Extraction method and application of effective components of Gentiana macrophylla

Abstract

This invention belongs to the field of Gentiana macrophylla extraction technology, specifically a method for extracting the effective components of Gentiana macrophylla. The method includes pulverizing dried Gentiana macrophylla raw material and passing it through a 40-80 mesh sieve to obtain Gentiana macrophylla powder; performing compound enzymatic hydrolysis pretreatment; feeding the powder into an ultra-high pressure microfluidic homogenizer; circulating the powder 1-3 times under a pressure of 150-250 MPa, controlling the temperature during the treatment process to be below 40℃; centrifuging the treated material, collecting the supernatant, concentrating under reduced pressure, and drying to obtain the Gentiana macrophylla effective component extract. This method can simultaneously and efficiently extract multiple major medicinal components with different polarities, avoiding the cumbersome step-by-step extraction process, simplifying the process, and improving efficiency. The invention also includes the application of this method for extracting effective components, including iridoid glycosides and alkaloids, using the aforementioned method. These effective component extracts can be applied to products including face creams, essences, and masks.

Description

Technical Field

[0001] This invention relates to the field of Gentiana macrophylla extraction technology, specifically to the extraction method and application of the effective components of Gentiana macrophylla. Background Technology

[0002] Gentiana macrophylla is a plant of the Gentianaceae family. Its active ingredients mainly include iridoid glycosides such as gentiopicrin and swertiamarin, as well as alkaloids such as gentianine A, which have important pharmacological activities such as anti-inflammatory, analgesic, and antirheumatic effects.

[0003] Currently, the main extraction methods for the effective components of Gentiana macrophylla include solvent reflux extraction, ultrasonic-assisted extraction, and microwave-assisted extraction.

[0004] Solvent reflux extraction involves high temperatures and long extraction times, easily leading to the decomposition and inactivation of heat-sensitive components, and also consumes a lot of energy. Although ultrasonic and microwave methods have improved efficiency to some extent, they still suffer from bottlenecks such as low extraction selectivity, incomplete cell wall disruption in herbs with tough cell walls, and difficulty in further improving the extraction rate of active ingredients. Therefore, this paper proposes an extraction method and application of active ingredients from Gentiana macrophylla to solve the above problems. Summary of the Invention

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] The extraction method of the effective components of Gentiana macrophylla includes the following steps:

[0007] 1) The dried Gentiana macrophylla raw material is pulverized and passed through a 40-80 mesh sieve to obtain Gentiana macrophylla powder;

[0008] 2) Compound enzymatic hydrolysis pretreatment

[0009] Mix Gentiana macrophylla powder with a buffer solution of pH 4.8-5.2 at a liquid-to-solid ratio of 10 mL:1 g to 20 mL:1 g, and preheat to 45-55℃. Then add 0.8%-1.5% by mass of a mixed enzyme of pectinase and β-mannanase, with a mass ratio of pectinase to β-mannanase of 1:0.5-1, and stir at low speed for 20-40 min for enzymatic hydrolysis. Next, add 1.0%-2.0% by mass of a complex cell wall lysin relative to the Gentiana macrophylla powder. The complex cell wall lysin consists of cellulase, xylanase, arabinofuranylase, and β-glucanase at a mass ratio of 4-5:2-3:1-2:1. Maintain the temperature at 50-55℃ and continue to stir at low speed for 50-80 min for enzymatic hydrolysis.

[0010] 3) Ethanol mixture

[0011] Mix the enzymatically hydrolyzed material from step 2) with an ethanol solution of 65%-75% by volume, and adjust the final liquid-solid ratio to 30 mL:1 g-50 mL:1 g to prepare a mixed slurry.

[0012] 4) Ultra-high pressure microjets extraction

[0013] The mixed slurry is fed into an ultra-high pressure micro-jet homogenizer and circulated 1-3 times under a pressure of 150-250 MPa, while controlling the temperature during the process to be below 40℃.

[0014] 5) Centrifuge the material after step 4), collect the supernatant, concentrate under reduced pressure, and dry to obtain the extract of effective components of Gentiana macrophylla.

[0015] A further technical solution is that the ultra-high pressure micro-jet homogenizer includes a power unit, a pressurization unit, and a diamond homogenization chamber. The power unit is a hydraulic pump, and the pressurization unit is a booster pump.

[0016] In a further technical solution, the power unit is used to continuously drive the plunger rod in the booster pump to reciprocate and draw in the material, so that the material enters the diamond homogenization chamber at high speed under high pressure and passes through multiple microporous channels with fixed geometric shapes. The material is subjected to the dynamic action of strong shearing, high-speed impact, cavitation explosion and instantaneous pressure release in a small space, which reduces the particle size of the material to obtain micron-sized or nano-sized material particles.

[0017] In a further technical solution, the ultra-high pressure micro-jet homogenizer adopts a Y-type or Z-type diamond interactive cavity with a microchannel aperture of 100-200 μm.

[0018] A further technical solution involves the following steps: In step 3) ultra-high pressure microjets extraction, the mixture pretreated by the compound enzymatic hydrolysis in step 2) is directly fed into an ultra-high pressure microjets homogenizer and circulated 1-2 times at a pressure reduced to 80-150 MPa. The microjets homogenizer employs a Y-shaped diamond interactive cavity with multi-stage tapered microchannels and a pore size of 75-150 μm. The mixture undergoes a combination of ultra-high shear rate, controllable cavitation effect, and instantaneous pressure release within the cavity. The temperature of the mixture is precisely controlled between 25-35°C throughout the process using an integrated closed-loop cooling system.

[0019] A further technical solution involves the spontaneous formation of nanoparticles containing fat-soluble active ingredients, using enzymatically hydrolyzed polysaccharide fragments as carriers, in the extract obtained by ultra-high pressure microfluidic extraction in step 3). The nanoparticles have a particle size distribution of 100-300 nm.

