 0.2% / 0.25%), as detailed below: Example 1 An enzymatic reaction system is provided for the enzymatic hydrolysis of glycoside precursors in Platycladus orientalis leaf extract. The system comprises Platycladus orientalis leaf extract and an enzyme activator, wherein the enzyme activator is a β-glucosidase derived from Aspergillus niger, and the Aspergillus niger-derived β-glucosidase accounts for 0.20% of the mass percentage of the enzymatic reaction system. The mass ratio of the Aspergillus niger-derived β-glucosidase to the Platycladus orientalis leaf extract is 1:15. The Platycladus orientalis leaf extract accounts for 45% of the mass percentage of the enzymatic reaction system.

[0046] A method for enzymatically hydrolyzing Platycladus orientalis leaf extract using an enzymatic reaction system includes the following steps: mixing Platycladus orientalis leaf extract with the aforementioned enzymatic reaction system, reacting under enzymatic hydrolysis conditions to obtain the enzymatic hydrolysis product; wherein the enzymatic hydrolysis conditions include: an enzymatic hydrolysis temperature of 38°C and an enzymatic hydrolysis time of 4 hours.

[0047] (1) Effect based on enzyme activator content Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the β-glucosidase from Aspergillus niger accounts for 0.15% of the mass of the enzymatic reaction system, while all other conditions are the same.

[0048] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the β-glucosidase derived from Aspergillus niger accounts for 0.25% of the mass of the enzymatic reaction system, while all other conditions are the same.

[0049] By testing the 4-hour precursor conversion efficiency of Examples 1 and Comparative Examples 1-2, it was found that when the added amount (mass percentage) of β-glucosidase from Aspergillus niger in the enzymatic reaction system was 0.15%, the enzyme amount was insufficient, resulting in a conversion efficiency of 78%. When the mass percentage of β-glucosidase from Aspergillus niger in the enzymatic reaction system was 0.2%, the enzyme and precursor concentrations were well matched, and the conversion efficiency was 88%, with the active ingredient concentration meeting the standard. When the mass percentage of β-glucosidase from Aspergillus niger in the enzymatic reaction system was 0.25%, the conversion efficiency only increased by 2%. Therefore, the optimal enzyme addition amount in the enzymatic reaction was determined to be 0.2% (mass ratio of 1:15 with Platycladus orientalis leaf extract).

[0050] (2) Based on the effect of temperature in enzymatic hydrolysis conditions: Example 2 Compared with Example 1, the difference is that the enzymatic hydrolysis temperature is 40°C, while all other conditions are the same.

[0051] Example 3 Compared with Example 1, the difference is that the enzymatic hydrolysis temperature is 28°C, while all other conditions are the same.

[0052] Example 4 Compared with Example 1, the difference is that the enzymatic hydrolysis temperature is 25°C, while all other conditions are the same.

[0053] Comparative Example 3 Compared with Example 1, the difference is that the enzymatic hydrolysis temperature is 41°C, while all other conditions are the same.

[0054] By testing the 4-hour precursor conversion efficiency of Examples 1-4 and Comparative Example 3, it was found that good enzymatic activity could be maintained within the range of 25-40℃. The peak activity range in vitro was 38-40℃, at which the enzyme's active site structure was stable, and the conversion efficiency was ≥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 that the enzyme maintains high activity at scalp physiological temperature (25-40℃, close to the actual effective temperature after contact with the scalp at room temperature), with a conversion efficiency ≥85%. No additional heating is required, perfectly suited for home use at room temperature. When the enzymatic hydrolysis temperature is 41℃ or higher, the enzyme protein spatial structure is destroyed, activity rapidly declines, and the quercetin degradation rate is ≥10%. Therefore, the core effective temperature range in 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, conforming to daily care habits.

[0055] (3) Effect of enzymatic hydrolysis time on enzymatic hydrolysis conditions Comparative Example 4 Compared with Example 1, Comparative Example 4 differs in that the enzymatic hydrolysis time is 3 hours, while all other conditions are the same.

