Method for preparing penthorum chinense pursh glycoside A through liquid nitrogen combined gradient temperature difference steady state and application of penthorum chinense pursh glycoside A

Abstract

The invention belongs to the technical field of food science and engineering, and discloses a method for preparing penthorum chinense pursh glycoside A by combining liquid nitrogen with gradient temperature difference steady state and application, and the method comprises the following steps: treating fresh penthorum chinense pursh with liquid nitrogen, adding a compound protective agent, and ball-milling and crushing the penthorum chinense pursh to obtain frozen penthorum chinense pursh powder; synthesizing a choline chloride-alpha-L-rhamnose natural deep eutectic solvent; mixing the frozen penthorum chinense pursh powder with the natural eutectic solvent; and placing in a gradient water bath to complete gradient temperature difference steady-state preparation of penthorum chinense pursh glycoside A. The process is simple, and the penthorum chinense pursh glycoside A prepared at low temperature is high in yield and stable. Compared with a traditional high-temperature preparation method, the preparation time is remarkably shortened, the extraction temperature is lowered, and the stability of penthorum chinense pursh glycoside A is guaranteed. The gradient temperature difference process can improve the extraction efficiency and reduce the energy consumption and mechanical loss, and is beneficial to reducing the cost in actual production. The yield of the penthorum chinense pursh glycoside A is 1.56 times that of a traditional heating extraction method, and the stability of the penthorum chinense pursh glycoside A is improved by 526%.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of food science and engineering, and particularly relates to a method for preparing chases grass glycoside A by combining liquid nitrogen with gradient temperature difference steady state and application thereof. BACKGROUND

[0002] Chases grass is mainly distributed in China, and the main producing area is Gulin County in Sichuan Province. Chases grass is widely used as a herb for treating inflammatory diseases such as hepatitis and enteritis. Studies have shown that chases grass has many health benefits, including anti-inflammatory, antioxidant and anticancer effects. The bioactive compounds such as flavonoids, alkaloids and organic acids in chases grass are closely related to health benefits. Flavonoids are the main bioactive compounds in chases grass, and mainly have antioxidant and anti-inflammatory effects. Among them, chases grass glycoside A is a unique flavonoid in chases grass. There is evidence that chases grass glycoside A shows liver protection, antioxidant, diuresis and anti-tumor effects. Therefore, it is necessary to carry out corresponding research on the preparation method of chases grass glycoside A.

[0003] Solid-liquid extraction is a traditional method for extracting flavonoids from plant raw materials. The most commonly used solvent in traditional extraction is an organic solvent (such as methanol, ethanol and acetone). Although these solvents are widely used, they may pose health risks and environmental challenges, resulting in increased health and pollution remediation costs. In line with the principles of green extraction, natural eutectic solvents developed in 2011 combine biodegradable natural metabolites together, and have great application potential in the food, cosmetics and pharmaceutical industries.

[0004] Traditional high-temperature hydrothermal extraction is a commonly used extraction method, which strengthens the extraction process through high-temperature effect, and the driving force of extraction is mainly controlled by temperature. The flavonoid structure of chases grass glycoside A is easily degraded at high temperature, affecting its extraction rate and biological activity. In order to ensure the stability of chases grass glycoside A during the extraction process, there is an urgent need for a low-temperature steady-state extraction process. It is well known that the fluidity and collision between the solvent and the plant material help to accelerate the dissolution of the target compound. The most easily realized form of fluidity and collision in the extraction system is the circulation flow caused by the temperature gradient. In this case, the circulation flow induced by cold stimulation can be used as the driving force of extraction to accelerate the extraction process at low temperature with less energy. However, traditional high-temperature extraction often only focuses on the diffusion effect of high temperature, ignoring its destructive effect on chases grass glycoside A.

[0005] More specifically:

[0006] (1) Toxicity and environmental risk of traditional organic solvent extraction method: The solid-liquid extraction method (such as solvent extraction) widely used in the prior art highly depends on organic solvents such as methanol, ethanol, acetone. These solvents can effectively dissolve flavonoids, but they are toxic, volatile and easy to remain in the extract, which poses a potential threat to the safety of the final product (health risk). At the same time, their production, use and subsequent waste liquid treatment process can easily cause environmental pollution, increase environmental governance costs and do not meet the requirements of modern green chemistry and sustainable development. The present application clearly proposes to follow the principle of green extraction, avoiding the use of toxic organic solvents and significantly reducing health and environmental risks.