[0020] A further technical solution is that the effective components in the Gentiana macrophylla extract include iridoid glycosides and alkaloids; the iridoid glycosides include gentiopicrin, swertiamarin, and loganic acid; the alkaloids include gentianine A.

[0021] Another object of the present invention is to provide an application of a method for extracting the effective components of Gentiana macrophylla, comprising using the above-described method for extracting effective component extracts including iridoid glycosides and alkaloids, which are used in applications including face creams, essences and masks.

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] 1. The compound enzyme pretreatment can specifically and gently degrade the dense structures such as cellulose and pectin in the cell wall of Gentiana macrophylla, greatly reducing the difficulty and energy consumption of subsequent microfluidic disruption. The microfluidic flow provides strong mechanical force to completely break down the cell wall weakened by enzymatic hydrolysis, achieving almost complete release of intracellular components and significantly improving the extraction rate of key components such as gentiopicrin and gentianine A. The entire process is carried out at a low temperature below 55°C, completely avoiding the damage of high temperature to heat-sensitive components such as iridoid glycosides and alkaloids, and maximizing the preservation of the natural activity and efficacy of the components. The method of this invention can simultaneously and efficiently extract multiple major medicinal components with different polarities, avoiding the cumbersome process of stepwise extraction, simplifying the process and improving efficiency. The method of this invention uses relatively little solvent, and the ethanol can be recycled. The compound enzyme is a biocatalyst, which is environmentally friendly and in line with the development direction of green processing.

[0024] 2. In the compound enzymatic pretreatment, β-mannanase, xylanase, and arabinofuranase are introduced. These enzymes are designed to target key components of hemicellulose in the cell wall of Gentiana macrophylla, such as glucomannan, xyloglucan, and arabinoxylan. Existing technologies mostly use general cellulase and pectinase, while this invention is the first to explicitly propose using a compound enzyme that specifically targets hemicellulose based on the chemical composition analysis of Gentiana macrophylla cell wall, achieving a leap from "general hydrolysis" to "precise deconstruction".

[0025] The method of "softening first, then deconstructing" first uses pectinase and mannanase to "dismantle" the "cement" between cells and the outer barrier; then a complex enzyme is used to attack the main structure of the cell wall. This temporal optimization simulates the natural process of biodegradation and can more efficiently and thoroughly deconstruct the cell wall. Its effect is far superior to the conventional method of adding all enzymes at once.

[0026] Furthermore, the introduction of low-frequency ultrasound assistance, but the key lies in its extremely low power density (below 100 W / L) and intermittent mode, which micro-disturbs the liquid flow, breaks the concentration boundary layer between the enzyme and the substrate, and increases the collision probability; it causes the enzyme molecules to vibrate slightly, which may make their conformation more conducive to binding with the substrate; it avoids the cell disruption and heat generation effects commonly found in ultrasound extraction; and it makes the synergy of "enzyme hydrolysis-ultrasound" a pure enhancement of biochemical reaction, rather than physical extraction, which is strictly different from existing ultrasound-assisted extraction technologies and perfectly meets the requirement of "deconstruction under mild conditions";

[0027] The cell wall is more thoroughly deconstructed, the number of subsequent microfluidic steps is reduced, and energy consumption will be further reduced, thereby improving the extraction rate of effective components of Gentiana macrophylla and the extraction rate of components deeply wrapped in the cell wall.

[0028] 3. The core function of the secondary cell wall lysin combination is to attack the load-bearing skeleton of the cell wall. A high proportion of endonuclease randomly endonucleates cellulose chains, shortening them; xylanase hydrolyzes xylan interwoven with cellulose microfibrils; the addition of β-glucosidase can hydrolyze cellobiose on the one hand, preventing product inhibition, and on the other hand, it can attack certain specific β-glycosidic bonds. Working synergistically with endonuclease, it "chiseles" a large number of micropores and weak points in the dense cellulose-hemicellulose network, greatly reducing the mechanical strength of the cell wall, making it "full of holes" but still largely intact in shape.

[0029] Because the cell wall has been deeply etched, its shear strength has decreased dramatically, and the pressure required for subsequent microfluidic homogenization can be reduced from the conventional pressure of over 200 MPa to 80-120 MPa. Low-pressure operation means less equipment wear, lower energy consumption, and less heat generation, better protection of heat-sensitive components, and significantly improved production safety. This results in a significant reduction in the working pressure of the microfluidic process.

[0030] Materials that have not undergone such specific enzymatic hydrolysis typically require 3-4 cycles of microfluidic treatment to be completely broken down; however, materials treated in step one, which specifically dissociates the intercellular layer and the outer layer of the primary cell wall, and in step two, which deeply etches the inner layer of the primary cell wall and the secondary cell wall, are extremely fragile and can achieve the same or even higher breakage rate in just 1-2 cycles, thus multiplying the extraction efficiency; achieving a reduction in the number of microfluidic treatments and a doubling of efficiency;

[0031] After specific dissociation of the intercellular layer and the outer layer of the primary cell wall in step one, and deep etching of the inner layer of the primary cell wall and the secondary cell wall in step two, the extract component spectrum is more complete and the activity is higher. In terms of physical effects, the low-pressure, short-time microjet treatment produces a smaller instantaneous temperature rise, maximizing the protection of heat-sensitive components. In terms of biochemical effects, this gentle fragmentation method avoids the damage that high-intensity shear forces may cause to the structure of small molecule compounds such as certain aglycones and alkaloids, while also reducing the opportunity for intracellular oxidases such as polyphenol oxidase to come into contact with the substrate, thereby better preserving the spectrum of active ingredients of the original medicinal material, reducing the generation of oxidation byproducts, and resulting in a lighter color and higher activity of the extract.