[0056] Comparative Example 5 Compared with Example 1, Comparative Example 5 differs in that the enzymatic hydrolysis time is 5 hours, while all other conditions are the same.

[0057] By testing the conversion efficiency of the precursors in Examples 1, 4, and 5 over 4 hours, it was found that the enzymatic hydrolysis time was 3 hours. Within 3 hours, the reaction did not reach saturation, the precursor conversion was insufficient, and the conversion efficiency was ≤75%. When the enzymatic hydrolysis time was 4 hours, the reaction reached saturation, the conversion efficiency was 88%, and the concentration of active ingredients reached its peak. Extending the time to 5 hours only increased the conversion efficiency by 1.5%, and the active ingredients slightly decreased due to oxidation. Considering both efficiency and cost, the enzymatic hydrolysis time was determined to be 4 hours.

[0058] II. Enzymatic reaction system using at least one of malt extract and citrus fruit fermentation broth as enzyme activators (1) Using malt extract as an enzyme activator The concentration of Platycladus orientalis leaf extract was fixed at 45% (based on the total mass ratio of the enzymatic hydrolysate / enzymatic reaction system). The addition of malt extract was set in gradients: 2%, 4%, 6%, 8%, 10%, and 12% (based on the total mass of the enzymatic hydrolysate). The control group consisted of unenzymatically hydrolyzed Platycladus orientalis leaf extract (45%, with deionized water as the enzymatic hydrolysate in the same proportion). Three replicates were set for each group, and the average result was taken. Details are as follows: Example 5 An enzymatic reaction system is provided for the enzymatic hydrolysis of glycoside precursors in Platycladus orientalis leaf extract, comprising Platycladus orientalis leaf extract and an enzyme activator, wherein the enzyme activator is malt extract, and the malt extract accounts for 6% by mass of the enzymatic reaction system. The Platycladus orientalis leaf extract accounts for 45% by mass of the enzymatic reaction system.

[0059] A method for enzymatically hydrolyzing Platycladus orientalis leaf extract using an enzymatic reaction system includes the following steps: mixing Platycladus orientalis leaf extract with the aforementioned enzymatic reaction system, reacting under enzymatic hydrolysis conditions to obtain the enzymatic hydrolysis product; wherein the enzymatic hydrolysis conditions include: an enzymatic hydrolysis temperature of 37°C and an enzymatic hydrolysis time of 5 hours.

[0060] Example 6 Compared with Example 5, the difference is that the malt extract accounts for 4% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0061] Example 7 Compared with Example 5, the difference is that the malt extract accounts for 8% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0062] Comparative Example 6 Compared with Example 5, the difference is that deionized water is used instead of malt extract in the enzymatic reaction system, and the deionized water accounts for 3% of the mass percentage of the enzymatic reaction system, while the other conditions are the same.

[0063] Comparative Example 7 Compared with Example 5, the difference is that the malt extract accounts for 2% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0064] Comparative Example 8 Compared with Example 5, the difference is that the malt extract accounts for 10% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0065] Comparative Example 9 Compared with Example 5, the difference is that the malt extract accounts for 12% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0066] Performance tests were conducted on Examples 5-7 and Comparative Examples 6-9, and the test results are shown in Table 2 below: Table 2 Performance test results of Examples 5-7 and Comparative Examples 6-9 As shown in Table 2, the optimal addition amount of malt extract for enzymatic hydrolysis is 6% (based on the total mass of the enzymatic hydrolysis reaction system), with a content range of 4%-8%. The precursor conversion efficiency is ≥58.3%, the quercetin content is ≥78.5mg / kg of raw material, and the 5α-reductase inhibition rate is ≥59.6%. If the addition amount is less than 4%, the precursor conversion efficiency is low, and if the addition amount is more than 8%, there is no additional efficacy improvement and the production cost is increased.