[0007] (2) Degradation and inactivation of Chasesu Glycoside A caused by high-temperature extraction process: The high-temperature hydrothermal extraction method commonly used in the prior art accelerates the mass transfer process by increasing the temperature (usually significantly higher than room temperature). However, Chasesu Glycoside A, as an active ingredient with a specific flavonoid structure, is sensitive to high temperatures. In a high-temperature environment (such as traditional water extraction or organic solvent reflux), Chasesu Glycoside A is prone to thermal degradation, oxidation or isomerization, resulting in a decrease in its extraction rate, destruction of its chemical structure, and loss or weakening of its inherent biological activity (such as liver protection, antioxidant, anti-tumor efficacy). The core of the present application is low-temperature steady-state extraction (30°C), and the introduction of liquid nitrogen ultra-low temperature pretreatment and protective agents (β-cyclodextrin + L-ascorbic acid) isolates oxygen, metal ions and free radicals at the molecular level, fundamentally avoiding degradation caused by high temperature and ensuring the structural integrity and biological activity of Chasesu Glycoside A in the extract.

[0008] (3) Lack of efficient low-temperature mass transfer driving force mechanism: Although the existing low-temperature extraction method (such as room temperature soaking) can avoid high-temperature degradation, it mainly relies on passive diffusion, which has low extraction efficiency and takes a long time, making it difficult to meet the requirements of industrial production in terms of efficiency and cost. Traditional methods have failed to effectively utilize or construct a physical driving force mechanism that can significantly enhance the mass transfer (flow and collision) between the solvent and the plant material at low temperatures. Although it is recognized that temperature-induced circulating flow can accelerate extraction, existing technologies (especially low-temperature technologies) generally lack effective utilization of this mechanism. The present application innovatively designs a gradient temperature-induced circulating flow as the core driving force of low-temperature extraction. By precisely controlling the temperature gradient (such as using liquid nitrogen pre-cooling followed by warming or external circulation temperature control), a strong natural convection or microcirculation is actively induced in the system at low temperature (30°C), greatly enhancing the solvent penetration, solubility and diffusion rate, thereby significantly improving the extraction efficiency and speed while ensuring low-temperature stability, solving the bottleneck problem of low-temperature extraction efficiency.

[0009] The application chases the grass through liquid nitrogen ultra-low temperature treatment, and beta-cyclodextrin forms a "molecular capsule" through molecular inclusion to isolate the oxygen molecules and metal ions from chases the grass, and L-ascorbic acid is used as an antioxidant to remove free radicals and prevent oxidative degradation. The beta-cyclodextrin inclusion structure can be destroyed by heating to 30 DEG C, and the protective agent can be removed. The low-temperature steady-state extraction mechanism of the gradient temperature difference combined with the preparation method of chases the grass can create a more effective and novel preparation method in the field. SUMMARY

[0010] The application aims to overcome the deficiencies in the prior art and provide a method for preparing chases the grass A by liquid nitrogen combined with gradient temperature difference steady state and application.

[0011] The application solves the technical problems by adopting the technical solutions as follows:

[0012] A method for preparing chases the grass A by liquid nitrogen combined with gradient temperature difference steady state, comprising the following steps:

[0013] (1) treating fresh chases the grass with liquid nitrogen, adding a compounded protective agent, ball milling and crushing the chases the grass to obtain frozen chases the grass powder;

[0014] (2) synthesizing choline chloride-alpha-L-rhamnose natural eutectic solvent;

[0015] (3) mixing the frozen chases the grass powder with the natural eutectic solvent;

[0016] (4) placing the mixture of step (3) in a gradient water bath to complete the preparation of chases the grass A by gradient temperature difference steady state.

[0017] Further, in step (1), the liquid ratio mL: g of liquid nitrogen to fresh chases the grass is 10-20:1.

[0018] Further, in step (1), the compounded protective agent is a mixture of beta-cyclodextrin and L-ascorbic acid with a mass ratio of 3:1, and the addition amount of the compounded protective agent is 0.5-1.5% of the mass of the chases the grass.

[0019] Further, in step (1), the rotation speed during ball milling is 150-200 r / min, the ball-to-material ratio g:g is 25-30:1, and the ball milling time is 6-8 min.

[0020] Further, the natural eutectic solvent is prepared from choline chloride and alpha-L-rhamnose, and the mass ratio of choline chloride to alpha-L-rhamnose is 0.5-2:1.