[0032] After specific dissociation of the intercellular layer and the outer layer of the primary cell wall in step one, and deep etching of the inner layer of the primary cell wall and the secondary cell wall in step two, a nanoscale self-assembled carrier system is formed. Water-soluble polysaccharide fragments such as oligosaccharides and oligosaccharides produced by enzymatic hydrolysis and lipid-soluble components such as some alkaloids and volatile oils after microfluidic nano-sizing may spontaneously form a unique plant-derived nanocarrier system during high-speed collision. That is, water-soluble polysaccharides act as carriers to encapsulate lipid-soluble active ingredients, which greatly improves the bioavailability of these poorly soluble components. This is a novel structure and effect that cannot be produced by simply mixing two technologies and is completely unexpected.

[0033] 4. After specific enzymatic hydrolysis by a mixture of pectinase and β-mannanase, and a complex cell wall lysin composed of cellulase, xylanase, arabinofuranase, and β-glucanase, the mechanical structure of the Gentiana macrophylla cell wall is biochemically pre-set, becoming fragile and porous. The ultra-high pressure microfluidic extraction section of this application does not use violent crushing, but rather utilizes relatively mild low-pressure conditions to precisely trigger and assemble the pre-set fragile structure in situ. Through the precise coupling of low-pressure, low-temperature, and few-time microfluidic conditions with pre-deep enzymatic hydrolysis, the triple goals of energy saving and high efficiency, ultimate protection, and functional creation are achieved. The synergistic cell wall breaking strategy of biological enzymatic hydrolysis and physical microfluidic hydrolysis is not a simple superposition of two technologies, but rather a qualitative change is triggered by redefining the operating parameters of the microfluidic hydrolysis and transforming the role from a disruptor to a trigger and assembler. Attached Figure Description

[0034] Figure 1 Statistical analysis of the extraction of various effective components of Gentiana macrophylla using experimental groups 1-4 and control groups 1-3;

[0035] Figure 2 Statistical analysis of the extraction of effective components from Gentiana macrophylla using experimental groups 1-4 and control groups 1-3;

[0036] Figure 3 Statistical analysis of the average particle size of the effective components of Gentiana macrophylla in experimental groups 1-4 and control groups 1-3;

[0037] Figure 4 Statistical analysis of DPPH scavenging rate of active ingredients in Gentiana macrophylla using experimental groups 1-4 and control groups 1-3;

[0038] Figure 5 Statistical analysis of the total phenol content of the effective components of Gentiana macrophylla in experimental groups 1-4 and control groups 1-3;

[0039] Figure 6 The graph shows the statistical analysis of the total flavonoid content of the effective components of Gentiana macrophylla in experimental groups 1-4 and control groups 1-3. Detailed Implementation

[0040] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1

[0042] The extraction method of the effective components of Gentiana macrophylla includes the following steps:

[0043] 1) The dried Gentiana macrophylla raw material is pulverized and passed through a 40-80 mesh sieve to obtain Gentiana macrophylla powder;

[0044] 2) Compound enzymatic hydrolysis pretreatment

[0045] Mix Gentiana macrophylla powder with a buffer solution of pH 4.8-5.2 at a liquid-to-solid ratio of 10 mL:1 g to 20 mL:1 g, and preheat to 45-55℃. Then add 0.8%-1.5% by mass of a mixed enzyme of pectinase and β-mannanase, with a mass ratio of pectinase to β-mannanase of 1:0.5-1, and stir at low speed for 20-40 min for enzymatic hydrolysis. Next, add 1.0%-2.0% by mass of a complex cell wall lysin relative to the Gentiana macrophylla powder. The complex cell wall lysin consists of cellulase, xylanase, arabinofuranylase, and β-glucanase at a mass ratio of 4-5:2-3:1-2:1. Maintain the temperature at 50-55℃ and continue to stir at low speed for 50-80 min for enzymatic hydrolysis.

[0046] Therefore, β-mannanase, xylanase, and arabinofuranase were introduced. These enzymes are designed to target key components of hemicellulose in the cell wall of Gentiana macrophylla, such as glucomannan, xyloglucan, and arabinoxylan. Existing technologies mostly use general cellulase and pectinase, while this invention is the first to explicitly propose using a complex enzyme that specifically targets hemicellulose based on the chemical composition analysis of Gentiana macrophylla cell wall, achieving a leap from "general hydrolysis" to "precise deconstruction".

[0047] 3) Ethanol mixture

[0048] Mix the enzymatically hydrolyzed material from step 2) with an ethanol solution of 65%-75% by volume, and adjust the final liquid-solid ratio to 30 mL:1 g-50 mL:1 g to prepare a mixed slurry.

[0049] 4) Ultra-high pressure microjets extraction

[0050] The mixed slurry is fed into an ultra-high pressure micro-jet homogenizer and circulated 1-3 times under a pressure of 150-250 MPa, while controlling the temperature during the process to be below 40℃.

[0051] 5) Centrifuge the material after step 4), collect the supernatant, concentrate under reduced pressure, and dry to obtain the extract of effective components of Gentiana macrophylla.

[0052] The compound enzyme pretreatment can specifically and gently degrade the dense structures such as cellulose and pectin in the cell wall of Gentiana macrophylla, greatly reducing the difficulty and energy consumption of subsequent microfluidic disruption. The microfluidic flow provides strong mechanical force to completely break down the cell wall weakened by enzymatic hydrolysis, achieving near-complete release of intracellular components and significantly improving the extraction rate of key components such as gentiopicrin and gentianine A. The entire process is carried out at a low temperature below 55°C, completely avoiding the damage of heat-sensitive components such as iridoid glycosides and alkaloids caused by high temperatures, and maximizing the preservation of the natural activity and efficacy of the components. The method of this invention can simultaneously and efficiently extract multiple major medicinal components with different polarities, avoiding the cumbersome process of stepwise extraction, simplifying the process and improving efficiency. The method of this invention uses relatively little solvent, and the ethanol can be recycled. The compound enzyme is a biocatalyst, which is environmentally friendly and in line with the development direction of green processing.

[0053] Example 2

[0054] The only difference between this embodiment and Embodiment 1 is that step 2) of the compound enzymatic hydrolysis pretreatment in Embodiment 1 is improved.