[0067] (2) Citrus fruit fermentation broth as enzyme activator The concentration of Platycladus orientalis leaf extract was fixed at 45% (based on the total mass ratio of the enzymatic hydrolysate / enzymatic reaction system). The addition rates of citrus fruit fermentation broth were set at gradients: 3%, 6%, 9%, 12%, 15%, and 18% (based on the total mass of the enzymatic hydrolysate). The control group consisted of unenzymatically hydrolyzed Platycladus orientalis leaf extract (45%, with deionized water used as the enzymatic hydrolysate in the same proportion). Three replicates were set for each group, and the average result was taken. Details are as follows: Example 8 An enzymatic reaction system is provided for the enzymatic hydrolysis of glycoside precursors in Platycladus orientalis leaf extract. The system comprises Platycladus orientalis leaf extract and an enzyme activator, wherein the enzyme activator is citrus fruit fermentation broth, and the citrus fruit fermentation broth accounts for 9% of the mass percentage of the enzymatic reaction system. The Platycladus orientalis leaf extract accounts for 45% of the mass percentage of the enzymatic reaction system.

[0068] A method for enzymatically hydrolyzing Platycladus orientalis leaf extract using an enzymatic reaction system includes the following steps: mixing Platycladus orientalis leaf extract with the aforementioned enzymatic reaction system, reacting under enzymatic hydrolysis conditions to obtain the enzymatic hydrolysis product; wherein the enzymatic hydrolysis conditions include: an enzymatic hydrolysis temperature of 36°C and an enzymatic hydrolysis time of 5 hours.

[0069] Example 9 Compared with Example 8, the difference is that the citrus fruit fermentation broth accounts for 6% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0070] Example 10 Compared with Example 8, the difference is that the citrus fruit fermentation broth accounts for 12% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0071] Comparative Example 10 Compared with Example 8, the difference is that deionized water is used instead of citrus fruit fermentation broth in the enzymatic reaction system, and the deionized water accounts for 3% of the mass percentage of the enzymatic reaction system, while the other conditions are the same.

[0072] Comparative Example 11 Compared with Example 8, the difference is that the citrus fruit fermentation broth accounts for 3% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0073] Comparative Example 12 Compared with Example 8, the difference is that the citrus fruit fermentation broth accounts for 15% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0074] Comparative Example 13 Compared with Example 8, the difference is that the citrus fruit fermentation broth accounts for 18% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0075] Performance tests were conducted on Examples 8-10 and Comparative Examples 10-13, and the test results are shown in Table 3 below: Table 3 Performance test results of Examples 8-10 and Comparative Examples 10-13 As shown in Table 3, the optimal addition amount of citrus fruit fermentation broth for enzymatic hydrolysis (based on the total mass of the enzymatic hydrolysis reaction system) is 9%, with a content range of 6%-12%. Within this range, the precursor conversion efficiency is ≥52.3%, the quercetin content is ≥68.5 mg / kg of raw material, and the 5α-reductase inhibition rate is ≥55.2%. When the addition amount is less than 6%, the enzyme activity is insufficient, and when it is more than 12%, the storage stability decreases and the irritation increases slightly due to excessive enzyme content.

[0076] (3) Using a combination of malt extract and citrus fruit fermentation broth as an enzyme activator. Based on the optimal results for single raw materials, the arborvitae leaf extract was fixed at 45% (the total mass percentage of the enzymatic hydrolysate / enzymatic reaction system). Using the mass percentages of malt extract (A: 4%, 6%, 8%) and citrus fruit fermentation broth (B: 6%, 9%, 12%) in the enzymatic reaction system as factors, an L9(3) model was designed. 2 Orthogonal experiment. The core evaluation index is the precursor conversion efficiency. Details are as follows: Example 11 An enzymatic reaction system is provided for the enzymatic hydrolysis of glycoside precursors in Platycladus orientalis leaf extract. The system comprises Platycladus orientalis leaf extract and an enzyme activator, wherein the enzyme activator is a combination of malt extract and citrus fruit fermentation broth. The malt extract accounts for 6% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 9% of the mass percentage of the enzymatic reaction system. The Platycladus orientalis leaf extract accounts for 45% of the mass percentage of the enzymatic reaction system.