[0021] Further, after mixing choline chloride and alpha-L-rhamnose, the choline chloride-alpha-L-rhamnose natural eutectic solvent is prepared by a magnetic stirrer at 80-85 DEG C with a stirring rate of 200-400 rpm for 30 min.

[0022] Further, in step ⑶, the liquid-to-powder ratio mL: g of the natural eutectic solvent to the frozen Desmodium caudatum is 20-25:1.

[0023] Further, in step ⑷, the gradient temperature difference steady-state preparation of Desmodium caudatum glycoside A is as follows: 1) 30℃ water bath for 3-5 min, 5000 rpm centrifugation to remove the protective agent; 2) 15℃ cold water bath for 3-5 min; 3) 4℃ water bath for 3-5 min.

[0024] Further, in step ⑷, Desmodium caudatum glycoside A has the function of regulating intestinal flora homeostasis, and the yield of Desmodium caudatum glycoside A is 1.56 times that of the traditional heating extraction method, and the stability of Desmodium caudatum glycoside A is improved by 526%.

[0025] The method as described above is applied in the preparation of Desmodium caudatum glycoside A.

[0026] The advantages and positive effects obtained by the present application are as follows:

[0027] 1. The method of the present application is simple, and the yield of Desmodium caudatum glycoside A prepared at low temperature is high and stable. Compared with the traditional high-temperature preparation method, the preparation time is significantly reduced and the extraction temperature is lowered, which ensures the stability of Desmodium caudatum glycoside A. The gradient temperature difference process can improve the extraction efficiency and reduce energy consumption and mechanical loss, which is beneficial to reduce the cost in actual production. The yield of Desmodium caudatum glycoside A obtained by the method of the present application is 1.56 times that of the traditional heating extraction method, and the stability of Desmodium caudatum glycoside A is improved by 526%.

[0028] 2. The liquid nitrogen combined with the gradient temperature difference steady-state preparation method of the present application can effectively solve the problems of energy consumption and heat production in traditional high-temperature extraction, and has important significance for building a green, low-carbon and sustainable modern society.

[0029] 3. The choline chloride-alpha-L-rhamnose natural eutectic solvent designed in the method of the present application can be recycled. Liquid nitrogen is combined with the gradient temperature difference steady-state preparation of Desmodium caudatum glycoside A. The Desmodium caudatum is treated by liquid nitrogen ultra-low temperature, the beta-cyclodextrin forms a "molecular capsule" through molecular inclusion, isolates Desmodium caudatum glycoside A from oxygen molecules and metal ions, L-ascorbic acid acts as an antioxidant to scavenge free radicals, and cooperates to prevent oxidative degradation. The beta-cyclodextrin inclusion structure can be destroyed by heating to 30℃, and the protective agent can be removed. The circulatory flow induced by gradient cold stimulation can be used as an extraction driving force to accelerate the extraction process at low temperature with less energy, thereby reducing costs. At the same time, the problem of unstable degradation of Desmodium caudatum glycoside A due to high-temperature heat production is reduced, which is suitable for industrial application.

[0030] 4、The method of the application uses choline chloride as a hydrogen bond acceptor and alpha-L-rhamnose as a hydrogen bond donor, with a mass ratio of 0.5-2:1, to synthesize an extraction solvent, which is then mixed with frozen Desmodium caudatum powder. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 A yield comparison chart of Desmodium caudatum glycoside A prepared by different extraction methods in the application;

[0032] Figure 2 A degradation rate comparison chart of Desmodium caudatum glycoside A prepared by different extraction methods in the application;

[0033] Figure 3 A model chart of Desmodium caudatum glycoside A and choline chloride-alpha-L-rhamnose deep eutectic solvent within 50 ps in the application;

[0034] Figure 4 A comparison chart of average hydrogen bond number of different extraction methods within 5-50 ps in the application;

[0035] Figure 5 An effect chart of Desmodium caudatum glycoside A prepared by different extraction methods in the application on intestinal microbial instability index of mice;

[0036] Figure 6 An effect chart of Desmodium caudatum glycoside A prepared by different extraction methods in the application on intestinal microbial health index of mice;

[0037] Figure 7 An effect chart of Desmodium caudatum glycoside A prepared by different extraction methods in the application on intestinal microbial alpha-diversity (Sobs index) of intestinal microbes of mice. DETAILED DESCRIPTION

[0038] The application will be further described below in conjunction with examples, which are descriptive rather than limiting, and cannot be used to limit the protection scope of the application.