[0055] Step 2) of the compound enzymatic hydrolysis pretreatment includes the following steps:

[0056] Step 1: Softening of the intercellular matrix

[0057] Gentian root powder was mixed with a citric acid and disodium hydrogen phosphate buffer solution at pH 4.8-5.2 at a liquid-to-solid ratio of 12 mL:1 g to 15 mL:1 g, stirred until homogeneous, and preheated to 48-52℃. Then, a mixed enzyme of pectinase and β-mannanase was added at a relative mass of 0.8%-1.5% of the gentian root powder, with a mass ratio of pectinase to β-mannanase of 1:0.5-1. The mixture was stirred at low speed for 20-40 min for enzymatic hydrolysis. This step aims to preferentially degrade pectin in the intercellular layer connecting cells and mannan in the cell wall, softening the tissue and opening channels for subsequent enzymes.

[0058] Step 2: In-depth deconstruction of the cell wall

[0059] Add 1.0%-2.0% by mass of a complex cell wall lysin directly to the mixture from step one. The complex cell wall lysin is composed of cellulase, xylanase, arabinofuranase, and β-glucanase in a mass ratio of 4-5:2-3:1-2:1. Maintain the temperature at 50-55℃ and continue to stir at low speed for 50-80 minutes for enzymatic hydrolysis.

[0060] Step 3: Gentle Physical Field Assistance

[0061] Simultaneously with step two, low-frequency ultrasound of 20-40 kHz is applied to the reaction system, with the ultrasonic power density controlled below 100 W / L, using an intermittent mode of 2 seconds of operation followed by an 8-second pause. This extremely low-power ultrasound is only used to enhance mass transfer and enzyme-catalyzed reaction efficiency, and will not cause significant thermal effects or mechanical breakage. The system temperature is strictly kept below 55°C.

[0062] In step 2), the pretreatment with compound enzymes involves "softening first, then deconstructing" by first using pectinase and mannanase to "dismantle" the "cement" between cells and the outer barrier; then using compound enzymes to comprehensively attack the main structure of the cell wall. This temporal optimization simulates the natural process of biodegradation, which can more efficiently and thoroughly deconstruct the cell wall. Its effect is far superior to the conventional method of adding all enzymes at once.

[0063] Furthermore, the introduction of low-frequency ultrasound assistance, but the key lies in its extremely low power density (below 100 W / L) and intermittent mode, which micro-disturbs the liquid flow, breaks the concentration boundary layer between the enzyme and the substrate, and increases the collision probability; it causes the enzyme molecules to vibrate slightly, which may make their conformation more conducive to binding with the substrate; it avoids the cell disruption and heat generation effects commonly found in ultrasound extraction; and it makes the synergy of "enzyme hydrolysis-ultrasound" a pure enhancement of biochemical reaction, rather than physical extraction, which is strictly different from existing ultrasound-assisted extraction technologies and perfectly meets the requirement of "deconstruction under mild conditions";

[0064] The cell wall is more thoroughly deconstructed, the number of subsequent microfluidic steps is reduced, and energy consumption will be further reduced, thereby improving the extraction rate of effective components of Gentiana macrophylla and the extraction rate of components deeply encapsulated in the cell wall.

[0065] Example 3

[0066] The only difference between this embodiment and Embodiment 1 is that step 2) of the compound enzymatic hydrolysis pretreatment in Embodiment 1 is improved.

[0067] Step 2) of the compound enzymatic hydrolysis pretreatment includes the following steps:

[0068] Step 1: Specific dissociation of the middle lamella and the outer layer of the primary cell wall

[0069] Gentian root powder was mixed with an acetic acid and sodium acetate buffer solution with a pH of 5.0-5.5 at a liquid-solid ratio of 10 mL:1 g to 15 mL:1 g, and preheated to 45-50℃. Then, 1.0%-1.8% of primary wall dissociation enzyme synthesizer (composed of pectinase, β-mannanase, and arabinofuranosides in a mass ratio of 3-4:2-3:1) was added relative to the mass of Gentian root powder. The mixture was then stirred at 200 rpm for 40-60 min.

[0070] The enzyme specifically targets the pectin-rich intercellular layer at the corners of the cell and the hemicellulose network of the primary cell wall, which is rich in glucomannan and arabinoxylan side chains. The addition of arabinofuranase is key, as it can efficiently cleave the main and side chain connections of arabinoxylan, greatly weakening the stability of the hemicellulose network. It precisely dismantles the "reinforced concrete" between cells and the external "scaffolding", making the cell structure loose but not broken.

[0071] Step 2: Deep etching of the primary wall inner layer and secondary wall

[0072] After completing step one, without changing the pH and temperature of the system, directly add 1.5%-2.5% of the secondary wall lysin mixture relative to the mass of Gentiana macrophylla powder. The secondary wall lysin mixture consists of endo-β-1,4-glucanase, xylanase, and β-glucosidase in a mass ratio of 5-6:3-4:1. Continue stirring at low speed for 60-90 minutes.

[0073] The core function of the secondary cell wall lysin combination is to attack the load-bearing skeleton of the cell wall. A high proportion of endonuclease randomly endonucleates cellulose chains, shortening them; xylanase hydrolyzes xylan interwoven with cellulose microfibrils; the addition of β-glucosidase can hydrolyze cellobiose on the one hand, preventing product inhibition, and on the other hand, it can attack certain specific β-glycosidic bonds. Working synergistically with endonuclease, it "chiseles" a large number of micropores and weak points in the dense cellulose-hemicellulose network, greatly reducing the mechanical strength of the cell wall, making it "riddled with holes" but still largely intact in shape.

[0074] Because the cell wall has been deeply etched, its shear strength has decreased dramatically, and the pressure required for subsequent microfluidic homogenization can be reduced from the conventional pressure of over 200 MPa to 80-120 MPa. Low-pressure operation means less equipment wear, lower energy consumption, and less heat generation, better protection of heat-sensitive components, and significantly improved production safety. This results in a significant reduction in the working pressure of the microfluidic process.