[0077] A method for enzymatically hydrolyzing Platycladus orientalis leaf extract using an enzymatic reaction system includes the following steps: mixing Platycladus orientalis leaf extract with the aforementioned enzymatic reaction system, reacting under enzymatic hydrolysis conditions to obtain the enzymatic hydrolysis product; wherein the enzymatic hydrolysis conditions include: an enzymatic hydrolysis temperature of 38°C and an enzymatic hydrolysis time of 5 hours.

[0078] Example 12 Compared with Example 11, the difference is that the malt extract accounts for 4% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 6% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0079] Example 13 Compared with Example 11, the difference is that the malt extract accounts for 4% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 9% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0080] Example 14 Compared with Example 11, the difference is that the malt extract accounts for 4% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 12% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0081] Example 15 Compared with Example 11, the difference is that the malt extract accounts for 6% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 6% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0082] Example 16 Compared with Example 11, the difference is that the malt extract accounts for 6% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 12% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0083] Example 17 Compared with Example 11, the difference is that the malt extract accounts for 8% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 6% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0084] Example 18 Compared with Example 11, the difference is that the malt extract accounts for 8% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 9% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0085] Example 19 Compared with Example 11, the difference is that the malt extract accounts for 9% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 12% of the mass percentage of the enzymatic reaction system, while all other conditions are the same.

[0086] Performance tests were conducted on Examples 11-19, and the test results are shown in Table 4 below: Table 4 Performance test results of Examples 11-19 As shown in Table 4, the optimal ratio of malt extract to citrus fruit fermentation broth is 6%:9% (45% Platycladus orientalis leaf extract, based on the total mass of the enzymatic hydrolysate), with a compound content range of 4%-8%:6%-12%. When the malt extract constitutes 6% of the enzymatic reaction system and the citrus fruit fermentation broth constitutes 9%, a synergistic enzymatic activity effect is achieved, resulting in a precursor conversion efficiency of 82.6%, a quercetin content of 118.5 mg / kg of raw material, and a 5α-reductase inhibition rate of 82.5%. The enzymatic hydrolysis mechanisms are complementary (the β-glucosidase of the malt extract targets and hydrolyzes flavonoid glycosides, while the pectinase of the citrus fruit fermentation broth disrupts the cell walls of Platycladus orientalis leaves).

[0087] III. Enzymatic reaction system using a combination of Aspergillus niger-derived β-glucosidase, malt extract, and citrus fruit fermentation broth as enzyme activators. Example 20 An enzymatic reaction system is provided for the enzymatic hydrolysis of glycoside precursors in Platycladus orientalis leaf extract. The system comprises Platycladus orientalis leaf extract and an enzyme activator. The enzyme activator consists of β-glucosidase derived from Aspergillus niger, malt extract, and citrus fruit fermentation broth. The β-glucosidase from Aspergillus niger accounts for 0.20% of the mass of the enzymatic reaction system, and the mass ratio of the β-glucosidase from Aspergillus niger to Platycladus orientalis leaf extract is 1:15. The malt extract accounts for 6% of the mass of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 9% of the mass of the enzymatic reaction system. The Platycladus orientalis leaf extract accounts for 45% of the mass of the enzymatic reaction system.

[0088] A method for enzymatically hydrolyzing Platycladus orientalis leaf extract using an enzymatic reaction system includes the following steps: mixing Platycladus orientalis leaf extract with the aforementioned enzymatic reaction system, reacting under enzymatic hydrolysis conditions to obtain the enzymatic hydrolysis product; wherein the enzymatic hydrolysis conditions include: an enzymatic hydrolysis temperature of 38°C and an enzymatic hydrolysis time of 5 hours.

[0089] Performance tests were conducted on Example 20, and the results are shown in Table 5 below: Table 5 Performance test results of Example 20 IV. An enzymatic reaction system in which β-glucosidase and cellulase complex from almonds, or their complex, replace β-glucosidase from Aspergillus niger, and is combined 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.

[0090] 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.