[0039] The various experimental operations involved in the specific examples are all conventional techniques in the art, and the parts not specifically annotated in this document can be implemented by referring to various commonly used toolbooks, scientific and technical literature or related instructions, manuals, etc. before the application date of the application.

[0040] Choline chloride (analytical pure), α-L-rhamnose (analytical pure), ethanol (analytical pure), formic acid (HPLC grade), acetonitrile (HPLC grade) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd.; The standard sample of Chamaesarachin A was purchased from Shanghai Maikelin Biochemical Technology Co., Ltd. GL323-1SCN thousandth gram precision electronic balance, Germany Sartorius company; Centrifuge 5804 table type high speed refrigerated centrifuge, Germany Eppendorf company; UV-8000 ultraviolet visible spectrophotometer, Shanghai Yuanzhi instrument Co., Ltd.; LC-2050 type high performance liquid chromatograph, Japan Shimadzu company; UPLC-Triple-TOF / MS liquid chromatograph-mass spectrometer system, American AB SCIEX company; ACQUITY UPLC HSS SB-C18 column (100mm×2.1mm×1.7μm), American Waters company.

[0041] A method for preparing Chamaesarachin A by liquid nitrogen combined gradient temperature difference steady state, comprising the following steps:

[0042] (1) treating fresh Chamaesarachin with liquid nitrogen, adding a compound protective agent, ball milling and crushing Chamaesarachin to obtain frozen Chamaesarachin powder;

[0043] (2) synthesizing choline chloride-α-L-rhamnose natural deep eutectic solvent;

[0044] (3) mixing the frozen Chamaesarachin powder with the natural deep eutectic solvent;

[0045] (4) placing the mixture of step (3) in a gradient water bath to complete the preparation of Chamaesarachin A by gradient temperature difference steady state.

[0046] Preferably, in step (1), the liquid ratio mL: g of liquid nitrogen to fresh Chamaesarachin is 10-20:1.

[0047] Preferably, in step (1), the compound protective agent is a mixture of β-cyclodextrin and L-ascorbic acid with a mass ratio of 3:1, and the addition amount of the compound protective agent is 0.5-1.5% of the mass of Chamaesarachin.

[0048] Preferably, in step (1), the rotation speed during ball milling is 150-200 r / min, the ball-to-material ratio g:g is 25-30:1, and the ball milling time is 6-8 min.

[0049] Preferably, the natural deep eutectic solvent is prepared by choline chloride and α-L-rhamnose, and the mass ratio of choline chloride to α-L-rhamnose is 0.5-2:1.

[0050] Preferably, the choline chloride and alpha-L-rhamnose are mixed and heated by a magnetic stirrer at 80-85°C at a speed of 200-400 rpm for 30 min to prepare the choline chloride-alpha-L-rhamnose natural eutectic solvent.

[0051] Preferably, in step ⑶, the liquid-to-solid ratio of the natural eutectic solvent to the frozen Bunge vinegar grass powder is 20-25:1.

[0052] Preferably, in step ⑷, the gradient temperature difference steady-state preparation of Bunge vinegar grass saponin A is performed according to the following steps: 1) 30°C water bath for 3-5 min, 5000 rpm centrifugation to remove the protective agent; 2) 15°C cold water bath for 3-5 min; and 3) 4°C water bath for 3-5 min.

[0053] Preferably, in step ⑷, the Bunge vinegar grass saponin A has the function of regulating intestinal flora homeostasis, and the yield of Bunge vinegar grass saponin A is 1.56 times that of the traditional heating extraction method, and the stability of Bunge vinegar grass saponin A is improved by 526%.

[0054] The method described above is applied to the preparation of Bunge vinegar grass saponin A.

[0055] Specifically, the related preparation and detection are as follows:

[0056] Example 1

[0057] A method for preparing Bunge vinegar grass saponin A by liquid nitrogen combined with gradient temperature difference steady-state, the specific steps are as follows:

[0058] (1) Freeze the fresh Bunge vinegar grass in liquid nitrogen, and the liquid-to-solid ratio of liquid nitrogen to fresh Bunge vinegar grass is 10:1;

[0059] (2) The protective agent is a mixture of β-cyclodextrin and L-ascorbic acid with a mass ratio of 3:1;

[0060] (3) Add the compounded protective agent to the Bunge vinegar grass treated by liquid nitrogen, and the addition amount is 0.5% of the mass of the Bunge vinegar grass, and the ball milling speed is 200 r / min, the ball-to-material ratio g:g is 25:1, and the ball milling time is 8 min, and the Bunge vinegar grass freeze-dried powder is obtained by ball milling;