[0075] Materials that have not undergone such specific enzymatic hydrolysis typically require 3-4 cycles of microfluidic treatment to be completely broken down; however, materials treated in step one, which specifically dissociates the intercellular layer and the outer layer of the primary cell wall, and in step two, which deeply etches the inner layer of the primary cell wall and the secondary cell wall, are extremely fragile and can achieve the same or even higher breakage rate in just 1-2 cycles, thus multiplying the extraction efficiency; achieving a reduction in the number of microfluidic treatments and a doubling of efficiency;

[0076] After specific dissociation of the intercellular layer and the outer layer of the primary cell wall in step one, and deep etching of the inner layer of the primary cell wall and the secondary cell wall in step two, the extract component spectrum is more complete and the activity is higher. In terms of physical effects, the low-pressure, short-time microjet treatment produces a smaller instantaneous temperature rise, maximizing the protection of heat-sensitive components. In terms of biochemical effects, this gentle fragmentation method avoids the damage that high-intensity shear forces may cause to the structure of small molecule compounds such as certain aglycones and alkaloids, while also reducing the opportunity for intracellular oxidases such as polyphenol oxidase to come into contact with the substrate, thereby better preserving the spectrum of active ingredients of the original medicinal material, reducing the generation of oxidation byproducts, and resulting in a lighter color and higher activity of the extract.

[0077] After specific dissociation of the intercellular layer and the outer layer of the primary cell wall in step one, and deep etching of the inner layer of the primary cell wall and the secondary cell wall in step two, a nanoscale self-assembled carrier system is formed. Water-soluble polysaccharide fragments such as oligosaccharides and oligosaccharides produced by enzymatic hydrolysis and lipid-soluble components such as some alkaloids and volatile oils after microfluidic nano-sizing may spontaneously form a unique plant-derived nanocarrier system during high-speed collision. That is, water-soluble polysaccharides act as carriers to encapsulate lipid-soluble active ingredients, which greatly improves the bioavailability of these poorly soluble components. This is a novel structure and effect that cannot be produced by simply mixing two technologies.

[0078] Example 4

[0079] This embodiment adds the following compared to Embodiment 1:

[0080] The ultra-high pressure micro-jet homogenizer includes a power unit, a pressurization unit, and a diamond homogenization chamber. The power unit is a hydraulic pump, and the pressurization unit is a booster pump.

[0081] Furthermore, the power unit continuously drives the plunger rod in the booster pump to reciprocate, causing the material to enter the diamond homogenization chamber at high speed (hundreds of meters per second, or even more than 300 m / s) under high pressure, and pass through multiple microporous channels with fixed geometric shapes. The material is subjected to dynamic forces including strong shearing, high-speed impact, cavitation explosion and instantaneous pressure release in a small space, which reduces the particle size of the material to obtain micron-sized or nano-sized material particles.

[0082] Furthermore, the ultra-high pressure micro-jet homogenizer adopts a Y-type or Z-type diamond interactive cavity with a microchannel aperture of 100-200 μm.

[0083] Example 5

[0084] The only difference between this embodiment and Embodiment 1 is that step 3) of ultra-high pressure microjets extraction in Embodiment 1 is improved.

[0085] In step 3), the ultra-high pressure microjets extraction process involves directly feeding the mixture pretreated by the compound enzymatic hydrolysis in step 2) into an ultra-high pressure microjets homogenizer. The mixture is then circulated 1-2 times at a pressure reduced to 80-150 MPa. The microjets homogenizer employs a Y-shaped diamond interactive cavity with multi-stage tapered microchannels and a pore size of 75-150 μm. The mixture undergoes a combination of ultra-high shear rate (greater than 10^6 s⁻¹), controllable cavitation effect, and instantaneous pressure release within the cavity. The temperature of the mixture is precisely controlled between 25-35℃ throughout the process using an integrated closed-loop cooling system.

[0086] After specific enzymatic hydrolysis by a mixture of pectinase and β-mannanase, and a complex cell wall lysin composed of cellulase, xylanase, arabinofuranase, and β-glucanase, the mechanical structure of the Gentiana macrophylla cell wall is biochemically pre-set, becoming fragile and porous. The ultra-high pressure microfluidic extraction section of this application does not use violent crushing, but rather utilizes relatively mild low-pressure conditions to precisely trigger and assemble the pre-set fragile structure in situ. Through the precise coupling of low-pressure, low-temperature, and few-time microfluidic conditions with pre-deep enzymatic hydrolysis, the triple goals of energy saving and high efficiency, ultimate protection, and functional creation are achieved. The synergistic cell wall breaking strategy of biological enzymatic hydrolysis and physical microfluidic hydrolysis is not a simple superposition of two technologies, but rather a qualitative change is triggered by redefining the operating parameters of the microfluidic flow and transforming the microfluidic flow from a disruptor to a trigger and assembler.

[0087] The low-pressure and low-temperature (25-35℃) operating conditions almost completely eliminate the risk of thermal damage to extremely heat-sensitive components such as certain volatile oils and highly active enzymes; the gentle shear force tends to "tear" rather than "crush" the cell wall that has been weakened by enzymatic hydrolysis, which can release the cell contents more completely and reduce the phenomenon of inactivation due to the breakage of active molecular chains of macromolecular polysaccharides and proteins caused by excessive shearing.

[0088] Furthermore, in the extract obtained by ultra-high pressure microjets in step 3), nanoparticles containing lipid-soluble active ingredients are spontaneously formed, with enzymatically hydrolyzed polysaccharide fragments as carriers. The particle size distribution is 100-300 nm, and the in vitro dissolution rate of gentianine A can reach more than 90% within 15 min.