[0091] Example 21 An enzymatic reaction system is provided for the enzymatic hydrolysis of glycoside precursors in Platycladus orientalis leaf extract, comprising an enzyme activator and 0.01 mmol / L Mn. 2+ (Activator), the enzyme activator is composed of almond-derived β-glucosidase, malt extract, and citrus fruit fermentation broth; the almond-derived β-glucosidase accounts for 0.20% of the mass percentage of the enzymatic reaction system, the malt extract accounts for 6% of the mass percentage of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 9% of the mass percentage of the enzymatic reaction system. The arborvitae leaf extract accounts for 45% of the mass percentage of the enzymatic reaction system, with the remainder being deionized water.

[0092] A method for enzymatically hydrolyzing Platycladus orientalis leaf extract using an enzymatic reaction system includes the following steps: mixing Platycladus orientalis leaf extract with the aforementioned enzymatic reaction system, reacting under enzymatic hydrolysis conditions to obtain the enzymatic hydrolysis product; wherein the enzymatic hydrolysis conditions include: pH 5.2~5.6, enzymatic hydrolysis temperature of 40℃, and enzymatic hydrolysis time of 5 hours.

[0093] Example 22 An enzymatic reaction system is provided for the enzymatic hydrolysis of glycoside precursors in Platycladus orientalis leaf extract. The system includes an enzyme activator composed of a cellulase complex, malt extract, and citrus fruit fermentation broth. The cellulase complex comprises 0.40% by mass of the enzymatic reaction system, the malt extract comprises 6% by mass, and the citrus fruit fermentation broth comprises 9% by mass. The Platycladus orientalis leaf extract comprises 45% by mass, with the remainder being deionized water.

[0094] A method for enzymatically hydrolyzing Platycladus orientalis leaf extract using an enzymatic reaction system includes the following steps: mixing Platycladus orientalis leaf extract with the aforementioned enzymatic reaction system, reacting under enzymatic hydrolysis conditions to obtain the enzymatic hydrolysis product; wherein the enzymatic hydrolysis conditions include: pH 4.8~5.3, enzymatic hydrolysis temperature of 42℃, and enzymatic hydrolysis time of 5 hours.

[0095] Example 23 An enzymatic reaction system is provided for the enzymatic hydrolysis of glycoside precursors in Platycladus orientalis leaf extract, comprising an enzyme activator and 0.01 mmol / L Mn. 2+ (Activator), the enzyme activator is composed of almond-derived β-glucosidase, cellulase complex enzyme, malt extract, and citrus fruit fermentation broth. The almond-derived β-glucosidase accounts for 0.10% of the mass of the enzymatic reaction system, the cellulase complex enzyme accounts for 0.20% of the mass of the enzymatic reaction system, the malt extract accounts for 6% of the mass of the enzymatic reaction system, and the citrus fruit fermentation broth accounts for 9% of the mass of the enzymatic reaction system. The arborvitae leaf extract accounts for 45% of the mass of the enzymatic reaction system, with the remainder being deionized water.

[0096] A method for enzymatically hydrolyzing Platycladus orientalis leaf extract using an enzymatic reaction system includes the following steps: mixing Platycladus orientalis leaf extract with the aforementioned enzymatic reaction system, reacting under enzymatic hydrolysis conditions to obtain the enzymatic hydrolysis product; wherein the enzymatic hydrolysis conditions include: pH 5.0~5.4, enzymatic hydrolysis temperature of 40℃, and enzymatic hydrolysis time of 5 hours.

[0097] Performance tests were conducted on Examples 21-23, and the test results are shown in Table 6 below: Table 6 Performance test results of Examples 21-23 The test results in Table 6 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.

[0098] 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. An enzymatic reaction system for enzymatically hydrolyzing glycoside precursors in Platycladus orientalis leaf extract, characterized in that, It contains arborvitae leaf extract and 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.

2. The enzyme-catalyzed reaction system according to claim 1, characterized in that, The enzyme activator is β-glucosidase derived from Aspergillus niger, and the β-glucosidase derived from Aspergillus niger accounts for 0.20-0.23% of the mass of the enzyme-catalyzed reaction system.