[0061] (4) Choline chloride is used as the hydrogen bond acceptor, and alpha-L-rhamnose is used as the hydrogen bond donor. After mixing choline chloride and alpha-L-rhamnose according to a mass ratio of 0.5:1, the mixture is heated by a magnetic stirrer at 80°C at a speed of 200 rpm for 30 min to form a choline chloride-alpha-L-rhamnose natural eutectic solvent. The mass ratio of the components of the natural eutectic solvent is 0.5:1 for choline chloride to alpha-L-rhamnose.

[0062] (5) the natural deep eutectic solvent is mixed with the dried and powdered Buchomeria fortunei in a liquid-to-material ratio of 20:1 mL:g uniformly;

[0063] (6) the mixture obtained in step (5) is treated by gradient temperature difference, and Buchomeria fortunei glycoside A is prepared by gradient temperature difference steady state, in the order of: 1) 30℃ warm water bath for 5 min, centrifuged at 5000 rpm to remove the protective agent; 2) 15℃ cool water bath for 3 min; 3) 4℃ water bath for 3 min.

[0064] Example 2

[0065] A method for preparing Buchomeria fortunei glycoside A by liquid nitrogen combined with gradient temperature difference steady state, the specific steps are as follows:

[0066] (1) freeze fresh Buchomeria fortunei in liquid nitrogen, the liquid-to-material ratio of liquid nitrogen to fresh Buchomeria fortunei is 15 mL / g;

[0067] (2) the protective agent is compounded by β-cyclodextrin and L-ascorbic acid in a mass ratio of 3:1, and the compounded protective agent is a mixture of β-cyclodextrin and L-ascorbic acid;

[0068] (3) add the compounded protective agent to the Buchomeria fortunei treated by liquid nitrogen, the addition amount is 1% of the mass of Buchomeria fortunei, the ball milling speed is 175 r / min, the ball-to-material ratio g:g is 27:1, and the ball milling time is 7 min, to obtain Buchomeria fortunei freeze-dried powder;

[0069] (4) take choline chloride as the hydrogen bond acceptor and α-L-rhamnose as the hydrogen bond donor, mix choline chloride and α-L-rhamnose in a mass ratio of 1:1, heat and stir at 82℃ by a magnetic stirrer at a speed of 300 rpm for 30 min to form a choline chloride-α-L-rhamnose natural deep eutectic solvent, and the mass ratio of the components of the natural deep eutectic solvent is choline chloride:α-L-rhamnose = 1:1;

[0070] (5) the natural deep eutectic solvent is mixed with the dried and powdered Buchomeria fortunei in a liquid-to-material ratio of 23:1 mL:g uniformly;

[0071] (6) the mixture obtained in step (5) is treated by gradient temperature difference, and Buchomeria fortunei glycoside A is prepared by gradient temperature difference steady state, in the order of: 1) 30℃ warm water bath for 4 min, centrifuged at 5000 rpm to remove the protective agent; 2) 15℃ cool water bath for 4 min; 3) 4℃ water bath for 4 min.

[0072] Example 3

[0073] A method for preparing Buchomeria fortunei glycoside A by liquid nitrogen combined with gradient temperature difference steady state, the specific steps are as follows:

[0074] (1) freeze fresh Buchomeria fortunei in liquid nitrogen, the liquid-to-material ratio of liquid nitrogen to fresh Buchomeria fortunei is 20 mL / g;

[0075] (2) The protective agent is compounded with β-cyclodextrin and L-ascorbic acid in a mass ratio of 3:1;

[0076] (3) The compound protective agent is added to the liquid nitrogen-treated Desmodium caudatum, and the added amount is 1.5% of the mass of Desmodium caudatum. The ball milling speed is 150 r / min, the ball-to-material ratio g:g is 30:1, and the ball milling time is 6 min. The ball milling is crushed to obtain Desmodium caudatum freeze-dried powder;

[0077] (4) Choline chloride is used as a hydrogen bond acceptor, and α-L-rhamnose is used as a hydrogen bond donor. After mixing choline chloride and α-L-rhamnose in a mass ratio of 2:1, heating and stirring are carried out at 85°C for 30 min by a magnetic stirrer at a speed of 400 rpm to form a choline chloride-α-L-rhamnose natural deep eutectic solvent. The mass ratio of each component of the natural deep eutectic solvent is choline chloride:α-L-rhamnose=2:1;

[0078] (5) The natural deep eutectic solvent is mixed with the Desmodium caudatum freeze-dried powder in a liquid-to-material ratio of 25:1;

[0079] (6) The mixture obtained in step (4) is treated by gradient temperature difference to prepare Desmodium caudatum glycoside A. The steps are as follows: 1) 30°C water bath for 3 min, 5000 rpm centrifugation to remove the protective agent; 2) 15°C cold water bath for 5 min; 3) 4°C water bath for 5 min.