[0089] Functional polysaccharide fragments (such as oligosaccharides and arabinoxylan fragments) produced by specific enzymatic hydrolysis using a mixture of pectinase and β-mannanase, as well as a complex cell wall lysin composed of cellulase, xylanase, arabinofuranase, and β-glucanase, along with lipid-soluble active ingredients (such as Gentiana macrophylla alkaloids and terpenes) released from cells, undergo in-situ self-assembly under the influence of a unique multi-level physical field (high shear, cavitation, and collision) within the microfluidic chamber. The hydrophilic polysaccharide fragments, acting as natural carriers, spontaneously encapsulate hydrophobic active molecules through hydrophobic interactions and hydrogen bonds, forming uniformly sized (100-300 nm) and structurally stable "plant-derived nanoliposomes / micelles." This in-situ formed nanocarrier system significantly improves the bioavailability of poorly soluble active ingredients and achieves synergistic effects. This novel structure and function, which cannot be achieved by enzymatic hydrolysis or microfluidics alone, represents a breakthrough in the innovation of traditional Chinese medicine extract dosage forms.

[0090] Example 6

[0091] This embodiment adds the following compared to Embodiment 1:

[0092] The active ingredients in the Gentiana macrophylla extract include iridoid glycosides and alkaloids; the iridoid glycosides include gentiopicrin, swertiamarin, and loganic acid; the alkaloids include gentianine A.

[0093] Example 7

[0094] Another object of the present invention is to provide an application of a method for extracting the effective components of Gentiana macrophylla, comprising using the above-described method for extracting effective component extracts including iridoid glycosides and alkaloids, which are used in applications including face creams, essences and masks.

[0095] Experimental Protocol: Creative Verification of the Synergistic Extraction of Active Components from Gentiana macrophylla by Compound Enzymatic Hydrolysis and Microfluidic Flow

[0096] I. Experimental Objective

[0097] By systematically comparing the differences between the "compound enzymatic hydrolysis-microfluidic synergistic method" and traditional extraction methods in terms of extraction efficiency, component retention, and energy consumption, its technological innovation and significant advantages are demonstrated.

[0098] II. Experimental Standards

[0099] Methods for content determination under the Gentiana macrophylla entry in the 2020 edition of the Chinese Pharmacopoeia;

[0100] GB / T 35887-2018 General Technical Requirements for Ultrafine Grinding Equipment;

[0101] GB 5009.3-2016 National Food Safety Standard - Determination of Moisture in Food;

[0102] III. Experimental Materials and Equipment

[0103] 3.1 Experimental Materials

[0104] Gentiana macrophylla (from the same batch, all tested and certified as qualified);

[0105] Cellulase (enzyme activity ≥10,000 U / g);

[0106] Pectinase (enzyme activity ≥30,000 U / g);

[0107] β-glucanase (enzyme activity ≥20,000 U / g);

[0108] Ethanol (chromatographic grade);

[0109] Methanol (chromatographic grade);

[0110] Sodium dihydrogen phosphate, citric acid (analytical grade);

[0111] 3.2 Experimental Equipment

[0112] Ultra-high pressure micro-jet homogenizer (PSI-20 model);

[0113] High performance liquid chromatograph (Agilent 1260 Infinity II);

[0114] Laser particle size analyzer (Malvern Mastersizer 3000);

[0115] UV-Vis spectrophotometer;

[0116] Biochemical incubator;

[0117] Vacuum concentration unit;

[0118] IV. Experimental Methods

[0119] 4.1 Sample Pretreatment

[0120] Take the same batch of Gentiana macrophylla medicinal materials, wash them, dry them at 60℃ to constant weight, pulverize them and pass them through a 60-mesh sieve, mix them evenly and seal them for later use.

[0121] 4.2 Experimental Group Design

[0122] Experimental group 1 uses a combined enzymatic hydrolysis-microfluidic synergistic method, specifically Example 1, which involves accurately weighing 100.0g of Gentiana macrophylla powder for extraction;

[0123] Experimental group 2 uses a combined enzymatic hydrolysis-microfluidic synergistic method, specifically Example 2, which involves accurately weighing 100.0g of Gentiana macrophylla powder for extraction;

[0124] Experimental group 3 uses a combined enzymatic hydrolysis-microfluidic synergistic method, specifically Example 3, which involves accurately weighing 100.0g of Gentiana macrophylla powder for extraction;

[0125] Experimental group 4 uses a combined enzymatic hydrolysis-microfluidic synergistic method, specifically Example 5, which involves accurately weighing 100.0g of Gentiana macrophylla powder for extraction.

[0126] Comparison Group 1, Traditional Reflux Method

[0127] 1) Accurately weigh 100.0 g of Gentiana macrophylla powder;

[0128] 2) Add 4000 mL of 70% ethanol;

[0129] 3) Reflux extraction at 80℃ twice, 2 hours each time;

[0130] 4) Combine the extracts and filter;

[0131] 5) Concentrate under reduced pressure at 50℃;

[0132] 6) Freeze-dry to obtain the extract.

[0133] Comparison Group 2, Microjets Only

[0134] 1) Accurately weigh 100.0 g of Gentiana macrophylla powder;

[0135] 2) Add 4000 mL of 70% ethanol;

[0136] 3) Microfluidic treatment (200 MPa, 3 cycles);

[0137] 4) Same as the subsequent processing in Example 1;

[0138] Control group 3, enzymatic hydrolysis only

[0139] 1) Same as the enzymatic hydrolysis steps in Example 1;

[0140] 2) After enzymatic hydrolysis, directly centrifuge and concentrate;

[0141] 3) Same as the subsequent processing in Example 1;

[0142] 4.3 Detection Indicators and Methods

[0143] 4.3.1 Determination of extract yield

[0144] The yield was determined according to the method in GB 5009.3-2016: Yield = (weight of extract / weight of raw material) * 100%;

[0145] 4.3.2 Determination of active ingredient content

[0146] According to the 2020 edition of the Chinese Pharmacopoeia:

[0147] Column: Agilent ZORBAX SB-C18 (4.6×250mm, 5μm);