3. The enzyme-catalyzed reaction system according to claim 1, characterized in that, The mass ratio of the β-glucosidase derived from Aspergillus niger to the extract of Platycladus orientalis leaves is 1:(10-20).

4. The enzyme-catalyzed reaction system according to claim 1, characterized in that, The enzyme activator is selected from at least one of malt extract and citrus fruit fermentation broth; when the enzyme activator contains malt extract, the malt extract accounts for 4%-8% of the mass percentage of the enzyme-catalyzed reaction system; when the enzyme activator contains citrus fruit fermentation broth, the citrus fruit fermentation broth accounts for 6%-12% of the mass percentage of the enzyme-catalyzed reaction system.

5. The enzyme-catalyzed reaction system according to claim 1, characterized in that, The enzyme activator is composed of β-glucosidase from Aspergillus niger, malt extract, and citrus fruit fermentation broth. The β-glucosidase from Aspergillus niger accounts for 0.20-0.23% of the mass of the enzyme reaction system, the malt extract accounts for 4%-8% of the mass of the enzyme reaction system, and the citrus fruit fermentation broth accounts for 6%-12% of the mass of the enzyme reaction system.

6. The enzyme-catalyzed reaction system according to claim 1, characterized in that, The enzyme activator is composed of almond-derived β-glucosidase, malt extract, and citrus fruit fermentation broth. The almond-derived β-glucosidase accounts for 0.10-0.20% of the mass of the enzyme-catalyzed reaction system, the malt extract accounts for 4%-8% of the mass of the enzyme-catalyzed reaction system, and the citrus fruit fermentation broth accounts for 6%-12% of the mass of the enzyme-catalyzed reaction system.

7. The enzyme-catalyzed reaction system according to claim 1, characterized in that, The enzyme activator is composed of a cellulase complex, malt extract and citrus fruit fermentation broth. The cellulase complex accounts for 0.20-0.40% of the enzymatic reaction system by mass, the malt extract accounts for 4%-8% of the enzymatic reaction system by mass, and the citrus fruit fermentation broth accounts for 6%-12% of the enzymatic reaction system by mass.

8. The enzyme-catalyzed reaction system according to claim 1, characterized in that, The enzyme activator is composed of almond-derived β-glucosidase, cellulase complex enzyme, malt extract, and citrus fruit fermentation broth. The almond-derived β-glucosidase accounts for 0.10-0.20% of the mass of the enzyme-catalyzed reaction system, the cellulase complex enzyme accounts for 0.20-0.40% of the mass of the enzyme-catalyzed reaction system, the malt extract accounts for 4%-8% of the mass of the enzyme-catalyzed reaction system, and the citrus fruit fermentation broth accounts for 6%-12% of the mass of the enzyme-catalyzed reaction system.

9. The enzyme-catalyzed reaction system according to claim 1, characterized in that, The arborvitae leaf extract accounts for 3-45% of the mass of the enzymatic reaction system.

10. A method for enzymatically hydrolyzing an extract from Platycladus orientalis leaves, characterized in that, Includes the following steps: The extract of Platycladus orientalis leaves is mixed with the enzymatic reaction system according to any one of claims 1-9, and the reaction is carried out under enzymatic hydrolysis conditions to obtain the enzymatic hydrolysis product.

11. The method for enzymatically hydrolyzing Platycladus orientalis leaf extract according to claim 10, characterized in that, The enzymatic hydrolysis conditions include: an enzymatic hydrolysis temperature of 25-42℃ and an enzymatic hydrolysis time of 4-7 hours.

12. An enzymatic hydrolysis product of Platycladus orientalis leaves, characterized in that, It is prepared by the method of enzymatic hydrolysis of Platycladus orientalis leaf extract as described in any one of claims 10 or 11.

13. The application of the enzymatic hydrolysate of Platycladus orientalis leaves as described in claim 12 in the preparation of cosmetics with anti-hair loss function.

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