[0080] Comparative Example 1

[0081] The other conditions are the same as in Example 1, except that only β-cyclodextrin is added.

[0082] Comparative Example 2

[0083] The other conditions are the same as in Example 1, except that only L-ascorbic acid is added.

[0084] Comparative Example 3

[0085] The other conditions are the same as in Example 1, except that no compound protective agent is added.

[0086] Comparative Example 4

[0087] The other conditions are the same as in Example 1, except that the natural deep eutectic solvent and Desmodium caudatum mixture are treated by a hot water bath, the water bath temperature is 85°C, the extraction time is 15 min, and no gradient temperature difference extraction is performed.

[0088] Comparative Example 5

[0089] Other conditions are the same as example 1, the difference is that the grinding powder is directly dried in the 80℃ oven without liquid nitrogen treatment.

[0090] The product obtained above or the product of the intermediate step is detected on the quality index, specifically as follows:

[0091] The extract of chases the grass glycoside A obtained in step (6) is analyzed by UPLC-Triple-TOF / MS, molecular dynamics simulation system, and mouse intestinal microbial model;

[0092] UPLC-Triple-TOF / MS detection:

[0093] Mobile phase composition: 0.1% formic acid solution (A), 0.1% formic acid acetonitrile solution (B); flow rate: 0.5 mL / min; elution gradient: 0-10 min mobile phase B 0%~12%, 10-19 min mobile phase B 12%~45%, 19-25 min mobile phase B 45%~95%, 25-32 min mobile phase B 95%~5%; column: Waters ACQUITY UPLC HSS SB-C18 column (100 mm x 4.6 mm x 1.8 μm); detection wavelength: 370 nm; column temperature: 40℃; injection volume: 5 μL. Cationic scanning mode is used: scanning range: m / z 100-1500; atomizing gas (GS1): 35 psi; atomizing gas (GS2): 50 psi; gas curtain gas (CUR): 35 psi; ion source temperature (TEM): 300℃; ion source voltage (IS): 4500V; collision energy: 30 eV.

[0094] Molecular dynamics test:

[0095] The three-dimensional structures of chases the grass glycoside A, choline chloride, α-L-rhamnose, ethanol and water molecules are generated in Chem3D software. The amorphous box model is built by using the Amorphous Cell module of Materials studio 2020 software. Forcite-Geometry Optimization is selected, the accuracy is set to Fine, and the force field is COMPASS II to optimize the geometry of the solvent model. Forcite-Dynamics is selected, the NVT system is set, the van der Waals force cutoff radius The cutoff radius of hydrogen bond Molecular dynamics simulation analysis within 50 ps is completed.

[0096] Mouse intestinal microbial model:

[0097] Experimental 20 male C57BL / 6J mice (7-8 weeks old) provided by Sanyou Biotechnology (Suzhou) Co., Ltd. were randomly divided into three groups: Example 1, Comparative Example 1, Comparative Example 2 and Comparative Example 3. All animal experiments were approved by the Lishui University Animal Ethics Committee (Approval No. : LSU20240014) and were carried out in strict accordance with the Lishui University Experimental Animal Guidelines. After 7 days of adaptive feeding, the mice were given 0.3 mL of Example or Comparative Example Desmoside A solution by gavage daily. After 14 days of continuous gavage, the mouse fecal samples were collected and entrusted to Shanghai Meiji Biological Co., Ltd. for 16S rRNA sequencing analysis.

[0098] The results are as follows:

[0099] (1) Preparation of Desmoside A by different extraction methods

[0100] As shown in Figure 1 , the extraction method of the present application can greatly improve the yield of Desmoside A. The yield of Desmoside A in Example 1 was increased by 39%, 67%, 93%, 106% and 133% compared with Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5, respectively. At the same time, by comparing Example 1, Comparative Example 3 and Comparative Example 4, it can be seen that the two steps of compounding the protective agent in step (3) and preparing Desmoside A by gradient temperature difference in step (6) in the method of the present application have a synergistic effect, which can synergistically improve the yield of Desmoside A prepared. By comparing Example 1, Comparative Example 4 and Comparative Example 5, it can be seen that the two steps of treating Desmoside A with liquid nitrogen in step (1) and preparing Desmoside A by gradient temperature difference in step (6) in the method of the present application have a synergistic effect, which can synergistically improve the yield of Desmoside A prepared.