[0148] Mobile phase: methanol-water gradient elution;

[0149] Detection wavelengths: 270nm (gentiopicrin), 240nm (gentiopicrin A);

[0150] Column temperature: 30℃;

[0151] Flow rate: 1.0 mL / min;

[0152] 4.3.3 Particle size distribution determination

[0153] According to GB / T 35887-2018 method:

[0154] Instrument: Malvern Mastersizer 3000;

[0155] Dispersion medium: deionized water;

[0156] Measurement range: 0.01-3500μm;

[0157] 4.3.4 Antioxidant Activity Assay

[0158] DPPH free radical scavenging capacity determination: Scavenging rate (%) = (1 - Asample / Acontrol) * 100%;

[0159] V. Experimental Results and Data Analysis

[0160] 5.1 Comparison of extraction efficiency, detailed in Table 1 below;

[0161] Table 1

[0162] Group Extract yield (%) Gentianoside extraction rate (%) Gentianaemine A extraction rate (%) Total effective ingredient content (mg / g) Experimental group 1 33.5 93.5 87.5 190.2 Experimental group 2 34.2 94.3 88.9 191.3 Experimental group 3 34.3 94.2 88.7 191.1 Experimental group 4 33.8 93.7 88.5 190.7 Comparison Group 1 25.3 75.9 69.4 144.5 Comparison Group 2 29.1 87.1 81.6 166.8 Comparison Group 3 18.7 61.7 54.5 112.8

[0163] 5.2 Comparison of physical properties, detailed in Table 2 below;

[0164] Table 2

[0165] Group Average particle size (nm) D90(μm) Specific surface area (m² / g) Experimental group 1 160.8 0.45 12.8 Experimental group 2 155.3 0.45 13.2 Experimental group 3 153.2 0.45 13.4 Experimental group 4 154.8 0.45 13.1 Comparison Group 1 1250.6 15.2 1.8 Comparison Group 2 385.2 2.1 5.2 Comparison Group 3 985.4 8.7 2.5

[0166] 5.3 Comparison of activity retention, detailed in Table 3 below;

[0167] Table 3

[0168] Group DPPH scavenging rate (IC50, μg / mL) Total phenol content (mg GAE / g) Total flavonoid content (mg RE / g) Experimental group 1 28.5 45.6 32.8 Experimental group 2 28.9 45.4 32.5 Experimental group 3 28.2 45.1 32.1 Experimental group 4 28.4 45.5 32.7 Comparison Group 1 44.4 32.1 24.3 Comparison Group 2 34.1 39.4 29.5 Comparison Group 3 48.9 28.7 21.6

[0169] 5.4 Energy consumption and efficiency analysis, detailed in Table 4 below;

[0170] Table 4

[0171] Group Total time (min) Energy consumption (kWh) Solvent usage (mL / g) Experimental group 1 75 1.8 40 Experimental group 2 74 1.7 40 Experimental group 3 75 1.8 40 Experimental group 4 75 1.8 40 Comparison Group 1 240 5.2 80 Comparison Group 2 15 1.2 40 Comparison Group 3 120 0.8 40

[0172] Through Tables 1-4, and Figures 1-6 It can be known that:

[0173] SEI = (Experimental group extraction rate) / [(Control group 2 extraction rate + Control group 3 extraction rate) / 2]

[0174] Experimental group 1: Gentianoside SEI = 93.5 / [(87.1 + 61.7) / 2] = 1.26

[0175] Gentian alkaloid A SEI = 87.5 / [(81.6 + 54.5) / 2] = 1.29

[0176] Experimental group 2: Gentianoside SEI = 94.3 / [(87.1 + 61.7) / 2] = 1.27

[0177] Gentian alkaloid A SEI = 88.9 / [(81.6 + 54.5) / 2] = 1.30

[0178] Experimental group 3: Gentianoside SEI = 94.2 / [(87.1 + 61.7) / 2] = 1.27

[0179] Gentian alkaloid A SEI = 88.7 / [(81.6 + 54.5) / 2] = 1.30

[0180] Experimental group 4: Gentianoside SEI = 93.7 / [(87.1 + 61.7) / 2] = 1.26

[0181] Gentian root alkaloid A SEI = 88.5 / [(81.6 + 54.5) / 2] = 1.30

[0182] The SEI values ​​of experimental groups 1 through 4 were all significantly greater than 1, demonstrating a clear synergistic effect.

[0183] Furthermore, the extraction rate of effective components in experimental groups 1-4 was 8.6-12.3% higher than that of the best single method (comparison 1-3); the activity retention was enhanced, and the antioxidant activity was increased by 20-40%; energy consumption was reduced and time was shortened; and product quality was improved: smaller particle size and higher bioavailability.

[0184] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0185] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for extracting the effective components of Gentiana macrophylla, characterized in that, Includes the following steps: 1) The dried Gentiana macrophylla raw material is pulverized and passed through a 40-80 mesh sieve to obtain Gentiana macrophylla powder; 2) Compound enzymatic hydrolysis pretreatment Mix Gentiana macrophylla powder with a buffer solution of pH 4.8-5.2 at a liquid-to-solid ratio of 10 mL:1 g to 20 mL:1 g, and preheat to 45-55℃. Then add 0.8%-1.5% by mass of a mixed enzyme of pectinase and β-mannanase, with a mass ratio of pectinase to β-mannanase of 1:0.5-1, and stir at low speed for 20-40 min for enzymatic hydrolysis. Next, add 1.0%-2.0% by mass of a complex cell wall lysin relative to the Gentiana macrophylla powder. The complex cell wall lysin consists of cellulase, xylanase, arabinofuranylase, and β-glucanase at a mass ratio of 4-5:2-3:1-2:

1. Maintain the temperature at 50-55℃ and continue to stir at low speed for 50-80 min for enzymatic hydrolysis. 3) Ethanol mixture Mix the enzymatically hydrolyzed material from step 2) with an ethanol solution of 65%-75% by volume, and adjust the final liquid-solid ratio to 30mL:1g-50mL:1g to prepare a mixed slurry. 4) Ultra-high pressure microjets extraction The mixed slurry is fed into an ultra-high pressure micro-jet homogenizer and circulated 1-3 times under a pressure of 150-250 MPa, while controlling the temperature during the process to be below 40℃. 5) Centrifuge the material after step 4), collect the supernatant, concentrate under reduced pressure, and dry to obtain the extract of effective components of Gentiana macrophylla.

2. The method for extracting the effective components of Gentiana macrophylla according to claim 1, characterized in that, In step 2), the compound enzymatic hydrolysis pretreatment includes the following steps: Step 1: Softening of the intercellular matrix Mix Gentiana macrophylla powder with a citric acid and disodium hydrogen phosphate buffer solution with a pH of 4.8-5.2 at a liquid-solid ratio of 12 mL:1 g-15 mL:1 g, stir well, and preheat to 48-52℃; then add a mixed enzyme of pectinase and β-mannanase at a relative mass of 0.8%-1.5% of the Gentiana macrophylla powder, with a mass ratio of pectinase to β-mannanase of 1:0.5-1, and stir at low speed for 20-40 min for enzymatic hydrolysis. Step 2: In-depth deconstruction of the cell wall Add 1.0%-2.0% by mass of a complex cell wall lysin directly to the mixture from step one. The complex cell wall lysin is composed of cellulase, xylanase, arabinofuranase, and β-glucanase in a mass ratio of 4-5:2-3:1-2:

1. Maintain the temperature at 50-55℃ and continue to stir at low speed for 50-80 minutes for enzymatic hydrolysis. Step 3: Gentle Physical Field Assistance Simultaneously with step two, low-frequency ultrasound of 20-40 kHz is applied to the reaction system, with the ultrasonic power density controlled below 100 W / L, using an intermittent mode of 2 seconds of operation followed by an 8-second pause.

3. The method for extracting the effective components of Gentiana macrophylla according to claim 1, characterized in that, In step 2), the compound enzymatic hydrolysis pretreatment includes the following steps: Step 1: Specific dissociation of the middle lamella and the outer layer of the primary cell wall Gentiana macrophylla powder was mixed with an acetic acid and sodium acetate buffer solution with a pH of 5.0-5.5 at a liquid-solid ratio of 10 mL:1 g to 15 mL:1 g, and preheated to 45-50℃. Then, 1.0%-1.8% of primary wall dissociation enzyme synthesizer relative to the mass of Gentiana macrophylla powder was added. The primary wall dissociation enzyme synthesizer consisted of pectinase, β-mannanase, and arabinofuranosides at a mass ratio of 3-4:2-3:

1. The mixture was then stirred at a low speed of 200 rpm for 40-60 min. Step 2: Deep etching of the primary wall inner layer and secondary wall After completing step one, without changing the pH and temperature of the system, directly add 1.5%-2.5% of the secondary wall lysin mixture relative to the mass of Gentiana macrophylla powder. The secondary wall lysin mixture consists of endo-β-1,4-glucanase, xylanase, and β-glucosidase in a mass ratio of 5-6:3-4:

1. Continue stirring at low speed for 60-90 min.

4. The method for extracting the effective components of Gentiana macrophylla according to claim 1, 2, or 3, characterized in that, The ultra-high pressure micro-jet homogenizer includes a power unit, a pressurization unit, and a diamond homogenization chamber. The power unit is a hydraulic pump, and the pressurization unit is a booster pump.

5. The method for extracting the effective components of Gentiana macrophylla according to claim 4, characterized in that, The power unit is used to continuously drive the plunger rod in the booster pump to pump back and forth, so that the material enters the diamond homogenization chamber at high speed under high pressure and passes through multiple microporous channels with fixed geometric shapes. The material is subjected to the dynamic action of strong shearing, high-speed impact, cavitation explosion and instantaneous pressure release in a small space, which reduces the particle size of the material to obtain micron-sized or nano-sized material particles.

6. The method for extracting the effective components of Gentiana macrophylla according to claim 5, characterized in that, The ultra-high pressure micro-jet homogenizer adopts a Y-type or Z-type diamond interactive cavity with a microchannel aperture of 100-200 μm.

7. The method for extracting the effective components of Gentiana macrophylla according to claim 1, 2, or 3, characterized in that, In step 3), the ultra-high pressure microjets extraction process involves directly feeding the mixture pretreated by the compound enzymatic hydrolysis in step 2) into an ultra-high pressure microjets homogenizer. The mixture is then circulated 1-2 times at a pressure reduced to 80-150 MPa. The microjets homogenizer employs a Y-shaped diamond interactive cavity with multi-stage tapered microchannels and a pore size of 75-150 μm. The mixture undergoes a combination of ultra-high shear rate, controllable cavitation effect, and instantaneous pressure release within the cavity. The temperature of the mixture is precisely controlled between 25-35°C throughout the process using an integrated closed-loop cooling system.

8. The method for extracting the effective components of Gentiana macrophylla according to claim 7, characterized in that, In the extract obtained by ultra-high pressure microjets in step 3), nanoparticles spontaneously form, which encapsulate lipophilic active ingredients using enzymatically hydrolyzed polysaccharide fragments as carriers, with a particle size distribution of 100-300 nm.

9. The method for extracting the effective components of Gentiana macrophylla according to claim 1 and its application, characterized in that, The active ingredients in the Gentiana macrophylla extract include iridoid glycosides and alkaloids; the iridoid glycosides include gentiopicrin, swertiamarin, and loganic acid; the alkaloids include gentianine A.

10. An application of a method for extracting the effective components of Gentiana macrophylla, characterized in that, This includes extracting an effective ingredient extract containing iridoid glycosides and alkaloids using the extraction method of Gentiana macrophylla as described in any one of claims 1-9, and applying the effective ingredient extract to fields including face creams, essences and masks.

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