[0101] (2) Degradation rate of Desmoside A prepared by different extraction methods

[0102] As shown in Figure 2 , the extraction method of the present application can greatly reduce the degradation rate of Desmoside A during extraction and improve the stability of Desmoside A. The degradation rate of Desmoside A in Example 1 was 203%, 247%, 311%, 526% and 387% lower than that of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5, respectively. At the same time, by comparing Example 1, Comparative Example 3 and Comparative Example 4, it can be seen that the two steps of compounding the protective agent in step (3) and preparing Desmoside A by gradient temperature difference in step (6) in the method of the present application have a synergistic effect, which can synergistically reduce the degradation rate of Desmoside A prepared during extraction. By comparing Example 1, Comparative Example 4 and Comparative Example 5, it can be seen that the two steps of treating Desmoside A with liquid nitrogen in step (1) and preparing Desmoside A by gradient temperature difference in step (6) in the method of the present application have a synergistic effect, which can synergistically reduce the degradation rate of Desmoside A prepared during extraction.

[0103] (3) 50 ps chase Huangcao glycoside A and choline chloride-α-L-rhamnose eutectic solvent model

[0104] As shown in Figure 3 , the amorphous complex system model of chase Huangcao glycoside A and choline chloride-α-L-rhamnose eutectic solvent was constructed by molecular dynamics simulation in Materials Studio software. After optimization by simulation, it can be seen that the extraction solvent and chase Huangcao glycoside A molecules form a dense hydrogen bond interaction network. This network significantly enhances the intermolecular forces between solute and solvent through the directional recognition of solvent anions, sugar hydroxyl groups and phenolic hydroxyl groups of glycosides, thereby effectively improving the solubility of chase Huangcao glycoside A in eutectic solvent and stabilizing its molecular conformation through hydrogen bond coordination.

[0105] (4) 5-50 ps average number of hydrogen bonds of different extraction methods

[0106] As shown in Figure 4 , the average number of hydrogen bonds formed in Example 1 (180) is 40%, 48%, 61%, 76% and 120% more than that of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5, respectively.

[0107] (5) Effect of chase Huangcao glycoside A prepared by different extraction methods on the intestinal microbial instability index of mice

[0108] As shown in Figure 5 , the chase Huangcao glycoside A prepared by the present application can prevent intestinal microbial instability and reduce the intestinal microbial instability index. The intestinal microbial instability index of mice treated with chase Huangcao glycoside A of Example 1 is-0.96, which is 201%, 225%, 246%, 298% and 360% lower than that of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5, respectively. At the same time, by comparing Example 1, Comparative Example 3 and Comparative Example 4, it can be seen that the complexing protective agent in step (3) and the gradient temperature difference steady-state preparation of chase Huangcao glycoside A in step (6) in the method of the present application have a synergistic effect, which can synergistically improve the related performance of the prepared chase Huangcao glycoside A. By comparing Example 1, Comparative Example 4 and Comparative Example 5, it can be seen that the liquid nitrogen treatment of chase Huangcao in step (1) and the gradient temperature difference steady-state preparation of chase Huangcao glycoside A in step (6) in the method of the present application have a synergistic effect, which can synergistically improve the related performance of the prepared chase Huangcao glycoside A.

[0109] (6) Effect of chase Huangcao glycoside A prepared by different extraction methods on the intestinal microbial health index of mice

[0110] As shown in Figure 6As shown, the chrysanthemum glycoside A prepared by the present invention can regulate intestinal microorganisms and maintain the stability of intestinal flora. The intestinal microbial health index of mice treated with the chrysanthemum glycoside A of Example 1 is 1.28, which is 39%, 44%, 51%, 78% and 113% higher than that of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5, respectively. At the same time, by comparing Example 1, Comparative Example 3 and Comparative Example 4, it can be seen that the compound protective agent in step (3) of the method of the present invention and the gradient temperature difference steady-state preparation of chrysanthemum glycoside A in step (6) have a synergistic effect, which can synergistically improve the relevant properties of the prepared chrysanthemum glycoside A. By comparing Example 1, Comparative Example 4 and Comparative Example 5, it can be seen that the liquid nitrogen treatment of chrysanthemum glycoside in step (1) of the method of the present invention and the gradient temperature difference steady-state preparation of chrysanthemum glycoside A in step (6) have a synergistic effect, which can synergistically improve the relevant properties of the prepared chrysanthemum glycoside A.

[0111] (7) Effects of different extraction methods of guanyloside A on the α-diversity of mouse intestinal microorganisms (Sobs index)

[0112] like Figure 7 As shown, the chrysanthemum glycoside A prepared by the present invention can improve the diversity of intestinal microorganisms and maintain the number and type of intestinal flora. The α-diversity (Sobs index) of the intestinal microorganisms of mice treated with the chrysanthemum glycoside A of Example 1 was 756, which was 26%, 31%, 43%, 75% and 104% higher than those of Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4 and Comparative Example 5, respectively. At the same time, by comparing Example 1, Comparative Example 4 and Comparative Example 5, it can be seen that there is a synergistic effect between the treatment of chrysanthemum glycoside with liquid nitrogen in step (1) of the method of the present invention and the steady-state preparation of chrysanthemum glycoside A by gradient temperature difference in step (6), which can synergistically improve the relevant properties of the prepared chrysanthemum glycoside A.

[0113] The present invention utilizes a method for the steady-state preparation of chrysanthemum glycoside A using liquid nitrogen combined with a gradient temperature difference. This method is simple, and the low-temperature preparation yields high and stable chrysanthemum glycoside A. Compared to traditional high-temperature preparation methods, this method significantly reduces preparation time and extraction temperature, ensuring the stability of chrysanthemum glycoside A. The gradient temperature difference process improves extraction efficiency and reduces energy consumption and mechanical loss, thus lowering actual production costs.

[0114] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing huangcaoside A in a steady state by combining liquid nitrogen with a gradient temperature difference, characterized in that: The steps include: (1) Treat fresh chrysanthemum with liquid nitrogen, add compound protective agent, and grind the chrysanthemum with ball mill to obtain frozen chrysanthemum powder; ⑵Synthesis of choline chloride-α-L-rhamnose natural deep eutectic solvent; (3) Mixing the frozen yellow grass powder with a natural deep eutectic solvent; (4) Place the mixture of step (3) in a gradient water bath to complete the gradient temperature difference steady-state preparation of chaetocene glycoside A.

2. The method according to claim 1, wherein: In step (1), the liquid nitrogen and fresh yellow grass liquid ratio mL: g is 10-20:

1.

3. The method according to claim 1, wherein: In step (1), the compound protective agent is a mixture of β-cyclodextrin and L-ascorbic acid in a mass ratio of 3:1, and the added amount of the compound protective agent is 0.5-1.5% of the mass of the yellow grass.

4. The method according to claim 1, wherein: In the step (1), the rotation speed during ball milling is 150-200 r / min, the ball-to-material ratio g:g is 25-30:1, and the ball milling time is 6-8 min.

5. The method according to claim 1, wherein: The natural deep eutectic solvent is prepared by choline chloride and α-L-rhamnose, and the mass ratio of choline chloride to α-L-rhamnose is 0.5-2:

1.

6. The method according to claim 5, characterized in that: Choline chloride and α-L-rhamnose are mixed, and the mixture is stirred and heated at 80-85° C. at a rate of 200-400 rpm for 30 minutes using a magnetic stirrer to prepare a choline chloride-α-L-rhamnose natural deep eutectic solvent.

7. The method according to claim 1, wherein: In step (3), the liquid-to-material ratio of the natural deep eutectic solvent to the frozen yellow grass powder is 20-25:1 in mL:g.

8. The method according to claim 1, wherein: In step (4), the gradient temperature difference steady-state preparation of chaete glycoside A is as follows: 1) 30°C warm water bath for 3-5 min, 5000 rpm centrifugation to remove the protective agent; 2) 15°C cold water bath for 3-5 min; 3) 4°C water bath for 3-5 min.

9. The method according to any one of claims 1 to 8, characterized in that: In step (4), guanyloside A has the function of regulating the homeostasis of intestinal flora. The yield of guanyloside A is 1.56 times that of the traditional heating extraction method, and the stability of guanyloside A is improved by 526%.

10. Use of the method according to any one of claims 1 to 8 in the preparation of chrysanthemumin A.

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